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FOOD & DIETARY SUPPLEMENTSDirectory of Services

800-675-8375 • covance.com/nutri 1

INTRODUCTION

Advancements in the way food and dietary supplements are developed, packaged and prepared require flexibility, innovation and a broad scientific understanding of those implications on analytical data. Covance has over 80 years of experience in food analysis and has built a reputation for scientific and regulatory expertise.

Understanding the key drivers in analytical testing—placing the customer first, creating processes to drive efficiency and providing high-quality data—has positioned Covance as a leader in outsourced food and dietary supplement testing services. This directory provides a description of our analytical chemistry assays, quality processes, service descriptions and ordering information. We invite you to visit our facilities, speak with our staff and experience a level of personal commitment to helping you reach your product development goals while remaining focused on regulatory compliance and food safety.

TABLE OF CONTENTS

Quick Reference .............................................................................................................................................................................2–19

Assays ..............................................................................................................................................................................................20–81

Ordering Information ................................................................................................................................................................82–84

Experience New Potential with SampleKinect® ............................................................................................................. 83

Sampling ....................................................................................................................................................................................... 84

Importing Samples to Covance ............................................................................................................................................ 84

ORDERING INFORMATION

Sample Submittal/Shipping

Please order via your SampleKinect account at samplekinect.covance.com or our Analysis Request Form available from your Customer Service Associate.

http://covance.com/nutri

samplekinect.covance.com

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3800-675-8375 • covance.com/nutri

NL-Proximate (moisture, ash, protein)

Calories (by calculation)

Calories from fat (by calculation) *Requires fat, moisture, protein and ash testing

Total fat [sum of fatty acids (saturated, monounsaturated, polyunsaturated, trans)]

Cholesterol

Total carbohydrates (by calculation) *Requires fat, moisture, protein and ash testing

Total dietary fiber

Sugars HPLC GC Either HPLC or GC will be used, depending on the sample matrix.

Vitamin A

beta Carotene If there is a source of beta carotene, the assay for beta carotene should be used instead of (or in addition to) vitamin A to obtain total vitamin A activity for nutrition labels.

Vitamin C

Calcium, iron, sodium (ICP)

Mandatory Nutrient PackageThis package includes all nutrients, with sugars assayed by GC, minerals by ICP and total fat as the sum of the fatty acids calculated as triglycerides.

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NUTRITION LABELING MANDATORY NUTRIENT LIST

Regulatory initiatives such as the Nutrition Labeling and Education Act have had a profound impact on the food industry. The adoption of standardized content and format guidelines for nutrition labeling have provided consumers with the tools to plan a balanced diet, as well as given producers data to distance their product from the competition.


4 Pricing available from a Covance representative. For updates to assays, refer to our online catalog at covance.com/nutri.

AstaxanthinBiotinCarotenoids Option 1: alpha Carotene, beta Carotene, Lycopene Option 2: Cryptoxanthin, Lutein, Zeaxanthin Option 3: Lutein onlyCholine (chemical)Choline (enzymatic)Cyanocobalamin (vitamin B12)Folate (vitamin B9)InositolL-Ascorbyl-2-PhosphateNiacin (vitamin B3)Pantothenic acid (vitamin B5)Pyridoxine (vitamin B6)

Riboflavin (vitamin B2)Thiamin (vitamin B1)Tocopherols (alpha, beta, gamma and delta)Tocotrienols (alpha, beta, gamma and delta)Vitamin A modified USP for dietary supplements (HPLC)Vitamin A, retinolVitamin B profile (HPLC)Vitamin B profile (HPLC-MS/MS)Vitamin CVitamin DVitamin E (alpha tocopherol) Vitamin E (in feeds and forages)Vitamin K1

VITAMIN ANALYSIS

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CholesterolConjugated linoleic acid (CLA)Fatty acid profile (C4 - C24) (including Omega-3 and Omega-6 isomers, and trans fatty acids)Fatty acid profile (C4 - C24) (including Omega-3 and Omega-6 isomers, not including fatty acids)Free fatty acids (total by titration)Free fatty acids (by GC)

GlycerolHexanalIodine valuep-Anisidine Peroxide valueSterolsTBA value

Acid detergent fiberAspartame, Acesulfame K, DKP and saccharine beta GlucanCrude fiberGalactooligosaccharides (GOS) content in Infant FormulaGalactooligosaccharides (GOS) content in Raw MaterialInsoluble and soluble fiber (Lee Method) Insoluble, soluble and total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid

chromatography (McCleary)Inulin (fructan), HPLCInulin (fructan), spectrographicLactuloseLigninNeutral detergent fiber (enzymatic)PolydextroseResistant starchStarch

Sucralose (finished products)Sucralose (packets, pure material) (USP Method)Sugar alcoholsSugar profile after acid hydrolysisSugar profile by GCSugar profile, low levelsSugar profile by HPLCSugar profile by ICTotal dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid

chromatography (McCleary) Total dietary fiber containing supplemented resistant maltodextrin (Fibersol)Total dietary fiber (Lee or Prosky)

LIPIDS

CARBOHYDRATES



Total amino acid profile (AOAC) Alanine, arginine, aspartic acid (including asparagine),

cystine, glutamic acid (including glutamine), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine

Total amino acid profile (HPLC) Alanine, arginine, aspartic acid (including asparagine),

cystine (including cysteine), glutamic acid (including glutamine), glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, tryptophan

Free amino acid profile (AOAC) Alanine, arginine, aspartic acid (including asparagine),

cystine, glutamic acid (including glutamine), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine

Free amino acid profile (HPLC) L-aspartic acid, L-glutamic acid, L-serine, L-histidine,

glycine, L-threonine, L-arginine, L-alanine, L-tyrosine, L-cystine, L-valine, L-methionine, L-phenylalanine, L-isoleucine, L-leucine, L-lysine, proline, L-asparagine, L-glutamine, L-citrulline, L-tryptophan, hydroxyproline

5-Hydroxytrytophan (5-HTP)beta AlanineCarnitineCysteineCystine and Methionine (AOAC) GABA (gamma aminobutyric acid)Monosodium glutamate (as glutamic acid) N-Acetyl-L-CysteineOrnithineTaurine (total and free)TheanineTryptophan (AOAC)Tryptophan (HPLC) (total and free)

AMINO ACIDS

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Organic Acid Profiles Option 1: Benzoic acid (sodium benzoate), sorbic acid (potassium sorbate)

Option 2: Citric acid, malic acid, lactic acid, acetic acid, oxalic acid, pyruvic, quinic acid Option 3: Tartaric acid, formic acid, succinic acid, fumaric acid Option 4: Propionic acid

ORGANIC ACIDS



Acid insoluble ashAsh Available carbohydrates (direct determination)Bomb calorimetryDensityFat extractions Acid hydrolysis

Methanol/chloroform Roese-Gottlieb Soxhlet

Fat (sum of fatty acids)Loss on drying (LOD)

Moisture (Karl Fischer)Moisture (vacuum oven)pHProtein (Dumas)Protein (Kjeldahl)Proximate package (moisture, ash, protein and fat) Calories (by calculation)

Total carbohydrates (by calculation)Residue on ignition (ROI)Titratable acidityWater activity

Alcohol (added) Alcohol (residual)BHA, BHT and TBHQ (antioxidants)Caffeine, theobromine and theophyllineCapsacin/capsacinoid (Scoville heat)DissolutionEthanol and MethanolNucleosidesNucleotides

OsmolalityParabens (methyl, ethyl, propyl, butyl, isopropyl, isobutyl)PolysorbatesResidual solventsSoy isoflavones (genistein, daidzein, glycitein)Species Identification by ELISA (beef, poultry or pork)Sulfites (Monier-Williams) TBHQViscosity (Brookfield or Bostwick)

Allergens Almond

Brazil Nut Casein Cashew Kernel Celery Coconut Crustaceae Shellfish Egg Gluten Hazelnut Lupine Macadamia Nut Mustard Peanut Pistachio Sesame Soy Allergen Total Milk Walnut

Other Contaminants 3-MCPD (3-Chloro-1,2-Propanediol)

4-MEI Acrylamide by LC-MS/MS Aflatoxins by HPLC (B1, B2, G1, G2) Aflatoxins (B1, B2, G1, G2), Ochratoxin A and Zearalenone by HPLC

Aflatoxins M1 by HPLC Arsenic Speciation Bisphenol A (BPA) Cyanuric acid by LC-MS/MS Heavy metals by ICP (arsenic, cadmium, lead, mercury) Heavy metals by ICP-MS (arsenic, cadmium, lead, mercury) Melamine by LC-MS/MS Melamine and analogs by UHPLC-MS/MS Mycotoxins in infant formula (Aflatoxin B1, B2, G1, G2, M1, M2, deoxynivalenol, Ochratoxin A, Zearalenone,

Fumonisin B1, B2, HT-2, T-2 toxin)Polycyclic aromatic hydrocarbons (PAH) Residual solvents

PROXIMATE

OTHER ASSAYS

NATURAL CONTAMINANTS

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ICP Aluminum Barium Beryllium Boron Cadmium Calcium Chromium Cobalt Copper Iron Magnesium Manganese Molybdenum Nickel Phosphorus Potassium Sodium Strontium Vanadium Zinc

ICP-MS Antimony Arsenic Barium Beryllium Bismuth Cadmium Caesium Calcium Chromium Cobalt Copper Gallium Germanium Iodine Iron Lanthanum Lead Lithium Magnesium Manganese

Mercury Molybdenum Nickel Palladium Platinum Rubidium Ruthenium Selenium Silver Strontium Sulfate* Sulfur Thallium Tin Titanium Tungsten Vanadium Zinc

INDIVIDUAL ELEMENTS

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INORGANIC ANALYSISThe method used for the analysis of various elements in food and dietary supplement samples is dependent on both the concentration and the sample matrix. For most elements the precision using any of these techniques is less than 5% RSD.

Inductively Coupled Plasma Spectroscopy (ICP) is used for macro-levels inorganic constituents in all matrices. Usually 10g of sample is either dry or wet ashed, or samples are analyzed directly. The limit of quantitation is based on the individual analyte and matrix, but generally yields detection in the PPM range. A common scan is for calcium, iron and sodium used for nutritional labeling.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is the method of choice when analyzing trace level analytes. It is applicable to all matrices. Usually 0.5 g of sample is wet ashed in a closed-vessel microwave digestion, although other ashing techniques are used. Limits of quantitation are lower than ICP and are dependent on the analyte and matrix, but generally yield detection in the PPB range. A common scan is for arsenic, cadmium, lead and mercury used for heavy metal analysis.

Atomic Absorption Spectrospcopy (AA) is used for single analyte measurements using flame, hydride-generation or graphite furnace techniques. This technology, in general, has been surpassed by the advances made in ICP and ICP-MS techniques. Please see the details in the assay section of the catalog for specifics.

Flame AA Silicon

Titanium Other analytes available on request include calcium, chromium (in fortified supplements), cobalt, copper, iron, lead, magnesium, manganese, molybdenum, potassium, selenium, sodium and zinc.

Graphite Furnace AA Aluminum

Other analytes, such as chromium, available on request

Inorganic Speciation IC-ICP-MS Arsenic

Miscellaneous AA Selenium (hydride)

* Calculated from total sulfur by ICP-MS



Inorganics by ICP Emission Spectrometry Packages Option 1: ICP3–calcium, iron, sodium (for nutrition

labeling) Option 2: ICPL–calcium, copper, iron, magnesium,

manganese, phosphorus, potassium, sodium, zinc Option 3: ICPN–Option 2 plus aluminum, barium, boron,

chromium*, molybdenum*, strontium Option 4: ICPS–Option 3 plus beryllium, cadmium, cobalt,

nickel, vanadium* May need to perform an alternate test for certain matrices.

Inorganics by Wet Chemistry Chloride Fluoride (chemical) Fluoride (ISE) Iodine (chemical) Iodine (ISE) Nitrate/nitrite by HPLC Nitrate/nitrite by IC and VIS Heavy metals as lead by USP <231>

INORGANIC ANALYSIS

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MICROBIOLOGY SERVICES

Feeling challenged by the demands for increased food safety in today’s market place? Covance is ready to help you produce safe products and keep you compliant and competitive with our broad array of services that address consumer and regulatory requirements.

In anticipation of your needs, we have dedicated two ISO accredited laboratories (Battle Creek, Michigan and Madison, Wisconsin) to delivering microbiological results you can use to base food safety decisions. Along with testing capabilities, Covance offers microbiology consulting, customized research and training services.

Contact Us Today (1.855.83MICRO or 1.855.33MICRO)

Discover why Covance is your optimum partner in food safety services

CONSULTING• Investigations to help

identify, isolate and eliminate microbial harborage points

• Responses to regulatory actions

• Expert witness services• Food safety program

development and review • GFSI audit preparation

RESEARCH• White paper, laboratory and

in-plant process validations• Customized challenge studies

based on NACMCF guidelines• Pathogen- and spoilage-based

microbial shelf-life evaluations• Confirmation and/or validation of

cooking/baking instructions

TRAININGGeneral education sessions or

customized courses, held in our classrooms or delivered at your facility

• International HACCP Alliance accredited training classes and instructors

• Prerequisite programs• Internal audits• Sampling technique• Sampling site selection• Laboratory techniques• Other topics upon request

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MICROBIOLOGY ASSAYS

Food, Ingredients and Environmental Swabs

Quantitative – Counts or MPN

Aerobic plate Lactic Acid Bacteria

Bacillus cereus Lactobacillus - Heterofermentative

Chronobacter sakazakii Mesophilic aerobic spores

Coliform Mesophilic anaerobic spores

Coliform MPN Staphylococcus species

E. coli Staphylococcus species – Coagulase (+)

E. coli MPN Yeasts and molds

Enterobacteriaceae Yeasts and molds – Osmophilic

Additional charges for samples requiring dilution beyond 1/1000.

Qualitative – Presence/Absence

E. coli O157:H7 Listeria species

E. coli – STEC Listeria monocytogenes

Salmonella species

Additional charges for sample sizes > 100 g and for compositing of individual samples.

Confirmation/Identification

Gram stain Biochemical – VITEK and API

Dietary Supplements and Nutritional Products

USP

Verification of assay suitability Clostridia species

Total aerobic microbial count (TAMC) E. coli

Total combined yeasts and molds count (TYMC) Salmonella species

Bile-tolerant Gram negative bacteria Staphylococcus aureus

Candida albicans

Additional charges for dilutions beyond 1/10 and sample neutralization.

Non-USP

Probiotic enumerations Enumeration of microencapsulated microorganisms

Shelf-life viability Simulated gastric dissolution

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CONTAMINANTS IN FOODS, FEEDS AND DIETARY SUPPLEMENTS

Global distribution of food, feed and dietary supplement products into new regions requires a broad understanding of the interplay between growers, manufacturers and regulators. First-hand experience developing new testing methods, global regulatory knowledge and the scientific expertise to develop an appropriate testing program is critical to meeting your specific product needs. Our scientists have designed and managed hundreds of testing programs for heavy metals, acrylamide, melamine and pesticide residues in support of Proposition 65, response to economic adulteration, routine supplier verification and Infant Formula EU Directive 2006/141/EC, respectively.

Our Center of Excellence for Contaminants in Greenfield, Indiana, offers a broad portfolio of contaminant testing services. The 10,000 sq ft state-of-the-art facility in Greenfield provides clients the most up-to-date analysis of chemical residues and contaminants as well as the development of new analytical methods.

We approach the analysis of contaminants based on analytical quantitation using highly selective technology including ICP-MS, LC- and GC-MS/MS. As a true partner in food and dietary supplement analysis, we have the resources to meet your requirements for quality, flexibility, value and customer service.

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Multi-Residue Analysis (MRA) by GC-MS/MS and LC-MS/MS (300+ compounds)

Botanicals, Spices and Other Specialty Commodities and Products All analyses reported to 10 ppb or LOQ, whichever is higher.

Finished Food and Raw Ingredients All analyses reported to 10 ppb or LOQ, whichever is higher.

This offering includes: ▶ Fruits and vegetables ▶ Grains and cereals ▶ Fats and oils ▶ High-fat food products ▶ Finished processed foods, including meals

Infant Formula All analytes are reported to 10 ppb (or lower if required). EU Directive 2006/141/EC (10 ng/g or less) offering is available.

Matrix-Based Quantitation Additional analyses (after initial analysis) to perform Method of Standard Addition, which provides the most

accurate quantitation.

Individual Pesticides or Subgroups Individual pesticides, subgroups of or additions to the MRA.

Carbamates by LC-MS/MS



USP/EP Analysis (99 Analytes)

USP 561 USP/EP Pesticide Full Reporting Package Report at the USP/EP limits. Full multipoint calibration for all analytes by GC-MS/MS and LC-MS/MS. Includes EBDCs

(dithiocarbamates) by GC, inorganic bromide by GC and matrix-based quantitation.*

* Matrix-Based Quantitation: additional analyses (after initial analysis) to perform Method of Standard Addition, which provides the most accurate quantitation.

Ala Cart Analysis

GC-MS/MS and LC-MS/MS Multi-Residue Analysis (select 1 to 90+ analytes)Matrix-based quantitationEBDCs (dithiocarbamates) by GCBromide, inorganic

COMPENDIAL TESTING SERVICES

Raw Material/Ingredient Analysis

At Covance, we have been performing monograph analyses of raw materials and ingredients for over 30 years. In accordance with many global regulations, our database of over 400 different raw materials and capacity to set up more provides you with the analytical services you need for your raw materials testing.

To expedite your analysis, please provide the following information in advance:▶ Quarterly or yearly list of raw materials▶ Notice of shipment▶ Analysis request form (available online or from Client Services)

This testing is compliant with GMP regulations (21 CFR Part 111) for ingredients in dietary supplements. For assays compliant with 21 CFR Part 211 for human drug ingredients, please contact us.

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CONTAMINANTS IN FOODS, FEEDS AND DIETARY SUPPLEMENTS



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STABILITY STUDIES: STABILITY AND SHELF-LIFE EVALUATION

Covance provides comprehensive analytical services related to stability and shelf-life of products for the food and dietary supplement industries. Stability is defined as the extent to which a product retains, within specified limits throughout its period of storage and use, the same properties and characteristics possessed at the time of packaging. Understanding product stability is an essential measure of the quality of a food or dietary supplement. Covance can custom design your stability study from start to finish.

Study Design - Essential Information for Stability Studies

▶ Packaging materials▶ Region the product is being sold▶ Product specifications▶ Type of study (accelerated or real time)▶ Shelf-life anticipated▶ Regulatory requirements (if applicable)▶ Method applicability

Testing Services for Stability Studies

▶ Chemical analysis (nutrients, active compounds)▶ Physical properties (moisture, water activity, disintegration,

hardness, etc.)▶ Organoleptic evaluation (appearance, taste, smell, color)▶ Microbiological evaluation (safety assessment,

antimicrobial preservative effectiveness)▶ Monograph testing (USP or International)

Goals of the Stability Study

▶ Ensure the product is stable over its shelf-life▶ Establish or verify the shelf-life of the product▶ Aid in product research and development▶ Provide data to support the quality of the product over time▶ Fulfill any regulatory requirements

Due to the custom nature of designing a stability study, please inquire on pricing and storage availability.

Typical Stability Studies

Testing Intervals Storage Study Condition (ICH guidelines)Real Time 25°C/60% RH 3, 6, 9, 12, 18, 24 monthsAccelerated 40°C/75% RH 3, 6 monthsAlternate 30°C/65% RH 6, 9, 12 months

PHYTOCHEMICALS

The terms phytochemicals and phytonutrients have been adopted as descriptors for certain organic components of plants that are thought to promote human health. Unlike the traditional nutrients, these compounds are not “essential” for life from a traditional standpoint but are being increasingly recognized as having beneficial health characteristics. As the popularity of these products has increased with mainstream consumers, the use of a variety of dietary supplements, herbals/botanicals and functional foods has expanded and resulted in an intense and competitive marketplace. The ability to scientifically substantiate claims for quality, purity, consistency, stability, safety and efficacy is an essential marketing tool and frequently a regulatory requirement.



METHOD DEVELOPMENT, VALIDATION AND TRANSFER

Responding to changes in market or regulatory requirements demands new or improved high-quality scientific methods engineered for greater efficiency, lower limits of detection and improved reproducibility. As a partner in developing solutions to method challenges, Covance offers the services of our Technical Development team on a contract basis. You can leverage the team’s expertise across a wide range of matrices and technologies, including LC-MS/MS and UHPLC, to address your analytical challenges. Projects are priced on a time/materials or project basis.

Typical projects we have completed include:

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SPECIAL SERVICES

Colors

Artificial Colors

FD&C Red No. 2 (Amaranth)FD&C Red No. 3 (Erythrosin B)FD&C Red No. 4 (Ponceau SX)FD&C Red No. 40 (Allura Red)

FD&C Green No. 3 (Fast Green FCF)FD&C Blue No. 1 (Erigluacine)FD&C Blue No. 2 (Indigo Carmine)Acid Orange 12 (Crocein Orange G)

FD&C Yellow No. 5 (Tartrazine)FD&C Yellow No. 6 (Sunset Yellow FCF)FD&C Yellow No. 10 (Quinoline Yellow)

▶ New method development▶ Compendial/journal method setup▶ Matrix extensions (incl. validation)

▶ Client method validation▶ Existing method modernization

▶ New technology application▶ Client method variability resolution

Japanese Export Testing

Sennosides (UV)Fenfluramine, N-nitroso-fenfluramine (HPLC)p-Hydroxybenzoates (methyl, ethyl, propyl, butyl, isoproyl, isobutyl)Thryoxine, triiodothyronine (HPLC)Benzoic and sorbic acids

Prices for these tests are all product-dependent—specific pricing for your supplement product can be obtained by calling us at 800-675-8375.



BOTANICALS

For centuries, people have used herbs and other botanicals for their healthful effects, possibly due to their phytochemical composition, and in recent years, the use of these products has experienced a rise in popularity among consumers. This has resulted in corresponding regulatory initiatives and the accelerated need for analytical methods for botanical products. Several regulatory initiatives, including changes to current Good Manufacturing Practices (cGMP), address identification testing for botanical ingredients.

FOOD CONTACT MATERIALS (MIGRATION COMPLIANCE)

The potential for migration of indirect additives and contaminants from food contact material is a concern for both food products and dietary supplements. As the design and use of innovative packaging methods change, there is a corresponding effect on the regulatory and experimental guidelines for determining the impact of packaging components on the safety of food and dietary products. In the case of dietary supplements and botanicals, migration could potentially have a negative effect on the efficacy of the active ingredient. When evaluating the safety of packaging materials there are three key aspects to be considered: the source of the materials, the nature of the manufacturing process and the conditions of use.

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▶ Total and chloroform-soluble extractables from food packaging

▶ Recycled and degradable packaging

▶ Retort pouch barrier integrity

▶ Pigment and dye migration from consumer products

▶ Benzene in polymeric packaging materials

▶ Microwave packaging

▶ End use testing verifying compliance with 21 CFR Parts 172-181

▶ Conditions of use A-J

▶ Experience with FCN and EU recommendations

For guidance, please refer to: www.packaginglaw.com



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DIETARY SUPPLEMENTS

Covance has over 80 years of experience in food analysis and has built a reputation for scientific and regulatory expertise, responsiveness to customer needs and the capacity to turn around samples quickly and efficiently. As a full-service laboratory, we analyze food, dietary supplement and biotechnology products.

Product/Analyte ....................................................................................................................................... Testing Method

5-Hydroxytryptophan (5-HTP) .......................................................................................................................................... HPLC

Acetyl Cysteine ................................................................................................................................................................... HPLC

Allicin (garlic) ..................................................................................................................................................................... HPLC

alpha Lipoic Acid (USP) .................................................................................................................................................... HPLC

Anthocyanins ..................................................................................................................................................................... HPLC

Anthocyanins (bilberry, elderberry, cranberry, fruits) ....................................................................................................... VIS

Astaxanthin ........................................................................................................................................................................ HPLC

beta-Glucan .......................................................................................................................................................................... VIS

Black Currant Oil (linoleic acid and GLA) ..........................................................................................................................GC

Caffeine, Theobromine, Theophylline ............................................................................................................................. HPLC

Capsaicin/Capsaicinoid (Scoville Heat) ........................................................................................................................... HPLC

Catechins (EGCG, GCG, catechin, EGC, EC, ECG, GC, CG) + gallic acid .................................................................... HPLC

Chlorogenic Acid ............................................................................................................................................................... HPLC

Chondroitin Sulfate by CPC (USP) ................................................................................................................................Titration

Citrus Bioflavanoids .......................................................................................................................................................... HPLC

Coenzyme Q10 ................................................................................................................................................................... HPLC

Conjugated Linoleic Acid (CLA) ..........................................................................................................................................GC

Cranberry (quinic, malic, citric acids) .............................................................................................................................. HPLC

Creatine .............................................................................................................................................................................. HPLC

Cryptoxanthin .................................................................................................................................................................... HPLC

DHEA or 7-KETO DHEA

(Dehydroepiandrosterone) or (3-acetyl-7-oxo-dehydropiandrosterone) ...................................................................... HPLC

Dong Quai (ferulic acid) .................................................................................................................................................... HPLC

Echinacea spp. (caftaric, chlorogenic, echinacoside, chicoric) ......................................................................................... HPLC

Echinacea spp. [total phenolics as gallic acid equivalents (GAE)] ...................................................................................... VIS

Elagic Acid .......................................................................................................................................................................... HPLC

Ephedrine Alkaloids

Ephedra or Ma Huang ................................................................................................................................................LC-MS/MS

Evening Primrose Oil (linoleic acid and GLA) ...................................................................................................................GC

Fo-ti Powder (trans-resveratrol) ........................................................................................................................................ HPLC

Fructan (AOAC 997.08) ............................................................................................................................................... HPAEC-PAD

Fructan (AOAC 999.03) ....................................................................................................................................................... VIS

Furfural............................................................................................................................................................................... HPLC

gamma Aminobutyric Acid (GABA) ................................................................................................................................ HPLC

gamma Linoleic Acid (GLA) .................................................................................................................................................GC



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DIETARY SUPPLEMENTS (CONTINUED)


Garcinia cambogia (hydroxycitric acid) ............................................................................................................................. HPLC

Garlic (allicin) ..................................................................................................................................................................... HPLC

Ginkgo Biloba Flavonoids ................................................................................................................................................. HPLC

Ginkgo Biloba Terpenoids ................................................................................................................................................. HPLC

Ginseng, Panax or Korean (ginsenosides) ....................................................................................................................... HPLC

Glucosamine .......................................................................................................................................................... HPLC/HPAEC-PAD

Glycerol (glycerine) ...............................................................................................................................................................GC

Goldenseal (hydrastine and berberine) ............................................................................................................................ HPLC

Grape Seed Extract

Catechins ......................................................................................................................................................................... HPLC

Total Polyphenols (Folin-Ciocalteu Method) ................................................................................................................. VIS

Proanthocyandins (polyphenols - catechins) ............................................................................................................ VIS/HPLC

trans-Resveratrol ............................................................................................................................................................. HPLC

Green Tea Catechins .......................................................................................................................................................... HPLC

Griffonia Seed Extract (5-Hydroxytryptophan) 5-HTP ..................................................................................................... HPLC

Guarana (theobromine, theophylline, caffeine) ............................................................................................................... HPLC

Hydroxycitric Acid (garcinia extract) ................................................................................................................................ HPLC

Isoflavones (daidzein, glycitein, genistein, daidzin, glycitin, genistin) ......................................................................... HPLC

Kudzu (isoflavones) ........................................................................................................................................................... HPLC

Lutein .................................................................................................................................................................................. HPLC

Lycopene ............................................................................................................................................................................. HPLC

Marine Lipids (EPA/DHA) ...................................................................................................................................................GC

Methylsulfonylmethane (MSM) ...........................................................................................................................................GC

Milk Thistle (silychristin, silydianin, silibibin A & B) ..................................................................................................... HPLC

Omega-3 Fatty Acids .............................................................................................................................................................GC

ORAC, Total (includes hydrophilic and lipophilic) ............................................................................................................ FL

Oxalic acid .......................................................................................................................................................................... HPLC

PABA (para-aminobenzoic acid) ....................................................................................................................................... HPLC

Phenolic Acid (Phenylproponoids) Caffeic, p-Coumaric, Sinapic, and Ferulic Acids ................................................... HPLC

Phospholipids (Lecithin Phospholipids)

Phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol ... HPLC

Phytic Acid .......................................................................................................................................................................... HPLC

Phytosterols (stigmasterol, beta sistosterol, campesterol, brassicasterol) ........................................................................GC

Policosanol (octacosanol, triacontanol, tetracosanol, hexacosanol)...................................................................................GC

Polydextrose ........................................................................................................................................................... HPLC/HPAEC-PAD

Polygonum cuspidatum (trans-resveratrol) ........................................................................................................................ HPLC

Polyphenols (Total Polyphenols) Folin-Ciocalteu Method ................................................................................................ VIS

Polyphenols (Total) with % Solids

Tea and tea products for Tea Association members only .............................................................................................. VIS

Pueraria (isoflavones) ........................................................................................................................................................ HPLC

Punicalagins ....................................................................................................................................................................... HPLC


DIETARY SUPPLEMENTS (CONTINUED)


Pygeum (beta sitosterol) .......................................................................................................................................................GC

Pyruvate (pyruvic acid) ...................................................................................................................................................... HPLC

Quercetin ............................................................................................................................................................................ HPLC

Rose Hips (ascorbic acid) ..................................................................................................................................................... FL

Royal Jelly 10-HDA (10-hydroxy-2-decanoic acid) ............................................................................................................ HPLC

Rutin ................................................................................................................................................................................... HPLC

SAMe (S-adenosylmethionine) ......................................................................................................................................... HPLC

Saw Palmetto (phytosterols + fatty acids) ............................................................................................................................GC

Scoville Heat (capsaicin and capsaicinoid) ....................................................................................................................... HPLC

Soy Isoflavones ................................................................................................................................................................... HPLC

Stinging Nettles (5-hydroxytryptophan) ........................................................................................................................... HPLC

Theobroma cacoa (caffeine, theobromine, theophylline) ................................................................................................. HPLC

Tocotrienols (alpha, beta, gamma, delta) ......................................................................................................................... HPLC

Tomato (lycopene) ............................................................................................................................................................. HPLC

trans-Resveratrol ................................................................................................................................................................ HPLC

Ubiquinone (Coenzyme Q10) ........................................................................................................................................... HPLC

Valerian Extract (valerenic acid) ........................................................................................................................................ HPLC

Yerba Mate (caffeine, theobromine, theophylline) .......................................................................................................... HPLC

Zeaxanthin ......................................................................................................................................................................... HPLC


QUICK REFERENCE




ASSAYS


ASSAYS

3-CHLORO-1,2-PROPANEDIOL (3-MCPD)

Purpose: Applicable for the determination of 3-MCPD in hydrolyzed vegetable protein, soups and stocks, stock cubes, soy sauce, malt extract, salami, fish, cheese, flour, starch, cereals and bread.

Method facts: ▶ Sample size: 25 g ▶ Limit of quantitation: 0.025 ppm for most matrices▶ Precision: Varies with matrix▶ Method reference: AOAC 2000.01

Description: An internal standard is added to the sample followed by a salt solution. This is then blended to homogenize, sonicated, then purified. A portion of the purified extract is derivitized and analyzed by gas chromatography with mass spectrometric detection (GC-MS).

4-METHYLIMIDAZOLE (4-MEI) AND 2-METHYLIMIDAZOLE (2-MEI)

Purpose: Applicable for the determination of 4-MeI and 2-MeI in beverages and water soluble samples.

Method facts: ▶ Sample size: 25 g▶ Limit of quantitaion: Varies with matrix, as low as 1 ppb for

4-MeI and 2 ppb for 2-MeI▶ Precision: Varies with matrix▶ Method reference: Simultaneous quantitation of 2- and

4-methylimidazoles in carbonated drinks containing caramel coloring by ultrahigh-performance liquid chromatography tandem mass spectrometry, by Wang, J.; Liu, X.; Pohl, C. and Schnute, W., Food Quality Magazine, June/July 2011, 34-38.

Solid-phase extraction of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-1,2,3,4-tetrahydroxylbutyl-imidazole (THI) from foods and beverages with subsequent liquid chromatographic-electrospray mass spectrometric quantification. J. Sep. Sci. 2006, 29, 378-384.

Reversed-phase high-performance liquid chromatographic/mass spectrometric method for separation of 4-methylimidazole and 2-acetyl-4-tetrahydroxybutyl imidazole at pg levels. Analytic Chimica Acta 477 (2003) 49-58.

Description: Sample is weighed and an internal standard is added. Sample is diluted with purified water and analyzed using HPLC-MS/MS.

5-HYDROXYTRYPTOPHAN (5-HTP)

Analyzed with free amino acid procedure

ACESULFAME K

Analyzed with aspartame: see Aspartame

ACETIC ACID

see Organic acid profiles — option 2

ACID DETERGENT FIBER

see Detergent fiber, acid

ACRYLAMIDE

Purpose: Applicable for the determination of acrylamide in various foods.

Method facts:▶ Sample size: 5 g▶ Limit of quantitation: 10 ppb▶ Precision: Varies with matrix▶ Method reference: United States Food and Drug

Administration, Center for Food Safety and Applied Nutrition Office of Plant & Dairy Foods and Beverages, “Detection and Quantitation of Acrylamide in Foods” (2002)

Description: Acrylamide is extracted from food samples with 0.1% formic acid. If product contains fat, petroleum ether is used prior to extraction with 0.1% formic acid. The extract is purified through a solid phase extraction (SPE cartridge). Acrylamide in the sample extract is determined using LC-MS/MS. The acrylamide is determined using least square linear regression with 13C3-labeled acrylamide as an internal standard. Ions monitored for acrylamide are m/z 55, 44 and 27 and for the internal standard m/z 58. The ratio of peak areas for m/z 55 (acrylamide) and m/z 58 (internal standard) are compared to those for standards over the standard curve range.

AFLATOXINS

Purpose: Applicable for the determination of aflatoxins B1, B2, G1 and G2 in a wide range of matrices including premixes, raw materials, food ingredients and finished feeds.

Method facts:▶ Sample size: 50 g▶ Limit of quantitation: 0.5 ppb for each aflatoxin compound

21



▶ Precision: NA▶ Method reference: AOAC 991.31

Description: The sample is extracted with a mixture of methanol:water. The extract is diluted with water and a portion is applied to an antibody affinity column. The column is washed first with water to remove major interferences. The aflatoxins are eluted with acetonitrile and the eluent is then dried under a stream of nitrogen. The aflatoxins are derivitized with acid to form the highly fluorescent hemi-acetal compounds. The sample extract is then quantified utilizing HPLC by comparison to standards of known concentration.

AFLATOXIN (M1)

Purpose: This method is applicable to the analysis of aflatoxin M1 in fluid milk, infant formulas and other matrixes.

Method facts: ▶ Sample size: 20 g of powder or 100 mL of liquid ▶ Limit of Quantitation: 0.1 ppb for M1 compound ▶ Precision: NA ▶ Method reference: Official Methods of Analysis for

AOAC International 2000.08

Description: The sample is extracted with a buffer solution. The extract is diluted and a portion is applied to an antibody affinity column. The column is washed first with water to remove major interferences. The aflatoxins are eluted with acetone and the eluent is then dried under a stream of nitrogen. The aflatoxins are derivatized acid to form the highly fluorescent hemi-acetal compounds. The sample extract is then quantified utilizing high performance liquid chromatography by comparison to standards of known concentration.

AFLATOXIN, OCHRATOXIN A AND ZEARALENONE BY HPLC

Purpose: Applicable for the analysis of aflatoxins B1, B2, G1, G2, ochratoxin A and zearalenone in a wide range of foods, feeds and nutriceutical products.

Method facts:▶ Sample size: 50 g▶ Limit of quantitation: 0.5 ppb for each aflatoxin compound,

1.0 ppb for ochratoxin A and 10 ppb for zearalenone▶ Precision: NA▶ Method reference: AOAC 2005.08, AOAC 993.17, AOAC

999.07

Description: The mycotoxins are extracted from the sample with a methanol/sodium bicarbonate mix. Following extraction, the samples are cleaned with liquid-liquid partition and antibody affinity solid phase extraction columns. The mycotoxins are determined by HPLC-fluorescence detection, utilizing post column photochemical derivitization to determine aflatoxin B1 and G1.

ALACHLOR

see USP 561 and EP pesticides

ALDRIN AND DIELDRIN


ALLICIN (GARLIC)

see Garlic

ALMOND PROTEIN BY ELISA

Purpose: Allergens are proteins in food that can create an immune response in sensitive individuals. Clear ingredient labeling of food products, by manufacturers, aids in protection from accidental ingestion by those individuals. Testing for the presence of these proteins ensures the food is free of the allergen at a potentially harmful level.

Method facts: ▶ Sample size: 50 g ▶ Limit of quantitation: 2.5 ppm ▶ Method reference: Veratox® Quantitative Almond Allergen

Kits Neogen Corporation

Description: Allergen protein is extracted with a buffered salt solution, allowed to settle then placed in an antibody (capture) coated microwell to bind if present. All unbound protein is washed away before a detector antibody (enzyme labeled) is added. The detector antibody then binds to bound protein if present. After another wash, and the addition of a substrate, color develops as a result of bound detector antibodies. After the addition of a stop reagent, the test is read with a Stat Fax reader. Optical densities of the controls form a standard curve which samples are plotted against to obtain concentrations of allergen protein in ppm.

ALPHA CAROTENE

see beta Carotene

ASSAYS


ASSAYS


ALPHA LIPOIC ACID (USP)

Purpose: Applicable for the determination of alpha lipoic acid in tablets, capsules, premixes and raw material.

Method facts:▶ Sample size: 5 g▶ Limit of quantitation: Most matrices - 0.025%▶ Precision: On a drink preparation, the RSD is 6.37%▶ Method reference: The United States Pharmacopoeia

Description: Sample extraction is performed by mechanical shaking. A portion of the extract is analyzed by HPLC and UV detection, then quantitated by comparing the response to a standard curve of known concentration.

ALPHA TOCOPHEROL

Purpose: Applicable to the determination of tocopherols and tocotrienols in foods, feeds and nutritional products. This method may also be used at the supervisor’s discretion for matrices outside of the intended scope.

Method facts: ▶ Sample size: 30 g in food products, 10 g in supplements

and premixes, 50 g in feeds▶ Limit of quantitation: 0.500 mg/100 g, but may vary with

sample matrix▶ Precision: On an infant formula matrix, the RSD is 6.10%▶ Method reference: Speek, A.J., Schijver, J. and Schreurs,

W.H.P., Journal of Food Science, 50: 121-124 (1985) (modified)

Cort, W.M., Vincente, T.S., Waysek, E.H. and Williams, B.D., Journal of Agricultural Food Chemistry, 31: 1330-1333 (1983) (modified)

McMurray, C.H., Blanchflower, W.J. and Rice, D.A., Journal of the Association of Official Analytical Chemists, 63: 1258-1261 (1980) (modified)

Description: The product is typically saponified to break down the fat and release the vitamins. The digest is then extracted with organic solvent. Additionally, alternate extraction procedures which do not require saponification may be utilized for specified matrices. The tocopherols and tocotrienols are quantitated by ultra or high performance liquid chromatography (UHPLC or HPLC) using fluorescence detection.

ALUMINUM (ICP)

see Inorganic analysis by ICP

ALUMINUM (GRAPHITE FURNACE)

Purpose: Applicable for the determination of aluminum in foods, feeds, dietary supplements and biological materials.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.5 ppm▶ Precision: On a rice flour sample matrix with a certified

value of 4.4 ppm, the RSD is 5.85%▶ Method reference: Standard Methods for the Examination

of Water and Wastewater, 20th Ed., Method 3113: 3-20-3-27, American Public Health Association, Washington, D.C. (1998) (modified)

Description: An appropriately sized sample is wet-ashed with nitric acid, hydrofluoric acid, and 30% hydrogen peroxide using open vessel microwave digestion. The amount of aluminum is determined by comparing the signal of the unknown sample, measured by the graphite furnace atomic absorption spectrophotometer, with the signal of the standard solutions. A rhodium/magnesium matrix modifier is used in the analysis.

AMINO ACID PROFILE (AOAC), TOTAL

Purpose: Applicable to all samples for the following amino acids: Alanine, arginine, aspartic acid (including asparagine), cystine, glutamic acid (including glutamine), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, valine. (An accurate protein value is recommended to perform these analyses.)

Method facts:▶ Sample size: 5 g▶ Limit of quantitation: Most matrices - 0.1 mg/g▶ Precision: On an infant formula matrix, examples of

typical RSDs are as follows: leucine - 1.9%, aspartic - 1.6%, glutamic - 1.5%, alanine - 1.7%

▶ Method reference: AOAC 982.30

Description: The sample is hydrolyzed in hydrochloric acid (HCl) and adjusted to pH 2.2 for all amino acids except tryptophan. Tryptophan samples are hydrolyzed in sodium hydroxide and adjusted to pH 5.2. Individual amino acids are determined using an automated amino acid analyzer. Cystine and methionine (AOAC) onlyTryptophan (AOAC) only


ASSAYS


AMINO ACID PROFILE (AOAC), FREE BY AAA

Purpose: Applicable to all samples for the following amino acids: alanine, arginine, aspartic acid (including asparagine), cystine (including cysteine), glutamic acid (including glutamine), glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.1 mg/g▶ Precision: NA▶ Method reference: AOAC Method 982.30

Description: The sample is extracted in a mild acid. Determination is by an amino acid analyzer.

AMINO ACID PROFILE, FREE BY HPLC

Purpose: Applicable to all samples for the following amino acids: L-aspartic acid, L-glutamic acid, L-serine, L-histidine, glycine, L-threonine, L-arginine, L-alanine, L-tyrosine, L-cystine, L-valine, L-methionine, L-phenylalanine, L-isoleucine, L-leucine, L-lysine, proline, L-asparagine, L-glutamine, L-citrulline, L-tryptophan and hydroxyproline.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.1 mg/g▶ Precision: On a corn sample, examples of typical RSDs

are as follows: proline 4.8%, glutamic acid 5.0%, histidine 4.6%, aspartic acid 3.1%.

▶ Method reference: Henderson, J.W., Ricker, R.D., Bidlingmeyer, B.A., Woodward, C., “Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA columns and the Agilent 1100 HPLC,” Agilent Publication, 2000

R. Schuster, “Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivitization and HPLC,” J. Chromatogr., 1988, 431, 271-284

Description: The sample is extracted in acid. Determination is by high-performance liquid chromatography (HPLC) with fluorescence or diode array detection. The primary amino acids are derivitized with fluorenylmethyl chloroformate before injection.

AMINO ACID PROFILE (HPLC), TOTAL

Purpose: Applicable to all samples for the following amino acids: alanine, arginine, aspartic acid (including asparagine), cystine (including cysteine), glutamic acid (including glutamine), glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.1 mg/g▶ Precision: On an infant formula, examples of typcial RSDs

are as follows: leucine 1.4%, aspartic acid 1.8%, tyrosine 2.5%, methionine 2.9%.

▶ Method reference: Henderson, J.W., Ricker, R.D., Bidlingmeyer, B.A., Woodward, C., “Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA columns and the Agilent 1100 HPLC,” Agilent Publication, 2000

Barkholt and Jenson, “Amino Acid Analysis: Determination of Cystine plus Half-Cystine in Proteins after Hydrochloric Acid Hydrolysis with a Disulfide Compound as Additive,” Analytical Biochemistry, 1989, 177, 318-322

R. Schuster, “Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivitization and HPLC,” J. Chromatogr., 1988, 431, 271-284

Description: The samples are hydrolyzed in 6 N hydrochloric acid for 24 hours at approximately 110°C. Phenol is added to the 6 N hydrochloric acid to prevent halogenation of tyrosine. Cystine and cysteine are converted to S-2-carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan is hydrolyzed from proteins by heating at approximately 110°C in 4.2 N sodium hydroxide. The samples are analyzed by HPLC after pre-injection derivitization. The primary amino acides are derivitized with o-phthalaldehyde (OPA) and the secondary amino acids are derivitized with fluorenylmethyl chloroformate (FMOC) before injection.

P-ANISINIDE VALUE

Purpose: Applicable for the determination of p-anisinide value in all normal fats and oils. This method determines the amount of aldehydes in animal and vegetable oils.


ASSAYS


Method facts:▶ Sample size: 20 g▶ Limit of quantitation: NA▶ Precision: NA▶ Method reference: AOCS Cd 18-90

Description: The sample is diluted in isooctane and read on a spectrophotometer. The sample is then spiked and re-read on a spectrophotometer to determine the amount of aldehydes in the sample.

ANTHOCYANINS, TOTAL

Purpose: Applicable for the determination of total anthocyanin content in blueberry, cranberry, bilberry and other fruit extracts in tablets, capsules, premixes and raw material.

Method facts:▶ Sample size: 5 g▶ Limit of quantitation: <0.01%▶ Precision: Varies with matrix▶ Method reference: Givisti, M.M., Molar Absorptivity

and Color Characteristics of Acylated and Non-Acylated Pelrgonidin-based Anthocyanins, J. Agri and Food Chemistry, 47(11):4631-4637 (1999)

Description: Sample extraction is performed with water and sonication. Portions of that extract are subjected to buffers with differing pH. Aliquots are analyzed for absorbance on a spectrophotometer. The difference in absorbance and the molar absorbtivity coefficient of cyanidin-3-glucoside determine total anthocyanin content.

ANTHOCYANINS BY HPLC

Purpose: Applicable for the determination of anthocyanin content in blueberry, cranberry, bilberry and other fruit extracts in tablets, capsules, premixes and raw material.

Method facts:▶ Sample size: 5 g▶ Limit of quantitation: Most matrices- <10 ppm▶ Precision: Varies with matrix▶ Method reference: Indena Method 29/04/LRA1-00

Description: Samples are extracted in an acidic methanol solution with sonication. Separation is achieved with reversed-phase HPLC and a C18 column with detection set at 535 nm. Twenty-one anthocyanins and anthocyanidins are identified and quantified by comparing to cyanidin chloride and cyanidin-3-O-glucoside, respectively. The final result for each compound is then converted by the molecular weight ratio.

ARSENIC

see Inorganic analysis by ICP-MS

ARSENIC SPECIATION

Purpose: This method is applicable to the determination of organic and inorganic arsenic, as well as the most common individual species in rice, rice products and juice. Both of the highly toxic inorganic species arsenite (As(+3)) and arsenate (As(+5)), and the organic species dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) can be determined. Of the less common organic species in rice and juice, arsenocholine (AsC) is able to be determined individually, while arsenobetaine (AsB) and trimethylarsine oxide (TMAO) both elute in the void volume together and are not able to be determined individually*.

* Note: While the sum of organic arsenic will include the presence of any species that elute at this time, they will be calculated using AsB as the reference standard.

Method facts:▶ Sample size: 2 g▶ Limit of quantitation: 10.0 ppb in rice and products

containing rice 2.00 ppb in juices▶ Precision: In rice products the RSD is 4.8%. In juices the

RSD is 3.3%.▶ Method reference: FDA Elemental Analysis Manual

[Internet]. Silver Spring (MD): Food and Drug Administration (US); Section 4.11 [Version 1.1; 2012 November] Arsenic Speciation in Rice and Rice Products Using High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Determination

Kutscher, D., McSheehy, S., Wills, J., Jensen, D. IC-ICP-MS Speciation Analysis of As in Apple Juice using the Thermo Scientific iCAP Q ICP-MS. Thermo Scientific Application Note 43099, 2012

Description: Various arsenic species are extracted from samples in plastic vessels with dilute nitric acid using oven digestion and then filtered prior to analysis. The amount of any given arsenic species is determined using an ion chromatography - inductively coupled plasma - mass spectrometer (IC-ICP-MS) by comparing the peak area generated for the samples to those generated by standards of known concentration.


ASSAYS

ASCORBIC ACID

see Vitamin C

L-ASCORBYL-2-PHOSPHATE

(2-phospho-L-ascorbic acid) Purpose: Applicable to the quantitation of L-ascorbyl-2-phosphate in foods, feeds and premixes. Also applicable to the various salts of the acid. This method does not determine the di- or tri-phosphate forms of ascorbic acid.

Method facts:▶ Sample size: 25 g▶ Limit of quantitation: 50 mg/kg▶ Precision: 2.25% on a dry dog food control▶ Method reference: Simultaneous HPLC Analysis of

L-Ascorbic Acid, L-Ascorbyl-2-Sulfate, and L-Ascobyl-2-Polyphosphate, Journal of Liquid Chromatography and Related Technology, 19(19):3105-3118 (1996), (modified)

Description: The sample is extracted with an aqueous phosphate buffer solution. The extract is then filtered, pH adjusted and injected onto an HPLC system equipped with a Phenomenex, Inertsit ODS-2, 250 mm x 4.6 mm, 5 µL column, held in an oven set at 30.0°C. The analyte is eluted with a phosphate buffer, acetonitrile and ethanol mobile phase. The amount of L-ascorbyl-2-phosphate is detected using an ultraviolet absorbance detector.

ASH

Purpose: Applicable for the determination of ash in most foods and feeds.

Method facts: ▶ Sample size: 5 g▶ Limit of quantitation: Most matrices - 0.1%▶ Precision: On a dog food matrix, the RSD is 5.0%▶ Method reference: AOAC 923.03

Description: Organic matter is burned off by igniting the sample at 550°C in an electric furnace. The remaining material is determined gravimetrically and referred to as ash.

ASPARTAME AND ACESULFAME K, DKP AND SACCHARINE

Purpose: Applicable for the determination of aspartame, diketopiperazine (DKP), acesulfame K and saccharine in food and beverages. It may not be applicable for samples with a high protein content.

Method facts:▶ Sample size: 25 g▶ Limit of quantitation: Most matrices - 50 ppm▶ Precision: Varies with matrix▶ Method reference: Prodolliet, J., and Bruelhart, M.,

Determination of Aspartame and its Major Decomposition Products in Foods. J. of AOAC, 76(2) (1993)

Description: Samples are weighed, then dissolved in a phosphate buffer/methanol solution. The solutions are injected on an HPLC equipped with a UV detector. Samples are calculated against standards of known concentration.

ASTAXANTHIN

Purpose: This method is applicable to the determination of free or total astaxanthin (sum of free and esterified) in nutritional supplements and fish.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: 0.200 mg/100 g for softgels, and

0.600 mg/100 g for salmon flesh▶ Method reference: Britton, G., Liaaen-Jensen, S., Pfander,

H. (1995). Carotenoids Volume 1A: Isolation and Analysis. Basel, Switzerland: Birkhäuser Verlag

Fuji Chemical Industry Co., LTD., “Spectrophotometric and HPLC Analysis Method for Determining Astaxanthin Content in AstaREAL®-P2AF” [online] Ver. June 1918, Http://www.fujihealthscience.com, Fuji Health Science Inc., (accessed 29 Dec. 2011)

Description: Esterified astaxanthin is hydrolyzed (de-esterified) by an enzymatic procedure to yield all free astaxanthin. The sample is then injected on a normal phase HPLC (high-performance liquid chromatograph) system with visible (VIS) light detection. Results are quantified using an internal standard and linear regression analysis.



ASSAYS

BHA, BHT AND TBHQ (ANTIOXIDANTS)

Purpose: Applicable for the determination of Butylated Hydroxyanisole (BHA)/Butylated Hydroxytoluene (BHT)/tert-Butylhydroquinone.

Method facts:▶ Sample size: 25 g▶ Limit of quantitation: Varies with matrix▶ Precision: 5% for most matrices▶ Method reference: Official Methods of Analysis of AOAC

INTERNATIONAL (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 968.17 (modified)

Description: The lipid is extracted from food and feed samples and diluted. BHA, BHT and TBHQ are quantified by gas liquid chromatography (GLC).

BENZENE

Purpose: Applicable for the determination of benzene in most matrices.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.02 mg/100 g▶ Precision: Varies with matrix▶ Method reference: Bertsch, Brian, “Developing One

Universal Method for Residual Solvents Using the New Teledyne Tekmar HT3 Headspace Sample Introduction System,” Application Note (Teledyne Tekmar), Document #HT3-001, September 2005

Description: The samples are introduced to a gas chromatograph-mass spectrometer (GC-MS) via a Tekmar HT3 purge-and-trap headspace autosampler.

BENZOIC ACID (SODIUM BENZOATE), SORBIC ACID (POTASSIUM SORBATE)

see Organic acid profiles - option 1

BERBERINE

see Goldenseal

BETA CAROTENE

Purpose: Applicable for the determination of alpha and beta carotene in food, feeds and dietary supplements.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: Most matrices - 0.02 mg/100 g▶ Precision: On an infant formula matrix, the RSD is 7.4%▶ Method reference: AOAC 941.15 Quackenbush, F. W., Journal of Liquid Chromatography,

10:643-653 (1987)

Description: Food products are de-esterified with potassium hydroxide and extracted with hexane. Juice products are blended with alcohol and extracted. Nutraceutical products are enzymatically treated and then extracted. The extracts are injected on a reverse-phase HPLC system equipped with UV detection and compared to a standard curve.

BETA-GLUCAN

Purpose: Applicable for the determination of beta-glucan in food products, with the exception of yeast and mushroom products.

Method facts:▶ Sample size: 2 g▶ Limit of quantitation: Most matrices - 0.5%▶ Precision: On a cereal sample matrix, the RSD is 4.81%▶ Method reference: AOAC 995.16

Description: beta-Glucan is hydrolyzed to oligosaccharides by lichenase. These oligosaccharides are reduced to glucose by beta glucosidase. The glucose is reacted with a glucose oxidase/peroxidase reagent mixture, and the reaction product is determined spectrophotomerically at 510 nm.

BILBERRY

see Anthocyanins, total see Anthocyanins by HPLC

BIOTIN

Purpose: Applicable for the determination of biotin in most foods, feeds and dietary supplements.

Method facts:▶ Sample size: 2 g▶ Limit of quantitation: Most matrices - 0.005 mcg/g▶ Precision: On an infant formula matrix, the RSD is 7.1%



ASSAYS

▶ Method reference: Wright & Skeggs, Procedures of the Society of Experimental Biology and Medicine, 56:95 (1944)

Schiner & Deritter, “Biotin Content of Feed Stuffs,” Journal of Agricultural Food Chemistry, 23:1157-1162 (1975)

Methods of Analysis for Infant Formulas, Infant Formula Council (1985)

Journal of the AOAC, 49:882, (1996)

Description: The sample is extracted with either water, dilute sodium hydroxide (NaOH), or sulfuric acid (H

2SO

4). The

amount of biotin is determined by comparing the growth response of the sample, using the bacteria Lactobacillus plantarum, with the growth response of a biotin standard. This response is measured turbidimetrically.

BISPHENOL A

Purpose: Applicable to analysis of Bisphenol A (BPA) in infant formula, infant formula concentrates, baby food and various food matrices. BPA as part of a packaging material analysis is available upon request.

Method facts:▶ Sample size: 10 g▶ Limit of quantitation: 0.5 ppb in infant formula;

LOQ varies by sample matrix▶ Precision: NA▶ Method reference: Determination of Bisphenol A in Liquid

Infant Formula by Solid Phase Extraction with Acetic Anhydride Derivatization and Gas Chromatography-Mass Spectrometry, Minister of Health Canada (2008).

Description: The sample is diluted with water and acetonitrile, then mixed and centrifuged. The supernatant is diluted with a phosphate buffer and passed through a solid phase extraction column (Varian 1210-2052, 500 mg C18, 6 mL). The column is eluted with an aqueous solution of acetonitrile and the extract concentrated. The sample is derivatized and extracted into iso-octane and MTBE, then concentrated to near dryness and diluted with toluene. The BPA is quantified and confirmed by GC/MS on an HP-5ms column.

BLACK CURRANT OIL (LINOLENIC ACID AND GLA)

see Fatty acid profiles

BOMB CALORIMETRY

Purpose: Applicable for the determination of total calories in foods and feeds.

Method facts:▶ Sample size: 2 g for solids, 7 g for liquids▶ Limit of quantitation: Most matrices - 5calories/g▶ Precision: On a benzoic acid matrix, the RSD is 0.67%▶ Method reference: Metals Energy Mining Agriculture

Geology, AC-350 Instruction Manual, LECO Corporation, St. Joseph, MI, pp 5-26 through 5-29 (2002)

Description: Calories are determined by burning a weighed sample in an atmosphere of oxygen inside a bomb submerged in a measured quantity of water. The temperature rise of the water, resulting from the combustion of the sample, is used to calculate the number of calories liberated.

BROMIDE, INORGANIC

Bromine containing fumigants determined as total inorganic bromide

Purpose: Applicable to the determination of inorganic bromide in dietary supplements (raw materials, ingredients and soft gels), high fat matrices and plant materials.

Method facts: ▶ Sample size: 25 g ▶ Limit of quantitation: Dry samples - 5 µg/g, moist

or liquid samples - 1 µg/g ▶ Method reference: Community Reference Laboratory for

Single Residue Methods, CVUA, Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany

T. Stijve, Gas Chromatographic Determination of Inorganic Bromide Residues - a Simplified Procedure, Dtsch. Lebenm. Rundsch. 77 99-101 (1981)

Deutsche Forschungsgemeinschaft (DFG), Manual of Pesticide Residue Analysis, Volume I, by Verlag Chemie, 1987. The bromide method has the code S 18. ISBN 3-527-27010-8

Description: The samples are homogenized and suspended in an acidified aqueous solution of propylene oxide, with bromide being simultaneously extracted and derivatized into 1-bromo-2-propanol and 2-bromo-1-propanol. The derivatives are partitioned into ethyl acetate and determined by GC-ECD.



ASSAYS

CADMIUM (ICP OR ICP-MS)

see Inorganic analysis by ICP or ICP-MS

CAFFEIC ACID

see Phenolic acid

CAFFEINE, THEOBROMINE, THEOPHYLLINE

Purpose: Applicable for the determination of caffeine, theobromine and theophylline in various food and supplements.

Method facts:▶ Sample size: 25 g▶ Limit of quantitation: Most matrices - 50 ppm▶ Precision: On a cocoa powder matrix, the RSD is 5.42% for

caffeine and 3.62% for theobromine▶ Method reference: Journal of Food Science,

48:745-747 (1983)

Description: The sample is extracted with boiling water. A portion of the extract is injected onto an HPLC system with UV detection set at 272 nm. The areas of the peaks are determined and compared with those obtained from injected standards.

CALCIUM (ICP)


CALORIES, BY CALCULATION

Purpose: Used to calculate calories in foods and feeds.

Method facts:▶ Sample size: NA▶ Limit of quantitation: <1.7 calorie▶ Precision: NA▶ Method reference: 21 CFR 101.9

Description: Calories = (Protein(g) x 4) + (Carbohydrates(g) x 4) + (Fat(g) x 9) or calculated by specific Atwater factors or other corrections by request.

CALORIES FROM FAT, BY CALCULATION

Purpose: Used to calculate calories from fat.

Method facts:▶ Sample size: NA▶ Limit of quantitation: <0.9 calorie from fat

▶ Precision: NA▶ Method reference: 21 CFR 101.9

Description: Calories from fat = Fat(g) x 9

CAPSAICIN/CAPSAICINOID (SCOVILLE HEAT)

Purpose: Applicable for the determination of pungency levels in capsicums and their oleoresins.

Method facts:▶ Sample size: 30 g▶ Limit of quantitation: Most matrices ~10 ppm▶ Precision: On a powder matrix, the RSD is 5.40%▶ Method reference: AOAC 995.03

Description: The sample is extracted in warm ethanol. The extract is filtered and then injected using HPLC equipped with an ultraviolet detector. Results are typically expressed in Scoville units.

CARBAMATE PESTICIDES (LC-MS/MS)

Purpose: Applicable but not limited to, the analysis of selected carbamate pesticides and related metabolites in food and dietary supplement products and raw materials.

Method facts:▶ Sample size: 25 g▶ Limit of quantitation: Listed in the table to the right▶ Precision: Varies with matrix▶ Method reference:

1 AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate

2 CEN Standard Method EN 15662: Food of plant origin – Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE – QuEChERS method

3 Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. Journal of AOAC INTERNATIONAL 2003, 86, 412-431

4 Lehotay, S.J.; Mastovska, K; Lightfield, A.R. Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. Journal of AOAC INTERNATIONAL 2005, 88, 615-629



ASSAYS

5 Lehotay, S.J.; Mastovska, K.; Yun, S. J. Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes. Journal of AOAC INTERNATIONAL 2005, 88, 630-638

6 Mastovska, K.; Dorweiler, K.; Lehotay, S.J.; Wegscheid, J.; Szpylka, K. Pesticide multiresidue analysis in cereal grains using modified QuEChERS method combined with automated direct sample introduction GC-TOFMS and UPLC-MS/MS techniques. J. Agric. Food Chem., 2010, 58, 5959-5972

Description: The sample extraction and clean-up are based on the QuEChERS method.1-6 The final extract is analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Typical reporting limits are listed in the table below. Other reporting limits (at or above LOQ for a given carbamate-matrix combination) can be used based on client requests:

CARBOHYDRATES, TOTAL, BY CALCULATION

Purpose: Applicable for the determination of carbohydrates in foods and feeds.

Method facts:

▶ Sample size: NA

▶ Limit of quantitation: Most matrices - <0.4%

▶ Precision: NA

▶ Method reference: 21 CFR 101.9

Description: Carbohydrate = 100 - [protein(g) + fat(g) + ash(g) + moisture(g)]

L-CARNITINE

Purpose: Applicable for the quantitation of L-carnitine in infant formulas, powders and premixes.

Method facts:

▶ Sample size: 25 g

▶ Limit of quantitation: 0.50 mg/100 g

▶ Precision: 4.15% on an infant formula control

▶ Method reference: Starey et al.; Journal of AOAC INTERNATIONAL vol. 91, No. 1, 2008 (modified)

Description: The sample is diluted in water and a deuterated analog of carnitine is added as an internal standard. It is then filtered and injected onto a Waters Symmetry C8, 50 mm x 2.1 mm, 3.5 µm column, held in an oven set at 30.0°C. Three mobile phases are used in a gradient consisting of 0.1% heptafluorobutyric acid (HFBA) in water, 0.1% HFBA in methanol and 80% water/20% methanol. The amount of L-carnitine is determined using HPLC-MS/MS.

CAROTENOIDS

Option 1: alpha Carotene, beta Carotene, Lycopene Option 2: Cryptoxanthin, Lutein, Zeaxanthin Option 3: Lutein only

Purpose: Applicable to the determination for the following carotenoids in foods, feeds, premixes, pharmaceutical and nutrition supplements: lycopene, α and β-carotene, free lutein, lutein esters, β-cryptoxanthin, or zeaxanthin. Vitamin A from carotenes may be calculated.

Method facts:

▶ Sample size: 25 g for food or feed products; 2 g for supplements and premixes

Carbamate Pesticide LOQ (mg/kg)

Aldicarb 0.01

Aldicarb sulfone 0.01

Aldicarb sulfoxide 0.01

Bendiocarb 0.01

Butocarboxim 0.02

Butoxycarboxim 0.01

Carbaryl 0.01

Carbofuran 0.01

Carbofuran, 3-hydroxy- 0.01

Chlorpropham 0.01

Dioxacarb 0.01

Ethiofencarb 0.01

Fenobucarb 0.01

Fenoxycarb 0.01

Indoxacarb 0.02

Iprovalicarb 0.01

Isoprocarb 0.01

Methiocarb 0.01

Methiocarb sulfone 0.01

Methiocarb sulfoxide 0.01

Methomyl 0.02

Metolcarb 0.01

Oxamyl 0.01

Promecarb 0.01

Propham 0.01

Propoxur 0.01

Thiofanox 0.05



ASSAYS

▶ Limit of quantitation: 0.0200 mcg/100 g, but can vary by sample size

▶ Precision: Varies with matrix

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL, Current Ed., Method 2005.07, AOAC INTERNATIONAL, Gaithersburg, MD, USA (modified)

Official Methods of Analysis of AOAC INTERNATIONAL, Current Ed., Methods 941.15, 2005.07, AOAC INTERNATIONAL, Gaithersburg, MD, USA (modified)

Quackenbush, F. W., Reverse Phase HPLC Separation of cis- and trans-Carotenoids and its Application to Beta Carotenes in Food Materials,” Journal of Liquid Chromatography, 10: 643-653 (1987) (modified)

Description: Low-fat samples are extracted with alcohol and/or tetrahydrofuran. Samples with a higher level of fat are saponified and extracted with hexane. Each sample is then injected on a reverse phase high-performance liquid chromatography system (HPLC) with ultraviolet (UV) detection. Quantitation is achieved with a linear regression analysis.

CATECHINS

Epigallocatechin, catechin, epigallocatechin gallate, gallocatechin gallate, gallic acid, epicatechin, epicatechin gallate, gallocatechin, catechin gallate.

Purpose: Applicable for the determination of catechins in various matrices including extracts (e.g. grape seed), tea, fortified products and premixes.

Method facts:


▶ Limit of quantitation: Most matrices-100 ppm, liquids-1 ppm

▶ Precision: On a green tea matrix, the RSD is 4.09%

▶ Method reference: Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., and Kanazawa, K., “Simultaneous Determination of All Polyphenols in Vegetables, Fruits, and Teas,” Journal of Ag Food Chem., 51(3):572-580 (2003)

Description: Sample extraction is performed using a buffered solution. A portion of that extract is analyzed on a HPLC using UV detection. Analytes are then quantitated by comparing the response of a standard of known concentration.

CHLORIDE

Purpose: Applicable for the determination of acid-soluble chloride in feeds, plants, food products and tissues.

Method facts:


▶ Limit of quantitation: Most matrices - 200 ppm

▶ Precision: On a cereal matrix, the RSD is 1.01%

▶ Method reference: AOAC 963.05, 969.10, 971.27 (modified)

Description: Samples are weighed, double-deionized water is added and the solution is mixed thoroughly and made acidic with nitric acid. Chloride is determined potentiometrically by titrating with a silver nitrate solution to a predetermined endpoint.

CHLOROGENIC ACID

Purpose: Applicable for the determination of chlorogenic acid in raw material, extracts and dietary supplement preparations.

Method facts:


▶ Limit of quantitation: 50 ppm, 1 ppm for liquids

▶ Precision: 5.02%

▶ Method reference: Journal of Ag Food Chem, 51(3):572-580

Description: The sample is extracted in a variety of ways depending upon the matrix. A portion of the extract is injected into an HPLC using UV detection. Chlorogenic acid is quantified by comparing the response to a known standard.

CHLOROPHYLL

Purpose: Applicable to the determination of total chlorophyll content derived from plants.

Method facts:


▶ Limit of quantitation: Most matrices - 0.01 mg/g

▶ Precision: NA

▶ Method reference: AOAC Official Methods of Analysis of AOAC International (2006) 18th Edition, AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 942.04. (modified)



ASSAYS

Description: Chlorophyll is extracted from the sample with acetone. The sample extract is then washed with ethyl ether in a separatory funnel. The chlorophyll migrates to the ethyl ether layer and the remaining acetone is removed through a series of water rinses. The ethyl ether extract is then read at 642.5 and 660 nm on a UV-VIS spectrophotometer to determine the total chlorophyll content.

CHOLESTEROL

Purpose: Applicable for the determination of cholesterol in most matrices including foods, feeds and fecal samples.

Method facts:


▶ Limit of quantitation: 1 mg/100 g

▶ Precision: On a ground waffle matrix, the RSD is 6.0%


Description: The sample is saponified using ethanolic potassium hydroxide. The unsaponifiable fraction that contains cholesterol and other sterols is extracted with toluene. The toluene is evaporated to dryness and the residue is dissolved in dimethylformamide (DMF). The samples are derivitized to form trimethylsilyl ethers. The derivitized cholesterol is quantitatively determined by gas chromatography using 5 a-cholestane as an internal standard.

CHOLINE (CHEMICAL)

Purpose: Applicable for the determination of choline in premixes, infant formula, dietary supplements and feeds.

Method facts:


▶ Limit of quantitation: Most matrices - 15.1 mg/100 g; liquid samples - 3.3 mg/100 g

▶ Precision: On a powdered infant formula matrix, the RSD is 6.31%

▶ Method reference: Glick, Journal of Biological Chemistry, 156:643 (1944)

Description: The sample is hydrolyzed with mild acid. The resulting solution is purified on an ion exchange column. The concentration of choline is determined spectrophotometrically from a reineckate coupling reaction.

CHOLINE (ENZYMATIC)

Purpose: Applicable for the determination of choline in milk, infant formula, foods and beverages.

Method facts:


▶ Limit of quantitation: Most matrices - 25 mg/100 g

▶ Precision: On an infant formula matrix, the RSD is 3.78%


Description: The product is hydrolyzed at 70°C to release bound choline. Following pH adjustment, residual choline phospholipids are cleaved with phospholipase D and free choline is subjected to choline oxidase with liberation of peroxide. In the presence of peroxidase, phenol is oxidized and a quinoneimine chromiphore is formed with 4-aminoantipyrine. Absorbance is measured and choline content calculated by interpolation from a multilevel calibration.

CHONDROITIN SULFATE BY CPC

Purpose: Applicable for the determination of chondroitin sulfate in tablets, premixes, capsules and other dietary supplements. Sources may include porcine, bovine or shark.

Method facts:


▶ Limit of quantitation: 25 mg/g

▶ Precision: On a tablet, the RSD is 1.87%

▶ Method reference: USP

Description: Sample extraction is performed with water and agitation. Extracts are analyzed on an auto-titrator equipped with a light emitting phototriode. Samples are calculated against a standard curve of known concentrations.

CHROMIUM (ATOMIC ABSORPTION)

Purpose: Applicable for the determination of chromium in feeds, animal tissues, food products, plants, dietary supplements, water, soils and minerals.

Method facts:



▶ Precision: Based on a powdered drink control, the RSD is 1.90%



ASSAYS

▶ Method reference: Analytical Methods for Atomic Absorption Spectrophotometry, Perkin-Elmer: Norwalk, CT (2000)

AOAC 974.27

Description: The sample can either be dry-ashed, wet-ashed or read directly. If dry-ashed, the sample is ashed at 500°C ±50° until ashing is complete. The resulting ash is treated with concentrated hydrochloric acid, dried and redissolved in hydrochloric acid solution. If wet-ashed, the sample is digested on a hot plate with nitric acid, hydrochloric acid and/or hydrogen peroxide.

The amount of chromium is determined by comparing the signal of the unknown sample, measured by the atomic absorption (AA) spectrophotometer, with the signal of the standard solutions.

CHROMIUM (ICP OR ICP-MS)

see Inorganic analysis by ICP or Inorganic analysis by ICP-MS

CITRIC ACID


CITRUS BIOFLAVONOIDS

Purpose: This method is applicable to the determination of common citrus bioflavonoids: rutin, narirutin, naringin, hesperidin, neohesperidin, quercetin, naringenin, hesperetin, nobiletin and tangeretin in dietary supplements (tablets, capsules and powders).

Method facts:

▶ Sample size: 10g

▶ Limit of quantitation: 1.2 mg/g

▶ Method reference: Covance developed method

Description: Each sample is sonicated in dimethyl sulfoxide, methanol is added and the mixture is again sonicated. The extract is analyzed by ultra high performance liquid chromatography (UHPLC) with variable wavelength detection.

COBALT (ICP OR ICP-MS)


COENZYME Q10

Purpose: Applicable for the determination of coenzyme Q10 in dietary supplements and raw material.

Method facts:



▶ Precision: Based on a powdered drink control, the RSD is 1.90%


Description: Coenzyme Q10 is extracted using alcohol and hexane. The extract is then injected on a reverse-phase HPLC system using UV detection for quantitation.

COLORS, ARTIFICIAL

Purpose: This method is designed to demonstrate the absence of food coal tar colorants:

▶ FD&C Red No. 2 (Amaranth)

▶ FD&C Red No. 3 (Erythrosin B)

▶ FD&C Red No. 4 (Ponceau SX)

▶ FD&C Red No. 40 (Allura Red)

▶ FD&C Green No. 3 (Fast Green FCF)

▶ FD&C Blue No. 1 (Erigluacine)

▶ FD&C Blue No. 2 (Indigo Carmine)

▶ Acid Orange 12 (Crocein Orange G)

▶ FD&C Yellow No. 5 (Tartrazine)

▶ FD&C Yellow No. 6 (Sunset Yellow FCF)

▶ FD&C Yellow No. 10 (Quinoline Yellow)

Method facts:


▶ Limit of quantitation: 5 ppm

▶ Precision: NA

▶ Method reference: Canadian Food Inspection Agency; HPLC Identification and Quantification of Synthetic Food Colours in Aqueous Media; LCAQ-016-05; 2009 (modified)

Description: Samples are extracted in a basic solution and then injected on an HPLC equipped with a UV detector capable of reading at multiple wavelengths. Samples are then calculated against standards of a known concentration. Analytes detected: FD&C Red No. 2, FD&C Red No. 3, FD&C



ASSAYS

Red No. 4, FD&C Red No. 40, FD&C Green No. 3, FD&C Blue No. 1, FD&C Blue No. 2, Acid Orange 12, FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Yellow No. 10.

CONJUGATED LINOLEIC ACID (CLA)

Purpose: This procedure is used to quantitate conjugated linoleic acids in foods and feeds.

Applicable for foods and feed containing at least 0.2% lipids. The method detects fatty acids having 8 to 22 carbon atoms as methyl esters. The conditions specified in this method are not suitable for determining epoxy or oxidized fatty acids that have been polymerized.

Method facts:

▶ Sample size: 4 g, 1 g if oil

▶ Limit of quantitation: Most matrices - 0.1%

▶ Precision: The RSD value for 18:1 conjugated in an oil control sample is 2.76%.

▶ Method reference: AOAC 996.06/AOCS Ce 1k-07/ Ce 2-66

Description: The lipid is extracted, saponified with 0.5 N methanolic sodium hydroxide, and methylated with 14% BF3-methanol. The resulting methyl esters of the fatty acids are extracted with heptane containing an internal standard. The methyl esters of the fatty acids are analyzed by gas chromatography using external standards for quantitation.

COPPER (ICP OR ICP-MS)


COUMARIC ACID

see Phenolic acids

CRANBERRY (QUINIC, MALIC, CITRIC ACIDS)

see Organic acid profiles

CRANBERRY


CREATINE

Purpose: Applicable for the determination of creatine and creatinine in dietary supplements, sports nutrition products, etc.

Method facts:


▶ Limit of quantitation: 0.5%

▶ Precision: On a creatine capsule, the RSD is 1.87%

▶ Method reference: Analytical Biochemistry, 214, pp278-283

Description: The sample is extracted with water. The extract is injected into an HPLC using UV detection. Results are calculated against a standard of known concentration.

CRYPTOXANTHIN

see Carotenoids

CYANOCOBALAMIN

see Vitamin B12

CYANURIC ACID (LC-MS/MS)

Purpose: Applicable for the determination of cyanuric acid using LC-MS/MS in food products.

Method facts:


▶ Limit of quantitation: 0.5 ppm Cyanuric Acid

▶ Precision: 5% RSD at 0.5 ppm

▶ Method reference: United States Food and Drug Administration, Interim Method for Determination of Melamine and Cyanuric Acid Residues in Food using LC-MS/MS: Version 1.0 Laboratory Information Bulletin No. 4422 (October 2008)

Description: Cyanuric acid is extracted from tissue and infant formula with a 50:50 acetonitrile:water extraction solution, followed by centrifugation and SPE cleanup. The compound is analyzed using a zwitterionic HILIC LC column. Electrospray ionization is used in the negative ion (cyanuric acid) mode. This procedure has been validated by the FDA for the determination of cyanuric acid in tissues. The method has been validated in infant formula powder by Covance.



ASSAYS

Two multiple reaction monitoring (MRM) transitions are monitored for the compound. Isotope internal standards are used to correct for any matrix effects. Fortified test portions were within 85-105% recovery for the infant formula powder validation.

CYSTEINE, FREE

see Amino acid profile, free by AAA

CYSTINE (AOAC)

see Amino acid profile (AOAC)

CYSTINE, FREE

see Amino acid profile, free

DHEA OR 7-KETO DHEA

Dehydroepiandrosterone or 3-acetyl-7-oxo-dehydropiandrosterone

Purpose: Applicable for the determination of DHEA or 7 KETO DHEA in premixes and dietary supplements.

Method facts:



▶ Precision: 4.2%

▶ Method reference: Journal of AOAC INTERNATIONAL, 83(4):847-857

Description: Extraction is conducted by sonicating with methanol. Samples are analyzed by HPLC and quantified by comparison with a known standard.

DENSITY

Purpose: Applicable for the determination of specific gravity in liquid samples.

Method facts:

▶ Sample size: 100 mL

▶ Limit of quantitation: NA

▶ Precision: On a water matrix, the RSD is 0.3%

▶ Method reference: USP <841>

Description: A known volume of sample is weighed. The weight of the sample per unit volume is calculated.

DETERGENT FIBER, ACID (ADF)

Purpose: Applicable for the determination of acid detergent fiber in forages and feeds with low carbohydrate contents.

Method facts:



▶ Precision: On a dog food matrix, the RSD is 14.3%

▶ Method reference: United States Department of Agriculture Forage and Fiber Analysis, Handbook #379.8, United States Department of Agriculture, Washington, D.C. (1970)

Description: The protein, carbohydrate and ash content is removed by treating samples with a boiling detergent solution and filtering. The fats and pigments are removed via an acetone wash leaving lignocellulose fraction in a frit, which is determined gravimetrically.

DETERGENT FIBER, NEUTRAL (NDF)

Purpose: Applicable for the determination of neutral detergent fiber in forages and feeds.

Method facts:



▶ Precision: On commercial dog food matrix, the RSD is 11.1%

▶ Method reference: AACC 32.20

United States Department of Agriculture, Forage and Fiber Analysis, Agriculture Handbook #379.8, United States Department of Agriculture, Washington, D.C. (1970)

Description: The protein, carbohydrate, enzyme, and ash content is dissolved by a boiling detergent solution and filtered off. The fats and pigments are removed via an acetone wash leaving hemicellulose, cellulose, and lignin fractions in a frit and determined gravimetrically.

DIGESTIBLE FAT

Purpose: Applicable to fat and saturated fat in Olestra products, pure Olestra and blended Olestra.

Method facts:





ASSAYS

▶ Precision: On a potato chip the RSD is 9.28%

▶ Method reference: “Capillary Gas Chromatographic Determination of Fat in Olestra Savory Snack Products,” Guererra III, Frank P., Pharmline Inc., personal correspondence, April 1996

Description: Samples are extracted with chloroform-methanol solution, yielding a lipid extract that contains the total fat and olestra. The extracted lipid is hydrolyzed by lipase, yielding unreacted olestra and fatty acid from the fat. The fatty acids are precipitated as calcium soaps. Olestra is extracted from the insoluble soaps with hexane and discarded. The isolated soaps are converted back to fatty acids with hydrochloric acid and extracted into hexane. These fatty acids are converted to methyl esters with boron trifluoride-methanol solution and quantified by gas chromatography using an internal standard.

DISSOLUTION PREPARATION (USP)

Purpose: This test is provided to determine compliance with the dissolution requirements where stated in the individual monograph for a tablet or capsule dosage form, except where the label states that the tablets are to be chewed.

Method facts:

▶ Sample size: 25 tablets or capsules

▶ Method reference: United States Pharmacopeia, <2040>, United States Pharmacopeial Convention, Inc.: Rockville, Maryland (current edition)

Description: Unless otherwise stated in the individual monograph, 6 dosage units are tested as directed under Dissolution <2040>. A stated volume of Dissolution Medium is placed into each vessel of the dissolution apparatus specified in the individual monograph. Once the Dissolution Medium is equilibrated to 37 ± 0.5°C, 1 tablet or 1 capsule is placed into each of 6 vessels. The apparatus is immediately operated at the rate specified in the individual monograph. Within the time interval specified, a specimen is withdrawn from a specific zone of each vessel. Equal volumes from each vessel are filtered and pooled into a single container.

DONG QUAI (FERULIC ACID)

see Phenolic acids

EBDCs (DITHIOCARBAMATES)

Purpose: Applicable for the determination of EBDC residues in milk, eggs, nuts, feeds, crops and formulations. This method screens for EBDC compounds using standards from mancozeb, maneb, metiram, thiram, nabam, ziram and zineb.

Method facts:


▶ Limit of quantitation: Most matrices - 2000 ppb

▶ Precision: Based on spike recovery; most matrices are 70-120%

▶ Method reference: The Analyst, 6:782-787 (July 1981)

Description: EBDCs are quantitatively decomposed to carbon disulfide using a headspace solvent layer procedure. The sample is weighed into a 125 mL serum bottle and a hydrochloric acid-stannous chloride solution is added. Iso-octane is volumetrically pipetted onto the sample and the serum bottle is sealed with a crimp top cap fitted with a Teflon-coated silicone septum. The sample is heated at approximately 80°C in a water bath for 1 hour and allowed to cool to room temperature. The sample is then centrifuged to separate it from the iso-octane layer. The iso-octane layer is then pipetted off the top of the sample and analyzed on a gas chromatograph using a flame photometric detector in sulfur mode.

ECHINACEA SPP.

Caftaric, chlorogenic, echinacoside, chicoric

Purpose: Applicable for the determination of echinacosides in raw material or dietary supplements.

Method facts:


▶ Limit of quantitation: 50 ppm (1 ppm for liquids)


▶ Method reference: Journal of Ag Food Chem., 51(3): 572-580

Description: Extraction technique may vary with matrix. The extract is injected into an HPLC with UV detection. The echinacosides are quantified by comparison to known standards.



ASSAYS

ELDERBERRY


EPHEDRINE ALKALOIDS

Ephedra or Ma Huang

Purpose: Applicable to the analysis of the ephedrine-type alkaloids norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine. This method is applicable to dietary supplements, raw ephedra herb, ephedra extracts, ephedra capsules and high-protein drink mix.

Method facts:

▶ Sample size: 20 tablets, 20 capsules or 10 g

▶ Limit of quantitation: 1 ppm (most matrices)

▶ Precision: NA

▶ Method reference: Sullivan D. et al., “Determination of Ephedra Alkaloids by Liquid Chromatography/Tandem Mass Spectrometry” Journal of AOAC INTERNATIONAL, 3:86

Description: The ephedrine-type alkaloids are extracted from dietary supplements with methanol:water (80:20). The amount of ephedrine-type alkaloids present in dietary supplements is determined by liquid chromatography using tandem mass selective detection (LC-MS/MS).

EGG PROTEIN BY ELISA


Method facts:

▶ Sample Size: 50 g

▶ Limit of quantitation: 2.5 ppm

▶ Method reference: Veratox® Quantitative Egg Allergen Kits Neogen Corporation

Description: Allergen protein is extracted with a buffered salt solution, allowed to settle then placed in an antibody (capture) coated microwell to bind if present. All unbound protein is washed away before a detector antibody (enzyme labeled) is

added. The detector antibody then binds to bound protein if present. After another wash, and the addition of a substrate, color develops as a result of bound detector antibodies. After the addition of a stop reagent, the test is read with a Stat Fax reader. Optical densities of the controls form a standard curve which samples are plotted against to obtain concentrations of allergen protein in ppm.

ERYTHRITOL

see Sugar alcohols

EVENING PRIMROSE OIL

Linolenic acid and GLA


ETHANOL AND METHANOL

Purpose: This method is applicable to the determination of residual alcohols in most matrices. Other analytes commonly requested are acetone, 2-propanol and ethyl acetate.

Method facts:


▶ Limit of quantitation: 10 ppm in most matrices

▶ Method reference: Anthony, Robert M.; Sutheimer, Craig A.; Sunshine, Irving, Acetaldehyde, Methanol, and Ethanol Analysis by Headspace Gas Chromatography, Journal of Analytical Toxicology, Volume 4, Number 1, January 1980, pp. 43-45(3) (modified)

Description: Liquid samples can be analyzed directly or diluted with water prior to analysis. Solid samples should be weighed and diluted to a volume similar to that of the standards.

Samples are incubated at 80°C for approximately 18 minutes and analyzed by gas chromatography with flame ionization detection (GC-FID)

FAT (ACID HYDROLYSIS)

Purpose: Applicable for the determination of fat in most products.

Method facts:




▶ Method reference: AOAC 922.06, 925.32, 933.05, 954.02



ASSAYS

Description: The sample is hydrolyzed with hydrochloric acid. The fat is extracted using ether and hexane. The extract is filtered through a sodium sulfate column. The solvent is evaporated from the remaining extract and the fat is dried and weighed.

FAT (METHANOL/CHLOROFORM)

Purpose: Applicable for the determination of lipids in foods and feed. This method is preferred when the sample contains fish, shellfish or their products.

Method facts:





Description: A known amount of sample is enzymatically hydrolyzed and extracted using chloroform and methanol. An aliquot of the extract is transferred to a tared flask and evaporated to dryness under a stream of nitrogen gas. The lipid residue is dried and weighed and the percent lipid is calculated.

FAT (SOXHLET)

Purpose: Applicable for the determination of fat in meats, seeds and nuts. It may also be used for other foods and feeds by client request.

Method facts:



▶ Precision: On a peanut matrix, the RSD is 0.47%; on a meat matrix, the RSD is 3.69%

▶ Method reference: AOAC 960.39, 948.22

Description: The sample is weighed into a cellulose thimble containing sand or sodium sulfate. The thimble is dried to remove excess moisture. Pentane is dripped through the sample to remove the fat. The extract is then evaporated, dried and weighed.

FAT, TOTAL (ROESE-GOTTLIEB)

Purpose: Applicable for the determination of fat in milk (liquid or powder), buttermilk, half and half, cream and ice cream.

Method facts:



▶ Precision: 4.7% on a dry milk matrix

▶ Method reference: AOAC 989.05, 920.11, 932.06

Description: The sample is hydrolyzed in a water bath using concentrated ammonium hydroxide. The fat is extracted using ether and hexane. The extract is evaporated, dried and weighed.

FAT, TOTAL (NLEA)


FATTY ACID PROFILES

Purpose: This procedure is used for traditional fatty acid profile analysis, including Omega-3 (EPA and DHA) and Omega-6, but not trans fatty acids. The method detects fatty acids having 4 to 24 carbon atoms as methyl esters. The conditions specified in this method are not suitable for determining epoxy or oxidized fatty acids that have been polymerized.

Method facts:



▶ Precision: On a butter matrix, the RSDs for fatty acids are 1-5%

▶ Method reference: AOCS methods Ce 1b-89, Ce 1-62, Ce 1e-91, Ce 1e-07 and Ce 2-66

Description: The extraction can vary depending upon the type of product being analyzed. For some products, the lipid is extracted, saponified and subsequently derivitized. For other products a direct saponification is performed followed by derivatization. The methyl esters of the fatty acids are analyzed by gas chromatography using external standards for quantitation.

FATTY ACID PROFILES (NLEA)

Purpose: This procedure is used for quantitation of NLEA total fat using fatty acid profile analysis to include trans fat, polyunsaturated fatty acids, monosaturated fatty acids, Omega-3 (EPA and DHA) and Omega-6 fatty acids. The method detects fatty acids having 4 to 24 carbon atoms as



ASSAYS

methyl esters. The conditions specified in this method are not suitable for determining epoxy or iodized fatty acids that have been polymerized.

Method facts:



▶ Precision: On a hydrogenated vegetable oil matrix, the RSDs for fatty acids are 1-5%

▶ Method reference: AOAC 996.06, AOCS Ce 1j-07 and Ce 1h-05

Description: The extraction can vary depending upon the type of product being analyzed. For some products, the lipid is extracted, saponified and subsequently derivitized. For other products, a direct saponification is performed followed by derivatization. The methyl esters of the fatty acids are analyzed by gas chromatography using external standards for quantitation.

FATTY ACIDS, FREE (TOTAL BY TITRATION)

Purpose: Applicable for the determination of free fatty acids in vegetable oils and marine oils, animal fats, dark sulfonated oils and other extracted lipids. Reported as oleic acid or acid value.

Method facts:

▶ Sample size: 10 g of food/feed, 5 g of oil


▶ Precision: NA

▶ Method reference: AOCS Ca 5a-40

USP <4017> fats and fixed oils

Description: Lipid is extracted from food and feed samples; oils are sampled directly. The free (uncombined) fatty acids are determined by titration with sodium hydroxide solution.

FATTY ACIDS, FREE (BY GC)

Purpose: Applicable for the determination of any free or extracted anhydrous fat. It is not applicable to acid hydrolysis fat.

Method facts:



▶ Precision: Varies for each fatty acid

▶ Method reference: AOCS Ce 1-62

Description: Free fatty acids in a fat are absorbed on anhydrous alkaline ion exchange resin. Lipid is washed from the resin with petroleum ether. The free fatty acids are converted to methyl esters directly on the resin by treatment with HCl methanol. The methyl esters of the free fatty acids are then determined by gas-liquid chromatography.

FERULIC ACID

see Phenolic acids

FIBER, CRUDE

Purpose: Applicable for the determination of crude fiber in foods, feeds, grains, meals, flours, plant material and other fiber-bearing material from which the fat can be extracted, leaving a workable residue.

Method facts:


▶ Limit of quantitation: Varies with matrix



Description: Crude fiber is the loss on ignition of dried residue remaining after digestion of sample with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions under specific conditions. Note: for products containing fructan sources, a fructan analysis may need to be performed.

FIBER, INSOLUBLE AND SOLUBLE DIETARY (LEE)

Purpose: Applicable to most foods and feeds.

Method facts:

▶ Sample size: Solid matrices-5 g, liquid matrices-10 g


▶ Precision: On a commercial cereal sample, the RSD is 6.83% (IDF) and 13.9% (SDF)

▶ Method reference: AOAC 991.43 (modified)

Description: Duplicate food samples are gelatinized with alpha-amylase and digested with enzymes in a Mes-Tris buffer to break down starch and some protein. The digest is then filtered and the filtrate retained for soluble dietary fiber analysis. The residue (insoluble dietary fiber) is washed with ethanol and acetone to remove starch and protein degradation products and moisture. Residue is dried and weighed. Protein content is determined for one of the duplicates; ash content is



ASSAYS

determined for the other. The soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) contents of the sample are then calculated after adjustment for the protein and ash values. Note: for products containing fructan sources, a fructan analysis may need to be performed.

FIBER, TOTAL DIETARY (LEE)

Purpose: Applicable to most foods.

Method facts:

▶ Sample size: Solid matrices - 5 g/liquid matrices - 10 g




Description: Duplicate food samples are gelatinized with alpha-amylase and digested with enzymes in a Mes-Tris buffer to break down starch and some protein. Soluble dietary fiber in the aqueous enzyme digest is precipitated by treatment with ethanol. The digest is then filtered and the residue is washed with ethanol and acetone to remove starch, protein degradation products and moisture. The residue is dried and weighed. Protein content is determined for one of the duplicates; ash content is determined for the other. The total dietary fiber content of the sample is calculated after adjustment for the protein and ash values. Note: for products containing fructan sources, a fructan analysis may need to be performed.

FIBER, TOTAL DIETARY (PROSKY)

Purpose: Applicable to most foods.

Method facts:





Description: Duplicate samples are gelatinized with alpha-amylase and digested with enzymes in a phosphate buffer to break down starch and some protein. Ethanol is added to each sample to precipitate the soluble fiber. The samples are filtered, and the residue is rinsed with ethanol and acetone to remove starch and protein degradation products and moisture. Protein content is determined for one of the duplicates; ash content is determined for the other. The

total dietary fiber in the sample is calculated after adjustment for the protein and ash values. Note: for products containing fructan sources, a fructan analysis may need to be performed.

FIBER, TOTAL DIETARY (CODEX DEFINITION) BY ENZYMATIC-GRAVIMETRIC METHOD AND LIQUID CHROMATOGRAPHY (MCCLEARY)

Purpose: Applicable for the determination of total dietary fiber and low molecular weight soluble fiber (LMWSF) in food products containing dietary fiber, resistant starch and resistant oligosaccharides.

Method facts:



▶ Precision: Available on request


Description: Duplicate test portions are incubated with pancreatic α-amylase and amyloglucosidase (AMG) for 16 hours at 37°C. During this step, non-resistant starch is solubilized and hydrolyzed to glucose and maltose by the combined action of the two enzymes. Protein in the sample is digested with protease. For the measurement of high molecular weight dietary fiber, ethanol is added and the insoluble and high molecular weight soluble dietary fiber is captured, washed with ethanol and acetone, dried and weighed. One of the duplicate residues is analyzed for protein and the other for ash. The low molecular weight soluble dietary fiber in the filtrate is concentrated on a rotary evaporator, desalted through ion exchange resins, further concentrated and finally analyzed by size exclusion HPLC equipped with a refractive index detector. Results are reported for total dietary fiber (CODEX definition), total resistant oligosaccharides and sum of measured fibers.

FIBER, INSOLUBLE, SOLUBLE, AND TOTAL DIETARY (CODEX DEFINITION) BY ENZYMATIC-GRAVIMETRIC METHOD AND LIQUID CHROMATOGRAPHY (MCCLEARY)

Purpose: This method is applicable for the determination of Insoluble, Soluble and Total Dietary Fiber (IDF, SDF and TDF, respectively) inclusive of resistant starch (RS) and dietary fiber that is not precipitated in 4 parts alcohol, 1 part water (SDFS/LMWSDF) of degree of polymerization (DP) > 3 consistent with the CODEX definition adopted in 2009



ASSAYS

(ALINORM 09/32/REP). This method is applicable to plant material, foods and food ingredients.

Method facts:


▶ Limit of Quantitation: Most matrices - 0.1%

▶ Precision: On a fortified breakfast cereal, the RSD for Total Dietary Fiber is 5.09%

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 2011.25. (modified)

Description: Duplicate test portions are incubated with pancreatic α-amylase and amyloglucosidase (AMG) for 16 hours at 37°C in sealed 250 mL bottles in a shaking water bath while mixing with sufficient vigor to maintain continuous suspension. During this step, non-resistant starch is solubilized and hydrolyzed to glucose and maltose by the combined action of the two enzymes. The reaction is terminated by pH adjustment and temporary heating. Protein in the sample is digested with protease. For the measurement of IDF, the digestate is filtered and the IDF is determined gravimetrically after correction for any protein or ash in the residue. For the measurement of the water soluble, but water:alcohol insoluble dietary fiber (SDFP/HMWSF), ethanol is added to the filtrate of the IDF; the precipitated SDFP is captured by filtration and determined gravimetrically after correction for any protein or ash in the precipitate. Nonprecipitable, water:alcohol soluble dietary fiber (SDFS/LMWSF) in the filtrate is recovered by concentrating the filtrate, deionizing through ion exchange resins, concentrating and quantitating by LC.

FIBER, TOTAL DIETARY (CODEX DEFINITION) BY ENZYMATIC-GRAVIMETRIC METHOD AND LIQUID CHROMATOGRAPHY (FIBERSOL)

with total dietary fiber

Purpose: Applicable for the determination of food products containing resistant maltodextrins.

Method facts:

▶ Sample size: Powder samples 5 g, liquid samples 20 g




Description: Duplicate samples are gelatinized with alpha-amylase and digested with enzymes to break down starch and protein. Ethanol is added to each sample to precipitate the soluble fiber. The samples are filtered, and the filtrate is purified and then concentrated. The sample is then analyzed using an HPLC system with refractive index detection.

FLAVONOIDS IN TEA

With % solids tea and tea products for Tea Association members only

see Polyphenols, total

FLUORIDE (CHEMICAL)

Purpose: Applicable for the determination of total fluoride in a variety of samples, including oils, raw materials, plant tissues, bones, water and many types of foods.

Method facts:


▶ Limit of quantitation: Most matrices - <2.0 ppm

▶ Precision: On a feed sample matrix, the RSD is 6.19%


Description: The sample is weighed into a platinum dish along with a small amount of calcium hydroxide and phenolphthalein to ensure the sample is alkaline. The sample is mixed with double-deionized water to form a slurry. It is then dried on a hot plate, and ashed for at least 4 hours at 550°C in a muffle furnace. The ashed portion is then transferred into a distillation apparatus where the fluoride is steam distilled with perchloric acid. Fluoride levels are then determined titrametrically.

FLUORIDE (ION SELECTIVE ELECTRODE — ISE)

Purpose: Applicable for the determination of fluoride in drinking water, surface water, saline water and domestic and industrial wastes. This method is also applicable to some foods, infant formula and pharmaceutical grade reagents.

Method facts:

▶ Sample size: 20 mL

▶ Limit of quantitation: Most matrices - 0.2 ppm

▶ Precision: On a commercially manufactured mouth formula rinse, the RSD is 5.50%



ASSAYS

▶ Method reference: Standard Methods for the Examination of Water and Wastewater, 17th Edition, Method 4500-F-C., APHA, AWWA, WPCF, Washington, DC (1989) (modified)

Food Chemical CODEX, Sixth Ed. pp 1077-1078, National Academy Press, Washington, D.C. (2009) (modified)

Description: Fluoride is determined potentiometrically by the use of an ion selective electrode. An ionic strength buffer is used to eliminate the effects of other ionic species present in the sample. The concentration of fluoride is calculated using a standard curve.

FOLATE

see Folic acid

FOLIC ACID/FOLATE

Purpose: Applicable for the determination of folic acid in most foods, dietary supplements and feeds.

Method facts:


▶ Limit of quantitation: Most matrices - 0.06 mcg/g


▶ Method reference: AOAC 960.46 & 992.05

Description: The sample is hydrolyzed in potassium phosphate buffer. Following hydrolysis, the sample is treated with a chicken-pancreas enzyme and incubated for approximately 18 hours. Utilizing the bacteria Lactobacillus rhamnosus (casei), the amount of folic acid is determined turbidimetrically by comparing the growth response of a sample against the growth response of a folic acid standard.

FORMIC ACID


FO-TI POWDER

see trans-Resveratrol

FREE AMINO ACIDS

see Amino acid profile, free by HPLC see Amino acid profile, free by AAA

FREE FATTY ACIDS

see Fatty acids, free (by titration) see Fatty acids, free (by GC)

FRUCTAN (HPLC)

Purpose: Applicable for the determination of fructans, which includes inulin and fructo-oligosaccharides (FOS). Well suited for high-level fructan samples.

Method facts:





Description: Fructans are extracted from the matrix with water. The extract is centrifuged, filtered and an appropriate dilution is injected for the analysis of free fructose and sucrose. An aliquot of the filtrate is also subjected to treatment by enzymes to liberate fructose from the fructans. The net fructose content is determined on an HPAEC using PAD and compared against known standards.

FRUCTAN (SPECTROPHOTOMETRIC)

Purpose: Applicable for the determination of fructans, which includes: fructo-oligosaccharides (FOS) and inulin.

Method facts:



▶ Precision: On a spiked flour matrix, the RSD is 2.04%


Description: The sample is extracted with water. The extract is then treated with enzymes to hydrolyze and remove sucrose, starch and maltosaccharides. An aliquot of the filtrate is also subjected to treatment by enzymes to liberate fructose and glucose from fructan. The amount of these reducing sugars from fructan is determined by spectrophotometric methods.

FRUCTOSE

see Sugar profile by GC see Sugar profile by HPLC see Sugar profile by IC see Sugar profile, low levels



ASSAYS

FRUITS

see Anthocyanins, total

FUMARIC ACID


FURFURAL

Purpose: Applicable to the determination of free furfurals.

Method facts:



▶ Precision: 1.7%

Description: The sample is extracted with 4% TCA and injected into an HPLC. Furfural is quantified against standards of known concentration.

FURY (ZETA CYPERMETHRIN)

Information available on request

GALACTOOLIGOSACCHARIDES (GOS) CONTENT IN INFANT FORMULA

Purpose: This method is applicable for the determination of galactooligosaccharides (GOS) in infant formula by Intact Fingerprint Analysis using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

Method Facts:

▶ Sample required: 25 g

▶ Level of quantitation: 0.1%

▶ %RSD: 1.7

▶ Method reference: Covance Internal Method

Description: Powdered infant formula is extracted in water with low heat. The extract from the powdered infant formula or liquid (ready to feed) infant formula is then diluted appropriately with water taking into account the final dilution with acetonitrile. An aliquot is finally treated with acetonitrile to precipitate protein, centrifuged and filtered into an injection vial to be injected onto a high performance anion exchange chromatography (HPAEC) system to separate and quantify 5 specific oligomer peaks that represent the total GOS concentration within the sample. The actual lot used to fortify the product is used as the reference material

to generate a calibration curve for quantification. A reference material is made from a mixture of GOS reference material lots from a specific supplier to generate a calibration curve for quantification if the supplier is known, but the actual lot of reference material used to fortify the product is unknown. The associated error between peak profile differences between individual lots is decreased with this procedure. The same concentration of the reference material is used to represent each of the 5 oligomer peaks separately. The final GOS content of the product is calculated from the average result of the 5 peaks.

GALACTOOLIGOSACCHARIDE (GOS) CONTENT IN RAW MATERIAL

Purpose: This method is applicable for the determination of galactooligosaccharide (GOS) purity in the raw material used to fortify a variety of products in the food industry by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

Method Facts:

▶ Sample required: 10 g

▶ Range of quantitation: 50-75% on a dry basis

▶ %RSD: 1.2

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL (2006) 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 2001.02 (modified)

Dionex Application Note 155: Determination of Trans-Galactooligosaccharides in Foods by AOAC Method 2001.02 (modified)

Description: A buffered extract of the GOS is analyzed directly for the inherent amounts of galactose, glucose and lactose. A separate aliquot of the buffered extract is treated with the enzyme β-galactosidase in order to hydrolyze the GOS oligomers and lactose to galactose and glucose. Appropriate dilutions of the untreated and treated extracts are injected onto a high performance anion exchange chromatography (HPAEC) system to separate and quantify the galactose, glucose and lactose which are detected and quantified by pulsed amperometric detection (PAD). The GOS content is calculated by subtracting the amount of free galactose and the galactose released from lactose from the total galactose (from free, lactose and GOS). A factor determined from the ratio of glucose and galactose released from GOS is applied that corrects for hydrolysis and the contribution from glucose.



ASSAYS

GAMMA AMINOBUTYRIC ACID (GABA)

Purpose: Applicable to all samples prepared for the analysis of gamma aminobutyric acid (GABA).

Method facts:


▶ Limit of quantitation: Varies within matrix

▶ Precision: 1.7%

▶ Method reference: Hischenhuber, C., Deutsche Lebensmittel-Rundschau, “High Performance Liquid Chromatographic and Thin-Layer Chromatographic Determination of Taurine in Infant Formulas,” /84, JAHRG /HEFT 4/1988 (modified)

Stuart, J.D.; Wilson, T.D.; Hill, D.W.; Walters, F.H.; Feng, S.Y., “High Performance Liquid Chromatographic Separation and Fluorescent Measurement of Taurine, a Key Amino Acid,” Journal of Liquid Chromatography, 2(6), 809-821 (1979)

Description: The sample is dissolved in warm water with trichloroacetic acid to precipitate proteins. GABA is then derivatized with orthophthalaldehyde and quantitated on a reverse-phase high-performance liquid chromatography (HPLC) system.

GAMMA LINOLENIC ACID (GLA)

Can be analyzed as part of the fatty acid profile. See Fatty acid profiles.

GARCINIA CAMBOGIA

see Hydroxycitric acid

GARLIC

Purpose: Applicable for the determination of allicin potential in powdered garlic and powdered garlic tablets or capsules.

Method facts:



▶ Precision: On a garlic tablet matrix, the RSD is 8.9%

▶ Method reference: Simultaneous Determination of Alliin and Allicin in Allium Plants and Their Products by Liquid

Chromatography, Journal of AOAC INTERNATIONAL, 80(5) (1997)

Description: Garlic powder is dissolved in a buffer solution. Alliin present in the sample reacts with alliinase enzyme to form allicin. The extract is then filtered and analyzed using HPLC.

GINKGO BILOBA FLAVONOIDS

Purpose: Applicable for the determination of the ginkgo flavones, quercetin, isorhamnetin and kaempferol, in ginkgo biloba or ginkgo biloba extracts.

Method facts:



▶ Precision: 4-7%

▶ Method reference: Journal of Chromatographic Science, 605: 41-48

Description: Samples are extracted with methanol:water. The extract is injected into an HPLC with ELSD detection. The flavones are quantified by comparison with known standards.

GINKGO BILOBA TERPENOIDS

Purpose: Applicable for the determination of ginkgo terpenes in ginkgo biloba or ginkgo biloba extracts.

Method facts:



▶ Precision: 6% for the major compounds

▶ Method reference: Journal of Chromatographic Science, vol 36, (April 1998)

Description: Samples are extracted with methanol/water. The extract is injected into an HPLC system equipped with an ELSD. Results are quantified by comparison to known standards.

GINSENG, PANAX OR KOREAN (GINSENOSIDES)

Purpose: Applicable for the determination of ginsenosides in powdered ginseng root and ginseng extracts.

Method facts:




ASSAYS

▶ Limit of quantitation: Varies with ginsenoside compound and matrix

▶ Precision: 1-6% depending upon the ginsenoside compound and matrix

▶ Method reference: Journal of Chromatographic Science, 736: 77-81

Description: Samples are extracted with methanol/water. The extract is injected into an HPLC system with ELSD. Results are quantified by comparison to known standards. Ginsenosides tested include Rf, Rg1, Re, Rb1, Rc, Rb2 and Rd.

GLUCOSAMINE

Purpose: Applicable for the determination of glucosamine compounds in premixes, tablet preparations, raw materials, dietary supplements and pet foods.

Method facts:

▶ Sample size: 5 g – 20 tablets


▶ Precision: 5.7%


Description: Glucosamine is extracted with water. Triethylamine is used to release glucosamine free base. The solution is derivitized and injected into an HPLC with UV detection. Results are quantified against known standards.

GLUCOSE


GLUTEN/GLIADIN

Purpose: Gluten is an allergen that is a mixture of prolamin and glutelin proteins from wheat (gliadin), rye (secalin) and barley (hordein). Celiac disease is a permanent intolerance to gluten that results in damage to the small intestine. Those individuals with this intolerance must avoid gluten, and rely upon the correct labeling of food to make appropriate food choices. Testing for the presence of gluten ensures food manufacturers that an unlabeled, and potentially dangerous, ingredient did not make its way into a food product.

Method facts:


▶ Limit of quantitation: 5 ppm gluten (2.5 ppm gliadin)

▶ Method reference: RIDASCREEN® Gliadin Kit r-biopharm AOAC RI License Number 120601, Official Method (SM) number 2012.01

Description: The basis of this test is the antigen-antibody reaction. Samples are extracted using a RIDA® Extraction Solution specific to this ELISA kit. After extraction and sample dilution, standards and samples are placed in microwells coated with specific antibodies against gliadins. Present gliadin will bind to the capture antibodies resulting in an antibody-antigen-complex. Components not bound are removed with a washing step. Antibody conjugated to peroxidase is added. This antibody-conjugate is bound to the Ab-Ag-complex (antibody-antigen) forming the “sandwich.” Any unbound enzyme conjugate is removed with another washing. Enzyme substrate and chromogen are added to the wells and incubated. A color reaction occurs where protein is present and results are measured with a Stat Fax reader. Optical densities of the standard control forms a curve which samples are plotted against to obtain concentrations of gluten/gliadin protein in ppm/ppb.

GLYCEROL (GLYCERINE)

Purpose: Applicable for the determination of glycerol in detergents, cosmetics, foods, feeds products and raw material.

Method facts:


▶ Limit of quantitation: Most matrices - <1.0%

▶ Precision: On a cereal bar matrix, the RSD is 5.58%

▶ Method reference: Sweely, Bentley, Makita and Wells, J.A.C.S. 85: 2495-2507 (1963)

Cierce, A. E., Silylation of Organic Compounds, Pierce Chemical

Description: The sample is extracted with methanol then filtered. Aliquots of the sample and of the standard solutions are derivitized with Tri-Sil followed by extraction into hexane. Samples and standards are then injected on a GC System equipped with a flame ionization detector (FID). Quantitation of glycerol is performed by comparing sample responses against standards of known concentrations.



ASSAYS

GOLDENSEAL

Purpose: Applicable for the determination of hydrastine and berberine in goldenseal extracts, tablets, capsules and other dietary supplement preparations.

Method facts:




▶ Method reference: Sakakibara, H.; Honda, Y.; Nakagawa, S.; Ashida, H.; and Kanazawa, K., “Simultaneous Determination of All Polyphenols in Vegetables, Fruits, and Teas,” Journal of Ag Food Chem, 51(3):572-580 (2003)

Description: Samples are extracted using the appropriate technique for the sample matrix. Aliquots are analyzed using HPLC with UV detection and quantified by comparison to known standards.

GRAPE SEED EXTRACT

see Catechins see Polyphenols, total see trans-Resveratrol

GREEN TEA

see Catechins see Polyphenols, total

GRIFFONIA SEED EXTRACT

see 5-Hydroxytryptophan (free amino acid profile)

HAZELNUT PROTEIN BY ELISA


Method facts:



▶ Method reference: Veratox® Quantitative Hazelnut Allergen Kits Neogen Corporation


HEAVY METALS AS LEAD (USP <231>)

Purpose: Applicable for the determination of metallic impurities (Ag, As, Bi, Cd, Cu, Hg, Mo, Pb, Sb, Sn) that are colored by the sulfide ion. Primarily applicable to foods, food additives, feeds and raw materials.

Method facts:



▶ Method reference: USP <231>

Description: The sample is digested with nitric acid and sulfuric acid on a hot plate, then placed in a muffle furnace at 500°C. Dilute hydrochloric acid is added to the cooled sample and taken to dryness on a hot plate. The pH is adjusted to remove iron impurities. The sample is filtered into a culture tube, thioacetomide solution is added and the color that develops is compared with that of lead standards.

HEAVY METALS BY ICP-MS


HEPTACHLOR


HEXACHLORBENZENE


HEXACHLOROCYCLOHEXANE ISOMERS




ASSAYS

HEXANAL

Purpose: Applicable for the determination of hexanal in foods and ingredients.

Method facts:



▶ Precision: Varies by matrix

▶ Method reference: Journal of Food Science, 67:1212-1218

Description: Samples are analyzed by headspace using gas chromatography.

HPTLC

Identity verification testing by high-performance thin-layer chromatography

Purpose: HPTLC is a versatile, high-throughput and cost-effective tool used to verify the quality and identity of botanical materials.

Method facts:


▶ Limit of quantitation: HPTLC for identity verification is not quantitative

▶ Precision: NA

▶ Method reference: Varies by matrix

Covance currently has the following reference materials available:

Common Name Species Name Plant Part

American Ginseng Panax quinquefolium Root

Bilberry Vaccinium myrtillus Fruit

Bitter Melon Momordica charantia Leaf

Bitter Melon Momordica charantia Fruit

Black Cohosh Actaea cimicifuga/racemosa Root

Carrot Daucus carota Root

Chaste Tree Vitex agnus-castus Berry

Chinese Ginseng Panax ginseng Root

Chinese Goldthread Coptis chinesis Root

Chinese Licorice Glycyrrhiza uralensis Root

Cinnamon Cinnamon cassia Bark

Fenugreek Trigonella foenum-graecum Seed

Gentian Root Gentiana lutea Root

Ginkgo Ginkgo biloba Leaf

Grape Seed Vitis vinifera Seed

Green Tea Camellia sinensis Leaf

Hibiscus Hibiscus rosa Flower

Hibiscus Hibiscus sabdariffa Flower

Hops Humulus lupulus Strobile

Indian Frankincense Boswellia serrata Resin

Isatis Root Isatis inidgotica Root

Licorice Glycyrrhiza glabra Root

Motherwort Leonura cardiaca Herb

Nettles Urtica dioica Root

Pumpkin Cucurbita pepo Seed

Pygeum Bark Prunus africana Bark

Red Sage Salvia miltiorrhiza Root

Reishi Mushroom (Black) Ganoderma sinense Fruiting body

Rosemary Rosmarinus off icinalis Leaf

Saw Palmetto Serenoa repens Fruit

Stevia Stevia rebaudiana Leaf

Turmeric Curcuma longa Rhizome

Description: Methods vary by matrix. Currently there are over 100 methods available at Covance for HPTLC identity verification testing. Proper documentation of the sample material by genus, species, part, extract and any additional information that can be provided to the lab is critical in providing HPTLC verification. In general, the method involves an extraction, application to a thin-layer stationary phase plate, application of a mobile phase, derivitization and



ASSAYS

exposure to the appropriate wavelength of light. Each sample separation is monitored for banding (Rf ) which indicates the migration of active ingredients in the raw material. To ensure the fastest possible turn around time of samples, please provide a certificate of analysis with each sample to verify the method that will be needed for the sample.

HYDRASTINE

see Goldenseal

HYDROXYCITRIC ACID (GARCINIA EXTRACT)

Purpose: Applicable for the determination of hydroxycitric acid in dietary supplement products.

Method facts:





Description: Samples are extracted with acid and clarified with filtration/centrifugation. The extract is injected into an HPLC system with UV detection. Results are quantified by comparison with known standards.

HYDROXYPROLINE

see Amino acid profile, free or total

INORGANIC ANALYSIS BY ICP

Purpose: Applicable for the determination of up to 20 elements (ppm levels) in virtually all matrices. See below for options. Options 3 and 4 are matrix dependent. Please call to verify if the package price is applicable to your sample.

Option 1: Calcium, iron, sodium (for nutrition labeling)

Option 2: Calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc

Option 3: Option 2 plus aluminum, barium, boron, chromium, molybdenum, strontium

Option 4: Option 3 plus beryllium, cadmium, cobalt, nickel, vanadium

Individual elements can be performed on request.

Method facts:


▶ Limit of quantitation: Element and matrix dependent

▶ Precision: For most elements, the RSD is <5%


Description: The sample can be either dry-ashed, wet-ashed or read directly. If dry-ashed, the sample is ashed at 500˚C ±50˚ until ashing is complete. The resulting ash is treated with concentrated hydrochloric acid, dried and redissolved in hydrochloric acid solution. If wet-ashed, the sample is digested on a hot plate with nitric acid, hydrochloric acid and/or hydrogen peroxide. The amount of each element is determined with an ICP spectrometer by comparing the emission of the unknown sample against the emission of each element from standard solutions.

INORGANIC ANALYSIS BY ICP-MS

Typical heavy metal scan: arsenic, cadmium, lead, mercury (antimony included upon request). Additional available elements: Ag, As, Ba, Be, Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Ge, Hg, I, La, Li, Mg, Mn, Mo, Ni, Pb, Pd, Pt, Rb, Ru, S, Sb, Sn, Sr, Ti, Tl, U, V, W, Zn (other non-routine elements available upon request. Sulfate (calculated from total sulfur by ICP-MS).

Purpose: Applicable for the determination of low level metals (ppb levels) in foods, food ingredients, feeds, biological materials and various other matrices.

Method facts:



▶ Precision: For most elements, the RSD is <5%


Description: The sample is wet-ashed with nitric acid using closed-vessel microwave digestion. Depending on matrix, alternate digestion techniques may include open-vessel microwave digestion or dry ashing. Samples that are readily soluble in water may be dissolved, acidified and then analyzed directly. Using ICP-MS, the amount of each element is determined by comparing the counts generated by the unknowns to those generated by standard solutions of known concentration.



ASSAYS

IODINE, HIGH LEVEL

Purpose: Applicable for the determination of total iodine in premixes, dietary supplements, salts and various raw materials.

Method facts:



▶ Precision: On a multivitamin matrix, the RSD is 11.1%


USP

Description: The sample is digested and fused. The sample is then filtered using a hot water wash into a 500 mL, wide-mouth Erlenmeyer flask until the volume is 300 mL. The sample is neutralized to methyl orange with 85% phosphoric acid (H

3PO

4), with 1 mL in excess. A slight excess

of bromine (as saturated bromine water) is then added, and the solution is boiled for 5 minutes after it has become colorless. Any excess bromine that remains is removed by the addition of salicylic acid. When the solution is cool, 1 mL of 85% H

3PO

4 and about 0.5 g of potassium iodide (KI)

are added. The resulting iodine is titrated with thiosulfate (Na

2S

2O

3) (0.005 N or 0.10 N) to a clear end point.

IODINE VALUE (TITRATION)

Purpose: The iodine value is used to measure the amount of unsaturation of fats and oils and is expressed in terms of the number of centigrams of iodine absorbed per gram of sample. It is a applicable to all normal fats and oils with iodine values in the range of 15 to 70. It is not applicable to products containing conjugated double bonds.

Method facts:



▶ Precision: NA

▶ Method reference: AOCS Cd 1b-87

Description: The sample is dissolved in cyclohexane and a Wijs solution, then titrated to an end point.

INOSITOL

Purpose: Applicable to the determination of inositol in foods, feeds, infant formulas, premixes and nutritional supplement products.

Method facts:




▶ Method reference: Methods of Analysis for Infant Formulas, Infant Formula Council, “Inositol” (modified) Atlanta, Georgia, Section C-7, (1985)

Tagliaferri, E.G.; Bonetti, G.; Casella, G.; Blake, C.J. Ion Chromatographic Determination of Inositol in Infant Formulae and Clinical Products for Enteral Feeding. J. Chromatogr. A. 2000, 879, 129-135. (modified)

Dionex Technical Note 20: Analysis of Carbohydrates by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD (modified)

Description: The inositol sample is extracted with water or acid hydrolyzed at a high temperature and filtered. Samples containing known levels of glycerol undergo enzyme phosphorylation with glycerol kinase. An appropriate dilution is injected onto a high-performance anion exchange chromatography (HPAEC) system equipped with a pulsed amperometric detector (PAD) for determination.

INULIN

see Fructan (spectrophotometric) see Fructan (HPLC)

IODIDE (ION SELECTIVE ELECTRODE — ISE)

Purpose: Applicable for the determination of iodide in dairy products, infant formulas and dietary supplements.

Method facts:

▶ Sample size: 10 g(solids)/50 mL (liquids)


▶ Precision: On a powdered infant formula, the RSD is 8.15%


Description: Samples are weighed and proteins are precipitated with addition of dilute acid. Samples are filtered and iodide is determined using an ion selective electrode and method of standard addition. Nickel nitrate is added to reduce interferences.



ASSAYS

IODINE (ICP-MS)

Purpose: Applicable for the determination of total iodine in a variety of matrices including foods (i.e. powders, liquids, dairy products, infant formulas), nutritional supplements and mineral premixes at levels ranging from low ppm to percent.

Method facts:



▶ Precision: On a milk powder matrix, the RSD is <5%

▶ Method reference: “Determination of Iodine in Nutritional Supplements by ICP-MS,” presented at the 123rd AOAC Annual Meeting, Philadelphia, PA (2009)

“Determination of Iodine by ICP-MS,” presented at the 122nd AOAC Annual Meeting, Dallas, TX (2008)

Description: The sample is weighed into a single-use polypropylene (pp) centrifuge tube. A purified water solution with a basic pH is added. The sample is digested by warming in a microwave to ensure complete extraction of any iodine present in the sample. A stabilizer is added to maintain iodine in the appropriate form. The digested sample is taken to volume with purified water, filtered, then diluted appropriately prior to analysis by ICP or ICP-MS.

IRON (ICP OR ICP-MS)


ISOFLAVONES

Daidzein, glycitein, genistein, daidzin, glycitin, genistin

Purpose: Applicable for the determination of the isoflavones daidzein, glycitein and genistein and their glucoside derivatives found in a variety of food products.

Method facts:



▶ Precision: On an infant formula matrix, the RSDs for daidzin, glycitin, genistin and the total glucosides are 4.7, 10.3, 8.3 and 6.2, respectively

▶ Method reference: Seo, A. and Morr, C. V., “Improved High-Performance Liquid Chromatographic Analysis of Phenolic acids and Isoflavonoids from Soybean Protein Products,” Journal of Agricultural and Food Chemistry, 32(3):530-533 (1984)

Petterson, H. and Kiessling, K.H., “Liquid Chromatographic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed,” Association of Official Analytical Chemists Journal, 67(3):503-506, (1984)

Description: The samples are extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The extract is brought to volume, diluted and centrifuged. An aliquot of the supernatant is placed onto a C18 solid-phase extraction column. The column is washed with 20% methanol and isoflavones are eluted with 80% methanol. The samples are analyzed by HPLC with UV detection against an external standard curve.

ISOMALT

see Sugar alcohols

JAPANESE EXPORT TESTING

▶ Sennosides (UV)

▶ Fenfluramine, HPLC

▶ p-Hydroxybenzoates (methyl, ethyl, propyl, butyl, isopropyl, isobutyl)

▶ Thyroxine, Triiodothyronine HPLC

▶ Benzoic acid, sorbic acid

▶ Artificial colors

▶ Polysorbate


KUDZU

see Isoflavones

LACTIC ACID


LACTITOL

see Sugar alcohols

LACTOSE




ASSAYS

LACTULOSE

Purpose: Applicable for the determination of lactulose in foods and infant formulas at levels below 0.1%.

Method facts:


▶ Limit of quantitation: Levels as low as 0.005% can be detected, large quantities of some sugars can interfere with the proper determination of lactulose.


▶ Method reference: Journal of AOAC INTERNATIONAL, Vol. 88, No. 3, 2005

Description: The sample is extracted with warm water. Lipids are removed with chloroform extraction and proteins are removed by centrifugation. The extract is filtered and appropriate dilutions are injected onto a high-performance anion exchange chromatograph (HPAEC) equipped with a pulsed amperometric detector (PAD).

LEAD (BY ICP-MS)

see Inorganic Analysis by ICP-MS

LIGNIN

Purpose: Applicable for the determination of lignin in forages and feeds with low carbohydrate contents.

Method facts:



▶ Precision: NA

▶ Method reference: United States Department of Agriculture, Forage and Fiber Analysis, Agriculture Handbook #379.8, United States Department of Agriculture, Washington, D.C. (1970)

Description: The protein, carbohydrate and ash content is removed by treating samples with a boiling detergent solution and filtered. The fats and pigments are removed via an acetone wash leaving the lignocellulose fraction in a frit. The cellulose is then dissolved with sulfuric acid leaving the lignin fraction, which is determined gravimetrically.

LINDANE


LOSS ON DRYING (LOD)

Purpose: Applicable for the determination of loss on drying in refined, reagent-grade materials.

Method facts:



▶ Precision: NA

▶ Method reference: USP or FCC

Description: The sample is dried to a constant weight at a temperature specified by the individual monograph.

LUTEIN

see Carotenoids

LYCOPENE

see Carotenoids

MA HUANG

see Ephedrine alkaloids

MAGNESIUM (ICP)


MALIC ACID


MALTITOL

see Sugar alcohols

MALTODEXTRIN, RESISTANT

see Resistant maltodextrin

MALTOSE




ASSAYS

MANDATORY NUTRIENT PACKAGE

This package includes all mandatory labeling nutrients.

Information available on request.

MANGANESE (ICP OR ICP-MS)


MANNITOL

see Sugar alcohols

MARINE LIPIDS (EPA/DHA)


MELAMINE (LC-MS/MS)

Purpose: Applicable for the determination of melamine using LC-MS/MS in food products.

Method facts:


▶ Limit of quantitation: 0.05 ppm melamine

▶ Precision: 6% RSD at 0.05 ppm

▶ Method reference: United States Food and Drug Administration, Interim Method for Determination of Melamine and Cyanuric Acid Residues in Foods Using LC-MS/MS: Version 1.0 Laboratory Information Bulletin No. 4422 (October 2008)

Description: Melamine is extracted from tissue and infant formula with a 50:50 acetonitrile:water extraction solution, followed by centrifugation and SPE cleanup. The compound is analyzed using a zwitterionic HILIC LC column. Electrospray ionization is used in the positive ion (melamine) mode. This procedure has been validated by the FDA for the determination of melamine in tissues. The method has been validated in infant formula powder by Covance. Two multiple reaction monitoring (MRM) transitions are monitored for the compound. Isotope internal standards are used to correct for any matrix effects. Fortified test portions were within 85-105% recovery for the infant formula powder validation.

MELAMINE AND ANALOGS BY UHPLC-MS/MS

Purpose: Applicable to the determination of melamine (MEL) and analogs in infant formula, starch based products and a variety of other foods and ingredients.

Method facts:


▶ Limit of quantitation: LOQ of 10 ppb for each analyte in sample

▶ Precision: 5.9% (RSD)

▶ Method reference: In-house developed method, based on a hydrophilic interaction chromatographic method

Description: Samples are extracted using ultra-pure water and acetonitrile and analyzed by UHPLC-MS/MS. The quantitation of melamine and analogs are performed with the internal standard of each analyte. Those analytes are: melamine, cyanuric acid, ammeline, ammelide, dicyandiamide (DCD).

MERCURY (ICP-MS)


METHIONINE (AOAC)

see Amino acid profile (AOAC), total

METHYLSULFONYLMETHANE (MSM)

Purpose: Applicable for the determination of MSM in tablets, capsules, premixes or raw materials.

Method facts:

▶ Sample size: 5 g raw material, 20 tablets or capsules


▶ Precision: Based on tablet preparation matrix, the RSD is 3.77%

▶ Method reference: Guedes De Pinho, et al., Journal of Agricultural and Food Chemistry, 51:727-732, (2003)

Menta, et al., Journal of Chromatography, 383:400-404 (1986)

Description: The sample is extracted with methanol containing an internal standard. Samples and standards are then injected on a GC System equipped with a flame ionization detector (FID). Quantitation of MSM is done by comparing sample responses against standards of known concentrations.



ASSAYS

MILK PROTEIN, TOTAL BY ELISA


Method facts:



▶ Method reference: Veratox® Quantitative Milk Allergen Kits Neogen Corporation

Description: Allergen Protein is extracted with a buffered salt solution, allowed to settle then placed in an antibody (capture) coated microwell to bind if present. All unbound protein is washed away before a detector antibody (enzyme labeled) is added. The detector antibody then binds to bound protein if present. After another wash, and the addition of a substrate, color develops as a result of bound detector antibodies. After the addition of a stop reagent, the test is read with a Stat Fax reader. Optical densities of the controls form a standard curve which samples are plotted against to obtain concentrations of allergen protein in ppm.

MILK THISTLE

Silychristin, silydianin, silibibin A & B

Purpose: Applicable for the determination of silymarins in dietary supplement products.

Method facts:




▶ Method reference: USP (modified)

Description: Samples are extracted with methanol, filtered, centrifuged and are separated using reverse-phase HPLC and UV detection. Silychristin, silydianin, silybin A&B, isosilybin A&B and dehydrosilybin are identified, the sum of these peak areas is considered total silymarin content of the sample (as silybin equivalents).

MINERAL PROFILES (ICP OR ICP-MS)


MOISTURE, 60˚C (VACUUM OVEN)

Purpose: Applicable for the determination of moisture in sugars, syrups, honey and high sugar products.

Method facts:



▶ Precision: On a syrup matrix, the RSD is 2.70%


Description: The sample is dried in a vacuum oven at 60˚C to a constant weight.


Purpose: Applicable for the determination of moisture in fruits and vegetables.

Method facts:



▶ Precision: On a ground carrot matrix, the RSD is 1.00%




Purpose: Applicable for the determination of moisture in foods and feeds, except for fruit, fruit products, vegetables, vegetable products, sugar and sugar products.

Method facts:








ASSAYS

MOISTURE (KARL FISCHER)

Purpose: Applicable for the determination of water content in most matrices involving a USP or FCC monograph.

Method facts:





Description: The sample is dissolved or suspended in 50 mL of solvent and titrated with Karl Fischer moisture reagent. Milli-Q® water is used to calibrate the instrument.

MOLYBDENUM (ATOMIC ABSORPTION)

Purpose: Applicable for the determination of molybdenum in feeds, animal tissues, food products, plants, vitamin tablets and supplements, water, soils and minerals.

Method facts:



▶ Precision: Determined on a spike recovery basis

▶ Method reference: Analytical Methods for Atomic Absorption Spectrophotometry, Perkin-Elmer: Norwalk, Connecticut, (2000)

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 974.27, AOAC INTERNATIONAL, Gaithersburg, MD, USA, (modified)

Description: The sample can be either dry-ashed, wet-ashed or dissolved and read directly. If dry-ashed, the sample is dried, pre-charred and ashed at approximately 500° C for 5 to 16 hours. The sample is removed from the muffle furnace, cooled, treated with nitric acid, re-ashed and dissolved in dilute hydrochloric acid solution. If wet-ashed, the sample is digested on a hot plate with nitric acid, hydrochloric acid and/or hydrogen peroxide.

Water samples are evaporated to dryness on a hot plate with nitric acid and repeated as necessary to complete ashing prior to dissolution with dilute hydrochloric acid.

The amount of molybdenum is determined by comparing the signal of the unknown sample, measured by the atomic absorption (AA) spectrophotometer, with the signal of the standard solutions.

MOLYBDENUM (ICP OR ICP-MS)


MONOSODIUM GLUTAMATE (AS GLUTAMIC ACID)

see Amino acid profile, free

MYCOTOXINS

Purpose: This method is applicable to the determination of deoxynivalenol, aflatoxin M1, aflatoxin M2, aflatoxin G1, aflatoxin G2, alfatoxin B1, aflatoxin B2, fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, ochratoxin A and zearalenone in ready-to-feed infant formulas and powders.

Method facts:

▶ Sample size: 20 ml

▶ Limit of quantitation:

Ready-to-Feed Infant Formula

(ng/g)

Powdered Infant Formula (ng/g)

Aflatoxin M1 0.01 0.08

Aflatoxin M2+ 0.04 0.3

Aflatoxin B1 0.04 0.3

Aflatoxin B2 0.04 0.3

Aflatoxin G1 0.04 0.3

Aflatoxin G2 0.04 0.3

Ochratoxin A 0.2 1.7

Fumonisin B1 2 16.3

Fumonisin B2 2 16.3

Deoxynivalenol 8.1 64.9

Zearalenone 2 16.2

T-2 Toxin 0.8 6.5

HT-2 Toxin 8.1 64.9

▶ Method reference: AOAC 971.22, Standards for Aflatoxins. Varga, E.; Glauner, T.; Koppen, R.; Mayer, K.; Sulyok, M.; Schumacher, R.; Krska, R. and Berthiller, F., “Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS,” Analytical and BioAnalytical Chemistry, 402:2675-2686 (2012)



ASSAYS

Description: Samples are processed using a two-step mixed solvent extraction and analyzed by ultra high-performance liquid chromatography-tandem mass spectroscopy. The mycotoxins are then quantified against standards of known concentration with stable isotope internal standard dilution.

N-ACETYLCYSTEINE

Purpose: Applicable for the determination of n-Acetylcysteine in raw material.

Method facts:




▶ Method reference: USP (modified)

Description: Prepare sample using a sodium bisulfate solution, dilute to volume and mix. Inject sample into an HPLC system equipped with UV detection.

NL-PROXIMATE (MOISTURE, ASH, PROTEIN)

see individual tests for descriptions

N-METHYL CARBAMATES

see Carbamate pesticides (LC-MS/MS)

NEUTRAL DETERGENT FIBER

see Detergent fiber, neutral

NIACIN

Purpose: Applicable for the determination of niacin in foods, feeds, infant formulas, premixes and pharmaceutical products.

Method facts:



▶ Precision: On a infant formula matrix, the RSD is 4.08%


USP, and Methods of Analysis for Infant Formulas, Infant Formula Council (1985)

Description: The sample is hydrolyzed with sulfuric acid (H

2SO

4) and the pH is adjusted to remove interferences. The

amount of niacin is determined by comparing the growth

response of the sample, using the bacteria Lactobacillus plantarum, with the growth response of a niacin standard. This response is measured turbidimetrically.

NICKEL (ICP-MS)


NITRATE/NITRITE BY HPLC

Purpose: Applicable for the determination of nitrates and nitrites in premixes, foods and dietary supplements.

Method facts:


▶ Limit of quantitation: Most matrices - 1 ppm NO2,

1 ppm NO3

▶ Precision: On a recovery basis

▶ Method reference: Journal of Chromatography, 804:157-160 (1998)

Description: Samples are extracted with water. Lipids are removed with chloroform extraction and proteins precipitated by centrifugation. The extract is filtered and appropriate dilutions are injected onto a high-performance anion exchange (HPAE) system equipped with an electrochemical detector using pulsed amperometric detection (PAD). Nitrates and nitrites are quantitated against standards of known concentration.

NITRATE/NITRITE BY IC AND VIS

Purpose: This method is applicable for the determination of nitrite and nitrate in food and beverages.

Method facts:


▶ Limit of quantitation: 0.50 ppm for nitrite and 1.00 ppm for nitrate. The LOQ for protein samples is 1.00 ppm for nitrite and 2.50 ppm for nitrate.

▶ Method reference: Casanova, J.; Gross, L.; McMullen, S.; and Schenck, F. “Use of Griess Reagent Containing Vanadium (III) for Post-Column Derivatization and Simultaneous Determination of Nitrite and Nitrate in Baby Food,” J. AOAC Int., 89(2): 447-451 (2006) (modified)

Gapper, L.; Fong, B.; Otter, D.; Indyk, H. and Woollard, D. “Determination of Nitrite and Nitrate in Dairy



ASSAYS

Products by Ion Exchange LC with Spectrophotometric Detection,” Int Dairy J., 14: 881-887 (2004) (modified)

George, S.; Ofitserova, M. and Pickering, M., “Simultaneous Determination of Nitrite and Nitrate in Processed Foods,” Method Abstract for Post-column Liquid Chromatography 123, Pickering Laboratories, Inc. (2011) http://www.pickeringlabs.com

(accessed 06 Mar 2013).

Griess Reaction Diagram, www.biotek.com (modified)

Description: Samples are extracted or dissolved with water or buffer. Protein samples are treated with an acetonitrile precipitation and clarified using centrifugation to remove most protein. The clarified protein extract is diluted with 60% (v/v) acetonitrile if necessary and vialed for injection. All other samples are extracted with chloroform and centrifuged to remove lipids. A reverse phase solid phase cartridge is then used to remove any remaining hydrophobic components that may shorten the lifetime of the column. The extract is diluted with water and vialed for injection. Nitrite and nitrate are separated by anion exchange with post column pump and reaction coil. The post column reagent contain Griess reagent and vanadium (III). Nitrite elutes first and is derivatized by the Griess reagent to an azo compound that is detected at 540 nm. When nitrate elutes, it is first reduced to nitrite by vanadium at 95°C. Once converted to nitrite it reacts with the Griess reagent to form the chromophore detected at 540 nm.

NITROSAMINES — VOLATILE IN FOODS

5-batch minimum for analysis.

Purpose: Applicable for the determination of N-nitrosodiemethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) in food products.

Method facts:


▶ Limit of quantitation: Using a 10-g sample, the lowest confidence level for this assay is 1.0 ppb


▶ Method reference: Nitrosamines/One Trap GC-TEA Mineral Oil Vacuum Distillation Analysis, USDA FSIS Method NTR-1 (July 1991)

Description: The sample is vacuum distilled and collected in a liquid nitrogen trap. After distillation, the trap is allowed to warm to room temperature, then extracted with methylene chloride. The methylene chloride extract is concentrated to 1 mL and analyzed on a GC system equipped with a thermal energy analyzer.

NITROSAMINES — VOLATILE IN RUBBER PRODUCTS

5-batch minimum for analysis.

Purpose: Applicable for the determination of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA), N-nitrosodipropylamine (NDPA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) in rubber products.

Method facts:


▶ Limit of quantitation: Using a 10-g sample, the lowest confidence level for this assay is 1.0 ppb



D.C., Havery, T. Fazio, Fd. Chem. Toxic., 20:939-944 (1982)

Description: Volatile N-nitrosamines are extracted from rubber products with methylene chloride. The solution is made alkaline. The methylene chloride and water are distilled and N-nitrosamines in aqueous phase are partitioned back into methylene chloride. The extract is concentrated to 1 mL and analyzed on a GC system equipped with a thermal energy analyzer.

NUCLEOSIDES

Purpose: Applicable for the determination of the nucleosides cytidine, uridine, inosine, guanosine and adenosine in infant formula and some food products.

Method facts:



▶ Precision: On a control sample the RSDs are as follows: cytidine - 5.2%, uridine - 8.1%, inosine - 6.9%, guanosine - 14.5%, adenosine - 4.5%



ASSAYS

▶ Method reference: Olivartes, J. and Verdys, M., “Isocratic High-Performance Liquid Chromatographic Method for Studying the Metabolism of Blood Plasma Pyrimidine Nucleosides and Bases: Concentration and Radioactivity Measurements,” Journal of Chromatography, pp 434 (1988)

Description: The proteins are removed from the sample matrix by acid precipitation and filtration. The filtrate is further cleaned on a phenylboronic acid (PBA) solid-phase extraction column and then eluted with a formic acid solution. The nucleosides are quantified on an HPLC system equipped with a UV absorbance detector against a known standard.

NUCLEOTIDES

Purpose: Applicable for the determination of the nucleotides cytidine-5’-monophosphate (5’CMP), uridine-5’-monophosphate (5’UMP), guanosine-5’-monophosphate (5’GMP), inosine-5’-monophosphate (5’IMP) and adenosine-5’-monophosphate (5’AMP) in infant formula and some food products.

Method facts:


▶ Limit of quantitation: Most matrices - 3.0 mg/100 g

▶ Precision: On an infant formula sample, the RSDs are as follows: 5’AMP - 1.85%, 5’IMP - 4.66%, 5’GMP - 2.24%, 5’CMP - 1.70%, 5’UMP - 4.58%

▶ Method reference: Paubert-Braquet M., Dupont CH. Paoletti R, Foods, Nutrition and Immunity, “Quantification of Nucleotides in Human Milk and their effects on Cytokine Production by Murine Fibroblasts, J774A1 Macrophages and Human Monocytes,” DYN Nutr Res Basel, Karger, 1:22-34 (1992)

Description: The nucleotides are extracted into an aqueous, protein-free filtrate; collected on a solid-phase extraction (SPE) strong anion exchange (SAX) column; and subsequently eluted with phosphate buffer. The eluted nucleotides are quantified using HPLC equipped with UV detection against a standard of known concentration.

ORAC

Total (includes hydrophilic and lipophilic)

Purpose: Applicable for the determination of oxygen radical absorbance capacity in a variety of food products.

Method facts:


▶ Limit of quantitation: 5 micromoles TE/g


▶ Method reference: Journal of Agricultural and Food Chemistry (2003) 3273-3279

Description: ORAC analysis is conducted by subjecting a portion of extracted sample into a system that consists of a fluorescent probe and a free radical generator. This system stimulates the in vivo conditions and quantitatively evaluates the potential of antioxidant protection against cellular damage from the free radical. This is accomplished by monitoring the fluorescence of the probe over time. The extraction of antioxidants can be performed on a hydrophilic basis by means of a single extraction. Results are expressed in terms of Trolox equivalents.

OMEGA 3 FATTY ACIDS


ORGANIC ACID PROFILES

Purpose: Applicable for the analysis of organic acids in foods and food products. Please refer to the following options:

Option 1: Sorbic and benzoic acid

Option 2: Citric acid, malic acid, lactic acid and acetic acid, oxalic acid, pyruvic acid and quinic acid

Option 3: Succinic acid and formic acid, fumaric acid and tartaric acid

Option 4: Propionic acid

Method facts: Option 1


▶ Limit of quantitation: Option 1: 5 ppm


▶ Method reference: Journal of the AOAC INTERNATIONAL, 70(5) (1987)



ASSAYS

Description: Samples are extracted with water and methanol. The organic acids are separated using reverse-phase HPLC and measured with UV detection.

Method facts: Options 2-4



▶ Precision for Option 2: 3.65%

▶ Precision for Options 3 & 4: determined on a recovery basis


Description: Samples are extracted with a weak H2SO

4

solution. Organic acids are separated using reverse-phase HPLC and measured with UV detection. Results are quantified by comparing with a known reference standard.

ORNITHINE

see Amino acid profile, free by AAA

OXALIC ACID


PABA (PARA-AMINOBENZOIC ACID)

Purpose: Applicable for the determination of p-aminobenzoic acid in fortified products, premixes and various preparations.

Method facts:


▶ Limit of quantitation: Most matrices - 150 ppm, liquids-5 ppm

▶ Precision: On a tablet matrix, the RSD is 6.87%


Description: Sample extraction is performed using an aqueous/acetonitrile/acetic acid solution. A portion of that extract is analyzed on an HPLC equipped with UV detection set at 270 nm. P-aminobenzoic acid is then quantitated by comparing the sample response to standards of known concentration.

PANTOTHENIC ACID

Purpose: Applicable for the determination of pantothenic acid in most foods, feeds and dietary supplements.

Method facts:





Description: The sample is diluted with water or treated with an enzyme mixture to liberate the pantothenic acid from co-enzyme A and the pH is adjusted to remove interferences. The amount of pantothenic acid is determined by comparing the growth response of the sample, using the bacteria Lactobacillus plantarum with the growth response of a calcium pantothenate standard. This growth response is measured turbidimetrically.

PARABENS

Methyl, ethyl, propyl, butyl, isopropyl, isobutyl, para-hydroxybenzoates

Purpose: Applicable for the determination of parabenzoates in most samples.

Method facts:




▶ Method reference: Journal of the AOAC INTERNATIONAL (1985)

Description: Samples are extracted in a 50% methanol and 50% Millipore water solution. The samples are filtered and then injected into an HPLC equipped with a UV detector.

PEANUT PROTEIN BY ELISA




ASSAYS

Method facts:



▶ Method reference: Veratox® Quantitative Peanut Allergen Kits Neogen Corporation AOAC RI License Number 030403


PEROXIDE VALUE

Purpose: Applicable for the determination of peroxide value in all normal fats and oils and extracted fats and oils. This method is highly empirical and any variation in procedure may result in variation in results.

Method facts:

▶ Sample size: 20 g of food/feeds, 5 g of oil


▶ Precision: NA

▶ Method reference: AOCS method Cd 8-53

AOAC extraction 983.23

USP <401> fats and fixed oils

Description: This method determines all substances, in terms of milli-equivalents of peroxide per kilogram of sample, that oxidize potassium iodide under the conditions of the test. These are assumed to be peroxides or other similar products of fat oxidation.

PESTICIDE MULTIRESIDUE ANALYSIS (300+ COMPOUNDS)

Purpose: Applicable for the determination of pesticides in various food, dietary supplement, infant formula and other related matrices.

Method facts:

▶ Sample size: 25 g (50 g for high-moisture samples, such as fruits and vegetables)

▶ Limit of quantitation: 0.01 mg/kg for most analytes and matrices


▶ Method reference: 1AOAC Official Method 2007.01

2CEN Standard Method EN 15662

3 Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. Journal of the AOAC INTERNATIONAL, 2003, 86, 412-431

4 Lehotay, S.J.; Mastovska, K; Lightfield, A.R. Journal of the AOAC INTERNATIONAL, 2005, 88, 615-629

5 Lehotay, S.J.; Mastovska, K.; Yun, S. J. Journal of the AOAC INTERNATIONAL, 2005, 88, 630-638

6 Mastovska, K.; Dorweiler, K.; Lehotay, S.J.; Wegscheid, J.; Szpylka, K. J. Agric. Food Chem., 2010, 58, 5959-5972

Description: The sample extraction and clean-up are based on the QuEChERS method.1-6 The final extract is split for the analysis of GC- and LC-amenable pesticides using gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. If requested, the results can be corrected to potential matrix effects using the method of standard addition, which provides the most accurate quantification.

PHTHALATES

Purpose: Applicable for the determination of phthalates in food or food supplement matrices.

Method facts:


▶ Limit of quantitation: 1 ppm, can vary based on matrix. If matrix suppression is observed, results will be reported as “estimated,” and can be confirmed using method of Standard Addition.


▶ Method reference: Test Method for Assay of Di(2-ethylhexyl) phthalate (DEHP) in Food, Taiwan Food and Drug Administration (modified)




ASSAYS

Description: Phthalates are extracted by sonication with methanol and dilution to volume with water. The methanol solution is analyzed and each phthalate is identified based on retention time and specific Multiple Reaction Monitoring (MRM) transitions, one for quantification, and one or more for confirmation of identity. Compounds analyzed include the following:

▶ Butylbenzyl phthalate (BBP)

▶ Dibutyl phthalate (DBP)

▶ Bis(2-ethylhexyl)phthalate (DEHP)

▶ Di-n-octyl phthalate (DNOP)

▶ Diisononyl phthalate (DINP)

▶ Diisodecyl phthalate (DIDP)

PHENOLIC ACIDS

Purpose: Applicable for the determination of ferulic, p-coumaric, sinapic acid and caffeic acids in grains and forages.

Method facts:




▶ Method reference: Hagerman, A. E., Nicholson, R. L., High-Performance Liquid Chromatographic Determination of Hydroxycinnamic Acid in Maize Mesocotyl, Journal of Agriculture Food Chemistry, 3(6):1098-1102 (1982)

Description: The ground sample is extracted using methanol followed by alkaline hydrolysis and buffering prior to injection on an analytical high performance liquid chromatography (HPLC) system for quantification of sinapic acid, p-coumaric acid, caffeic acid and ferulic acid by ultraviolet (UV) detection.

PHOSALONE


PHOSPHOLIPIDS

Lecithin phospholipids, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol

Purpose: Applicable for the determination of lecithin

phospholipids in oil-containing lecithins, deoiled lecithins and lecithin fractions. This method is not applicable for the determination of lyso-PC and lyso-PE.

Method facts:


▶ Limit of quantification: Most matrices - 1%


▶ Method reference: AOCS Ja 7b-91

Description: The sample is extracted with 65:25:1 chloroform:2-propanol:water. The extract is then analyzed on an HPLC system equipped with an evaporative light-scattering detector (ELSD). A calibration curve is used for quantification.

PHOSPHORUS (ICP)


P-HYDROXYBENZOATES

Methyl, ethyl, propyl, butyl, isopropyl, isobutyl

see Parabens

PHYTIC ACID

Purpose: Applicable for the determination of phytic acid in food products.

Method facts:




▶ Method reference: Lehrfeld, J., HPLC Separation and Quantitation of Phytic Acid and Some Inositol Phosphates in Foods, Journal of Agriculture and Food Chemistry, 42:2726-2731 (1994)

Lehrfeld, J., HPLC Analysis of Phytic Acid on a pH Stable, Macroporous Polymer Column, Journal of Cereal Chemists, 66:510-515 (1989)

Description: The sample is extracted using ultrasonication. Purification and concentration is done on a silica based anion exchange (SAX) column. Sample analysis is done on a macroporous polymer HPLC column and a refractive index detector.



ASSAYS

PHYTOSTEROLS

see Sterols

PIPERONYL BUTOXIDE


PIRIMIPHOS-METHYL


POLYAROMATIC HYDROCARBONS

Purpose: Determination of PAH and similar compounds in vegetable oil, fish and seafood as food, foods and feeds.

Method facts:


▶ Limit of quantitation: 2 to 16 ppb, can vary based on matrix


▶ Method reference: NOAA Technical Memo

Description: Samples are extracted with petroleum ether:ethyl ether and injected into a GC. Residues are quantitated using a GC-MS/MS. Compounds analyzed are as follows:

3-Methylcholanthrene Coronene

5-Methylchrysene Cyclopenta(c,d)pyrene

7,12-Dimethylbenz(a)anthracene Dibenz(a,h)anthracene

Acenaphthene Dibenzo(a,h)pyrene

Acenaphthylene Dibenzo(a,i)pyrene

Anthanthrene Dibenzo(a,l)pyrene

Anthracene Dibenzo(a,e)pyrene

Benz(a)anthracene Parathion-methyl

Benzo(a)pyrene Fluoranthene

Benzo(b)fluoranthene Fluorene

Benzo(b)fluorene Dichlorvos

Benzo(b)naphthol(2,1-d)thiophene Phosalone

Benzo(c)phenanthrene Indeno(1,2,3-c,d)pyrene

Benzo(e)pyrene Perylene

Benzo(g,h,i)perylene Phenanthrene

Benzo(j)fluoranthene Pyrene

Benzo(k)fluoranthene Naphthalen

Chysene

POLYDEXTROSE (HIGH LEVEL)

Purpose: Applicable for the determination of polydextrose in premixes, foods and raw materials.

Method facts:



▶ Precision: Based on a spike recovery

▶ Method reference: Journal of Chromatography, 299:288-292 (1984)

Description: The sample is extracted with a warm dilute sulfuric acid solution. The sample is then filtered, and an appropriate dilution is injected in an HPLC equipped with a resin column and a refractive index detector. Samples are then quantitated by comparing responses against a standard of known concentration.

POLYDEXTROSE, WITH ENZYMATIC PRE-TREATMENT

Purpose: Applicable for the determination of polydextrose in foods, food products, pet foods and feeds.

Method facts:




▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL (2005) 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 2000.11 (modified)

Description: Interfering fructans such as inulin, starches and maltodextrins are removed by treating the sample with the enzymes iso-amylase, amyloglucosidase and fructanase. An aliquot of the final extract is filtered through a 0.45 µm filter into an injection vial and then applied to a high-performance anion exchange chromatography (HPAEC) system with pulsed-amperometric detection (PAD). The amount of polydextrose present is determined by comparing the response of a known standard to that of the sample.


ASSAYS

POLYPHENOLS, TOTAL

Folin-Ciocalteu method

Purpose: Applicable for the determination of total polyphenol content of tea leaves and other vegetable products. Vitamin C will interfere with this assay.

Method facts:



▶ Precision: On a green tea sample, the RSD is 2.43%

▶ Method reference: Methods in Enzymology, Oxidants and Antioxidants Part A, 299: 152-178 (1999)

Description: Polyphenols are extracted from the sample with boiling methanol. The sample is reacted with Folin-Ciocalteu reagent (FCR) to produce a color that can be measured spectrophotometrically at 760 nm. Results are reported in units of mg/g gallic acid equivalents (GAE).

POLYSORBATES

Purpose: Applicable for the analysis of polysorbates in foods and supplements.

Method facts:




▶ Method reference: KATO, H.; Nagai, Y.; Yamamoso, K., and Sakabe, Y., “Determination of Polysorbates in Food by Colorimetry with Confirmation by Infrared Spectrophotometry, Thin-Layer Chromatography and Gas Chromatography,” Journal of the Association of Analytical Chemistry, 72(1): 27-29 (1989)

Description: Polysorbates are extracted from the sample with dichloromethane and ethanol. The extract is centrifuged and filtered, and a portion is subjected to a cleanup procedure using a silica column. After exchanging the sample into water, the polysorbates are shaken with dichloromethane and a complexing agent to produce a blue color, which is read on a spectrophotometer.

POTASSIUM (ICP)


POTASSIUM SORBATE


PROANTHOCYANIDINS

Total polyphenols and catechins

see Individual tests

PROPIONIC ACID


PROTEIN (DUMAS)

Purpose: Applicable for the determination of protein in most foods and feeds.

Method facts:





Description: Nitrogen is determined by a combustion-detection technique. Nitrogen is released at high temperatures into a pure oxygen atmosphere and measured by thermal conductivity. The percent nitrogen is converted to the protein using a factor of 6.25.

PROTEIN (KJELDAHL)

Purpose: Applicable for the determination of protein in most foods and feeds.

Method facts:




▶ Method reference: AOAC 955.04, 979.09 (modified)

Description: The protein and other organic nitrogen in the sample is converted to ammonia by digesting the sample with sulfuric acid containing a catalyst mixture. The acid digest is made alkaline, and the ammonia is distilled and titrated with standardized acid. The percent nitrogen is determined and converted to protein using the factor 6.25.



ASSAYS

PUERARIA (ISOFLAVONES)

see Isoflavones

PYGEUM (BETA-SITOSTEROL)

see Sterols

PYRIDOXINE

see Vitamin B6

PYRUVATE (PYRUVIC ACID)


QUERCETIN

see Citrus bioflavonoids

QUINIC ACID, MALIC ACID, CITRIC ACID (CRANBERRY)


REDUCING SUGARS

Purpose: Applicable for the determination of reducing sugars in vegetables, sugar products, beverages, dairy products (except cheese) and food mixtures. It is not applicable to samples with large amounts of ascorbic acid or free amino acids.

Method facts:





Description: The sugars are extracted with water or alcohol, and deproteinized. Reducing sugars are gravimetrically determined by the reaction with copper sulfate-alkaline tartrate, and the remaining copper oxide. An extract may also be treated with hydrochloric acid to invert the non-reducing agents for a total sugar determination. Sugars are calculated using the Hammond table from AOAC Official Methods of Analysis.

RESIDUAL SOLVENTS (USP <467> CLASS 1, 2A, 2B)

Purpose: Applicable for the screening of residual solvents in most matrices. They include, but are not limited to methanol, dichloromethane, hexane, 1,1,1-trichloroethane, cyclohexane, benzene, 1,2-dichloroethane, toluene, 1,1,2-trichloroethane, n-butyl ether and xylenes(o,m,p).

Please specify analytes of interest and required limit of detection when submitting samples.

Method facts:


▶ Limit of quantitation: Most matrices - 1 to 5 ppm


▶ Method reference: Bertsch, Brian, “Developing One Universal Method for Residual Solvents Using the New Teledyne Tekmar HT3 Headspace Sample Introduction System,” Application Note (Teledyne Tekmar), Document #HT3-001 (September 2005)

Description: The samples are introduced to a gas chromatography mass spectrometry system via a Tekmar HT3 purge-and-trap headspace autosampler. A single point standard at the tolerance level is normally used.

RESIDUAL SOLVENTS (USP<467> CLASS 1, 2A, 2B AND 3)

Purpose: This method is applicable to the determination of residual solvents in most matrices. They include USP <467> class 1, 2a, 2b and most class 3 compounds. Class 3 solvents that are not analyzed by this method are: acetic acid, formic acid and dimethyl sulfoxide and all class 2c analytes. The solvents that are a part of this method are tetralin, anisole, cumene, xylenes, ethylbenzene, chlorobenzene, butyl acetate, 2-hexanone, 1-pentanol, isobutyl acetate, toluene, isoamyl alcohol, pyridine, methylisobutylketone, propyl acetate, 1,4-dioxane, methylcyclohexane, trichloroethylene, 1-butanol, n-heptane, isopropyl acetate, 1,2-dichloroethane, 1,2-dimethoxyethane, benzene, carbon tetrachloride, cyclohexane, 1,1,1-trichloroethane, chloroform, tetrahydrofuran, isobutanol, 2-butanol, ethyl acetate, 2-butanone, 1,2-dichloroethene, nitromethane, 1-propanol, n-hexane, tert-butylmethyl ether, methylene chloride, methyl acetate, acetonitrile, isopropanol, ethyl formate, methanol, acetone, 1,1-dichloroethene, diethyl ether, ethanol, and n-pentane.



ASSAYS

Method facts:


▶ Limit of quantitation: Equal or less than USP <467> tolerance for most matrices.

▶ Precision: Pass/Fail

▶ Method reference: Residual solvents by USP <467> class 1, 2a, 2b and 3

Description: Solid and liquid samples are weighed into tared 20 mL headspace vials at a nominal mass of 0.05 g. The samples are dissolved/mobilized in 1 mL DMSO, then diluted with 5 mL of water.

RESISTANT STARCH

Purpose: Applicable for the determination of resistant starch in food products.

Method facts:

▶ Sample size: 0.1 g (most matrices)


▶ Precision: Available on request


Description: Nonresistant starch is eliminated by enzyme treatment. Resistant starch (RS) is recovered and hydrolyzed to glucose, which is then quantified spectrophotometrically at 510 nm. From this, the RS content is calculated.

RETINOL

see Vitamin A

RIBOFLAVIN (VITAMIN B2)

Purpose: Applicable for the determination of riboflavin in most foods and feeds.

Method facts:





The United States Pharmacopeia, 29th Ed., P. 1913

Description: The sample is hydrolyzed with dilute hydrochloric acid (HCl) and the pH is adjusted to remove

interferences. The amount of riboflavin is determined by comparing the growth response of the sample, using the bacteria Lactobacillus casei, against the growth response of riboflavin standard. This growth response is measured turbidimetrically.

ROSE HIPS (ASCORBIC ACID)

Purpose: Applicable for the determination of ascorbic acid as an indicator of rose hips. It measures both ascorbic acid and iso-ascorbic acid if present.

Method facts:



▶ Precision: On a cereal sample matrix, the RSD is 7.33%


Description: The vitamin C in the sample is extracted, oxidized and reacted with o-phenylenediamine to produce a fluorophor. The vitamin C content is determined by comparing the sample extract fluorescence to the fluorescence of known standards.

ROYAL JELLY 10-HDA

10-Hydroxy-2-decenoic acid

Purpose: Applicable for the determination of 10-Hydroxy-2-decenoic acid in royal jelly and some products containing royal jelly.

Method facts:




▶ Method reference: Guererra III, Frank P., Pharmline Inc., “HPLC Determination of 10-Hydroxy-2-decenoic Acid (10-HAD) in Royal Jelly,” personal correspondence (April 1996)

Description: Samples are dissolved in mobile phase using sonication and filtered into autosampler vials. The samples are then analyzed by HPLC equipped with UV detection and compared against an external standard.

RUTIN

see Citrus bioflavonoids



ASSAYS

SAME (S-ADENOSYLMETHIONINE)

Purpose: Applicable for the determination of S-adenosyl methionine in dietary supplements, concentrates and some food products.

Method facts:



▶ Precision: 8.9%

▶ Method reference: d’Avignon, A.; Reepmeyer, J.; Revelle, L.; Zerfine, C., “Stability-Indicating Proton Nuclear Magnetic Resonance Spectroscopic Method for Determination of S-Adenosyl-L-Methionine in Tablets,” 78(2):353 (1995)

Edwards, R., “Determination of S-Adenosyl-L-Methionine and S-Adenosyl-L-Homocysteine in Plants,” 6:25-30 (1995)

Description: The product is extracted with a solution of ammonium phosphate buffer mixed with methanol and analyzed for SAMe content utilizing reverse-phase HPLC system with UV detection.

SAW PALMETTO (STEROLS + FATTY ACIDS)

Purpose: This involves the use of two methods, one for sterols and one for fatty acids, which serve as indicators for the presence of saw palmetto.

Method facts:


▶ Limit of quantitation: Sterols - 0.001%; fatty acids - 0.1%

▶ Precision: Sterols - 3.5%; fatty acids - 2-5%

▶ Method reference: AOAC 994.10 (sterols), AOCS Ce 1-62, Ce 1e-91, Ce 1a-07

Description:

Sterols: The sample (campesterol, beta sitosterol, stigmasterol) is saponified using ethanolic potassium hydroxide. The unsaponifiable fraction that contains cholesterol and other sterols is extracted with toluene. The toluene is evaporated to dryness and the residue is dissolved in dimethylformamide (DMF). The samples are derivitized to form trimethylsilyl ethers. The derivitized sterols are quantitatively determined by GC using 5α-cholestane as an internal standard.

Fatty acids: The lipid is extracted, saponified with 0.5 N methanolic sodium hydroxide, and methylated with 14% BF3-methanol. The resulting methyl esters of the fatty acids are extracted with heptane containing an internal standard. The methyl esters of the fatty acids are analyzed by GC using external standards for quantitation.

SELENIUM BY ICP-MS

Purpose: Applicable for the determination of selenium in a variety of matrices including, but not limited to, infant and adult nutritional formulas, rice, wheat, soy and seeds (e.g. canola).

Method facts:



▶ Precision: The RSD is 3.32% on a non-fat milk powder, 2.08% on a wheat flour, 1.83% on a rice flour, 2.39% on a powdered infant formula, 1.80% on an infant/adult nutritional formula, 7.76% on soy meal and 1.24% on canola meal

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL 18th Ed., AOAC INTERNATIONAL, Gaithersburg , MD, USA, Official First Action Method 2011.19 (2011) (modified)

Description: The sample is digested with concentrated nitric acid and water using a closed-vessel microwave digestion system. After digestion, the sample is transferred to a single-use disposable plastic vessel, then brought to a final volume with water. To normalize the organic contribution between sample and standard, a dilution is prepared for analysis containing methanol. The amount of selenium is determined with inductively coupled plasma-mass spectrometry (ICP-MS) by comparing the counts generated by the unkowns to those generated by standard solutions.

SELENIUM (HYDRIDE GENERATION)

Purpose: Applicable for the determination of selenium in infant formulas, food products and supplements.

Method facts:



▶ Precision: On a rice flour matrix with a certified value of 0.38 ppm, the RSD is 3.78%

▶ Method reference: AOAC 986.15, 996.16, 996.17 (modified)

Perkin Elmer, Flow Injection mercury/Hydrite Analyses, Reccommended Analytical Conditions and General Information, Norwalk, CT (1994) (modified)

Description: The sample is digested with a mixture of nitric and perchloric acids prior to a reduction with hydrochloric



ASSAYS

acid. Following reduction, the samples are reacted with sodium borohydride to produce the volatile selenium hydride, which is measured by atomic absorption spectroscopy. The amount of selenium is determined by comparing the signal of the unknown to the signal of standard solutions.

SENNOSIDES

Purpose: Applicable to the determination of sennosides in food products, laxatives, weight loss pills and other nutritional and medicinal products.

Method facts:



▶ Precision: On a vegetable laxative sample the RSD is 1.70%


Description: A standard of a known concentration of sennosides and samples are extracted in a pH 7.0 buffer solution with sonication. An aliquot is diluted with a sodium borate solution. An aliquot of this solution is then reacted with a sodium dithionite solution in a steam bath in a nitrogen gas atmosphere to form a fluorophor with the sennosides. The intensities of the solutions are measured on a fluorometer at an λex = 390, λem = 500. A blank solution is treated similarly, but not reacted with sodium dithionite. Its fluorescence intensity is subtracted from that of the sample solution to correct for background fluorescence in the sample. Results are calculated in reference to the calibration standard.

SESAME PROTEIN BY BIOKITS


Method facts:


▶ Limit of quantitation: 6.25 ppm Sesame

▶ Method reference: BioKits Sesame Assay; Neogen Corporation

Description: Allergen Protein is extracted with high salt Tris buffer solution, allowed to settle then placed in antibody (capture) coated micowell to bind if present. All unbound

protein is washed away then a biotin specific to the allergen is added and incubated. After another wash step, Avidin Peroxidase Conjugate is added and incubated. It binds to already bound protein. After another wash, and the addition of a substrate, color develops as a result of bound detector antibodies. After the addition of a stop reagent, the test is read with a Stat Fax reader. Optical densities of the controls form a standard curve which samples are plotted against to obtain concentrations of allergen protein in ppm.

SILICON (ATOMIC ABSORPTION)

Purpose: Applicable for the determination of silicon in most matrices including feeds, food products, minerals, animal tissues, plants and water samples.

Method facts:




▶ Method reference: Perkin-Elmer, Analytical Methods for Atomic Absorption Spectrophotometry, Norwalk, CT (2000)

Description: Most samples are dried, precharred, ashed overnight at 500°C ±50°, then fused with a mixture of sodium borate and sodium carbonate. Finally, the sample is dissolved in dilute hydrochloric acid. The amount of silicon is determined by comparing the absorbance of the unknown sample, measured by the atomic absorption spectrophotometer, to the absorbance of the standard solutions.

SINAPIC ACID

see Phenolic acids

SILVER (ICP-MS)


SODIUM (ICP)

see Inorganic Analysis by ICP

SODIUM BENZOATE




ASSAYS

SORBIC ACID


SORBITOL

see Sugar alcohols

SOY ISOFLAVONES

see Isoflavones

SOY PROTEIN BY ELISA


Method facts:



▶ Method reference: Veratox® Quantitative Almond Allergen Kits Neogen Corporation

Description: Allergen Protein is extracted with a buffered salt solution, allowed to settle then placed in antibody (capture) coated micowell to bind if present. All unbound protein is washed away then a detector antibody (enzyme labeled) is added. It binds to already bound protein. After another wash, and the addition of a substrate, color develops as a result of bound detector antibodies. After the addition of a stop reagent, the test is read with a Stat Fax reader. Optical densities of the controls form a standard curve which samples are plotted against to obtain concentrations of allergen protein in ppm.

SPECIES IDENTIFICATION BY ELISA

Beef, Poultry, Pork

Purpose: Applicable for the determination of the presence of a particular meat species.

Method facts:


▶ Limit of quantitation: Economic limit is 1%; scientific limit is 150-250 ppm

▶ Precision: NA

▶ Method reference: ELISA-TEK Cooked Meat Species ELISA

Description: The sample is extracted with a saline solution. The extract is carried into the ELISA test.

STARCH

Purpose: Applicable for the determination of starch in food products, pet foods and feeds.

Method facts:



▶ Precision: On a starch recovery basis, the RSD is 3.73%


Description: The sample is extracted with alcohol to remove carbohydrates other than starches. The starches are hydrolyzed to glucose with α-amylase and amyloglucosidase. Glucose is oxidized with glucose oxidase to form a peroxide, which reacts with a dye in the presence of peroxidase to give a stable color proportional to glucose concentration. The glucose concentration is quantitated spectrophotomerically at 510 nm and used to calculate starch content.

STEROLS

beta Sitosterol, campesterol, stigmasterol, brassicasterol

Purpose: Applicable for the determination of sterols in most matrices including foods, feeds and dietary supplements.

Method facts:


▶ Limit of quantitation: 1 mg/100 g

▶ Precision: On a ground waffle matrix, the RSD is 3.5%


Description: The sample is saponified using ethanolic potassium hydroxide. The unsaponifiable fraction that contains cholesterol and sterols is extracted with toluene. The toluene is evaporated to dryness and the residue is dissolved in dimethylformamide (DMF). The sample is derivitized to form trimethysily ethers. The derivitized sterols are quantitatively determined by GC using 5α-cholestane as an internal standard.

SUCCINIC ACID




ASSAYS

SUCRALOSE (FINISHED PRODUCTS)

Purpose: Applicable for the determination of sucralose in dietary supplements and food products.

Method facts:




▶ Method reference: Journal of the AOAC INTERNATIONAL 71(5):934-937 (1988)

Description: Sucralose is extracted from the sample with water. A portion of the extract is subjected to solid-phase extraction (SPE) clean up. The extract is filtered and appropriate dilutions are injected onto a high-performance anion exchange (HPAE) system equipped with an electrochemical detector using pulsed amperometric detection (PAD). Sucralose is quantitated against standards of known concentration.

SUCRALOSE (PACKETS, PURE MATERIAL)

Purpose: Applicable for the determination of raw material or table top packets of sucralose.

Method facts:

▶ Sample size: 100 mg


▶ Precision: NA

▶ Method reference: USP, FCC

Description: Sample is diluted in water/acetonitrile and analyzed by HPLC equipped with RI detection.

SUCROSE


SUGAR ALCOHOLS

Purpose: Applicable to quantify sorbitol, xylitol, mannitol, maltitol, lactitol, erythritol and iso-maltitol.

Method facts:




▶ Method reference: Dionex Technical Note 20. Analysis of Carbohydrates by High-Performance Anion Exchange Chromatography with Pulsed Amperomeric Detection (HPAE-PAD)

Cataldi, T.R.I.; Campa, C.; Casella, I.G.; Bufo, S.A. Determination of Maltitol, Isomaltitol, and Lactitol by High-pH Anion-Exchange Chromatography with Pulsed Amperomeric Detection. J. Agric. Food Chem. 1999, 47, 157-163.

Description: A water extraction with low heat is used to extract the sugar alcohols. An appropriate dilution is injected onto a high-performance anion exchange chromatograph (HPAEC) system equipped with a pulsed amperomeric detector (PAD) for determination.

SUGAR PROFILE AFTER ACID HYDROLYSIS

Purpose: Applicable to the determination of fructose, galactose, glucose, sucrose, lactose, maltose, raffinose and stachyose in food products, beverages, syrups, feeds and grains.

Method facts:



▶ Precision: On a cereal matrix, the RSDs for fructose, glucose, sucrose and maltose are 5.8%, 9.0%, 3.6% and 4.3%, respectively. On a soybean matrix, the RSDs for raffinose and stachyose are 2.7 % and 4.5%, respectively.

▶ Method reference: Mason, B.S. and Slover, H.T., “A Gas Chromatographic Method for the Determination of Sugars in Foods,” Journal of Agricultural and Food Chemistry, 19(3): 551-554, (1971) (modified)

Brosbt, K., “Gas Liquid Chromatography of Trimethylsilyl Derivations,” Methods in Carbohydrate Chemistry, 6:3-8, Academic Press, New York, NY (1972) (modified)

Description: Sugars are extracted from the sample with sulfuric acid and heat. Aliquots are dried under inert gas and reconstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl-β-D-glucoside as the internal standard. The resulting oximes are converted to silyl derivatives with hexamethyldisilazane (HMDS) and trifluoracetic acid (TFA) treatment and analyzed by gas chromatography using a flame ionization detector.



ASSAYS

SUGAR PROFILE BY GC

Purpose: Applicable for the determination of fructose, galactose, glucose, sucrose, lactose and maltose in a variety of matrices.

Method facts:



▶ Precision: On a cereal matrix, the RSDs for fructose, glucose, sucrose and maltose are 2.22%, 1.99%, 1.19% and 6.01%, respectively

▶ Method reference: Mason, B.S. and Slover, H.T., “A Gas Chromatographic Method for the Determination of Sugars in Foods,” Journal of Agricultural and Food Chemistry, 19(3):551-554 (1971)

Brosbt, K., “Gas Liquid Chromatography of Trimethylsilyl Derivations,” Methods in Carbohydrate Chemistry, Academic Press, New York, NY, 6:3-8 (1972)

Description: Sugars are extracted from the sample with water and methanol. Aliquots are dried under inert gas and reconstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl-β-D-glucoside as the internal standard. The resulting oximes are converted to silyl derivatives with hexamethyldisilazane (HMDS) and trifluoracetic acid (TFA) treatment and analyzed by gas chromatography using a flame ionization detector.

SUGAR PROFILE BY HPLC

Purpose: Applicable for the determination of fructose, glucose, sucrose, maltose and lactose in a variety of matrices. It may not be applicable for samples that are high in salts (especially sodium chloride) or samples with high protein content.

Method facts:



▶ Precision: On a cereal matrix, the RSDs for fructose, glucose, and sucrose are 2.74%, 2.77% and 1.68%, respectively.


Description: Sugars are extracted from the sample with water and methanol. Aliquots are dried under inert gas and reconstituted with an acetonitrile-water solvent. The solutions are injected on an HPLC system equipped with a refractive index (RI) detector.

SUGAR PROFILE BY IC

Purpose: The method is applicable to the determination of fructose, glucose, sucrose, maltose and lactose in food products, beverages, syrups, feeds and grains. It is not applicable for samples containing galactose and lactulose.

Method facts:



▶ Precision: On a cereal matrix the RSDs for fructose, glucose, sucrose, maltose are 1.88%, 3.41%, 2.50% and 5.61% respectively. On a milk powder matrix the RSD for lactose is 0.76%.

▶ Method reference: Official Method No. 982.14, Official Methods of Analysis of AOAC INTERNATIONAL (modified), [Online] 2012, AOAC INTERNATIONAL http://www.eoma.aoac.org (accessed 02 Jan 2013)

Dionex Technical Note 20: Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

Description: Sugars in the sample are extracted with a mixture of equal parts of water and methanol, with the addition of an internal standard. Samples are diluted within the calibration range with water and filtered prior to analysis.

SUGAR PROFILE, LOW LEVELS

Purpose: Applicable for the determination of galactose, glucose, isomaltulose, fructose, sucrose, lactose and maltose in foods and infant formulas at levels below 0.1%.

Method facts:


▶ Limit of quantitation: Levels as low as 0.005% can be detected, large quantities of some sugars can interfere with the proper determination of neighboring sugars. The presence of lactulose may interfere with the determination of extremely low levels of lactose.


▶ Method reference: Dionex Chromatography Database 4.2.0, Record 755

Description: The sample is extracted with warm water. Lipids are removed with chloroform extraction and proteins are removed by centrifugation. The extract is filtered and appropriate dilutions are injected onto a high-performance anion exchange chromatograph (HPAEC) equipped with a pulsed amperometric detector (PAD).



ASSAYS

SULFITES (MONIER-WILLIAMS)

Purpose: Applicable for the determination of total sulfite in food products, plant tissue, and most other materials in the presence of other volatile sulfur compounds.

Method facts:





Description: The sulfite is extracted from the sample in a distillation flask containing hydrochloric acid, to which a condenser is connected. As the distillation progresses, nitrogen gas is used to carry the sulfite out the top of the condenser into receptacles containing hydrogen peroxide. The resultant sulfuric acid solution is titrated with sodium hydroxide to determine the sulfite content.

SULFUR (ICP-MS)


TARTARIC ACID


TAURINE

Purpose: Applicable for the determination of taurine in most food, dietary supplements and feeds.

Method facts:



▶ Precision: On a cat food matrix, the RSD is 3.5%

▶ Method reference: Henderson, J.W.; Ricker, R.D.; Bidlingmeyer, B.A.; Woodward, C., “Rapid, Accurate, Sensitive, and Reproducible HPLC Analaysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA columns and the Agilent 1100 HPLC,” Agilent Publication, 2000

R. Schuster, “Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivitization and HPLC,” Journal of Chromatography, 1988, 431, 271-284

AOAC 999.12

Description: The sample is hydrolyzed in hydrochloric acid and is quantitated using an automated amino acid analyzer.

THEOBROMA CACOA

see Caffeine, theobromine, theophylline

THEOBROMINE


THEOPHYLLINE


THIAMIN (VITAMIN B1)

Purpose: Applicable for the determination of thiamin in most foods, dietary supplements and feeds.

Method facts:



▶ Precision: On a commercial egg noodle sample, the RSD is 2.44%


Description: The sample is autoclaved in dilute acid to extract the thiamin. The resulting solution is incubated with a buffered enzyme solution to release bound thiamin and hydrolyze starch. The solution is purified on an ion-exchange column. An aliquot is taken and reacted with potassium ferricyanide to convert thiamin to thiochrome. The thiochrome is extracted into isobutyl alcohol and read on a fluorometer against a known standard.

THIOBARBITURIC ACID (TBA) VALUE

Purpose: Applicable for the determination of oxidation products of unsaturated fatty acids as a measure of rancidity in foods.

Method facts:


▶ Limit of quantitation: Most matrices - 1 mg/kg


▶ Method reference: American Oil Chemists’ Society, “Cd 19-90 2-Thiobarbituric Acid Value Direct Method,” Official Methods and Recommended Practices of the AOCS, Fifth Ed., American Oil Chemists’ Society, Champaign, IL (1997)



ASSAYS

Description: The sample is homogenized and distilled at pH 1.5. The 3 carbon fraction (malonaldehyde) derived from oxidation of monoeonic or polyeonic fatty acids is released from rancid foods through acidification and distillation. The malonaldehyde is collected and reacted with TBA to produce a red pigment. The quantity of pigment found is determined by measuring absorbance at 538 nm on a spectrophotometer.

TIN (ICP-MS)


TIN (GRAPHITE FURNACE)


TITANIUM (ATOMIC ABSORPTION)

Purpose: Applicable for the determination of titanium in most materials including feeds, animal tissue, food products, plants, vitamin tablets and supplements, water, soils, minerals and coals.

Method facts:




▶ Method reference: Perkin-Elmer, Analytical Methods for Atomic Absorption Spectrophotometer, Norwalk, CT, (2000)

Description: Most samples are dried, pre-charred, ashed overnight at 500°C ±50°, then fused with a mixture of sodium borate and sodium carbonate. Finally, the sample is dissolved in dilute hydrochloric acid. The amount of titanium is determined by comparing the absorbance of the unknown sample, measured by the atomic absorption spectrophotometer, to the absorbance of the standard solutions.

TOCOPHEROLS

alpha, beta, gamma, delta

Purpose: Applicable for the determination of alpha, beta, gamma and delta tocopherol in oils, foods, feeds and nutritional products.

Method facts:



▶ Precision: On a infant formula matrix, the RSD is 6.10%

▶ Method reference: Cort, W.M.; Vicente, T.S. and Williams, B.D., Journal of Agricultural Food Chemistry, 31:1330-1333 (1983)

Description: The product is extracted with an organic solvent followed by separation on an HPLC silica column, and quantitation using fluorescence detection.

TOCOTRIENOLS

alpha, beta, gamma, delta

Purpose: Applicable for the determination of alpha, beta, gamma and delta tocotrienols in oils, food, premixes and other dietary supplement preparations.

Method facts:




▶ Method reference: Cort, W.M.; Vincente, T.S.; Waysek, E.H. and Williams, B.D., Journal of Agricultural Food Chemistry, 31:1330-1333 (1983)

Speek, A.J.; Schijver, J. and Schreurs, W.H.P., Journal of Food Science, 50:121-124 (1985)

McMurray, C.H.; Blanchflower, W.J. and Rice, D.A., Journal of the Association of Official Analytical Chemists, 63:1258-1261 (1980)

Description: The product is extracted with organic solvent, separated on an HPLC silica column and measured using fluorescence detection.

TRANS FAT AND FATTY ACIDS, BY GC

see Fatty acids

TRANS-RESVERATROL

Purpose: Applicable for the determination of trans-Resveratrol in raw material, extracts, capsules, tablets or other dietary supplement preparations.

Method facts:






ASSAYS

▶ Method reference: Sakakibara, H.; Honda, Y.; Nakagawa, S.; Ashida, H. and Kanazawa, K.,“Simultaneous Determination of All Polyphenols in Vegetables, Fruits, and Teas,” J. Agric. Food Chem., 51(3):572-580 (2003)

Description: Sample extraction is performed utilizing the appropriate technique. A portion of that extract is analyzed by HPLC with fluorescence detection; results are then quantified by comparing the response to a standard of known concentration.

TRYPTOPHAN (AOAC)

see Amino acid profile (AOAC), total

TRYPTOPHAN (HPLC)

see Amino acid profile (HPLC), total

USP 561 AND EP PESTICIDES

Purpose: Applicable, but not limited to, dietary supplement products and raw materials for the analysis of pesticides listed in the US and European Pharmacopeias (USP and EP).1,2

Method facts:


▶ Limit of quantitation: Varies by compound; USP and EP have set limits


▶ Method reference: 1 USP34-NF29:561 Articles of Botanical Origin. In: The United States Pharmacopeia, 2011

2 EP 07/2008:20813 Pesticide Residues. In: The European Pharmacopoeia, 7th Edition, 2011

3 AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate

4 CEN Standard Method EN 15662: Food of plant origin – Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE – QuEChERS method

5 Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. Journal of AOAC INTERNATIONAL 2003, 86, 412-431

6 Lehotay, S.J.; Mastovska, K; Lightfield, A.R. Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. Journal of AOAC INTERNATIONAL 2005, 88, 615-629

7 Lehotay, S.J.; Mastovska, K.; Yun, S. J. Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes. Journal of AOAC INTERNATIONAL 2005, 88, 630-638

8 Mastovska, K.; Dorweiler, K.; Lehotay, S.J.; Wegscheid, J.; Szpylka, K. Pesticide multiresidue analysis in cereal grains using modified QuEChERS method combined with automated direct sample introduction GC-TOFMS and UPLC-MS/MS techniques. J. Agric. Food Chem., 2010, 58 5959-5972

9 Lehotay, S.J.; Ae Son, K.; Kwon, H.; Koesukwiwat, U.; Fu, W.; Mastovska, K.; Hoh, E.; Leepipatpiboon, N. Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables. J. Chromatogr. A 2010, 1217, 2548-2560

Description: The sample extraction and clean-up are based on the QuEChERS method.3-9 The final extract is split for the analysis of GC- and LC-amenable pesticides using gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The reporting limits are based on the limits specified in the USP and EP pesticide residue monographs:

Pesticide

USP/EP Limit (mg/kg)

Acephate 0.1

Alachlor 0.05

Aldrin and dieldrin (sum of ) 0.05

Azinphos-ethyl 0.1

Azinphos-methyl 1

Bromide, inorganic (calculated as bromide ion)*** 125

Bromophos-ethyl 0.05

Bromophos-methyl 0.05

Bromopropylate 3

Chlordane (sum of cis- and trans- isomers and

oxychlordane)

0.05

Chlorfenvinphos 0.5

Chlorpyrifos-ethyl 0.2

Chlorpyrifos-methyl 0.1

Chlorthal-dimethyl 0.01



ASSAYS

Cyfluthrin (sum of isomers) 0.1

Cyhalothrin, λ- 1

Cypermethrin (sum of isomers) 1

DDT (sum of o,p’-DDT, p,p’-DDT, o,p’-DDE, p,p’-DDE,

o,p’-DDD, and p,p’-DDD)

1

Deltamethrin 0.5

Diazinon 0.5

Dichlofluanid 0.1

Dichlorvos 1

Dicofol 0.5

Dimethoate and omethoate (sum of ) 0.1

Dithiocarbamates (expressed as CS2)*** 2

Endosulfan (sum of isomers and endosulfan sulfate) 3

Endrin 0.05

Ethion 2

Etrimphos 0.05

Fenchlorphos (sum of fenchlorphos and fenchlorphos-

oxon)

0.1

Fenitrothion 0.5

Fenpropathrin 0.03

Fensulfothion (sum of fensulfothion, fensulfothion-

oxon, fensulfothion-oxon sulfone and fensulfothion

sulfone)

0.05

Fenthion (sum of fenthion, fenthion-oxon, fenthion-

oxon sulfone, fenthion-oxon sulfoxide, fenthion

sulfone and fenthion sulfoxide)

0.05

Fenvalerate 1.5

Flucytrinate 0.05

Fluvalinate, τ- 0.05

Fonofos 0.05

Heptachlor (sum of heptachlor and cis- and trans-

heptachlor epoxides)

0.05

Hexachlorobenzene 0.1

Hexachlorocyclohexane isomers (other than γ) 0.3

Lindane (γ-hexachlorocyclohexane) 0.6

Malathion and malaoxon (sum of ) 1

Mecarbam 0.05

Methacriphos 0.05

Methamidophos 0.05

Methidathion 0.2

Methoxychlor 0.05

Mirex 0.01

Monocrotophos 0.1

Parathion-ethyl and paraoxon-ethyl (sum of ) 0.5

Parathion-methyl and paraoxon-methyl (sum of ) 0.2

Pendimethalin 0.1

Pentachloranisol 0.01

Permethrin (sum of isomers) 1

Phosalone 0.1

Phosmet 0.05

Piperonyl butoxide 3

Pirimiphos-ethyl 0.05

Pirimiphos-methyl (sum of pirimiphos-methyl and

N-desethyl-pirimiphos-methyl)

4

Procymidone 0.1

Profenophos 0.1

Prothiophos 0.05

Pyrethrum (sum of cinerin I, cinerin II, jasmolin I,

jasmolin II, pyrethrin I, and pyrethrin II)

3

Quinalphos 0.05

Quintozene [sum of quintozene, pentachloroaniline

and methyl pentachlorophenyl sulfide (MPCPS)]

1

S-421 0.02

Tecnazene 0.05

Tetradifon 0.3

Vinclozolin 0.4

***Bromide and dithiocarbamates (EBDCs) are determined using different methods.

UBIQUINONE (COENZYME Q10)

see Coenzyme Q10

UNSAPONIFIABLE MATTER

Purpose: Applicable for the determination of normal animal and vegetable fats and oils. It is not suitable for fats and oils containing excessive amounts of unsaponifiable matter such as marine oils.

Method facts:



▶ Precision: NA

▶ Method reference: American Oil Chemists’ Society, Ca 6a-40 Unsaponafiable Matter, Official Methods and Recommended Practices of the AOCS, Fifth Ed., American Oil Chemists’ Society, Champaign, IL (1997)

Description: The sample is saponified and then extracted multiple times using petroleum ether and ethyl alcohol. The solvent is evaporated from the remaining extract into a tared beaker. The residue is then weighed and then titrated to obtain the % unsaponifiable matter. Unsaponifiable matter



ASSAYS

includes substances that are frequently found dissolved in fats and oils that cannot be saponified by the caustic alkalies but are soluble in the ordinary fat solvents. Included are the higher aliphatic alcohols, sterols, pigments and hydrocarbons.

VALERIAN EXTRACT (VALERENIC ACID)

Purpose: Applicable for the determination of acetoxyvalerenic, hydroxyvalerenic and valerenic acid in extracts.

Method facts:




▶ Method reference: Sakakibara, H.; Honda, Y.; Nakagawa, S.; Ashida, H. and Kanazawa, K. “Simultaneous Determination of All Polyphenols in Vegetables, Fruits, and Teas,” J. Agric. Food Chem., 51(3):572-580 (2003)

Description: Samples are extracted using the appropriate technique for the sample matrix. Aliquots are analyzed using HPLC with UV detection. Standards are compared to samples to quantify concentrations.

VANADIUM (ICP OR ICP-MS)


VITAMIN A, BETA CAROTENE

see beta Carotene

VITAMIN A, RETINOL

Purpose: Applicable to the determination of retinol in foods, food products, infant formulas, premixes, pharmaceuticals, pet foods, feeds and other matrices. The method measures all-trans and 13-cis retinol isomers. It does not include contributions of vitamin A activity from pro-vitamin A carotenoids.

Method facts:

▶ Sample size: 30 g in food products, 10 g in supplements and premixes, 50 g in feeds

▶ Limit of quantitation: 1 IU/g but can vary with sample size

▶ Precision: Most matrices - 100I U/100 g

▶ Method reference: Official Methods of Analysis of AOAC International, Current Ed., Methods 992.04, 992.06 and 2001.13, AOAC INTERNATI0NAL, Gaithersburg, MD, USA (modified)

Description: The sample is saponified to break down fat and release the vitamins. The digest is extracted with an organic solvent. Vitamin A is quantitated directly as all-trans retinol and 13-cis retinol by ultra or high performance liquid chromatography (UHPLC or HPLC).

VITAMIN A, TOTAL, RETINOL AND BETA CAROTENE

see beta Carotene and Vitamin A, retinol

VITAMIN A IN DIETARY SUPPLEMENTS

Purpose: This method is for the determination of vitamin A esters in dietary supplements, premixes, liquid concentrates and raw materials. These include, but are not limited to, tablets, capsules, powders and liquids, which contain oil- and water-soluble vitamins with and without minerals. The method is not recommended for matrices with concentrations below 50 µg/g. This method quantifies the following vitamin forms: retinyl acetate and retinyl palmitate.

Method facts:


▶ Limit of quanititation: Most matrices - 100 IU/g

▶ Precision: the RSD is 5.65%

▶ Method reference: Covance developed method based upon the chemical principles of:

U.S. Pharmacopeial Convention, USP-NF [online], current version, Dietary Supplements, Vitamins, Oil- and Water- Soluble Vitamins with Minerals Tablets, Assay for Vitamin A, Method 2. http://www.uspnf.com (accessed 23 Jan 13).

U.S. Pharmacopeial Convention, USP-NF [online], current version, USP Monographs, Vitamin E Preparation

Description: The samples are prepared by one of the following procedures: Direct extraction dispersal with bicarbonate and ascorbate-pyrogallol solution followed by organic extraction or treatment with pancreatin followed by a subsequent phase extraction. Vitamin A esters and free and esterified. The esters are then analyzed using normal phase chromatography.



ASSAYS

VITAMIN B PROFILE BY HPLC

Purpose: Applicable for the determination of vitamins B1, B2, B3, B5 and B6 fortified products, premixes and various preparations.

Method facts:


▶ Limit of quantitation: Most matrices-150 ppm, liquids-5 ppm

▶ Precision: On a tablet matrix, the RSD ranges from 2 to 8%

▶ Method reference: The United States Pharmacopeia, Twenty-Eighth Revision, p. 2162, United States Pharmacopeial Convention, Inc.: Rockville, Maryland (2005)

Description: Sample extraction is done utilizing an aqueous/ acetonitrile/acetic acid solution. A portion of that extract is analyzed on an HPLC equipped with UV detection set at 210 and 270 nm. The analyte is then quantitated by comparing the sample response to a standard of known concentration. The result is then converted and reported in the appropriate form.

VITAMIN B PROFILE BY HPLC-MS/MS

Purpose: Applicable to the determination of up to seven B vitamins in infant formula, various food and dietary supplement products. Vitamins included are thiamin (B1), riboflavin (B2), niacin (B)3, pantothenic acid (B5), pyridoxine (B6), biotin (B7) and folic acid (B9).

Method facts:


▶ Limit of quantitation:

Vitamin Common Name LOQ (µg/g)

B1

Thiamin 0.05

B2

Riboflavin 0.025

B3

Niacin 0.375

B5

Pantothenic Acid 0.25

B6

Pyridoxine 0.05

B7

Biotin 0.025

B9

Folic Acid 0.025

▶ Precision: Ranging from 2 to 5%, depending upon the

vitamin

▶ Method reference: Covance internal method

Description: Samples are extracted with an acidic aqueous-solvent solution followed by addition of a small amount of alkaline solution to precipitate protein and aid with chromatography. Extracts are filtered and introduced to the HPLC-MS/MS system. Results are calculated against known reference standards. Stable Isotope internal standards of each B vitamin are included.

VITAMIN B2

see Riboflavin

VITAMIN B6 (PYRIDOXINE)

Purpose: Applicable for the determination of vitamin B6 (pyridoxine) in most foods, dietary supplements and feeds.

Method facts:





Description: The sample is hydrolyzed with dilute sulfuric acid in an autoclave, and then pH is adjusted to remove interferences. Utilizing the yeast Saccharomyces carlsbergenesis, the amount of B6 is determined turbidimetrically by comparing the growth response of a sample against the growth response of a B6 standard.

VITAMIN B12 (CYANOCOBALAMIN)

Purpose: Applicable for the determination of vitamin B12 in most foods, feeds and dietary supplements.

Method facts:





USP (modified)

Methods of Analysis for Infant Formulas, Infant Formula Council (1985)

Description: Vitamin B12 is extracted from the sample into a buffer by heating in an autoclave. Utilizing the bacteria Lactobacillus delbrueckii, the amount of B12 is determined turbidimetrically by comparing the growth response of a sample against the growth response of a B12 standard.



ASSAYS

VITAMIN C

Purpose: Applicable for the determination of vitamin C in most foods, dietary supplements and feeds. It measures both ascorbic acid and erythorbic acid if present.

Method facts:



▶ Precision: On a cereal sample matrix, the RSD is 7.33%


Description: The vitamin C in the sample is extracted, oxidized and reacted with o-phenylenediamine to produce a fluorophor. The vitamin C content is determined by comparing the sample extract fluorescence to the fluorescence of known standards.

VITAMIN C BY HPLC

Purpose: Applicable for the analysis of ascorbic acid in tablets, capsules, pre-mixes, juices and raw materials.

Method facts:


▶ Limit of quantitation: Most matrices - 2 mg/g

▶ Precision: On a multivitamin sample, the RSD is 2.44%


Asami, D.K. (2003). Journal of Agricultural and Food Chemistry. Vol. 51. p. 1237-1241

Description: The sample is extracted with a m-phosphoric acid and acetic acid solution. The extract is then analyzed on an HPLC system equipped with an UV detector. A calibration curve is used for quantification.

VITAMIN D BY HPLC

Purpose: Applicable to the determination of vitamins D2 or D3 in a wide variety of matrices. These include, but are not limited to, most food products, pet foods, feeds, infant formulas, premixes, multi vitamins and nutraceutical products.

Method facts:

▶ Sample size: 30 g in food products, 5 g in supplements and premixes, 50 g in feeds

▶ Limit of quantitation: 20 IU/100 g in food products and feeds, 14.4 IU/g in supplements and premixes

▶ Precision on an infant formula matrix, the RSD is 7.29%

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL (2005) 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 982.29, 2002.05 (modified)

Official Methods of Analysis of AOAC International, (2000) 17th Ed., Methods 979.24 and 980.26, AOAC INTERNATIONAL, Gaithersburg, MD, USA (modified)

Description: Samples with more than a negligible amount of lipids are saponified and extracted with hexane. The digests are fractionated by alumina open column chromatography and/or normal phase high-performance liquid chromatography (HPLC) to remove interferences. Once the purification of the extracts is complete, the samples are injected on a reverse phase HPLC system using ultraviolet (UV) detection. Some matrices, with adequate levels of vitamin D2 or D3, may not require purification prior to analysis.

VITAMIN D BY LC-MS/MS

Purpose: Applicable to pet food, cereals and other matrices.

Method facts:

▶ Sample size: 10 g per assay

▶ Limit of quantitation: D3 in pet food is 0.05 IU/g; D2 in pet food about 0.2 IU/g

▶ Precision: The RSD is 5%


Description: The sample is extracted into hexane using BHT as a preservative. A portion of the hexane is washed with ethanol/water and centrifuged. The solvent is taken to dryness and reconstituted in ACN/water and filtered through a 0.45 µm PTFE filter. Isotope internal standard is added for LC-MS/MS analysis. The response is compared to that of known standards.

VITAMIN D3 BY HPLC

Purpose: This method is applicable to research and commercial products of infant formulas and nutritional beverages for the determination of vitamin D3.

Method facts:

▶ Sample size: 10 g in powdered infant formulas and 30 g in liquid supplements and liquid infant formulas

▶ Limit of quantitation: 0.1 IU/g



ASSAYS


▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg , MD, USA, Official Method 992.26

Description: Samples with more than a negligible amount of lipids are saponified and extracted with hexane. The digests are fractionated by alumina open column chromatography and/or normal phase high-performance liquid chromatography (HPLC) to remove interferences. Once the purification of the extracts is complete, the samples are injected on a reverse phase HPLC system using ultraviolet (UV) detection. Some matrices, with adequate levels of vitamin D2 or D3, may not require purification prior to analysis.

VITAMIN E (ALPHA TOCOPHEROL)

Purpose: Applicable for the determination of vitamin E in foods, dietary supplements and premixes.

Method facts:


▶ Limit of quantitation: 0.5 IU/100 g

▶ Precision: On a corn oil matrix, the RSD is about 5%

▶ Method reference: Covance internal method

Description: The product is saponified with alcoholic KOH. The tocopherols are extracted with organic solvent, separated on an HPLC silica column and measured using fluorescence detection. Milligrams of alpha tocopherol are converted to international units (IU) by the following calculations:

1 mg d-alpha tocopherol = 1.49 IU of vitamin E (natural vitamin E)

1 mg dl-alpha tocopherol = 1.10 IU of vitamin E (synthetic vitamin E)

VITAMIN E (IN FEEDS AND FORAGES)

Purpose: Applicable for the determination of vitamin E in feeds.

Method facts:


▶ Limit of quantitation: Most matrices - 0.5 IU/100 g


▶ Method reference: Cort, W.M.; Vincente, T.S.; Waysek, E.H. and Williams, B.D., Journal of Agricultural Food Chemistry, 31:1330-1333 (1983)

Speek, A.J.; Schijver, J. and Schreurs, W.H.P., Journal of Food Science, 50:121-124 (1985)

McMurray, C.H.; Blanchflower, W.J. and Rice, D.A., Journal of the Association of Official Analytical Chemists, 63:1258-1261 (1980)

Description: The sample is saponified with alcoholic KOH. The digest is extracted with an organic solvent. The vitamin E is quantitated directly as alpha tocopherol by HPLC with fluorescence detection.

VITAMIN E IN DIETARY SUPPLEMENTS

Purpose: This method is for the determination of free and esterfied vitamin E in dietary supplements, premixes, liquid concentrates and raw materials. These include, but are not limited to, tablets, capsules, powders and liquids, which contain oil- and water-soluble vitamins with and without minerals. The method is not recommended for matrices with concentrations below 3 mg/g for vitamin E. This method quantifies the following vitamin forms: α-tocopherol, α-tocopheryl acetate and α-tocopheryl acid succinate.

Method facts:


▶ Limit of quanititation: Most matrices - 0.25 mg/g

▶ Precision: On multiple vitamin tablet matrix the RSD is 1.60%

▶ Method reference: Covance developed method based upon the chemical principles of:

US Pharmacopeial Convention, USP-NF [online], current version, Dietary Supplements, Vitamins, Oil- and Water- Soluble Vitamins with Minerals Tablets, Assay for Vitamin E, Method 2. http://www.uspnf.com (accessed 23 Jan 13).

US Pharmacopeial Convention, USP-NF [online], current version, USP Monographs, Vitamin E Preparation

Description: The samples are prepared by one of the following procedures: Direct extraction dispersal with bicarbonate and ascorbate-pyrogallol solution followed by organic extraction or treatment with pancreatin followed by



ASSAYS

a subsequent phase extraction. Free and esterified vitamin E are determined separately using High Performance Liquid Chromatography (HPLC) with UV detection. Free and esterified vitamin E are analyzed using reverse phase chromatography on a C18 column.

VITAMIN K1, LIPASE

Purpose: Applicable to the determination of total vitamin K1 in infant formula, certain nutritional supplements and high fat oil products.

Method facts:


▶ Limit of quantitation: 5.00 mcg/100 g

▶ Precision: 5.5%

▶ Method reference: Official Methods of Analysis of AOAC INTERNATIONAL Current Ed., Method 999.15, AOAC INTERNATIONAL, Gaithersburg, MD, USA (modified)

Description: Following enzymatic digestion of fat and precipitation of fatty acids, vitamin K1 is extracted with hexane. The sample is injected on a reverse phase high-performance liquid chromatography (HPLC) system with post-column reduction and fluorescence detection.

VITAMIN K1 (TRANS PHYTONADIONE)

Purpose: Applicable to the determination of total vitamin K1 in certain vitamin tablets, food products, agricultural products, infant formulas, drink mixes and high level concentrates.

Method facts:

▶ Sample size: 5 g/20 tablets

▶ Limit of quantitation: Most matrices - 5 mcg/100 g



Description: The sample is extracted with organic solvents and injected on reverse phase high-performance liquid chromatography (HPLC) system with post column reduction and fluorescence detection.

WATER ACTIVITY

Purpose: Applicable to the determination of water activity in most food products, which is a critical factor in determining product safety and quality.

Method facts:


▶ Limit of quantitation: <0.030aW

▶ Precision: On ground corn, the RSD is 2.0%

▶ Method reference: AOAC International, “978.18 Water Activity of Canned Vegetables” (modified), Official Methods of Analysis, (ed.) Patricia Cunniff, 18th Ed., AOAC International, Gaithersburg, MD (2005)

Description: The sample is placed in an AquaLab Series 3 instrument. The instrument measures the headspace humidity and calculates the water activity (a

w) of the sample.

The water activity is an indicator of perishability.

XYLITOL

see Sugar alcohols

YERBA MATE


ZEAXANTHIN

see Carotenoids

ZINC (ICP OR ICP-MS)






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SAMPLING

The analysis performed in the laboratory is only as good as the sample which is received at the laboratory for analysis. Care should be taken to ensure that the sample is representative of the lot or product you are trying to characterize. Additionally, enough sample should be sent in to ensure that an adequate amount of material is present to perform all of the testing that is requested. Lastly, the sample must be shipped under conditions so that it will not degrade in transit to the laboratory.

IMPORTING SAMPLES TO COVANCE

Import Registration

The FDA has made changes to the process for importing food products. The FDA requires all facilities, including international parties, to be registered and to provide prior notice to the FDA once their shipment is ready to leave for the US. These changes are a direct result of the Bioterrorism Act of 2002. Non-compliance to these changes results in the shipment being sent back to source or destroyed.Please make certain any facility shipping samples to Covance is registered with the FDA and has provided the FDA with prior notice for the materials. The registration must be completed by the facility sending the shipment. There is no cost to register and the registration takes 15 to 20 minutes. The Covance Madison FDA registration number is 14345226602. This registration number is needed while establishing prior notice.

FDA website: http://www.fda.govClick on “Register a Facility” or, once registered, “Prior Notice of Imports.”

International Broker

The shipment should be routed through Chicago, Illinois. All paperwork related to the shipment should be faxed to the import broker prior to shipping any samples. Please make certain that a complete ingredient list is provided for clearance.

Covance Import Broker:Scarbrough International Ltd 1350 Michael Drive, Suite C Wood Dale, IL 60191 Phone: 630-787-4502 Fax: 630-595-3430 Attn: Covance Import Coordinator [email protected]

Wire Transfer Information

Bank Name: PNC Bank Bank Address: Route 38 & East Gate Drive Moorestown, NJ 08057 Sort Code: ABA# 0312-07607 Account No: ACCT# 8005497809 Account Name: Covance Company Name: Covance Laboratories Inc. Company Address: 3301 Kinsman Blvd. Madison WI 53704 Company Phone: 608-241-4471 VAT Number: none

Please order via your SampleKinect account at samplekinect.covance.com or our Analysis Request Form available from your Customer Service Associate.



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Covance Inc., headquartered in Princeton, NJ, USA, is the drug development business of Laboratory Corporation of America Holdings

(LabCorp). COVANCE is a registered trademark and the marketing name for Covance Inc. and its subsidiaries around the world.

The Americas +1.800.675.8375Europe +44.1423.848864

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