Sampling, preparation and analysis of Heavy Metal in Foods

Sampling, preparation and analysis of Heavy Metal in

Foods

Wee Siew MoiChemical Contaminants Expert

Nestlé Quality Assurance Center, Asia Oceania Africa

CII, 11th Food Safety & Quality Summit 6-7 December 2016

A. Heavy (Toxic) metals analysis

B. Types of Equipment (Décision criterions)

C. Good Laboratory Practice (GLP) for sample preparation

D. Sample preparation

- digestion techniques/preparation of test solutions

E. Détermination

F . Quality Control

G. Method Performance

2

Outline of lecture

Heavy metals (Toxic metals)

Metal are elements that are typically hard, opaque, shiny, and has good electrical and

thermal conductivity. Metals are generally malleable, as well as fusible.

About 91 of the 118 elements in the periodic table are metals (some elements appear in

both metallic and non-metallic forms).

In food/feed, metals are usually

found in ionic or covalent form (not

metallic).

The species (oxididation degree,

inorganic versus organic) may

impact the toxicity, e.g.

Cr (III) versus Cr (VI)

As inorganic versus As organic

Toxic metals - food point of view

Metals can be

Essential macronutrients (Ca, Mg, K, Na)

Essential micronutrients (Cr, Co, Cu, Ni, Se)

Toxic metals

But some essential metals may become toxic!

«Heavy metals» is often used to describe toxic metals.

Strictly speaking it should be used only for element > 200Da.

In practice it refers to metals with high gravity and which have high attraction for

biological tissues.

5

Heavy Metal Analysis

Heavy

Metals

Analysis

Sampling and Sample

Preparation

Determination

Calibration

Digestion

Preparation of

Test solutions

• Avoid contamination when

preparing a test portion

• Obtain homogenized

representative test sample

• Use the appropriate digestion

program according to the type and

amount of sample.

• Avoid contamination of acids and dust

• Carry out blank test in parallel by the

same procedure

• Check if digestion is complete

• Prepare the test solution with similar

HNO3, HCl, Au and IPA concentrations as

those of calibrants depending on ICP-MS

technologies.

• ICP-MS instrument set-up

• Check ICP-MS performance

Critical Points






E. Détermination

F . Quality Control


6

Outline of lecture

B. Types of Equipment (Atomic Spectroscopy)

7

Flame AASGraphite

Furnace AAS

AAS instruments can be flame only, furnace only, or combined (switchable)

ICP-OES ICP-MS

Photos taken from Agilent Technologies as illustration purposes

Decision criterions in what to choose

8

Decision criterions in what to choose

9

Number of Analytes vs Detection Limits

ICP-MS versus Atomic Absorption Spectroscopy(AAS)

For higher sample throughput

10

Figure 2. ICP-MS detection limit ranges and orders of magnitude of

signal intensity compared to other atomic spectroscopic techniques

Figure 2 shows that ICP-MS is selected as the technique) than Flame

(FAA)

or Graphite Furnace (GFAA), and (Hydride Generation) Inductively

Coupled Plasma–Optical Emission Spectroscopy ((HG)-ICP-OES).

- Allow samples with varying analyte concentrations to be

analyzed together

due to its wide analytical working range (9 orders of magnitude):

Combination of wide analytical working range and excellent

sensitivity:

Provides short ICP-MS analysis times.

Can reduce sample-handling requirements.

Minimizes potential analytical errors.

Avoids to frequently recalibrate the system of choice to:

- Get better instrumental detection limits in solution (ng/L

Major limitations to use ICP-MS equipment is the high initial

investment and cost of consumable supplies / Gases / Power

compared to other atomic spectroscopic techniques

What is ICP-MS?

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ICP-MS stands for Inductively Coupled Plasma Mass Spectrometry.

ICP-MS Instrument comprises five basic analytical parts as shown in Figure 1:

Sample introduction generating an aerosol of the liquid (or solid) sample

Plasma source ionizing the aerosol

Sampling interface extracting ions from ICP

Ion optics and mass spectrometer focusing and separating ions

Ion detector converting ions into an electronic signal processed by

the data handling system

Figure 1- Analytical parts of ICP-MS instrumentation

Determination






E. Détermination

F . Quality Control


12

Outline of lecture

C. GLP for sample preparation

13

Food and beverage samples should be stored in their typical commercial

storage conditions (either frozen, refrigerated, or at room temperature)

until analysis. Samples should be analyzed within 6 months of

preparation.

If food or beverage samples are subsampled from their original storage

containers, ensure that containers are free from contamination for the

elements of concern.

Well-homogenized samples and small reproducible aliquots help

minimize interferences. Particle size and distribution should be

normalized using blenders, grinders and mixers.

Sampling and Sample

Preparation

C. GLP for sample preparation

14

• Management of analytical blanks (Cleanliness of laboratory environment)During sample preparations for trace element analysis by ICP-MS, the main requirement

is the management of analytical blanks.

Analytical blank s the measure of all external sources of elemental contamination and is used

to make a correction to the measured sample correction.

Contamination for trace analysis can occur from the:

• Laboratory environment (clean air facilities, air filters and flow, hoods and bench tops)

• Materials (plastic ware for storage and handling)

• Reagents (standard solutions, water, acids, hydrogen peroxide, TMAH as alkaline buffer,

enzymes, isopropanol as carbon buffer)

• Apparatus (auto-sampler, plastic ware, nebulizer, spray chamber and torch, peristaltic,

transfer and drain tubings, sampler and skimmer cones and lens)

• Analyst (bare hand, cosmetics, hair, wearing jewels and watches)

Sampling and Sample

Preparation






E. Détermination

F . Quality Control


15

Outline of lecture

D. Digestion techniques

16

For accurate decomposition (destruction of organic matter) of food sample.

Digestion

Why decomposition is required?

• Conversion from solids into liquids

• Destruction of matrix

• Separation of interfering substances

• Isoformation of sample and standard

• Homogenization

• Preconcentration of analytes

Pros:

Leads to a representative sample

Reduces problems in the

measurement step

Easy to standardize

Cons:

Labor intensive cost factor

Bottle neck in analytical process

Risk of contamination or losses of

analyte

Microwave

Digestion

High

Pressure

Asher

D. Digestion techniques

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Decomposition may be safe

1. Analytically accurate

2. Economically efficient

3. Safe and easy to perform

Digestion

Analytical accurate

No contamination

No loss of elements

Complete decomposition

Reliable equipment

Reproducible

analytical results

Economically efficient

Low consumption of

chemicals

Ease of handling

Low investment/operating

costs

Automation

Reduce cost of faulty

analytical results

Safety

Low amounts of

hazardous chemicals

Simple handling

Spontaneous reactions

Reduce operator error

Instrument safety

(design/Manufacture)

Safety First

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Preparation of sample solution

15

.09

.201

3

Reagent Blank The blank test must be carried out in parallel with the

determination by the same procedure but omitting the test

portion

Preparation of digested

samples

Check if the digestion of test portion is complete. Prepare the

test solution by diluting the digested solution with ultrapure

water to a known volume.

After diluting to volume, the test solution should be clear and

colorless to slightly yellow.

Turbidity and/or a deep color usually indicate an incomplete

digestion

Reagents High-purity reagents should always be used. Each reagent

lot should be tested and certified to be low in the elements of

interest before use.

Standard (Stock/Internal) Elements must be compatible and stable in solutions

together. Concentrations need to be verified before use.

For analysis of As, Cd, Pb, and Hg in food matrices, internal

standard solution of rhodium (Rh), indium (In), and thulium

(Tm) is recommended

Calibration standard Fresh calibration standards should be prepared every day, or

as needed.

Preparation

of Test

solutions






E. Détermination

F . Quality Control


19

Outline of lecture

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ICP-MS Instrument Setup/Optimization

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Determination

(1) Instrument startupTune ICP-MS instrument using suitable optimizing / tuning solutions and

according to manufacturer’s instructions

Note : Tuning of the ICP-MS instrument (i.e. optimization for best performance)

usually while aspirating a prescribed element solution called tuning solution is

started using default or recommended settings by manufacturer for plasma power,

sampling depth, gas flow rates and sample introduction rate (plasma gas flow rate

is set at a fixed value and not optimized at all). Ensure that ICPMS passes all

check criteria of tuning after stabilization of plasma during 30 min.

(2) OptimizationCheck mass resolution, mass calibration, sensitivity and stability of the system

- Adjust ICP-MS instrument daily with an optimizing solution to achieve

maximum ion signals and both low oxide rates (e.g. < 2 %) and low rates of

doubly charged ions (e.g. < 2 %).

Note: The optimizing solution should contain elements that cover the whole mass

range giving a high rate of oxides and doubly charged ions. The solutions

recommended by the manufacturer of the ICP-MS instrument may be used.

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Calibration1

5.0

9.2

01

3

A set of at least seven different calibration solutions including blank solution

(i.e. zero member compensation) with evenly spaced concentrations must be

used for external standard calibration so that the concentration range should

be chosen with respect to the concentrations expected in samples and with

respect to the linear dynamic range. It is important that the concentration of

acids and IPA in the calibration solutions and in the sample solutions are the

same.

Calibration

Prepare calibration solutions with similar HNO3 ,IPA, HCl (or Au) concentrations

as those of sample digests depending on ICP-MS / digestion system.

R2 must be > 0.995 using recommended weighted least squares

regression






E. Détermination

F . Quality Control


22

Outline of lecture

23

Quality Control

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3

• The correlation coefficients of the weighted-linear calibration curves

for each element must be ≥0.995 to proceed with sample analysis.

• The percent recovery of the ICV standard should be 90-110% for each

element being determined.

• Perform instrument rinses after any samples suspected to be high in

metals, and before any method blanks, to ensure baseline sensitivity

has been achieved.

• Run rinses between all samples in the batch to ensure a consistent

sampling method.

• Each analytical or digestion batch must have at least three

preparation (or method) blanks associated with it if method blank

correction is to be performed. The blanks are treated the same as the

samples and must go through all of the preparative steps. If method

blank correction is being used, all of the samples in the batch should

be corrected using the mean concentration of these blanks.

QC samples (certified, P-test, in-house reference samples or spiked

samples) must be regularly included and analysed in duplicate.

Quality

Control

24

Summary of quality control samples(Ref: AOAC Official Method 2015.01 Heavy Metals in Food)

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3

Quality

Control

25

Summary of quality control samples(Ref: AOAC Official Method 2015.01 Heavy Metals in Food)

15

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3

Quality

Control






E. Détermination

F . Quality Control


26

Outline of lecture

27

Method Performance (Ref: AOAC Official Method 2015.01 Heavy Metals in Food)

Performance

Characteristics

Definition

Linearity A coefficient of determination R2 ≥ 0.995 should be generally obtained for ten

standards using weighted linear regression

Limit of Detection (LoD) Limit of detection (LOD) and LOQ were determined through the analysis

method blanks. LOD was calculated as 3 times the SD of the results of the

blanks, and LOQ was calculated as 2 times the value of the LOD, except

where the resulting LOQ would be less than the lowest calibration point, in

which case LOQ was elevated and set at the lowest calibration point and LOD

was calculated as 1/3 of the LOQ. All LOQs achieved are ≤10 μg/kg for all

food matrices and ≤8 μg/kg for liquid matrices, such as infant formula. Limit of Quantification (LoQ)

Repeatability The absolute difference between two independent single test results obtained

using the same method on identical test material in the same laboratory by the

same operator using the same equipment within a short interval of time and

calculated as should not be greater than 20 % (25 % for values close to

PLOQ) which corresponds to the repeatability limit, r, at 95 % confidence

level.

Intermediate reproducibility The absolute difference between two independent single test results obtained

using the same method, on identical test material by different operators using

different equipments at different days for intermediate reproducibility test and

calculated as should not be greater than 35 % (40 % for values close to

PLOQ) which corresponds to the reproducibility limit, iR, at 95 % confidence

level.

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Method Performance (Ref: AOAC Official Method 2015.01 Heavy Metals in Food)

Sample-specific LOQs for several matrices, based on LOQs determined by

the default method, and adjusted for changes in sample mass for particular

samples, are shown. Values have been rounded up to the nearest

part-per-billion.

Sample-specific LOQs

Sample LOQ, µg/kg As Cd Pb Hg

Infant Formula 2 1 4 3

Chocolate 4 2 8 6

Rice Flour 4 2 8 6

Fruit juice 1 1 2 2

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In summary

Heavy

Metals

Analysis

Sampling and Sample

Preparation

Determination

Calibration

Digestion

Preparation of

Test solutions

• Avoid contamination when

preparing a test portion

• Obtain homogenized

representative test sample

• Use the appropriate digestion

program according to the type and

amount of sample.

• Avoid contamination of acids and dust

• Carry out blank test in parallel by the

same procedure

• Check if digestion is complete

• Prepare the test solution with similar

HNO3, HCl, Au and IPA concentrations as

those of calibrants depending on ICP-MS

technologies.

• ICP-MS instrument set-up

• Check ICP-MS performance

Critical Points

Thank you for your attention

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Documents

Sampling, preparation and analysis of Heavy Metal in Foods