Analysis of California Cannabis List of Pesticides, Mycotoxins, and Pyrethrins by LC-MS/MS and GC-MS/MS Using Ascentis^® Express Biphenyl and SLB^®-5ms Columns

Geoffrey Rule, Ph.D, Principal R&D Scientist, Katherine Stenerson, Product Manager, Cannabis & Pharma QC Reference Materials

Merck

Section Overview

• Cannabis Analysis for Pesticides, Mycotoxins, and Pyrethrins
• LC-MS/MS Analysis of Pesticides, mycotoxins, and Pyrethrins Using Ascentis^® Express Biphenyl Column
• GC-MS/MS Analysis of Pesticides Using the SLB^®-5ms Column
• Discussion: Improved Sensitivity and Resolution of Pesticides in Cannabis

Cannabis Analysis for Pesticides, Mycotoxins, and Pyrethrins

Clinical studies into medical use of cannabis have been limited by availability of federally approved high THC cannabis. Recently, several sites were given approval to produce high THC cannabis products for use in federally funded clinical studies. Not unexpectedly, these sites are also expected to provide high quality cannabis products for use in such studies.

To produce medical quality cannabis products there are a number of attributes necessary to demonstrate quality including potency, purity, and the absence of hazardous contaminants such as heavy metals, mycotoxins, and pesticides.

Here we show chromatography achieved with our Ascentis^® Express Biphenyl HPLC column, SLB^®-5ms GC column, and our newly developed Certified Reference Material (CRM) mixes for the California pesticide list. For the LC-MS  portion of the analysis figures are provided for the five mycotoxin standards, as well as six plant-derived pyrethrins run on the biphenyl column under the same chromatographic conditions. The biphenyl phase was selected for the superior peak shape provided for the analytes of interest. For the GC-amenable pesticides, the SLB^®-5ms column was chosen for its low bleed and inertness. 

LC-MS/MS Analysis of Pesticides, mycotoxins, and Pyrethrins Using Ascentis^® Express Biphenyl Column

Chromatographic Conditions and MS Parameters

Instrumentation: Agilent 1290 series HPLC and autosampler with 6460 QQQ

Table 1. LC conditions for pesticides, mycotoxins, and pyrethrins

Column:	Ascentis® Express Biphenyl, 15 cm x 2.1 mm, 2.7 um, 90 Å, (64068-U)
Mobile Phase:	[A] 2 mM NH4 formate, 0.1% formic acid in 2% acetonitrile in water; [B] 2 mM NH4 formate, 0.1% formic acid in 2% water in acetonitrile
Gradient:	Time (min) A (%) B (%) 1 100 0 3 58 42 15 18 82 17 2 98 19 2 98 20 100 0
Flow Rate:	0.4 mL/min
Max Pressure:	3200 psi
Injection:	10 µL
Column temperature:	40 °C
Detection:	MS/MS (see Table 2 for parameters)

Table 2. MS source parameters

Parameter	Value (+)
Gas Temp (°C)	320
Gas Flow (l/min)	12
Nebulizer (psi)	45
Sheath Gas Heater	320
Sheath Gas Flow	12
Capillary (V)	4000
V Charging	500
*For abamectin, the Gas Temp can be modified to 250 and Sheath Gas Heater to 220 for optimum sensitivity.

Figure 1. Analysis of 66 California pesticides in CRM mixes (5 ng/µL each compound in acetonitrile/water 70/30 v/v) using Ascentis^® Express biphenyl column.

Table 3. Retention times for pesticide, mycotoxin, and pyrethrins analysis by LC-MS
Note: Captan, methyl parathion, PCNB, and chlordane were not included as they are typically analyzed by GC-MS (see below). Spinetoram reference material was not available at the time of analysis.

Compound Name	Ret Time (min)	Compound Name	Ret Time (min)	Compound Name	Ret Time (min)
Daminozide	1.6	Aflatoxin B1	6.3	Pyrethrin II	9.7
Acephate	3.0	Chlorantraniliprole	6.7	Piperonyl butoxide	9.8
Oxamyl	4.1	Dimethomorph	6.7	Avermectin B1b	10.0
Methomyl	4.2	Fludioxonil	6.7	Clofentezine	10.0
Dimethoate	4.3	Methiocarb	6.7	Jasmolin II	10.1
Flonicamid	4.3	Spirotetramat	6.8	Coumaphos	10.3
Mevinphos I	4.4	Ethoprophos	6.9	Trifloxystrobin	10.4
Thiamethoxam	4.5	Fenhexamid	7.0	Avermectin B1a	10.5
Mevinphos II	4.7	Myclobutanil	7.0	Cinerin I	10.8
Acetamiprid	4.8	Spinosyn A	7.0	Chlorfenapyr	11.0
Imidacloprid	4.8	Tebuconazole	7.1	Etoxazole	11.0
Aldicarb	5.0	Ochratoxin	7.5	Fenpyroximate	11.1
Dichlorvos	5.1	Spinosyn D	7.5	Pyrethrin I	11.2
Imazalil	5.1	Boscalid	7.7	Chlorpyrifos	11.4
Thiacloprid	5.2	Phosmet	7.8	Hexythiazox	11.5
Aflatoxin G2	5.4	Propiconazole	7.9	Jasmolin I	11.6
Propoxur	5.5	Malathion	8.0	Spiromesifen	12.0
Carbofuran	5.6	Azoxystrobin	8.1	Pyridaben	12.3
Aflatoxin B2	5.8	Bifenazate	8.1	Cyfluthrin	13.1
Aflatoxin G1	5.8	Fenoxycarb	8.2	Cypermethrin	13.4
Carbaryl	5.8	Fipronil	8.3	Permethrin	13.6
Metalaxyl	5.8	Diazinon	8.5	Etofenprox	13.9
Paclobutrazol	6.0	Kresoxim-methyl	9.1	Bifenthrin	14.1
Spiroxamine	6.1	Cinerin II	9.3	Acequinocyl	14.8
Naled	6.2	Prallethrin II	9.4

Figure 2. Analysis of five mycotoxins under the same chromatographic conditions. (100 pg/µL Aflatoxin B1 and G1, 30 pg/µL Aflatoxin B2 and G2, and 500 pg/µL for Ochratoxin in acetonitrile/water 70/30 v/v).

Figure 3. Analysis of six pyrethrins (4 ng/µL each in acetonitrile/water 70/30 v/v ) under the same chromatographic conditions.

GC-MS/MS Analysis of Pesticides Using the SLB^®-5ms Column

Chromatographic Conditions and MS parameters

Instrumentation:  Agilent 7890 GC with 7000C series QQQ

Table 4. GC conditions for pesticide determination

Column:	SLB®-5ms, 30 m x 0.25 mm I.D., 0.25µm, Cat # 28471-U
Oven:	60°C (1 min), 40°C/min to 170°C, 10°C/min to 310°C (3 min)
Inj. temp.:	250°C
Carrier gas:	Helium, 1.2 mL/min, constant flow
Detectior:	MS, MRM
MS source/quad temps (Q1&Q2):	230°C / 150°C
Injection:	1 µL, pulsed splitless (50 psi until 0.75 min, splitter open at 0.75 min)
Liner:	4 mm I.D. FocusLiner™ with taper
Sample:	TraceCERT® California Pesticide Mixes 1,2,3,5,6 diluted to 200 ng/mL in acetonitrile

Figure 4. Analysis of TraceCERT^® California pesticide mixes using the SLB^®-5ms GC column.

Table 5. Retention data for pesticides on the SLB^®-5ms column

Peak #	Compound Name	Ret Time (min)	Peak #	Compound Name	Ret Time (min)	Peak #	Compound Name	Ret Time (min)
1	Dichlorvos	5.07	16	Chlordane I	11.75	31	Permethrin II	16.40
2	Mevinphos	6.03	17	Paclobutrazol	11.76	32	Coumaphos	16.46
3	Acephate	6.11	18	Chlordane II	11.99	33	Pyridaben	16.48
4	Methiocarb	6.96	19	Fludioxonil	12.08	34	Cyfluthrin I	16.80
5	Propoxur	7.33	20	Myclobutanil	12.32	35	Cyfluthrin II	16.89
6	Ethoprophos	7.56	21	Kresoxim-methyl	12.32	36	Cyfluthrin III	16.96
7	Naled	7.78	22	Chlorfenapyr	12.51	37	Cyfluthrin IV	17.00
8	Pentachloronitrobenzene	8.72	23	Trifloxystrobin	13.38	38	Cypermethrin I	17.13
9	Spiroxamine I	9.69	24	Fenhexamid	13.78	39	Cypermethrin II	17.23
10	Methyl Parathion	9.76	25	Piperonyl Butoxide	14.01	40	Cypermethrin III	17.29
11	Malathion	10.24	26	Spiromesifen	14.24	41	Boscalid	17.29
12	Chlorpyrifos	10.44	27	Phosmet	14.60	42	Cypermethrin IV	17.33
13	Fipronil	11.02	28	Etoxazole	14.66	43	Etofenprox	17.44
14	Prallethrin	11.45	29	Bifenthrin	14.76	44	Azoxystrobin	19.00
15	Captan	11.45	30	Permethrin I	16.28

Discussion: Improved Sensitivity and Resolution of Pesticides in Cannabis

Figures 1-4 illustrate chromatographic peak shape and retention times in the LC and GC analysis of the compounds in the TraceCERT^® California Pesticide mixes plus five mycotoxins. The Ascentis^® Express Biphenyl phase was selected for the LC-MS/MS analysis as it provided the best peak shape for the combination of pesticides, mycotoxins, and pyrethrins. The sharper peaks afforded by the Fused-Core^® particle technology of the Ascentis^® Express column line provide improved sensitivity and higher resolution for more accurate quantitation of these compounds. For the GC-MS/MS portion of the analysis, the SLB^®-5ms was chosen for its low bleed and high inertness characteristics. In addition, use of a FocusLiner™ in the GC inlet ensured minimal adsorption and excellent injection reproducibility.