Imidacloprid Hplc Analysis

7/27/2019 Imidacloprid Hplc Analysis

1/8

CALIFORNIA DEPT. OF FOOD & AGRICULTURE Method #: EM 12.7Cente r for Analytical Chemistry Original Date: October 1, 2002Environmental Monitoring Section Revised Date:3292 Meadowview Road Page 1 of 8Sacramento, CA 95832(916) 262-2080 FAX (916) 262-1572

HPLC Determ ination of Total Imidacloprid in VegetationScope: This method is for the determination of total imidacloprid in vegetation. The reporting limit forthis method is 0.05 ppm for oleander sprig and 1 O ppm for liquid amber sprig.Principle:

Sample is ground with ace tonitrile. The extract is then cleaned up with a series of solid phasecartridges. The final extract is analyzed by reverse-phase using HPLC w ith a W detector.Imidacloprid at these levels can be confirmed by LC/MSiM S using triple quadropole or ion trapmass spectrometer.Safety:All g eneral laboratory safety rules for sample preparation and analysis shall be followed.Interference:A clean-up procedure is necessary to eliminate interferences found in the material used tovalidate this method.Reagents, Equipm ent and Instrument:Reagents:

1. Imidacloprid Standard; 1 O mg/mL in methanol, obtained from CD FA Standard Repository2. Acetonitrile, HPLC grade3. Methanol, HPLC grade4. Methylene Chloride, HPLC5. Water, HPLC grade6. Sodiu m Chloride, granular, anhydrous

section; CAS registry number: 1 05827-78-9

Equipment:1.2.3.4.5.7.

Blender with stainless steel cupAmb er bottles with cap, 250 mLNitrogen evapora tor, N-EVA'?", Organomation Associates, Inc; Model 112Vortex mixer, Thermolyne, Model 37600Disposable filter, Acrodisc', Gelm an, 25 mm x 0.2 pmFunnel, long stem, 60', 100 mm diameter


2/8

HPLC Determination of Imidacloprid in Surface and Well Water page 2 of 8

Reagents, Equipment andInstrument: continued8. Conical test tubes, graduated, 15mL9. Ctg Bond-Elute cartridge10. N H 2 Bond-Elute cartridge11,Filter paper,E&K Scientific, sharkskin, 18.5 cm12 . Variable transformer, Superior, Powerstat

Instrument:1, HPLC: Hewlett-Packard 1050 Liquid Chromatograph w ith ChemStation and UV detector2. Analytical column: Beckman Ultrasphere, ODS, 5p x 4.6 mm x 25 cm

Standard Preparation:1. Dilute the 1 mg/mL im idacloprid standard with methanol to m ake up a series of standardsolutions. These standard solutions will be used for spiking, nstrumen t calibration andsam ple calculation.2. Keep all prepared standards in the designated refrigerator or freezer for storage when not inuse.3 . The expiration da te of each standard is six mon ths from the preparation date.

Samp le Preservation and Storage:1. Check the temperature of ten percent of the samplesupon am va l and record it.2. Sign the sample chain of custody and obtain an EMON number.3 . Store all sampleswaiting for extraction in the walk-in free zer.4. Store all samples waiting fo r analysis in a refrigerator.

Analysis:Sample Extraction:1, Bring sample to room temperature if frozen.2. Cut sam ple into small piece s (including stem and twigs asapplicable).

3 . Weigh-out 50.0 grams of sample and put in stainless teel blender cup.4. Add 20 0 mL acetonitrile.5. Blend at eighty setting on variable transformer dial for two minutes.6 . Filter the extract through sharkskin filter paper in to 250 mL bottles with 10 grams of sodium7. Shake vigorously for 2 minutes and let stand30 to 40minutes.Sample clean up or Oleander Sprig

chloride.

a) Take 10 mL aliquot of extract and concentrate to less than 0.1 mL on he N-EVAPTM.b) Suspend the extract in 2 to 3 mL of methanol.c) Condition a C ISBond-Elute cartridge by rinsing w ith 5.0 mL of methanol on a vacuumd) Load the extract uspension on the cartridge andcollect the eluant in a test tube.e) Rinse the est tubes 2 times w ith 3 mL methanol. Collect each of thecartridge rinses in

manifold.

the sam e test tube.


3/8

HPLC Determination of Imidacloprid in Surface and Well Water page 3 of 8

Analysis: continuedf) Concentrate the eluant to less than 0.1 mL on the N-EVAP TM.g) Suspend the e xtract n 2 mL of 1% methanol in methylene chloride.h) Condition aN H 2 Bond -Elute cartridge by rinsing with 5 mL of 1% methanol ini) Load the extract suspen sion on the cartridge and collect the eluant in a test tube.j ) Rinse the test tubes 2 times with 3 mL 1% methanol in methylene chloride. Collect each

methylene chloride on a vacuum manifold.of the cartridge rinsesn the same test tube.

Sample clean upfor Liquid Amber Spriga) Take 1 mL aliquot of extract and concentrate to less than 0.1 mL on the N-EVAPTM.b) Suspen d the extract in 2 to 3 mL of meth anol.c) Condition a Cl8 Bond-Elute cartridgewith 5.0 mL of methanolon a vacuum manifold.d) Load the extrac t suspens ion n the cartridgeand collect the eluant in a test ube.e) Rinse the test tubes times with 3 mL methanol. Collect each of the cartridgerinses inf) Concentrate the eluant to less than 0.1 mL on the N-EVAP m.g) Suspend the extract in 2 L of 10% methanol in methylene chloride.h) Condition aN H z Bond-Elute cartridgeby rinsing with 5 mL of 10 % methanol ini) Load the extract suspens ionon the cartridge and collect the eluant in a test tube.j) Rinse the test tubes2 times with 3 mL 1%meth anol in methylene chloride. Collect each

the same test tube.

methylene chloride on a vacuum manifold.

of the cartridge rinses in the same test tube.8) Concentrate the extract o less than 0.1 mL and bring to 1 O mL of methanol.9) Filter the extract through 0.2 pm AcrodiscO into an auto sampler vial.

Preparation of blanks and spikesBlank: Weigh out 50 g f the homogeneous background sample into a blender.Follow thesample extraction procedure outlinedbove.Spike: Weigh out 50 gof the homogeneous background sample into a blender. Spike a knownamount of imidacloprid into the samp le. Mix well and let stand for t least one minu te,them follow the sample extraction procedure outlined above.

Instrument Cond itionsInstrument:PLC: Hewlett-Packard 1050,ontrolled by ChemS tationColumn: ODS 5p x 4.6 mm x5mUV Detector:70 qmMobilehase:socratic (70 % Water - 30% Acetonitrile)Flow: 1O mLimin.Injectionolume: 20 pLRetentionime:midacloprid - 5.5inutes


4/8

HPLC Determination of Imidacloprid in Surface and Well Water page 4of 8

Ins t rument cal ibrat ion:Load the method and run a set of calibration standards (0.1ng/pL, 0.25 ng/pL, 0.5 ngipL, and1O ng/pL) to check system linearity.Method Performance:Quality Control:1. A 4-po int calibration curve of 0.1, 0.2 5, 0.5 and 1.0 qg /pL is btained at thebeginning and the end of each sam ple set. The chemstation software is used to calculatesample result in ppm.2. Each sample shall benjected two times to insure reliability of the analysis. If the signal of asample is greater than that of the highest standard in the calibration curve, dilute the sample.Re-inject the diluted sample twomore times together with standards.3 . Samp le storage: All field samples shall b ekept frozen at -10 "C.4. Sam ple ex tracts: All extracts shall be kept refrigerated at 5 "C until analyzed.5 . Refrigerator and freezer temperature sh all be monitored and recorded daily.6. For each set of samples, aminimum of one matrix blank and one matrix spike shall beincluded. Each set of samples shall ot contain more than welve samples.7. To avoid cross-contam ination, glassware shall be wash ed following Environmen talMon itoring standard operation procedure (SOP 502.6).8. At least ten percent of the sample esults shall be manually alculated to check instrumentresults. Hand c alculated results are based on a single calibration point using the followingequation:

(sample peak ht.)(std. conc., pg/mL)(std. vol. inj., vL)(sample final vol., mL.)PPm = (Std. peak ht.)(sample vol. injected, pL)(sample wt. , g)

Recovery Data:The method w as validated by preparing three sets of spike d samples. Originally, each setcontained a blank and three levels of spikes at 1.0ppm, 10ppm and 100ppm . Later, two morelevels of spike wereadded for oleander sprig at 0.1 ppm and 0.5 ppm . T he background plantmaterials were specified by the Department of Pesticide Regulation and samples collected fromthe Meadowview comp lex. Sets w ere processed through the entire analytical method on separatedays. Calculated recoveries for imidacloprid are tabulated in Appendix I.Method Detection Limit (MDL):The MD L efers to the minimum concentration of imidacloprid that can be etected in the

a) Prepare 7 replicates of imidacloprid at 0.1 ppm for each matrix.b) Process each sample through the entire method along with a blank.c) Calculate thepercent recovery for each sample.d) Calculate the standard deviationS) for thepercent recovery.c) Compute theMDL as follows:

material with 99% confidence. The MDL was compu ted based on the following procedure:


5/8

HPLC Determinationof Imidacloprid in Surface and Well Water page 5 of 8

Method Performance: continuedM D L = t x Swhere;

t is the Studen t t' value for the 9% confidenc e level with -1 degrees of freedom(n-1, 1 - C = 0.99) where n represents the number ofreplicates. For n=7, t=3.143,S denotes the standard deviation obtained from replicate analyses.

The results for the standard deviations nd MDL calculation s are tabulated in Appendix II.Reporting Limit (E):to determ ine the R L . In this meth od, the RL is set at 5 times or less the MD L. The imidaclopridFUis 0.05 ppm in oleande r spring nd 1.0 ppm in liquid amb er.

The RL refers to level above which quantitative resultsare reported. The MDL is used as a guide

Calculations:Samples are calculated using a multilevel ca libration curvef response versus concen tration.The multilevel calibration curve issed to confirm linearity over he calibration range.The methodof linear fit ignoring the origin is selectedo create the curve. Each calibration evel correspon ds to acalibration standardof known concentration. Calibration standards should berepared so that theimidacloprid concentration varies acrosshe range of concentrations expected in the samples. Theinstrument calculate resultsICR) are obtained using the following equation:

(sample peak. ht.) RF., pg/mL) (std. vol. injected, pL)FV., mL)(samp le vol. injected, pL) (sample wt., g)

ICR (pprn) =

andC[ ( std . conc.,, pg/mL) / (std. peak ht.")]

RF @g/mL) = nn = number of standardsFor oleander, the sampleweight is 2.5 grams and the final volum e s 1mL (if no dilution is mad e).Therefore , a multiplier actor of 0.4 mL/g is put on the sequen ce table toccount for final volum e ndsample weight on the ICR equation. For liquid amber, the sample weight is 0.25 grams and the final

volume is 1 mLif no dilution is mad e). The refore, a multiplier factorf 4.0 mL/g is put on thesequen ce table. If samp le has to be diluted, the equence table dilution colum n needs to bentered withthe proper dilution factor.

Acceptance Criteria:1. The standard curvesat the beginning and the end of each samp le set should not have an averag epercent change greater than 15%. The % change in response is calculated as follows:


6/8

HPLC Determination of Imidacloprid in Surfacend Well Water page 6 of 8

Acceptance Criteria: continued

2. The samp les are calculated based on the calibration curve before the samples are analyzed usingthe instrument chemstation software. If the results between the two duplicate injections differ byless than 10% either result can be reported. A change greater than 10 % with no known reasonrequires a third injection to check results agreement. Mo re work shall be done if needed toobtain reproducible results.Discussion:David Hirano developed this method. David did the method MDL and validation for the 1 ppm,10 ppm,10 0 ppm spike levels. Jorge L Hernandez did the validation 0.1 ppm and 0.5 ppm spikes

levels and wrote the meth od. It is our experience that imidacloprid is heat sensitive. To achieveacceptable recoveries, prolonged heating must be avoided and the recommen ded temperature mustbe followed during evaporation to prevent low recoveries. The im idacloprid calibration curve islinear in the selected range and any of the calibration points may beused for manual calculation.However, it is recommended for this calculation to use a standard with a peak height close to thepeak height of the calculated sample .References:1. Duc Tran., Determination of of Hexazinone in Plant Material by HPLC.Center fo r Analytival2. Ting, K.C., High Performance Liquid Ch roma tograph ic M ethodo r Determination of SixChemist, Enviromental Monitoring Method#1.3N-Methylcarbamates in Vegetables and Fruits, Bull. Environm ental Contamination & Toxicology,3. Keith, Lawrence H., etc., Principals ofEnvironmenta1 Analysis, Anal.Chem., 1983,55,2210-22181984, 33 , 538-547

WRITTEN BY:David Hirano

TITLE: Agricultural Chemist II

and Jorge L. Hernandez! '

Agricultural Chemist 1 1 0

RE/YIEWED BY : Catherine Cooper

TITLE: Laboratory SupervGorEnvironmental Monitoring


7/8

HPL C Determination of Imidacloprid in Surface and Well Waterag e 7of 8

Appendix I: Method Validation Results andRecoveries

ImidaclolOleander Sprig

Spike Level (ppm) Results (ppm) %0.1 0.0999.0I I 0.092 92.00.088 88.00.5 0.471 94.1I I 0.4316.2t 0.441 88.11 0.756 75.60.708 70.80.803 80.310 7.2622.6 7.2072.1 9.212 92.1100 82.831 82.878.179 78.288.651 88.7

i d TotalLiquid Amber Sprig

Resultsppm) %

0.901 90.11.050 l 0 5 . 01.015 101.59.4017.894 78.97.7527.586.7956.8 82.6132.6 70.398 70.4


8/8

HP LC Determination of Imidacloprid in Surface and Well Water

Appendix 11: MDL Determination~~~~ ~~Imida

Blank NDSpike 1 0.0808 80.8Spike 2 0.0795 79.5Spike 3 0.0896 89.6Spike 4 0.0763 76.3Spike 5 0.0798 79.8Spike 6 0.0743 74.3Spike 7 0.0699 69.9STDEV 0.0062 6.16MDL 0.0193RL 0.05

page 8 of 8

ioprid ~~~ ~ ~Liquid Amber Sprig

pp m %ND0.6120 61.20.6400 64.00.4680 46.80.5700 57.00.5980 59.80.6680 66.80.69309.30.0742 7.420.2331 o

Documents

Imidacloprid Hplc Analysis