As cannabis and hemp markets continue to mature, there has never been a higher demand for rapid turnaround time in cannabis pesticide and mycotoxin testing laboratories. Laboratories must continue to optimize their workflows to reduce turnaround time if they are to remain competitive and retain their clients.
The sheer complexity and range of reagents and consumables involved in cannabis and hemp pesticide residue testing means that currently, laboratories must invest significant time in method development, the procurement of reagents and consumables, storage of a range of shipments and the preparation of calibration and QC standards.
This time-consuming process is especially prevalent in high-volume laboratories running hundreds or even thousands of samples every month.
PerkinElmer has developed the ONE Pesticide420 TM ISO 17034 Reagent Kit (Part Number: COP-001KT) to help reduce the turnaround time, improve overall data quality and enhance efficiency in labs operating in line with Oregon, California, or comparable regulatory requirements.
This comprehensive kit provides laboratories with pre-aliquoted pesticide and mycotoxin calibration reagents that have been specifically designed for easy, streamlined use. The kit includes isotopically labeled quality control and internal standards.
This article investigates the amount of time saved by utilizing the ONE Pesticide420 TM ISO 17034 Reagent Kit workflow in comparison with an existing analytical workflow.
Results and discussion
This study involved a series of pesticide and mycotoxin testing laboratories comparing time to analysis via existing calibration and QC reagent preparation protocols with time to analysis using the ONE Pesticide420 TM Reagent Kit.
Laboratories reported that on average, using utilizing PerkinElmer‘s preconfigured kit resulted in up to three times faster calibration of standard preparation protocols than traditional workflows. Some laboratories reported that it took as little as 3 minutes to prepare the eight calibration vials for LC-MS/MS analysis.
Figure 1. Workflow comparison showing calibration standard preparation time using existing workflows and PerkinElmer’s ONE Pesticide420 TM Kit. Existing workflows require sourcing materials and reagents from multiple vendors while using the ONE Pesticide420 TM Kit only requires coordination with one vendor. Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions
Figure 1 illustrates a typical calibration standard preparation workflow in contrast with the ONE Pesticide420 TM Reagent Kit’s workflow.
Figure 2. The components of PerkinElmer ONE Pesticide420 TM reagent and consumable kit. Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions
The time and effort necessary for procuring a range of analytical standards, internal standards, solvents and consumables is also minimized when using the ONE Pesticide420 TM Reagent and Consumable Kit (Figure 2).
There is no longer any requirement to source these supplies from multiple vendors as the ONE Pesticide420 TM Reagent Kit provides standards and internal standards consumables for a wide range of sample pesticide and mycotoxin quantitation.
The time savings afforded by the ONE Pesticide420 TM Reagent Kit allow laboratories to divert employee time and energy towards more valuable activities rather than simply ordering and preparing reagents.
Generating calibration curves using ONE Pesticide420 TM Reagent Kit
Calibration curves for each pesticide and mycotoxin standard were prepared using LC-MS/MS to effectively demonstrate the performance of the ONE Pesticide420 TM Reagent Kit.
The figure below displays linearity and shows R2 values greater than 0.99 for all analytes – a figure in line with requirements from the California Bureau for Cannabis Control (BCC), which requires R2 values to be greater than 0.99.
Table 1. Observed R2 values for pesticides and mycotoxins generated using the eight point calibration curve provided in the ONE Pesticide420 TM Reagent Kit. Calibration curve levels are at 1, 5, 10, 25, 50, 100, 150 and 250 ppb. Source: PerkinElmer Cannabis & Hemp Testing Solutions
| Analyte |
R2 |
| Abamectin |
0.9965 |
| Acephate |
0.9980 |
| Acequinocyl |
0.9984 |
| Acetamiprid |
0.9988 |
| Aldicarb |
0.9996 |
| Azoxystrobin |
0.9996 |
| Bifenazate |
0.9987 |
| Bifenthrin |
0.9990 |
| Boscalid |
0.9992 |
| Carbaryl |
0.9996 |
| Carbofuran |
0.9991 |
| Chlorantraniliprole |
0.9992 |
| Chlorfenapyr |
0.9998 |
| Chlorpyrifos |
0.9995 |
| Cinerin-I |
0.9996 |
| Clofentezine |
0.9992 |
| Cyfluthrin |
0.9971 |
| Cypermethrin |
0.9994 |
| Daminozide |
0.9979 |
| Diazinon |
0.9991 |
| Dichlorvos |
0.9989 |
| Dimethoate |
0.9986 |
| Ethoprophos |
0.9994 |
| Etofenprox |
0.9994 |
| Etoxazole |
0.9994 |
| Fenoxycarb |
0.9989 |
| Fenpyroximate |
0.9988 |
| Fipronil |
0.9993 |
| Flonicamide |
0.9985 |
| Fludioxonil |
0.9994 |
| Hexythiazox |
0.9988 |
| Imazalil |
0.9979 |
| Imidacloprid |
0.9986 |
| Jasmolin-I |
0.9996 |
| Kresoxim-methyl |
0.9994 |
| Malathion |
0.9990 |
| Metalaxyl |
0.9991 |
| Methiocarb |
0.9996 |
| Methomyl |
0.9973 |
| Methyl parathion |
0.9978 |
| MGK-264 |
0.9995 |
| Myclobutanil |
0.9990 |
| Naled |
0.9989 |
| Oxamyl |
0.9980 |
| Paclobutrazol |
0.9991 |
| Permethrin |
0.9980 |
| Phosmet |
0.9997 |
| Piperonyl butoxide |
0.9995 |
| Prallethrin |
0.9992 |
| Propiconazole |
0.9996 |
| Propoxur |
0.9980 |
| Pyrethrin-I |
0.9994 |
| Pyrethrin-II |
0.9991 |
| Pyridaben |
0.9991 |
| Spinosad |
0.9985 |
| Spiromesifen |
0.9995 |
| Spirotetramat |
0.9986 |
| Spiroxamine |
0.9989 |
| Tebuconazole |
0.9997 |
| Thiacloprid |
0.9993 |
| Thiamethoxam |
0.9992 |
| Trifloxystrobin |
0.9991 |
| Trifloxystrobin |
0.9991 |
Quality control samples were compared at a known concentration of 75 ppb from generated calibration curves of each analyte (Table 2) in order to assess the accuracy of quantitation. A strict maximum deviation of 30% was achieved for all of the quality control samples investigated.
Table 2. Accuracy of quality control sample at 75 ppb of each analyte. To calculate accuracy, the observed ppb value was divided by the theoretical ppb value at 75 ppb and multiplied by 100. Source: PerkinElmer Cannabis & Hemp Testing Solutions
| Observed (ppb) |
Accuracy (%) |
R2 |
| Abamectin |
79.39 |
105.9 |
| Acephate |
75.75 |
101.0 |
| Acequinocyl |
76.41 |
101.9 |
| Acetamiprid |
74.71 |
99.6 |
| Aldicarb |
74.59 |
99.5 |
| Azoxystrobin |
75.43 |
100.6 |
| Bifenazate |
71.57 |
95.4 |
| Bifenthrin |
77.62 |
103.5 |
| Boscalid |
75.07 |
100.1 |
| Carbaryl |
74.31 |
99.1 |
| Carbofuran |
74.46 |
99.3 |
| Chlorantraniliprole |
73.92 |
98.6 |
| Chlorfenapyr |
81.74 |
109.0 |
| Chlorpyrifos |
73.47 |
98.0 |
| Cinerin-I |
76.10 |
101.5 |
| Clofentezine |
76.24 |
101.7 |
| Cyfluthrin |
79.50 |
106.0 |
| Cypermethrin |
77.42 |
103.2 |
| Daminozide |
72.69 |
96.9 |
| Diazinon |
78.50 |
104.7 |
| Dichlorvos |
71.81 |
95.7 |
| Dimethoate |
74.56 |
99.4 |
| Ethoprophos |
75.18 |
100.2 |
| Etofenprox |
76.42 |
101.9 |
| Etoxazole |
75.25 |
100.3 |
| Fenoxycarb |
74.52 |
99.4 |
| Fenpyroximate |
75.28 |
100.4 |
| Fipronil |
75.41 |
100.5 |
| Flonicamide |
73.88 |
98.5 |
| Fludioxonil |
71.54 |
95.4 |
| Hexythiazox |
74.42 |
99.2 |
| Imazalil |
72.17 |
96.2 |
| Imidacloprid |
75.56 |
100.7 |
| Jasmolin-I |
73.50 |
98.0 |
| Kresoxim-methyl |
72.80 |
97.1 |
| Malathion |
77.38 |
103.2 |
| Metalaxyl |
73.27 |
97.7 |
| Methiocarb |
77.13 |
102.8 |
| Methomyl |
73.81 |
98.4 |
| Methyl parathion |
73.74 |
98.3 |
| MGK-264 |
76.41 |
101.9 |
| Myclobutanil |
72.71 |
96.9 |
| Naled |
77.17 |
102.9 |
| Oxamyl |
73.34 |
97.8 |
| Paclobutrazol |
75.79 |
101.1 |
| Permethrin |
77.69 |
103.6 |
| Phosmet |
77.27 |
103.0 |
| Piperonyl butoxide |
77.09 |
102.8 |
| Prallethrin |
73.95 |
98.6 |
| Propiconazole |
75.41 |
100.5 |
| Propoxur |
76.88 |
102.5 |
| Pyrethrin-I |
74.60 |
99.5 |
| Pyrethrin-II |
76.15 |
101.5 |
| Pyridaben |
74.79 |
99.7 |
| Spinosad |
71.54 |
95.4 |
| Spiromesifen |
77.23 |
103.0 |
| Spirotetramat |
73.25 |
97.7 |
| Spiroxamine |
72.37 |
96.5 |
| Tebuconazole |
74.23 |
99.0 |
| Thiacloprid |
76.53 |
102.0 |
| Thiamethoxam |
73.21 |
97.6 |
| Trifloxystrobin |
74.68 |
99.6 |
The use of internal standards is advisable due to the prevalence of matrix effects in complex cannabis samples. The ONE Pesticide420 TM Reagent Kit includes a total of 30 internal standards with a view to improving quantitative analysis and overall recovery.
The use of internal standards can also address possible analyte loss during sample preparation.
Experimental
The PerkinElmer ONE Pesticide420 TM ISO 17034 Reagent Kit (Part Number: COP-001KT) has been designed to help improve laboratory efficiency and consistency. It includes the following calibration curve and QC standards:
- 8 x Calibrator vials including 77 pesticide standards, 5 mycotoxin standards and 30 internal standards from 1ng/mL to 250 ng/mL
- 1 x Intermediate standard mix at 1µg/mL for preparation of laboratory control samples (LCS)
- 3 x Quality Control Standard Vials
- 75 ng/mL initial calibration verification (ICV) standard - prepared from a second lot
- 15 and 175 ng/mL continuing calibration verification (CCV) standards
- 1 x Internal standard vial including 30 internal standards
The kit features a total of 77 certified pesticide reference standards specifically selected to help laboratories meet pesticide testing requirements in US states with comparable testing requirements to California and Oregon as of March 2021.
It is recommended that laboratories check regulations in their individual state for any further requirements.
Isotopically labeled reference internal standards are also included. These enable the straightforward and accurate quantitation of pesticides and mycotoxins in a cannabis or hemp matrix.
Alongside the Reagent Kit, PerkinElmer also supplies a Consumable Kit (Part Number: COP-001CT) which includes the full range of consumables required to prepare 500 cannabis or hemp samples.
The kit includes the following consumables:
- 200 x autosampler LC-MSMS vials for calibrators and quality controls
- 700 x autosampler LC-MSMS vial caps
- 500 x 2 mL amber vials
- 500 x nylon syringe filters
- 500 x disposable syringes
- 500 x 50 mL graduate conical tubes
Using the ONE Pesticide420 TM Reagent Kit
Using the ONE Pesticide420 TM Reagent Kit is simple and straightforward. The following steps are required:
- Bring eight vials of calibrator solution to room temperature
- Vortex these for 30 seconds to mix
- Transfer a total of 200 µL from each calibrator solution by pipetting each into a different empty LC-MS autosampler vial
- Ensure there are eight separate autosampler vials for each calibrator solution
- Transfer the autosampler vials to a mass spectrometer for final analysis
Table 3. Concentrations of reagents included in PerkinElmer’s ONE Pesticide420 TM Reagent Kit (Part Number COP-001KT). Calibrators are pre-mixed and already include addition of internal standards. Source: PerkinElmer Cannabis & Hemp Testing Solutions
| Part Number |
Quantity Included in Kit |
Description |
Concentration of Analytes (ng/mL or ppb) |
| COP- IntCal |
1 |
ONE Pesticide420™ - Initial Calibrator - 1 μg/mL in 2 mL ACN with 0.1% formic acid |
1000 |
| COP-Cal-01 |
1 |
ONE Pesticide420™ - Calibrator 1 - 1 ng/mL in 4 mL ACN with 0.1% formic acid |
1 |
| COP-Cal-02 |
1 |
ONE Pesticide420™ - Calibrator 2 - 5 ng/mL in 4 mL ACN with 0.1% formic acid |
5 |
| COP-Cal-03 |
1 |
ONE Pesticide420™ - Calibrator 3 - 10 ng/mL in 4 mL ACN with 0.1% formic acid |
10 |
| COP-Cal-04 |
1 |
ONE Pesticide420™ - Calibrator 4 - 25 ng/mL in 4 mL ACN with 0.1% formic acid |
25 |
| COP-Cal-05 |
1 |
ONE Pesticide420™ - Calibrator 5 - 50 ng/mL in 4 mL ACN with 0.1% formic acid |
50 |
| COP-Cal-06 |
1 |
ONE Pesticide420™ - Calibrator 6 - 100 ng/mL in 4 mL ACN with 0.1% formic acid |
100 |
| COP-Cal-07 |
1 |
ONE Pesticide420™ - Calibrator 7 - 150 ng/mL in 4 mL ACN with 0.1% formic acid |
150 |
| COP-Cal-08 |
1 |
ONE Pesticide420™ - Calibrator 8 - 250 ng/mL in 4 mL ACN with 0.1% formic acid |
250 |
| COP-QC-01 |
1 |
ONE Pesticide420™ - Quality Control - 01 - 15 ng/mL in 4 mL ACN with 0.1% formic acid |
15 |
| COP-QC-02 |
1 |
ONE Pesticide420™ - Quality Control - 02 - 75 ng/mL in 4 mL ACN with 0.1% formic acid (second) |
75 |
| COP-QC-03 |
1 |
ONE Pesticide420™ - Quality Control - 03 - 75 ng/mL in 4 mL ACN with 0.1% formic acid |
175 |
| COP-IntStd |
2 |
ONE Pesticide420™ Internal Standard - 4 mL ACN with 0.1% formic acid |
n/a |
Hardware and software
A PerkinElmer LC-MS/MS® LX50 UHPLC system was employed in the Chromatographic separation, and a PerkinElmer Q-Sight® 420 MS/MS detector with a dual ionization ESI and APCI source was employed in the detection of analytes.
This instrument’s sources operate independently with two separate inlets into the mass spectrometer.
The Simplicity 3Q™ software platform was utilized for instrument operation, data acquisition and data processing.
LC method and MS source conditions
Tables 4 and 5 display the LC method and MS source parameters.
Table 4. LC method conditions for ESI and APCI. Source: PerkinElmer Cannabis & Hemp Testing Solutions
| LC Conditions |
| LC Column |
PerkinElmer Quasar SPP Pesticides (4.6 × 100 mm, 2.7 μm) |
| Column Oven Temperature |
30 ºC |
| Auto sampler Temperature |
20 ºC |
| LC Conditions |
| Mobile Phase A |
2 mM ammonium formate + 0.1% formic acid in LCMS grade water |
| Mobile Phase B |
2 mM ammonium formate + 0.1 % formic acid in LCMS grade methanol |
| Mobile Phase Gradient |
A 18.5 min LC-MS/MS method with optimized gradient using ESI source was used for separation and analysis of 62 out of 66 pesticides and five mycotoxins residues regulated by California state in different cannabis matrices. |
| Injection Volume |
3.0 μL |
| APCI Method |
| Mobile Phase A |
LCMS grade water |
| Mobile Phase B |
LCMS grade methanol |
| Mobile Phase Gradient |
A fast 6 min. LC-MS/MS method with short gradient, optimum mobile phase composition and APCI source was used for measurement of remaining four pesticides regulated by California state in different cannabis matrices. |
| Injection Volume |
10 μL |
Table 5. MS source conditions for both ESI and APCI methods. Source: PerkinElmer Cannabis & Hemp Testing Solutions
| MS Source Conditions for ESI Source & APCI Source |
| Drying Gas |
150 arbitrary units |
| Nebulizer Gas |
350 arbitrary units |
| Detection Mode |
Time-managed MRM™ |
| ESI Method |
| Source Temperature |
315 ºC |
| HSID Temperature |
200 ºC |
| ESI Voltage (Positive) |
+5100 V |
| ESI Voltage (Negative) |
-4200 V |
| APCI Method |
| APCI Corona Discharge |
-3 μA |
| Source Temperature |
250 ºC |
| HSID Temperature |
180 ºC |
Conclusion
The study outlined here demonstrates the ONE Pesticide420 TM Reagent and Consumable Kits’ potential to saves testing labs considerable time in their procurement, reagent preparation and consumable handling.
As demonstrated by the calibration curves and quality control samples, this kit facilitates rapid, reliable quantitation of up to 77 pesticide and five mycotoxin residues in cannabis samples when used alongside mass spectrometry.
The kit also provides laboratories a single source for all pesticide, mycotoxin and internal standard reagents, as well as quality control reagents and laboratory consumable hardware, for example, vials and filters.
The easy-to-use kits are preconfigured to ISO 17034 CRM standards and reagents, not only improving data quality but also enabling closer alignment with ISO 17025 requirements.
Using the ONE Pesticide420 TM Reagent and Consumable Kit, it is possible to prepare calibration and QC standards in a matter of minutes, empowering laboratories to increase productivity and test more samples in less time than existing solutions.
Acknowledgments
Produced from materials originally authored by Avinash Dalmia, Jacob Jalali, Dominique B. Figueroa, and Toby Astill from PerkinElmer.
About PerkinElmer Cannabis & Hemp Testing Solutions
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