• Application Note

A Multi-Residue Method for the Analysis of Pesticides in Cannabis Using UPLC-MS/MS and APGC-MS/MS to Meet Canadian Regulatory Requirements

A Multi-Residue Method for the Analysis of Pesticides in Cannabis Using UPLC-MS/MS and APGC-MS/MS to Meet Canadian Regulatory Requirements

  • David James
  • Nichole Taylor
  • Christopher Stadey
  • Bloom Labs at Perennia Laboratories
  • Waters Corporation

Abstract

This application note presents the use of a simple sample extraction and d-SPE (dispersive solid phase extraction) cleanup where the resulting extract is analyzed by UPLC-MS/MS or APGC-MS/MS. 

Benefits

  • Sensitive and reproducible workflow for screening cannabis for the Canadian list of pesticides
  • Minimal sample preparation followed by rapid UPLC and GC separations
  • UPLC-MS/MS and APGC-MS/MS analysis of the same sample extracts on one tandem quadrupole mass spectrometer
  • Analysis of large suites of pesticides in a single injection per chromatographic inlet
  • Analysis of cannabis at legislatively relevant levels 

Introduction

Health Canada requires mandatory testing for the presence of pesticide residues in cannabis before the product can be sold to consumers.1,2 The regulations are present to ensure the highest safety and quality standards possible when it comes to the supply of cannabis for medical or recreational use. To adhere to testing requirements, licensed cannabis producers must demonstrate that no unauthorized pesticides have been used on their products and that there is no contamination of the products within the limits set out by Health Canada. Currently, the target list consists of 96 pesticides, with limits of quantitation as low as 20 ppb in dried cannabis. Tandem mass spectrometry is a sensitive and selective technique. When coupled with both gas (GC) and liquid chromatography (LC), it provides a comprehensive analysis for a wide range of pesticide residues with sufficient sensitivity to meet the Health Canada regulations. The advantage of ultraperformance liquid chromatography (UPLC) coupled with tandem quadrupole mass spectrometry (MS/MS) for multi-residue pesticide analysis is widely reported.3 More recently, the use of GC-MS/MS operated at atmospheric pressure (APGC) has been shown to offer significant improvements in performance over EI for challenging pesticides, in terms of selectivity, specificity and speed of analysis.4,5 Regulations for cannabis testing will most likely evolve and possibly become even more rigorous. The use of both LC-MS/MS and GC-MS/MS ensures system flexibility that can be adapted  in the event that more pesticides are regulated.

In this Application Note, we present the use of a simple sample extraction and d-SPE (dispersive solid phase extraction) cleanup where the resulting extract is analyzed by UPLC-MS/MS or APGC-MS/MS. A single workflow for the multi-residue analysis of pesticides in cannabis is demonstrated. Utilizing the universal source of the Waters Xevo TQ-S micro allows for LC and GC analyses to be completed on the same tandem quadrupole MS instrument. The performance of the method will be highlighted in terms of sensitivity, recovery, and linearity for both LC and GC analysis.

Experimental

Materials and reagents

1. Pesticide standards

Pesticide analytical standards were purchased from LGC Standards. Mix 1 consisted of 35 pesticides at 50 ppm in acetonitrile, Mix 2 consisted of 45 pesticides at 100 ppm in acetonitrile, and Mix 3 consisted of 14 pesticides at 100 ppm in toluene. Dimethomorph was also purchased from LGC Standards at 10 ppm in acetonitrile. Benzovindiflupyr was purchased separately from Chem Service at 100 ppm in methylene chloride solution. All 96 pesticides were combined in a 1 ppm stock solution of each.

2. Reagents

LC-MS-grade methanol, LC-MS-grade acetonitrile, and RO (reverse osmosis) water were all purchased from Fisher Scientific and were used as received. Formic acid was purchased from Waters (p/n: 186006691) and was used as received.

3. Miscellaneous

Helium and argon gases were obtained from Air Liquide. A Thermo Fisher Scientific vortex (0-3200 rpm), a Fisher Scientific accuSpin 400 centrifuge, a Fisher Scientific 60L gravity oven, and a Mettler Toledo AE50 analytical balance (0.1 mg) were all used in the sample preparation procedure.

Sample preparation

Preparation 

The representative samples were dipped in liquid nitrogen and frozen before grinding. After freezing, but before grinding, all stems and seeds were removed from the sample. The ground sample was equilibrated to room temperature. Several 0.5-g portions of ground cannabis were weighed. The initial mass was recorded. To ensure that all the liquid nitrogen had evaporated, and an accurate sample mass was obtained, the sample sat on the scale until there was <1 mg change in mass over a 10-minute period. 

Pesticide extraction

The 0.5-g samples of ground cannabis were placed in a 10-mL centrifuge tube and 5 mL of LC-MS/MS-grade acetonitrile was added. The sample was then vortexed for five minutes followed by centrifugation at 5000 rpm for five minutes. One milliliter of the supernatant was removed and used in the clean-up step. 

Clean-up

One milliliter of the supernatant from the pesticide extraction was placed in a d-SPE cartridge (150 mg MgSO4, 50 mg PSA, 50 mg C18, and 7.5 mg graphitized carbon black). The cartridge was shaken for one minute and centrifuged for five minutes at 5000 rpm. The resulting cannabis extracts were directly pipetted into clean 2-mL vials in preparation for analysis by LC-MS/MS and APGC-MS/MS. 

Calibration preparation

Calibration standards were made using a stock solution of 96 pesticides (1 ppm stock). Matrix-matched calibrations were used to ensure that the signals obtained in the analysis were representative of what the signal would be in cannabis samples. Standards ranging from 1–6400 ppb were made to accommodate the different ionization efficiencies of all analytes. Pesticides with low detection limits used the lower concentration standards and the pesticides with higher detection limits used higher concentration standards for their calibration curves. 

Instrumentation and software

A Waters ACQUITY UPLC H-Class PLUS System coupled with a Waters Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer (MS/MS) with electrospray as the ionization mode was used to carry out the analysis of 84 of the pesticides by LC-MS/MS (see Appendix A). An Agilent 7890B gas chromatograph (GC) coupled with a Waters Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer was used to carry out the analysis of the remaining 12 pesticides with APGC as the ionization mode. A nitrogen generator (Peak Scientific) was used as the source of the N2 gas. MassLynx MS Software v4.2 was used for data acquisition and processing for both LC-MS/MS and GC-MS/MS methods.

UPLC conditions

Separation mode:

Gradient

Column:

ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 100 mm

Solvent A:

Methanol

Solvent B:

Water

Solvent C:

2% formic acid in RO water

Flow rate:

0.500 mL/min

Column temp.:

60 °C

Sample temp.:

10 °C

Injection volume:

2 μL

Gradient table:

Time (min)

%A

%B

%C

0

2%

93%

5

8

95%

0%

5

9

95%

0%

5

9.1

2%

93%

5

12

2%

93%

5

Xevo TQ-S micro conditions

Ionization mode:

ESI+

Capillary voltage:

1.2 kV

Cone voltage:

30 V

Collision energy:

Various eV (see Appendix)

Desolvation temp.:

600 °C

Source temp.:

150 °C

Desolvation gas flow:

1000 L/hr

Cone gas:

50 L/hr

All MS/MS parameters including precursor ion (m/z), product ion (m/z), cone voltage (V), and collision energy (CE) for the 84 pesticides analyzed by LC-MS/MS can be found in Appendix A.

GC conditions

GC system:

Agilent 7890B

Column:

Agilent DB-5 MS (30 m × 0.250 mm × 0.25 μm)

Carrier gas:

Helium

Flow rate:

2 mL/min

Injection type:

Pulsed splitless

Injector temp.:

280 °C

Equilibration time:

1.5 min

Injection volume:

2 μL

Makeup gas:

Nitrogen at 350 mL/min

GC oven program

Rate (°C/min)

Temp. (°C)

Hold (min)

60

0.45

18.7

320

3.65

Total run time = 18.0 min

GC-MS/MS parameters

MS system:

Xevo TQ-S micro

Ionization mode:

APGC+

Corona:

2.0 μA

Transfer line temp.:

320 °C

Source temp.:

150 °C

Solvent delay:

3.5 min

Acquisition mode:

MRM

All MS/MS parameters including precursor ion (m/z), product ion (m/z), cone voltage (V), and collision energy (CE) for the 12 pesticides analyzed by GC-MS/MS can be found in Appendix B.

Method development and optimization

LC-MS/MS and GC-MS/MS data analysis 

The UPLC and GC parameters were optimized to ensure adequate separation of pesticide peaks with reduced background noise and optimum peak shapes. Upon completion of the sample run, a “multiplier” must be input into the UPLC and GC to account for the dilutions and sample mass weighed. The following formula is used to calculate the multiplier:

where Vextraction is the total volume of the extract used (5 mL) and Mass is the mass of the dried cannabis weighed for the extraction (0.5 g). This will convert all results in ppb in cannabis (µg of pesticide/g of cannabis).

Validation of method (sample spiking and recovery)

To validate the method, sample spikes were performed on ground cannabis prior to the extraction and clean-up. The pesticide mixes were spiked into 0.5 g of fresh ground “pesticide-free” cannabis samples. Extraction and clean-up were performed resulting in 2000, 1000, 500, 250, 100, 50, 25, 10, 5, and 2 ppb spiked samples. After applying the multiplier (described above), the concentration of the pesticides mentioned above are 10x higher in the cannabis sample.

The spiking procedure was performed at nine different spike concentrations for each pesticide to obtain the limit of quantification (LOQ) for each individual pesticide. Once the LOQ was established, three spikes of each analyte at their respective LOQ were performed to obtain average spike recoveries and relative standard deviations (RSD) for each pesticide individually. 

As shown in Table 1, spike recoveries for all pesticides at their LOQs averaged between 81.7% and 117.6%. The acceptable % recovery limits for method validation are between 70% and 120%. Low relative standard deviations (RSD) were also reported for all 96 spike recoveries (all <20%). The acceptable RSD for method validation is <20%. 

It should be noted that the recovery for daminozide is determined separately since it is strongly retained by the PSA sorbent. For spike recoveries and to test for the presence of daminozide in cannabis samples, a separate LC-MS/MS run is performed following sample extraction but before clean-up.  

Table 1. Spike recoveries for the 96 pesticides in dried cannabis sample.

Limits of quantification (LOQs)

The LOQs were calculated for all 96 pesticides. To determine the LOQs, pesticide-free cannabis samples were spiked with various concentrations of standards ranging from 1–2000 ppb. Sample spike recoveries between 80% and 120% were deemed acceptable. Once the lowest acceptable spike recoveries (lowest concentrated spike) were determined for each pesticide, three separate runs were performed and only after all three runs fell within the acceptable limits was the LOQ established. As shown in Table 2, all LOQ values are within Health Canada’s limits.

Table 2. Experimental limits of detection for all 96 pesticides using the LC-MS/MS and GC-MS/MS methods. 

Results and Discussion

Pesticides analysis by UPLC-MS/MS

Using the LC-MS/MS method, 84 pesticides were analyzed. The compounds analyzed by LC-MS/MS and the parameters used are listed in Table 2 and Appendix A. Representative MRM chromatograms for the pesticides acetamiprid (50 ppb), cyprodinil (25 ppb), fenoxycarb (25 ppb), and tetrachlorvinphos (25 ppb) in a pesticide-free extracted cannabis matrix are shown in Figure 1. 

Figure 1. Representative MRM chromatograms showing the primary quantification and the secondary qualifier transition for acetamiprid (A, 50 ppb), cyprodinil (B, 25 ppb), fenoxycarb (C, 25 ppb), and tetrachlorvinphos (D, 25 ppb) in pesticide-free cannabis extracted using the sample preparation protocol reported.

Matrix-matched calibration curves were generated using pesticide-free extracted cannabis. An example of the calibration curves for the pesticides acetamiprid, cyprodinil, fenoxycarb, and tetrachlorvinphos are shown in Figure 2. Linear calibration curves (R2>0.990) for all pesticides were obtained over the range tested as shown in the figure.

Figure 2. Representative examples of calibration curves for acetamiprid (A, 0.78–200 ppb), cyprodinil (B, 0.78–200 ppb), fenoxycarb (C, 0.78–1500 ppb), and tetrachlorvinphos (D, 0.78–800 ppb), demonstrating linearity over the ranges tested for these compounds.

Pesticides analysis by GC-MS/MS

Analysis of pesticide residues in cannabis also required the use of GC-MS/MS to meet the Canadian pesticide regulations. A complete list of compounds analyzed by GC-MS/MS and the parameters used is provided in Table 2 and Appendix B. Example chromatograms for endosulfan alpha and fenvalerate are shown in Figure 3.

Figure 3. Representative MRM chromatograms showing the primary quantification and the secondary qualifier transition for endosulfan alpha (100 ppb) and fenvalerate at a level and 400 ppb (ng/g) in pesticide-free cannabis extracted using the sample preparation protocol reported.

An example of the calibration curves for the pesticides endosulfan alpha and fenvalerate are shown in Figure 4. Linear calibration curves (R2>0.990) for both pesticides were obtained over the range tested, as shown in the figure.

Figure 4. Representative examples of calibration curves for endosulfan alpha and fenvalerate demonstrating linearity over the ranges tested for these compounds.

Conclusion

The simple sample extraction and d-SPE clean-up method followed by UPLC-MS/MS and GC-MS/MS analysis provides a rapid, sensitive, and robust workflow for the determination of the Canadian pesticide list in challenging cannabis matrices. Complex multi-residue pesticide analysis in a cannabis matrix was demonstrated using both UPLC and APGC  analysis on the same tandem quadrupole instrument (Xevo TQ-S micro) with detection at the maximum action levels for each of the 96 pesticides in the Canadian pesticide list. Having the flexibility of universal source architecture to provide access to both UPLC-MS/MS and GC-MS/MS on the same instrument, allows for an increase of laboratory efficiency, while maintaining required sensitivity and repeatability. This method meets the action levels for the Canadian pesticide list and mycotoxins in cannabis matrices. 

References

  1. Moulins, J. R.; Blais, M.; Montson, K.; Tully, J.; Mohan, W.; Gagnon, M.; McRitchie, T.; Kwong, K.; Snider, N.; Blais, D. R. Multiresidue Method of Analysis of Pesticides in Medical Cannabis. J. AOAC 2018, 1101 (6), 1948–1960.
  2. Health Canada. (2018) Mandatory Cannabis Testing for Pesticide Active Ingredients. https://www.canada.ca/content/dam/phac-aspc/documents/services/publications/drugs-health-products/cannabis-testing-pesticide-requirements/cannabis-testing-pesticide-requirements.pdf [accessed on June 11th 2019].
  3. Kovalczuk, T.; Jech, M.; Poustka, J.; Hajslova, J.; UPLC-MS/MS: A Novel Challenge in Multiresidue Pesticide Analysis in Food Analytica Chimica Acta 2006, 577.
  4. Tienstra, M.; Portoles, T.; Hernandez, F.; Mol, J. G. J. Fast Gas Chromatographic Residue Analysis in Animal Feed Using Split Injection and Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry J. Chromatogr,. A 2015, 1422.
  5. Cherta, L.; Portoles, T.; Beltran, J.; Pitarch, E.; Mol, J. G. J.; Hernandez, F. Application of Gas Chromatography – Mass Spectrometry with Atmospheric Pressure Chemical Ionization for the Determination of Multiclass Pesticides in Fruits and Vegetables 2013, 1314.

Appendix A.

MS/MS parameters for pesticides using UPLC.

Appendix B

MS/MS parameters for pesticides using GC.

720006711, November 2019

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