Analysis of California Cannabis List of Pesticides, Mycotoxins, and Pyrethrins Using Ascentis^® Express Biphenyl Column

Geoffrey Rule, Ph.D, Principal R&D Scientist

Merck

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 and our newly developed Certified Reference Material (CRM) mixes for the California pesticide list. In addition, figures are provided for the five mycotoxin standards, as well as six plant-derived pyrethrins, using the same column and chromatographic conditions. The biphenyl phase was selected for the superior peak shape provided for the analytes of interest.

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

Method Conditions
Column	Ascentis® Express Biphenyl, 15 cm x 2.1 mm, 2.7 um, 90 Å, Cat # 64068-U
Mobile Phase A	2 mm NH4 formate, 0.1% formic acid in 2% ACN in water
Mobile Phase B	2 mm NH4 formate, 0.1% formic acid in 2% water in ACN
Gradient	Time A B Flow mL/min 1 1.00 min 100.00 % 0.00 % 0.400 2 3.00 min 58.00 % 42.00 % 0.400 3 15.00 min 18.00 % 82.00 % 0.400 4 17.00 min 2.00 % 98.00 % 0.400 5 19.00 min 2.00 % 98.00 % 0.400 6 20.00 min 100.00 % 0.00 % ---
Injection	10 µL
Column temperature:	40 °C
Detection	MS/MS
Max Pressure:	3200 psi

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 using Ascentis® Express biphenyl column

Note: Captan, methyl parathion, PCNB, and chlordane were not included as they are typically analyzed by GC-MS. 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

Figure 3. Analysis of six pyrethrins under the same chromatographic conditions

Discussion: Improved Sensitivity and Resolution of Pesticides in Cannabis

Figures 1-3 illustrate chromatographic peak shape and retention times of the California pesticide mixes as well as the six pyrethrin compounds and the five mycotoxins that require monitoring. The analytes included were those amenable to LC-MS/MS analysis. Captan, methyl parathion, PCNB, and chlordane were not included as they are typically analyzed by GC-MS and reference material for spinetoram was not available at the time of analysis. The sharper peaks afforded by the Fused-Core particle technology of Ascentis^® Express provide improved sensitivity and higher resolution for more accurate quantitation of these compounds. The Ascentis^® Express Biphenyl phase was selected as it provided the best peak shape for the combination of pesticides, mycotoxins, and pyrethrins.