Workflow for Pesticide Analysis in Cannabis

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
1	Prepare 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.
2	Ensure that the solution is prepared in an amber volumetric flask as some pesticides are photosensitive and degrade with exposure to light.
3	Ensure 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

Step	Sample Preparation Instructions
1*	Weigh 1.0 ± 0.02 of cannabis sample and transfer into a 50 mL polypropylene centrifuge tube
2	Add homogenization beads, as appropriate for laboratory equipment.
3*	Add 15 mL of LC-MS grade acetonitrile (ACN)and cap
4	Shake the tube for 5-10 min on a vertical shaker at high speed
5	Prepare tube rack with C18 500 mg SPE cartridges (6 mL) and 50 mL centrifuge tubes for collection
6	Add 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)
7	Add 5 mL of additional ACN to the tube from step 4 and shake for 3-5 min at high speed
8	Transfer supernatant to SPE cartridge
9	Repeat step 7 with 5 mL of ACN and, again, pass through the SPE cartridge
10	Bring 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 Level	µg/g in hemp	µL of Working Solution	Solution
Cal 1	0.02	20	WS3
Cal 2	0.05	50	WS3
Cal 3	0.1	10	WS2
Cal 4	0.2	20	WS2
Cal 5	0.5	50	WS2
Cal 6	1	10	WS1
Cal 7	1.5	15	WS1
Cal 8	3	30	WS1
Cal 9	5	50	WS1

• 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)

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

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

Complete workflow for LC-MS/MS analysis

Mobile phase preparation

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

Chromatographic and MS Conditions

LC-MS/MS
Column	Ascentis RP-Amide, 2.1 mm x 10 cm, 3 um particles with a guard column
Detection	LC-MS/MS dMRM Acquisition Mode
Instrumentation	Agilent 1290 series HPLC and autosampler with 6460 QQQ
Mobile phase A	2 mm ammonium formate, 0.1% formic acid, 2% methanol in Milli-Q® ultrapure water
Mobile phase B	2 mm ammonium formate, 0.1% formic acid, 5% Milli-Q® ultrapure water in acetonitrile

Standard Injection of Pesticides in Hemp Extract

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

HPLC Gradient

Step	Time (min)	Mobile Phase A (%)	Mobile Phase B (%)	Flow rate (mL/min)
1	0.0	100	0	0.4
2	1.0	100	0	0.4
3	14.0	0	100	0.4
4	17.0	0	100	0.4
5	17.5	100	0	0.4
6	20.0	100	0	0.4

MS Parameters

Source Conditions	Value (+)
Gas Temp (°C)	200
Gas Flow (l/min)	10
Nebulizer (psi)	35
Sheath Gas Heat	200
Sheath Gas Flow	10
Capillary Voltage	4000
V Charging	500

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

Peaks	Compound	Retention Time (min)	MRM (m/z)	Collision Energy (eV)
1	Acephate	2.70	184.1 -> 143.1	0
2	Acequinocyl	15.27	402.3 -> 343.2	4
3	Acetamiprid	6.01	223.1 -> 126.1	20
4	Aldicarb	6.57	116.1 -> 89.1	4
5	Avermectin B1a	12.52	890.5 -> 305.3	28
6	Azoxystrobin	9.67	404.3 -> 372.2	10
7	Bifenazate	10.19	301.1 -> 198.2	5
8	Boscalid	10.00	343.2 -> 307.2	12
9	Carbaryl	8.34	202.2 -> 145.1	0
10	Carbofuran	7.70	222.2 -> 165.1	5
11	Chlorantraniliprole	9.30	484.0 -> 286.0	10
12	Chlorfenapyr	12.51	424.0 -> 368.0	12
13	Chlorpyrifos	12.46	349.9 -> 97.0	41
14	Clofentezine	11.44	303.2 -> 138.1	10
15	Coumaphos	11.31	363.2 -> 227.0	33
16	Daminozide	0.93	161.1 -> 143.0	8
17	Diazinon	10.96	305.2 -> 169.1	20
18	Dichlorvos	7.14	221.1 -> 109.1	12
19	Dimethoate	5.82	230.2 -> 199.1	0
20	Dimethomorph	9.01	388.1 -> 301.1	15
21	Ethoprophos	9.67	243.2 -> 97.0	30
22	Etofenprox	13.74	394.2 -> 177.2	10
23	Etoxazole	12.63	360.1 -> 141.1	28
24	Fenhexamid	10.64	302.2 -> 97.0	22
25	Fenoxycarb	10.39	302.2 -> 88.1	15
26	Fenpyroximate	12.52	422.3 -> 366.3	15
27	Flonicamid	4.77	230.2 -> 98.0	48
28	Fludioxonil	10.33	229.1 -> 158.1	18
29	Hexythiazox	12.49	353.2 -> 228.2	10
30	Imazalil	6.46	297.2 -> 159.1	20
31	Imidacloprid	5.67	256.2 -> 209.1	16
32	Kresoxim-methyl	10.67	314.2 -> 267.2	0
33	Malathion	10.15	331.2 -> 126.9	5
34	Metalaxyl	8.00	280.2 -> 220.2	10
35	Methiocarb	9.52	226.1 -> 169.1	0
36	Methomyl	4.27	162.9 -> 88.1	0
37	Mevinphos ǂ	5.25	225.2 -> 127.1	16
38	Myclobutanil	9.60	289.2 -> 70.0	15
39	Naled	8.71	380.8 -> 127.0	10
40	Oxamyl	4.03	237.2 -> 72.1	15
41	Paclobutrazol	9.25	294.1 -> 70.0	20
42	Phosmet	9.69	317.9 -> 160.0	10
43	Piperonyl butoxide	11.78	356.2 -> 177.1	5
44	Prallethrin II	11.40	301.2 -> 105.1	20
45	Propiconazole	10.29	342.2 -> 159.0	32
46	Propoxur	7.59	210.1 -> 111.1	10
47	Pyrethrins ǂǂ	11.45	373.3 -> 161.1	2
48	Pyridaben	13.23	365.2 -> 147.2	20
49	Spinetoram*	8.41	748.5 -> 142.1	26
50	Spinosad **	8.02	732.5 -> 142.1	28
51	Spiromesifen	13.00	388.3 -> 273.3	6
52	Spirotetramat	9.26	374.2 -> 302.2	12
53	Spiroxamine	7.15	298.2 -> 144.1	16
54	Tebuconazole	10.01	308.1 -> 70.0	40
55	Thiacloprid	6.84	253.2 -> 126.1	16
56	Thiamethoxam	4.88	292.2 -> 211.2	8
57	Trifloxystrobin	11.58	409.2 -> 186.1	12

‡ 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:

Peaks	Compound	R2	Recovery (%)	%RSD (n=3)	Category	Minimum Action Level µg/g
1	Acephate	0.998	90	< 5	II	0.1
2	Acequinocyl #	0.991	64	5.3	II	0.1
3	Acetamiprid	0.999	92	< 5	II	0.1
4	Aldicarb	0.999	85	< 5	I	0.1
5	Avermectin B1a	0.998	95	9.8	II	0.1
6	Azoxystrobin	0.996	92	< 5	II	0.1
7	Bifenazate	0.998	96	< 5	II	0.1
8	Boscalid	0.998	82	11.9	II	0.1
9	Carbaryl	0.997	92	< 5	II	0.5
10	Carbofuran	1.000	91	< 5	I	0.1
11	Chlorantraniliprole	0.998	71	6.1	II	10
12	Chlorfenapyr	0.996	88	5.7	I	0.1
13	Chlorpyrifos	0.992	116	30	I	0.1
14	Clofentezine	0.994	91	16	II	0.1
15	Coumaphos	0.998	92	12.8	I	0.1
16	Daminozide	0.997	12	5.4	I	0.1
17	Diazinon	0.998	94	< 5	II	0.1
18	Dichlorvos	0.999	97	12.1	I	0.1
19	Dimethoate	0.993	93	< 5	I	0.1
20	Dimethomorph	1.000	93	< 5	II	2
21	Ethoprophos	0.998	94	5.8	I	0.1
22	Etofenprox	0.995	97	4.8	I	0.1
23	Etoxazole	0.997	93	< 5	II	0.1
24	Fenhexamid	0.998	110	8.8	II	0.1
25	Fenoxycarb	0.996	92	< 5	I	0.1
26	Fenpyroximate	0.997	92	< 5	II	0.1
27	Flonicamid	0.996	101	14.8	II	0.1
28	Fludioxonil	0.991	100	29.1	II	0.1
29	Hexythiazox	0.995	96	9.3	II	0.1
30	Imazalil	1.000	85	< 5	I	0.1
31	Imidacloprid	0.998	89	6.6	II	5
32	Kresoxim-methyl	0.996	100	< 5	II	0.1
33	Malathion	0.995	88	10	II	0.5
34	Metalaxyl	0.999	93	< 5	II	2
35	Methiocarb	0.997	92	< 5	I	0.1
36	Methomyl	1.000	92	< 5	II	1
37	Mevinphos ǂ	0.999	96	6.1	I	0.1
38	Myclobutanil	0.998	93	< 5	II	0.1
39	Naled	0.998	110	13.5	II	0.1
40	Oxamyl	1.000	89	< 5	II	0.5
41	Paclobutrazol	0.998	91	< 5	I	0.1
42	Phosmet	0.998	107	16.8	II	0.1
43	Piperonyl butoxide	0.997	102	< 5	II	3
44	Prallethrin II	0.995	105	9.3	II	0.1
45	Propiconazole	0.998	96	10.5	II	0.1
46	Propoxur	0.999	93	< 5	I	0.1
47	Pyrethrins ǂǂ	0.996	92	9.1	II	0.5
48	Pyridaben	0.996	96	< 5	II	0.1
49	Spinetoram *	0.999	82	5.5	II	0.1
50	Spinosad **	0.999	84	< 5	II	0.1
51	Spiromesifen	0.996	91	< 5	II	0.1
52	Spirotetramat	0.998	87	9	II	0.1
53	Spiroxamine	0.999	81	< 5	I	0.1
54	Tebuconazole	0.998	94	6.7	II	0.1
55	Thiacloprid	0.999	93	< 5	I	0.1
56	Thiamethoxam	0.999	92	< 5	II	5
57	Trifloxystrobin	0.997	94	< 5	II	0.1

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

Step	Instructions
1	Weigh ~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).
2	Weigh ~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).
3	Add 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
4	Place 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
Column	SLB®- 5ms L × I.D. 30 m × 0.25 mm, df 0.25 μm
Detector	GC-MS/MS
Inlet	60 °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 Temperature	60 °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 gas	Helium
Liner	2 mm ID
Injection	2 µl – Solvent Vent Splitless injection with 0.2 uL Sandwich of Analyte Protectant Solution
Sample diluent	Acetonitrile
Standard solution	9 Matrix-Matched Calibration Standards of Pesticides Mix in Acetonitrile Extract

MS/MS Conditions
Tuning	AutoTune
Acquisition	MRM (EI mode)
Collision gas	Nitrogen @ 1.5 mL/min
Quench gas	Helium @ 2.25 mL/min
Solvent delay	3.5 min
MS source temperature	300 °C
Quad Temperature	150 °C
Electron Energy	70 eV
Dwell time	10 ms
Gain	10

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

Peaks	Compound	Retention Time (min)	MRM (m/z)	Collision Energy (eV)
1	Dichlorvos	5.178	184.9 -> 109.0	10
2	Mevinphos	6.151	127.0 -> 109.0	10
3	Acephate	6.312	136.0 -> 94.0	10
4	Methiocarb	7.097	168.0 -> 109.1	15
5	Propoxur	7.473	110.0 -> 63.0	25
6	Ethoprop	7.699	157.9 -> 97.0	15
7	Naled	7.915	145.0 -> 109.0	15
8	PCNB	8.847	248.7 -> 213.9	15
9	Parathion-methyl	9.405	125.0 -> 47.0	10
10	Spiroxamine I	9.836	100.0 -> 58.1	10
11	Spiroxamine II	10.288	100.0 -> 58.1	10
12	Malathion	10.393	126.9 -> 99.0	5
13	Chlorpyrifos	10.580	196.9 -> 169.0	15
14	Fipronil	11.213	366.8 -> 212.8	25
15	Prallethrin	11.63	123.0 -> 81.0	10
16	Captan	11.634	116.9 -> 82.0	30
17	Chlordane I	11.907	271.9 -> 236.9	15
18	Paclobutrazol	11.944	236.0 -> 125.1	10
19	Chlordane I & II	12.026	375.0 -> 265.8	25
20	Chlordane II	12.142	375.0 -> 265.8	25
21	Fludioxonil	12.325	248.0 -> 127.1	30
22	Kresoxim-methyl	12.479	116.0 -> 89.0	15
23	Myclobutanil	12.508	179.0 -> 125.1	10
24	Chlorfenapyr	12.672	246.9 -> 227.0	15
25	Trifloxystrobin	13.536	116.0 -> 89.0	15
26	Fenhexamid	13.969	177.0 -> 113.0	15
27	Piperonyl butoxide	14.161	176.1 -> 103.1	25
28	Spiromesifen	14.398	272.0 -> 254.2	5
29	Bifenthrin	14.616	181.2 -> 165.2	25
30	Phosmet	14.789	160.0 -> 77.1	20
31	Etoxazole	14.812	141.0 -> 63.1	30
32	Permethrin I	16.425	162.9 -> 91.1	15
33	Permethrin II	16.546	162.9 -> 91.1	15
34	Coumaphos	16.632	361.9 -> 109.0	15
35	Pyridaben	16.641	147.2 -> 117.1	20
36	Cyfluthrin I & II	17.151	198.9 -> 170.1	25
37	Cypermethrin I II III IV	17.359	181.0 -> 152.1	25
38	Boscalid	17.465	140.0 -> 112.0	10
39	Etofenprox	17.584	163.0 -> 107.1	20
40	Azoxystrobin	19.210	344.1 -> 329.0	15

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

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:

Peaks	Compound	R2	Recovery (%)	%RSD (n=3)	Category	Minimum Action Level µg/g
1	Dichlorvos	0.994	96	4.6	I	0.1
2	Mevinphos	0.998	102	4.4	I	0.1
3	Acephate	0.998	103	3.6	II	0.1
4	Methiocarb	0.985	103	3.3	I	0.1
5	Propoxur	0.997	102	4.3	I	0.1
6	Ethoprop	0.997	103	1.3	I	0.1
7	Naled	0.995	99	4.9	II	0.1
8	PCNB	0.999	100	3	II	0.1
9	Parathion-methyl	0.998	105	11.2	I	0.1
10	Spiroxamine I	0.996	105	2.5	I	0.1
11	Spiroxamine II	0.998	104	3.4	I	0.1
12	Malathion	0.999	101	2.4	II	0.5
13	Chlorpyrifos	0.999	102	0.6	I	0.1
14	Fipronil	0.999	99	1.1	I	0.1
15	Prallethrin	0.999	99	6	II	0.1
16	Captan	0.988	100	1	II	0.7
17	Chlordane I	0.995	104	6.8	I	0.1
18	Paclobutrazol	0.999	101	2.6	I	0.1
19	Chlordane I & II	0.998	103	0.6	I	0.1
20	Chlordane II	0.997	101	0.4	I	0.1
21	Fludioxonil	0.999	102	1.9	II	0.1
22	Kresoxim-methyl	0.999	101	1.8	II	0.1
23	Myclobutanil	0.999	102	3.8	II	0.1
24	Chlorfenapyr	0.999	98	5.3	I	0.1
25	Trifloxystrobin	0.999	101	1.8	I	0.1
26	Fenhexamid	0.998	100	1.7	I	0.1
27	Piperonyl butoxide	0.997	102	3.2	II	0.1
28	Spiromesifen	0.999	103	2.2	II	0.1
29	Bifenthrin	0.999	101	2	II	3
30	Phosmet	0.999	100	6	II	0.1
31	Etoxazole	0.999	102	1.7	II	0.1
32	Permethrin I	0.997	102	0.4	II	0.5
33	Permethrin II	0.998	100	2.2	II	0.5
34	Permethrin I & II	0.998	102	1.2	II	0.5
35	Coumaphos	0.998	98	1	I	0.1
36	Pyridaben	0.998	100	2.4	II	0.1
37	Cyfluthrin I & II	0.999	96	2.3	II	2
38	Cypermethrin I II III IV	0.997	99	2.1	II	1
39	Boscalid	0.999	101	3.3	II	0.1
40	Etofenprox	0.998	101	0.2	I	0.1
41	Azoxystrobin	0.998	102	7.5	II	0.1

Tabulated Results for All the Pesticides in the California List:

Analyte	GC-MS/MS MAL Met?	LC-MS/MS MAL Met?	Analyte	GC-MS/MS MAL Met?	LC-MS/MS MAL Met?
Dichlorvos	YES	YES	Kresoxim-methyl	YES	YES
Mevinphos	YES	YES	Myclobutanil	YES	YES
Acephate	YES	YES	Chlorfenapyr	YES	YES
Methiocarb	YES	YES	Trifloxystrobin	YES	YES
Propoxur	YES	YES	Fenhexamid	YES	YES
Ethoprop	YES	YES	Piperonyl butoxide	YES	YES
Naled	YES	YES	Spiromesifen	YES	YES
PCNB	YES	NO	Bifenthrin	YES	YES
Parathion-methyl	YES	NO	Phosmet	YES	YES
Spiroxamine I	YES	YES	Etoxazole	YES	YES
Spiroxamine II	YES	YES	Permethrins	YES	YES
Malathion	YES	YES	Thiacloprid	NO	YES
Chlorpyrifos	YES	YES	Pyrethrins	NO	YES
Fipronil	YES	YES	Coumaphos	YES	YES
Prallethrin	YES	YES	Pyridaben	YES	YES
Captan	YES	YES	Cyfluthrin I & II	YES	NO
Chlordane I	YES	NO	Cypermethrin I II III IV	YES	NO
Paclobutrazol	YES	YES	Boscalid	YES	YES
Chlordane I & II	YES	NO	Etofenprox	YES	YES
Chlordane II	YES	NO	Azoxystrobin	YES	YES
Fludioxonil	YES	YES	Acetamiprid	NO	YES
Tebuconazole	NO	YES	Diazinon	NO	YES
Carbofuran	NO	YES	Aldicarb	NO	YES
Clofentezine	NO	YES	Spirotetramat	NO	YES
Dimethomorph	NO	YES	Imidacloprid	NO	YES
Flonicamid	NO	YES	Chlorantraniliprole	NO	YES
Fenoxycarb	NO	YES	Bifenazate	NO	YES
Carbaryl	NO	YES	Methomyl	NO	YES
Avermectin	NO	YES	Propiconazole	NO	YES
Daminozide	NO	YES	Spinetoram J	NO	YES
Dimethoate	NO	YES	Spinoteram L	NO	YES
Hexythiazox	NO	YES	Spinosyn A	NO	YES
Imazalil	NO	YES	Oxamyl	NO	YES
Metalaxyl	NO	YES	Thiamethoxam	NO	YES
Fenproximate	NO	YES	Acequinocyl	NO	NO

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.

References

1. Available from: https://bcc.ca.gov/law_regs/cannabis_order_of_adoption.pdf

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