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HomePesticides and Mycotoxins in Cannabis/HempComplete Workflow for the Analysis of California List of Pesticides in Cannabis by LC-MS/MS and GC-MS/MS

Complete Workflow for the Analysis of California List of Pesticides in Cannabis by LC-MS/MS and GC-MS/MS

Geoffrey Rule, Ph.D., Rafael Acosta, Chris Leija, Ph.D., Romana Rigger, Ph.D., Stephan Altmaier, Dr., Uma Sreenivasan, Ph.D.

Merck

Introduction: Analysis of Pesticides in Cannabis

Recreational cannabis use has been legalized in 11 states and the District of Columbia with an additional 22 states allowing the use of medical marijuana.

This has led to mandatory testing for growers and processors of cannabis, and its derivative products, to show product safety according to the requirements of individual states. One of the requirements includes the testing of cannabis flowers for the presence of pesticides. This technical article demonstrates the use of Supelco® analytical standards, instrument consumables, and reagents to analyze low levels of pesticides in cannabis and, in particular, the 66 pesticides required by the State of California.1

In this workflow we present:

  • Complete details for the analysis of the California pesticides by GC-MS/MS and LC-MS/MS
  • Step-by-step procedures for calibrator and sample preparation
  • Methods for the evaluation of suppression and extraction recovery
  • Instructions for the preparation and use of analyte protectants in GC-MS/MS

Sample Preparation

Standardization & Calibration

GC-MS/MS & LC-MS/MS Analysis

Consistent Results

Preparation of Sample and Calibration Standards

Preparation of Pesticide Standard Working Solution (WS)

Calibration curves can be prepared using either a blank matrix extract that has gone through the extraction procedure or pure solvent. They can also be prepared by “spiking” plant material with a pesticide working solution (WS) in order to best imitate an actual plant sample. It is often desirable to perform each of these types of calibration experiments for different purposes, and the schemes outlined are provided for this purpose. The sets of calibration standards are then used for the evaluation of suppression or enhancement effects and for determining extraction recovery with a set of plant samples spiked prior to extraction.

Step

Standard Working Solution Preparation

1Prepare a 1 mL solution of a mix of 66 regulated pesticides at 100 (WS1), 10 (WS2), and 1 (WS3) µg/mL in LC-MS grade acetonitrile. Use positive displacement pipettors to ensure accuracy with organic solvents.
2Ensure that the solution is prepared in an amber volumetric flask as some pesticides are photosensitive and degrade with exposure to light.
3Ensure that working and stock solutions are stored in a subzero freezer when not in use and with minimal exposure to the environment.

Procedure for Sample Preparation

StepSample Preparation Instructions
1*

Weigh 1.0 ± 0.02 of cannabis sample and transfer into a 50 mL polypropylene centrifuge tube
2Add homogenization beads, as appropriate for laboratory equipment.
3*Add 15 mL of LC-MS grade acetonitrile (ACN)and cap
4Shake the tube for 5-10 min on a vertical shaker at high speed
5Prepare tube rack with C18 500 mg SPE cartridges (6 mL) and 50 mL centrifuge tubes for collection
6Add supernatant from step 4 to C18 SPE cartridge and allow it to pass through by gravity (if necessary, use a vacuum or positive pressure manifold)
7Add 5 mL of additional ACN to the tube from step 4 and shake for 3-5 min at high speed
8Transfer supernatant to SPE cartridge
9Repeat step 7 with 5 mL of ACN and, again, pass through the SPE cartridge
10Bring the final volume of centrifuge tube to 25 mL with ACN

Note: If preparing calibration standards in plant, Working Solutions can be added at step 1 or 3 above and as shown in Scheme I.

Preparation of Plant-Based Calibration Standards

Pre-Extraction Spiked Calibration Standards

Cal Levelµg/g in hempµL of Working SolutionSolution
Cal 10.0220WS3
Cal 20.0550WS3
Cal 30.110WS2
Cal 40.220WS2
Cal 50.550WS2
Cal 6110WS1
Cal 71.515WS1
Cal 8330WS1
Cal 9550WS1
  • Working solution (WS1): 100 µg/mL combined pesticides in LC-MS grade acetonitrile
  • Working solution (WS2): 10 µg/mL combined pesticides in LC-MS grade acetonitrile
  • Working solution (WS3): 1 µg/mL combined pesticides in LC-MS grade acetonitrile

How to prepare calibration standard solution example:

To make Cal 9 add 50 µL of WS1 solution as shown in step 1 (or 3) of sample preparation instructions (shown in 2.2 above)

Preparation of plant-based calibration standards

Scheme 1. Preparation of plant-based calibration standards; Pre-extraction spike

Post-Extraction Spiked and Solvent Calibration Standards

This scheme is used to prepare a set of calibration standards in either pure solvent or in a matrix extract that has been collected from blank, analyte free plant material

Level

Conc. in Hemp Flower

ug/g

Conc. in 25 mL Extract

ng/mL

µL

Solution

µL Blank Matrix Extract

Cal 1​

0.02

0.8​

50​

Cal 4​

450​

Cal 2​

0.05​

2​

10​

Cal 9​

990​

Cal 3​

0.1​

4​

20​

Cal 9​

980​

Cal 4​

0.2

8​

40​

Cal 9​

960​

Cal 5​

0.5

20​

50​

Cal 9​

450​

Cal 6​

1​

40​

100​

Cal 9​

400​

Cal 7​

1.5

60​

6​

WS2​

994​

Cal 8​

3​

120​

12​

WS2​

988​

Cal 9​

5

200​

20​

WS​2

980​

Working solution (WS2): 10 µg/mL Combined pesticides in LC-MS grade acetonitrile

  • To make Cal 9 take 20 µL of WS2 solution and add 980 µL of blank matrix extract,​ or acetonitrile, in a 2 mL amber autosampler vial
  • To make Cal 5 take 50 µL of Cal 9 solution and add 450 µL of blank matrix extract, or acetonitrile, in a 2 mL amber autosampler vial
  • To make Cal 1 take 50 µL of Cal 4 solution and add 450 µL of blank matrix extract, or acetonitrile, in a 2 mL amber autosampler vial
Preparation of plant-based calibration standards

Scheme 2.Preparation of plant-based calibration standards; Post-extraction spike

3 Evaluation of suppression and extraction recovery using pre-extraction, post-extraction, and solvent standards

An ideal calibration curve is generated using a set of calibration standards prepared in pure acetonitrile and against which the suppression/enhancement and extraction recovery studies are performed. The calibration standards are prepared as shown in scheme II.

To evaluate suppression and enhancement effects a set of calibrators are prepared from a “post-extraction” sample, as shown in scheme II. For this preparation, a “blank” or “control matrix” of analyte free cannabis is taken through the sample preparation procedure before adding the pesticide working solution. A comparison of the “post extract” regression (calibration curve) with the regression performed in pure acetonitrile allows the evaluation of either suppression or enhancement effects.

Note that suppression or enhancement effects can occur with both GC-MS and LC-MS instrumentation although the causes are somewhat different. The study of these effects can provide insight into possible remedies that may be used to improve assay performance.

To evaluate “extraction recovery” a set of plant-based calibration standards is prepared as shown in Scheme I. This set of standards is spiked with a working solution and then taken through the sample preparation procedure. A comparison of these calibrators with those prepared from the “Post-extraction” set allows for the evaluation of extraction recovery or losses that occur during the extraction procedure.

Matrix Enhancement, Suppression, and Recovery Evaluation

Matrix Enhancement, Suppression, and Recovery Evaluation

Complete workflow for LC-MS/MS analysis

Mobile phase preparation

Mobile PhasesInstructions
1 M ammonium formate
  1. Weigh out 630 mg +/- 10 mg on a balance.
  2. Transfer to 10 mL of 1:1 methanol:water mixture. Mix thoroughly to dissolve.
2 mM ammonium formate + 0.1% formic acid in 2% methanol
  1. Combine 20 mL of methanol with 980 mL of water.
  2. Add 1 mL formic acid and 2 mL 1 M ammonium formate.
  3. Mix and transfer to HPLC solvent bottle.
2 mM ammonium formate + 0.1% formic acid in 2% methanol
  1. Combine 50 mL of water with 950 mL of acetonitrile.
  2. Add 1 mL formic acid and 2 mL 1 M ammonium formate.
  3. Mix and transfer to HPLC solvent bottle.

Chromatographic and MS Conditions

LC-MS/MS
ColumnAscentis RP-Amide, 2.1 mm x 10 cm, 3 um particles with a guard column
DetectionLC-MS/MS dMRM Acquisition Mode
InstrumentationAgilent 1290 series HPLC and autosampler with 6460 QQQ
Mobile phase A2 mm ammonium formate, 0.1% formic acid, 2% methanol in Milli-Q® ultrapure water
Mobile phase B2 mm ammonium formate, 0.1% formic acid, 5% Milli-Q® ultrapure water in acetonitrile

Standard Injection of Pesticides in Hemp Extract

Standard injection of pesticides in hemp extract

Acquisition Parameters for LC-MS/MS Amenable Pesticides in the California List:

HPLC Gradient

StepTime (min)Mobile Phase A (%)Mobile Phase B (%)Flow rate (mL/min)
10.010000.4
21.010000.4
314.001000.4
417.001000.4
517.510000.4
620.010000.4

MS Parameters

Source ConditionsValue (+)
Gas Temp (°C)200
Gas Flow (l/min)10
Nebulizer (psi)35
Sheath Gas Heat200
Sheath Gas Flow10
Capillary Voltage4000
V Charging500

Acquisition Parameters for LC-MS/MS Amenable Pesticides in the California List:

PeaksCompoundRetention Time (min)MRM (m/z)Collision Energy (eV)
1Acephate2.70184.1 -> 143.10
2Acequinocyl15.27402.3 -> 343.24
3Acetamiprid6.01223.1 -> 126.120
4Aldicarb6.57116.1 -> 89.14
5Avermectin B1a12.52890.5 -> 305.328
6Azoxystrobin9.67404.3 -> 372.210
7Bifenazate10.19301.1 -> 198.25
8Boscalid10.00343.2 -> 307.212
9Carbaryl8.34202.2 -> 145.10
10Carbofuran7.70222.2 -> 165.15
11Chlorantraniliprole9.30484.0 -> 286.010
12Chlorfenapyr12.51424.0 -> 368.012
13Chlorpyrifos12.46349.9 -> 97.041
14Clofentezine11.44303.2 -> 138.110
15Coumaphos11.31363.2 -> 227.033
16Daminozide0.93161.1 -> 143.08
17Diazinon10.96305.2 -> 169.120
18Dichlorvos7.14221.1 -> 109.112
19Dimethoate5.82230.2 -> 199.10
20Dimethomorph9.01388.1 -> 301.115
21Ethoprophos9.67243.2 -> 97.030
22Etofenprox13.74394.2 -> 177.210
23Etoxazole12.63360.1 -> 141.128
24Fenhexamid10.64302.2 -> 97.022
25Fenoxycarb10.39302.2 -> 88.115
26Fenpyroximate12.52422.3 -> 366.315
27Flonicamid4.77230.2 -> 98.048
28Fludioxonil10.33229.1 -> 158.118
29Hexythiazox12.49353.2 -> 228.210
30Imazalil6.46297.2 -> 159.120
31Imidacloprid5.67256.2 -> 209.116
32Kresoxim-methyl10.67314.2 -> 267.20
33Malathion10.15331.2 -> 126.95
34Metalaxyl8.00280.2 -> 220.210
35Methiocarb9.52226.1 -> 169.10
36Methomyl4.27162.9 -> 88.10
37Mevinphos ǂ5.25225.2 -> 127.116
38Myclobutanil9.60289.2 -> 70.015
39Naled8.71380.8 -> 127.010
40Oxamyl4.03237.2 -> 72.115
41Paclobutrazol9.25294.1 -> 70.020
42Phosmet9.69317.9 -> 160.010
43Piperonyl butoxide11.78356.2 -> 177.15
44Prallethrin II11.40301.2 -> 105.120
45Propiconazole10.29342.2 -> 159.032
46Propoxur7.59210.1 -> 111.110
47Pyrethrins ǂǂ11.45373.3 -> 161.12
48Pyridaben13.23365.2 -> 147.220
49Spinetoram*8.41748.5 -> 142.126
50Spinosad **8.02732.5 -> 142.128
51Spiromesifen13.00388.3 -> 273.36
52Spirotetramat9.26374.2 -> 302.212
53Spiroxamine7.15298.2 -> 144.116
54Tebuconazole10.01308.1 -> 70.040
55Thiacloprid6.84253.2 -> 126.116
56Thiamethoxam4.88292.2 -> 211.28
57Trifloxystrobin11.58409.2 -> 186.112

‡ As Mevinphos I
‡‡ as Pyrethrin II
* as Spinetoram J
**as Spinosyn A

Consistent Results

Tabulated Results for LC-MS/MS Amenable Pesticides in the California List:

PeaksCompoundR2Recovery (%)%RSD (n=3)CategoryMinimum Action Level µg/g
1Acephate0.99890< 5II0.1
2Acequinocyl #0.991645.3II0.1
3Acetamiprid0.99992< 5II0.1
4Aldicarb0.99985< 5I0.1
5Avermectin B1a0.998959.8II0.1
6Azoxystrobin0.99692< 5II0.1
7Bifenazate0.99896< 5II0.1
8Boscalid0.9988211.9II0.1
9Carbaryl0.99792< 5II0.5
10Carbofuran1.00091< 5I0.1
11Chlorantraniliprole0.998716.1II10
12Chlorfenapyr0.996885.7I0.1
13Chlorpyrifos0.99211630I0.1
14Clofentezine0.9949116II0.1
15Coumaphos0.9989212.8I0.1
16Daminozide0.997125.4I0.1
17Diazinon0.99894< 5II0.1
18Dichlorvos0.9999712.1I0.1
19Dimethoate0.99393< 5I0.1
20Dimethomorph1.00093< 5II2
21Ethoprophos0.998945.8I0.1
22Etofenprox0.995974.8I0.1
23Etoxazole0.99793< 5II0.1
24Fenhexamid0.9981108.8II0.1
25Fenoxycarb0.99692< 5I0.1
26Fenpyroximate0.99792< 5II0.1
27Flonicamid0.99610114.8II0.1
28Fludioxonil0.99110029.1II0.1
29Hexythiazox0.995969.3II0.1
30Imazalil1.00085< 5I0.1
31Imidacloprid0.998896.6II5
32Kresoxim-methyl0.996100< 5II0.1
33Malathion0.9958810II0.5
34Metalaxyl0.99993< 5II2
35Methiocarb0.99792< 5I0.1
36Methomyl1.00092< 5II1
37Mevinphos ǂ0.999966.1I0.1
38Myclobutanil0.99893< 5II0.1
39Naled0.99811013.5II0.1
40Oxamyl1.00089< 5II0.5
41Paclobutrazol0.99891< 5I0.1
42Phosmet0.99810716.8II0.1
43Piperonyl butoxide0.997102< 5II3
44Prallethrin II0.9951059.3II0.1
45Propiconazole0.9989610.5II0.1
46Propoxur0.99993< 5I0.1
47Pyrethrins ǂǂ0.996929.1II0.5
48Pyridaben0.99696< 5II0.1
49Spinetoram *0.999825.5II0.1
50Spinosad **0.99984< 5II0.1
51Spiromesifen0.99691< 5II0.1
52Spirotetramat0.998879II0.1
53Spiroxamine0.99981< 5I0.1
54Tebuconazole0.998946.7II0.1
55Thiacloprid0.99993< 5I0.1
56Thiamethoxam0.99992< 5II5
57Trifloxystrobin0.99794< 5II0.1

ǂ As Mevinphos I
ǂǂ as Pyrethrin II
* as Spinetoram J
**as Spinosyn A

Complete Workflow for the GC-MS/MS Analysis

Preparation of Analyte Protectant Solutions

Analyte protectants reduce active sites in the GC inlet and sample-path to ensure reproducible and consistent results when analyzing pesticides at low ppb levels.2,3

StepInstructions
1Weigh ~500 mg of D-Sorbitol into a 10 mL volumetric flask and add 6 mL of LC-MS grade acetonitrile. Make up the volume using Milli-Q® ultrapure water (Solution A).
2Weigh ~500 mg of L-Gulonic acid γ-lactone into a 10 mL volumetric flask and add 5mL of LC-MS grade acetonitrile. Make up the volume using Milli-Q® ultrapure water (Solution B).
3Add 2 mL of solution A with 4 mL of solution B in a 10 mL volumetric flask and bring to volume with LC-MS grade acetonitrile
4Place into the appropriate autosampler vial for making sandwich injection with 0.2 μL of air gap above and 0.2 μL of the analyte protectant solution

GC-MS/MS Instrument Conditions

Gas Chromatograph Conditions
ColumnSLB®- 5ms L × I.D. 30 m × 0.25 mm, df 0.25 μm
DetectorGC-MS/MS
Inlet60 °C for 0.35 min. and then 600 °C/min to reach 300 °C; Solvent Vent Mode: 5psi until 0.3 min, split vent flow 50 mL/min at 1.5 min; Air Cooled Pressure and Temperature Programmable Inlet
Column Temperature60 °C for 1 minute, then 40 °C/min to reach 170 °C. Hold for 0 min. then 10 °C/min to reach 310 °C. Hold at 310 °C for 3 minutes
Flow 1.2 mL/min
Carrier gasHelium
Liner2 mm ID
Injection2 µl – Solvent Vent Splitless injection with 0.2 uL Sandwich of Analyte Protectant Solution
Sample diluentAcetonitrile
Standard solution9 Matrix-Matched Calibration Standards of Pesticides Mix in Acetonitrile Extract
MS/MS Conditions
TuningAutoTune
AcquisitionMRM (EI mode)
Collision gasNitrogen @ 1.5 mL/min
Quench gasHelium @ 2.25 mL/min
Solvent delay3.5 min
MS source temperature300 °C
Quad Temperature150 °C
Electron Energy70 eV
Dwell time10 ms
Gain10

Acquisition Parameters for GC-MS/MS Amenable Pesticides in California List:

PeaksCompoundRetention Time (min)MRM (m/z)Collision Energy (eV)
1Dichlorvos5.178184.9 -> 109.010
2Mevinphos6.151127.0 -> 109.010
3Acephate6.312136.0 -> 94.010
4Methiocarb7.097168.0 -> 109.115
5Propoxur7.473110.0 -> 63.025
6Ethoprop7.699157.9 -> 97.015
7Naled7.915145.0 -> 109.015
8PCNB8.847248.7 -> 213.915
9Parathion-methyl9.405125.0 -> 47.010
10Spiroxamine I9.836100.0 -> 58.110
11Spiroxamine II10.288100.0 -> 58.110
12Malathion10.393126.9 -> 99.05
13Chlorpyrifos10.580196.9 -> 169.015
14Fipronil11.213366.8 -> 212.825
15Prallethrin11.63123.0 -> 81.010
16Captan11.634116.9 -> 82.030
17Chlordane I11.907271.9 -> 236.915
18Paclobutrazol11.944236.0 -> 125.110
19Chlordane I & II12.026375.0 -> 265.825
20Chlordane II12.142375.0 -> 265.825
21Fludioxonil12.325248.0 -> 127.130
22Kresoxim-methyl12.479116.0 -> 89.015
23Myclobutanil12.508179.0 -> 125.110
24Chlorfenapyr12.672246.9 -> 227.015
25Trifloxystrobin13.536116.0 -> 89.015
26Fenhexamid13.969177.0 -> 113.015
27Piperonyl butoxide14.161176.1 -> 103.125
28Spiromesifen14.398272.0 -> 254.25
29Bifenthrin14.616181.2 -> 165.225
30Phosmet14.789160.0 -> 77.120
31Etoxazole14.812141.0 -> 63.130
32Permethrin I16.425162.9 -> 91.115
33Permethrin II16.546162.9 -> 91.115
34Coumaphos16.632361.9 -> 109.015
35Pyridaben16.641147.2 -> 117.120
36Cyfluthrin I & II17.151198.9 -> 170.125
37Cypermethrin I II III IV17.359181.0 -> 152.125
38Boscalid17.465140.0 -> 112.010
39Etofenprox17.584163.0 -> 107.120
40Azoxystrobin19.210344.1 -> 329.015

TIC MRM Chromatogram of GC-MS/MS Amenable Pesticides in California List Blank Sample Extract

TIC MRM chromatogram of GC-MS/MS amenable pesticides in California list blank sample extract

MRM Extracted Chromatogram of GC-MS/MS Amenable Pesticides in the California List - Standard at 200 ppb

MRM extracted chromatogram of GC-MS/MS amenable pesticides in the California list - Standard at 200 ppb

TIC MRM Chromatogram of GC-MS/MS Amenable Pesticides in the California List - Standard at 200 ppb

TIC MRM chromatogram of GC-MS/MS amenable pesticides in the California list - Standard at 200 ppb

Consistent Results

Tabulated Results for GC-MS/MS Amenable Pesticides in California List:

PeaksCompoundR2Recovery (%)%RSD (n=3)CategoryMinimum Action Level µg/g
1Dichlorvos0.994964.6I0.1
2Mevinphos0.9981024.4I0.1
3Acephate0.9981033.6II0.1
4Methiocarb0.9851033.3I0.1
5Propoxur0.9971024.3I0.1
6Ethoprop0.9971031.3I0.1
7Naled0.995994.9II0.1
8PCNB0.9991003II0.1
9Parathion-methyl0.99810511.2I0.1
10Spiroxamine I0.9961052.5I0.1
11Spiroxamine II0.9981043.4I0.1
12Malathion0.9991012.4II0.5
13Chlorpyrifos0.9991020.6I0.1
14Fipronil0.999991.1I0.1
15Prallethrin0.999996II0.1
16Captan0.9881001II0.7
17Chlordane I0.9951046.8I0.1
18Paclobutrazol0.9991012.6I0.1
19Chlordane I & II0.9981030.6I0.1
20Chlordane II0.9971010.4I0.1
21Fludioxonil0.9991021.9II0.1
22Kresoxim-methyl0.9991011.8II0.1
23Myclobutanil0.9991023.8II0.1
24Chlorfenapyr0.999985.3I0.1
25Trifloxystrobin0.9991011.8I0.1
26Fenhexamid0.9981001.7I0.1
27Piperonyl butoxide0.9971023.2II0.1
28Spiromesifen0.9991032.2II0.1
29Bifenthrin0.9991012II3
30Phosmet0.9991006II0.1
31Etoxazole0.9991021.7II0.1
32Permethrin I0.9971020.4II0.5
33Permethrin II0.9981002.2II0.5
34Permethrin I & II0.9981021.2II0.5
35Coumaphos0.998981I0.1
36Pyridaben0.9981002.4II0.1
37Cyfluthrin I & II0.999962.3II2
38Cypermethrin I II III IV0.997992.1II1
39Boscalid0.9991013.3II0.1
40Etofenprox0.9981010.2I0.1
41Azoxystrobin0.9981027.5II0.1

Tabulated Results for All the Pesticides in the California List:

AnalyteGC-MS/MS MAL Met?LC-MS/MS MAL Met?AnalyteGC-MS/MS MAL Met?LC-MS/MS MAL Met?
DichlorvosYESYESKresoxim-methylYESYES
MevinphosYESYESMyclobutanilYESYES
AcephateYESYESChlorfenapyrYESYES
MethiocarbYESYESTrifloxystrobinYESYES
PropoxurYESYESFenhexamidYESYES
EthopropYESYESPiperonyl butoxideYESYES
NaledYESYESSpiromesifenYESYES
PCNBYESNOBifenthrinYESYES
Parathion-methylYESNOPhosmetYESYES
Spiroxamine IYESYESEtoxazoleYESYES
Spiroxamine IIYESYESPermethrinsYESYES
MalathionYESYESThiaclopridNOYES
ChlorpyrifosYESYESPyrethrinsNOYES
FipronilYESYESCoumaphosYESYES
PrallethrinYESYESPyridabenYESYES
CaptanYESYESCyfluthrin I & IIYESNO
Chlordane IYESNOCypermethrin I II III IVYESNO
PaclobutrazolYESYESBoscalidYESYES
Chlordane I & IIYESNOEtofenproxYESYES
Chlordane IIYESNOAzoxystrobinYESYES
FludioxonilYESYESAcetamipridNOYES
TebuconazoleNOYESDiazinonNOYES
CarbofuranNOYESAldicarbNOYES
ClofentezineNOYESSpirotetramatNOYES
DimethomorphNOYESImidaclopridNOYES
FlonicamidNOYESChlorantraniliproleNOYES
FenoxycarbNOYESBifenazateNOYES
CarbarylNOYESMethomylNOYES
AvermectinNOYESPropiconazoleNOYES
DaminozideNOYESSpinetoram JNOYES
DimethoateNOYESSpinoteram LNOYES
HexythiazoxNOYESSpinosyn ANOYES
ImazalilNOYESOxamylNOYES
MetalaxylNOYESThiamethoxamNOYES
FenproximateNOYESAcequinocylNONO

MAL: Minimum Action Level

Conclusion: Viable method to Quantify Pesticides in Cannabis Using Both LC-MS/MS and GC-MS/MS

A method has been developed to quantify the California list of pesticides from dried cannabis, as per the state requirements, by using a combination of both LC-MS/MS and GC-MS/MS. A single flow through solid-phase extraction is used to prepare the samples for both instrumental techniques. Linearity, recovery, and precision are demonstrated using dried hemp, and schemes for performing the calibration, extraction recovery, and suppression/ enhancement studies are provided.

A total of 57 pesticides are reported using LC-MS/MS and 40 using GC-MS/MS. Due to the high levels of interfering CBDA, acequinocyl was not detected at minimum levels with the existing instrumentation. All other pesticides are reported using one or the other analytical technique to meet or exceed the current California regulatory limits for each category.

As seen from the illustrations and results, the use of Supelco® chromatography solvents, consumables, supplies, and analytical reagents in combination with GC-MS/MS and LC-MS/MS instrumentation provides an efficient way to analyze cannabis for the presence of pesticides. The minimum action levels of 0.1 µg/g (100 ppb) for California are easily achievable for most of the compounds. Obtaining consumables and reagents from one supplier ensures that the time is well spent in analyzing samples rather than evaluating multiple sources for analytical supplies.

The GC-MS/MS instrumentation is used here as a supplement to LC-MS/MS in order to detect all California cannabis pesticides at their required minimum action levels. Millipore Sigma does not endorse any particular instrumentation and aims to provide solutions for sample preparations and chromatographic challenges of laboratories regardless of the instrument used.

Conclusion

In this workflow we have presented:

  • Complete details for the analysis of the California pesticides by GC-MS/MS and LC-MS/MS
  • Step-by-step procedures for calibrator and sample preparation
  • Methods for the evaluation of suppression and extraction recovery
  • Instructions for the preparation and use of analyte protectants in GC-MS/MS

Approaches for Further Resolution Gains

This work was a continuation of our earlier studies examining the Oregon list of pesticides. It offers a solution for the issue of poor peak shape of weakly retained, early eluting analytes. However, there are other options available that may permit the use of our Ascentis® Express Fused-Core® columns to provide overall better peak shape and resolution. The issue for early eluting analytes is the high solvent strength of the sample following extraction, versus the solvent strength of the initial mobile phase. The approach taken here was to use a guard column to insert a porous bed into the flow path to provide a space for sample mixing with the solvent. This same approach can be used with our Fused-Core® column technology.

Approaches That Can Be Explored to Leverage the Fused-Core® Technology:

  • Coupling a high-resolution Ascentis Express analytical column with a porous particle guard column of lower hydrophobicity of which there are several options available.
  • Sample dilution with a small amount of water. This would entail trying a series of dilutions to determine which affords desirable improvement in the shape of early eluting peaks. In order to keep sample mass constant, the sample volume would need to be increased by the same factor as used in dilution. Additionally, be mindful of issues around analyte solubility as cannabinoids could precipitate out of solution. Utilizing more sensitive LC/MS/MS detection can also permit smaller sample volumes which can ameliorate the problem.

See also the Article in Analytix Reporter Issue 10 for more information on the infusion experiments determine in signal suppression.

Materials
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References

2.
Ma?tovská K, Lehotay SJ, Anastassiades M. 2005. Combination of Analyte Protectants To Overcome Matrix Effects in Routine GC Analysis of Pesticide Residues in Food Matrixes. Anal. Chem.. 77(24):8129-8137. https://doi.org/10.1021/ac0515576
3.
Anastassiades M, Ma?tovská K, Lehotay SJ. 2003. Evaluation of analyte protectants to improve gas chromatographic analysis of pesticides. Journal of Chromatography A. 1015(1-2):163-184. https://doi.org/10.1016/s0021-9673(03)01208-1
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