routine analysis of cannabis for pesticides and mycotoxins ... · dspe-2 mltube with 150 mg mgso4,...
TRANSCRIPT
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Routine Analysis of Cannabis for Pesticides and Mycotoxins
using UPLC-MS/MS and GC-MS/MSMarian Twohig, Ph.D.
Principal Scientist, Waters Corporation
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Overview• Challenges in pesticides testing
Regulatory requirements, number of compounds and action limits
• Sample preparation and cleanup optionsChallenges with cannabis matrix and strategies to minimize interferences
• Analytical workflow and application dataQuantitative and confirmatory analysis of LC and GC amenable pesticides
• Summary
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The Analytical Journey
Which direction to go in?
The true method of knowledge is
experiment
-William Blake
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The Analytical Journey
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Analysis of Oregon pesticide list using LC/MS/MS59 pesticides, 100 ppb to 2 ppm
+
• The action levels demanded by this state make it amenable to an LC-MS/MS only approach. • There are some challenging compounds to deal with but we were
able to see them at the required levels.
+
Waters App Note: 720006373EN TQ-S microAcquity H Class
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Pesticide ListsAbamectinAcephateAcequinocylAcetamipridAldicarbAzoxystrobinBifenazateBifenthrinBoscalidCaptanCarbarylCarbofuranChlorantraniliproleChlordane ChlorfenapyrChlorpyrifosClofentezineCoumaphosCyfluthrinCypermethrinDaminozideDDVP (Dichlorvos)
Diazinon
DimethoateDimethomorphEthoprop(hos)EtofenproxEtoxazoleFenhexamidFenoxycarbFenpyroximateFipronilFlonicamidFludioxonilHexythiazoxImazalilImidaclopridKresoxim-methylMalathionMetalaxylMethiocarbMethomylMethyl parathionMevinphos
MyclobutanilNaledOxamylPaclobutrazolPentachloronitrobenzene/QuintozinePermethrinPhosmetPiperonylbutoxidePrallethrinPropiconazolePropoxurPyrethrinsPyridabenSpinetoramSpinosadSpiromesifenSpirotetramatSpiroxamineTebuconazoleThiaclopridThiamethoxamTrifloxystrobin
CA Pesticide Residue List
Canada Additional Pesticides
AllethrinAzadirachtinBenzovindiflupyrBuprofezinClothianidinCyantranilipoleCyprodinilDeltamethrinDinotefuranDodemorphEndosulfan-alphaEndosulfan-beta
Endosulfan-sulfateEtridiazolFensulfothionFenthionFenvalerateFluopyramIprodioneKinopreneMethopreneMGK-264NovaluronPhenothrin
PirimicarbPyraclostrobinResmethrinSpirodiclofenTebufenozideTeflubenzuronTetrachlorvinphosTetramethrinThiophanate-methyl
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The Goal of The Analysis
• If we think about the challenging compounds that are to be analysedin the presence of the cannabis matrix, possibly in a variety ofmatrices. The ultimate goal is to be able to use a set of tools that willprovide the best success, given all of the variability that the differentstrains or matrices might present.
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Challenges
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Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
%
0
100 12.15
10.9310.14
11.70
12.27
XIC m/z 357
XIC m/z 315
THCA CBDA
THC CBD
Matrix Suppression and ComplexityMS Full Scan ESI-
MS Full Scan ESI+
Cannabis flower extract
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Matrix Suppression and ComplexityESI+ Red Trace
ESI- Green Trace
Pesticide MRM’s at ppb levels in extract
Cannabis flower extract
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100 ppb Standard 100 ppb Pre-spike
Not Detected
388 > 273
371 > 273
Matrix Suppression: Spiromesifen 100 ppb ESI
388 > 273
371 > 273
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Matrix Suppression: Spiromesifen 100 ppb APCI
100 ppb Pre-spike 100 ppb std 3
Time10.50 11.00 11.50 12.00
%
0
100
10.50 11.00 11.50 12.00
%
0
100APCI_05Oct2018_0031 53: MRM of 4 Channels AP+
388.2 > 273.1 (Spiromesifen)8.38e4
11.06
APCI_05Oct2018_0031 53: MRM of 4 Channels AP+ 388.2 > 255.1 (Spiromesifen)
1.34e411.06
100ppb spiked cannabis 1
Time10.50 11.00 11.50 12.00
%
0
100
10.50 11.00 11.50 12.00
%
0
100APCI_05Oct2018_0034 53: MRM of 4 Channels AP+
388.2 > 273.1 (Spiromesifen)7.86e4
11.05
APCI_05Oct2018_0034 53: MRM of 4 Channels AP+ 388.2 > 255.1 (Spiromesifen)
1.33e411.05
10.37
100 ppb Standard
388 > 273
371 > 273
388 > 273
371 > 273
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Spinetoram/Spinosad D Chromatographic ResolutionOR Gradient
1000 ppb std 3
Time10.00 10.25 10.50 10.75 11.00
%
0
100
10.00 10.25 10.50 10.75 11.00
%
0
100 10.70
10.71
CA Gradient
100ppb solvent std 3
Time9.00 9.50 10.00 10.50
%
0
100
9.00 9.50 10.00 10.50
%
0
10010Nov2018_12 71: MRM of 2 Channels ES+
746.5 > 142 (Spinosad D)6.36e5
9.71
10.02
10Nov2018_12 72: MRM of 2 Channels ES+ 748.5 > 142.1 (Spinetoram)
1.27e69.93
9.70
746.5 > 142
748.5 > 142
C42H67NO10Spinosad D
Spinetoram
C42H69NO10
748.5 > 142
746.5 > 142
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Sample Extraction, Clean Up and Analysis
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Some Natural Cannabis Constituents
THCATHC
CBDA
CBD
Myrcene
Cannabinoids – Potential Interferences
TerpenesGC Compounds
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Other Cannabis ConstituentsCommon to Most Plants
Chlorophyll
Contaminates GC Injectors
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Can I Get Rid of the Matrix?Physicochemical Properties Comparison
Pyrethrin I
MW 328.4Log P ~ 6
THC
MW 314.4Log P ~ 7
Separation of pesticide residues from cannabis resin will be difficult
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• Most pesticides are neutral or very weakly acidic
• Aflatoxins are neutral
• Ochratoxin is acidicCleanup with PSA sorbent will retain ochratoxin – reduced recovery for this compound
Pesticides and Mycotoxins
PSA silica structurepKa > 10 (diamine)
Aflatoxin B1
Ochratoxin ApKa > 3.2
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¡ dSPE Clean Up
– Shake dSPE tube 1min
– Centrifuge
– Supernatant to autosampler vial
dSPE - 2 mL tube with 150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg graphitized carbon black
Sample Preparation with dSPE for LC-MS/MS
• Initial Extraction
• 0.5 g ground cannabis bud weighed
into 15 mL centrifuge tube
• 5 mL acetonitrile added
• Geno Grinder for 5min @ 1500 rpm
• Remove 1 mL aliquot for dSPE
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Acetonitrile Extraction Only Versus dSPE
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Shaded area indicates compounds that co-elute with high abundance cannabis resin constituents
0
20
40
60
80
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Dami
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Metho
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% Matrix Effect no dSPE
% Matrix Effect dSPE
Matrix Suppression-Acetonitrile Extraction Versus dSPE0.2 µg/g Pre-Spiked Extract
Red bars = without dSPE Blue bars = with dSPE
120
0
60
% Suppression
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Recovery – Method Accuracy California
Recoveries for most of the pesticides were in the range of 80% to 120%
% Recovery of pesticides from cannabis matrix (n = 6)
020406080
100120140
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100 ug/g
500 ug/g
0
20
40
60
80
100
120
140
Flud
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0.1 µg/g0.5 µg/g
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Reproducibility
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Mevinp
hos
Methyl p
arathion
Fenhexam
id
Chlorpyrifos
Coumaphos
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omorph
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bin
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Spinetor
am
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A
Fipronil
%RSD (n=5) Pre-spike 0.10 µg/g
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LC Column Protection Products/Guard Columns
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Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
%
0
100Xbridge04Augus2018_30a 86: MRM of 2 Channels ES-
TIC (Fipronil)1.27e7
Workflow for Multi-Residue Pesticide AnalysisA high throughput detection of pesticides by UPLC-MS/MS and GC-MS/MS
LC pesticides
GC pesticides
UPLC/Xevo TQ-S micro
Xevo TQ-GC
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Workflow for Multi-Residue Pesticide AnalysisUPLC-MS/MS and GC-MS/MS
LCAmenablePesticides
GCAmenablePesticides
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Application Data – A Cannabis LC/MS/MS and GC/MS/MS Method to Meet California
Mycotoxin and Pesticide Residue Regulatory Requirements
Waters App Note: 720006465EN
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UPLC Method§ Acquity H Class coupled to TQ-S micro§ Column: XBridge C18, 2.1 x 150 mm, 2.5 µm@ 500C§ Mobile Phase A: Water + 2 mM ammonium acetate
with 0.02% formic acid§ Mobile Phase B: MeOH§ Flow rate: 0.40 mL/min§ Injection volume: 5 µl§ Gradient:
Experimental: Inlet Methods
Xevo TQ-GC§ Xevo TQ-GC§ Column: Rxi-5MS 20 m x 0.18 mm x 0.18 µm§ Carrier gas: Helium § Injector temp.: 280 OC§ Flow rate: 2 mL/min§ Injection volume: 1 µl§ Oven Program: Time % A % B
0 98 20.20 98 24.00 30 70
10.00 30 7012.00 1 9915.00 1 99
Rate (°C/min) Temperature (°C) Hold (min)
- 60 0.4518.70 330 2.25
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Method Generation: QuanpediaThree Clicks to an Analysis Method – 1, 2, ....
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QuanpediaThree Clicks to an Analysis Method – 3
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Quanpedia Generated Acquisition MethodMS acquisition method
MS processing method
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• QuanpediaEncyclopedia of pesticide and mycotoxin acquisition and processing methods
• IntelliStartAutomates optimization of new analytes, 1 or 2 at a time
• QuanOptimizeAutomates optimization of batches of new analytes, as many as necessary
Pesticides Analysis Now and in the FutureWhat If More Pesticides Get Added to the List?
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LC-MS/MS Analysis of Pesticides Spiked at 0.1µg/g in Cannabis Flower
min3.80 4.00 4.20 4.40 4.60 4.80
%
0
100
F10:MRM of 2 channels,ES+225.1 > 127.1
10Nov2018_16
1.004e+006
Mevinphos4.37
4.69*
min6.500 6.600 6.700 6.800 6.900
%
0
100
F28:MRM of 4 channels,ES+304.1 > 97.1
10Nov2018_16
6.669e+005
Fenhexamid6.70
min9.25 9.50 9.75 10.00 10.25 10.50 10.75
%
0
100
F72:MRM of 2 channels,ES+748.5 > 142.1
10Nov2018_16
7.588e+005
Spinetoram10.02
9.76
min6.200 6.300 6.400 6.500
%
0
100
F59:MRM of 2 channels,ES+388.1 > 301.1
10Nov2018_16
2.200e+006
Dimethomorph6.36
min7.200 7.300 7.400 7.500 7.600 7.700 7.800
%
0
100
F52:MRM of 3 channels,ES+365 > 229
10Nov2018_16
3.630e+005
Coumaphos7.47
min11.00 11.50 12.00 12.50
%
0
100
F47:MRM of 2 channels,ES+351.9 > 124.9
10Nov2018_16
3.020e+005
Chlorpyrifos11.73
Mevinphos
Fenhexamid
Spinetoram
Dimethomorph
Coumaphos
Chlorpyriphos
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100ppb spiked 3
Time4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80
%
0
100
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80
%
0
100
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80
%
0
100
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80
%
0
100
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80
%
0
100
10Nov2018_18 43: MRM of 5 Channels ES+ 331.1 > 245.1 (Aflatoxin G2)
4.43e44.63
10Nov2018_18 40: MRM of 4 Channels ES+ 329.1 > 243.1 (Aflatoxin G1)
1.14e54.78
10Nov2018_18 35: MRM of 4 Channels ES+ 315.1 > 287.1 (Aflatoxin B2)
3.02e44.94
10Nov2018_18 33: MRM of 4 Channels ES+ 313.1 > 285.1 (Aflatoxin B1)
6.30e45.07
10Nov2018_23 61: MRM of 3 Channels ES+ 404.1 > 239 (Orchatoxin A)
5.26e46.04
Mycotoxins Spiked at 0.02 µg/g in Cannabis Flower
Ochratoxin A
Aflatoxin G2
Aflatoxin G1
Aflatoxin B2
Aflatoxin B1
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Quantitation of Fenhexamid
Residuals < 2%
R2 = 0.99925-500 ppb in sample2.5-50 ppb in vial
25 ppb2.5 ppb in vial
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Quantitation of FenhexamidStandards (n=4)
Pre-spikes (n=6)
Matrix Matched Curve
Note in-sample concentrations listed in the Std. Conc. column
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Precision and Accuracy
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• System: Xevo TQ-GC• Column: Rxi-5MS 20m x 0.18mm x
0.18µm• Injection: Pulsed Splitless• Injector Temp: 280 ºC• Pulse Pressure: 45 psi• Flow: 2 mL/min• Oven Program:
60 ºC, hold for 0.45 min18.7 ºC/min to 330 ºC, hold 2.25 minTotal Run Time = 17.14 min
GC-MS/MS Analysis of Pesticides in Cannabis
¡Electron Energy: 70 eV¡Emission: 300 µA¡Source: 5.5 V¡Repeller: 44 V¡Extraction: 23 V¡Focus 1: 11 V¡Focus 2: 150 V¡Source Temp: 250¡GC Interface Temp: 300
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Residuals < 4%
R2 = 0.99925 – 1000 ppb (in sample)2.5 – 100 ppb (in vial)
25 ppb (2.5 ppbin vial)
GC-MS/MS Analysis of Pesticides in Cannabis-Methyl Parathion
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LC-MS/MS0.1 µg/g 5µL injection
GC-MS/MS0.1 µg/g 1µL injection
LC-MS/MS versus GC-MS/MS- Analysis of Pesticides in Cannabis-Methyl Parathion
min8.000 8.100 8.200 8.300 8.400 8.500 8.600 8.700 8.800
%
0
100
F16:MRM of 2 channels,EI+263 > 79
Cannabis_CA_9Nov18_016Matrix curve 100
1.750e+004Methyl Parathion
8.40
8.358.10 8.27
8.86
8.718.52
8.63 8.77
min
%
0
100
F16:MRM of 2 channels,EI+263 > 109
Cannabis_CA_9Nov18_016Matrix curve 100
7.338e+004Methyl Parathion
8.40
8.06
min5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90
%
0
100
F18:MRM of 3 channels,ES+264.15 > 232.1
10Nov2018_05 Smooth(Mn,2x2)100 ppb matrix match
3.845e+004Methyl parathion
6.03
5.96
5.915.755.17 5.22
6.46
6.19
6.62
min
%
0
100
F18:MRM of 3 channels,ES+264.15 > 125.1
10Nov2018_05 Smooth(Mn,2x2)100 ppb matrix match
4.440e+004Methyl parathion
6.03
5.64
100ppb spiked 1
Time5.00 6.00 7.00
%
0
100
5.00 6.00 7.00
%
0
100 S/N:PtP=63.79
Pre spike 100 1
Time8.00 8.50
%
0
100
8.00 8.50
%
0
100 S/N:PtP=74.97
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GC-MS/MS Analysis of Pesticides in Cannabis0.1 µg/g or 0.7 µg/g, 1µL injection
min11.050 11.100 11.150 11.200 11.250 11.300 11.350 11.400
%
0
100
F10:MRM of 2 channels,EI+181.1 > 165.1
Cannabis_CA_9Nov18_029Pre spike 100 4
1.966e+006
Bifenthrin11.16
11.02
1 2
3 4
min5.700 5.750 5.800 5.850 5.900 5.950 6.000 6.050 6.100 6.150 6.200 6.250
%
0
100
F7:MRM of 2 channels,EI+151 > 80
Cannabis_CA_9Nov18_029Pre spike 100 4
5.313e+004THPI5.95
min7.600 7.650 7.700 7.750 7.800 7.850 7.900
%
0
100
F15:MRM of 2 channels,EI+214 > 179
Cannabis_CA_9Nov18_027Pre spike 100 2
1.473e+004PCNB7.76
7.66
min9.300 9.350 9.400 9.450 9.500 9.550 9.600 9.650 9.700 9.750 9.800
%
0
100
F19:MRM of 2 channels,EI+373 > 266
Cannabis_CA_9Nov18_030Pre spike 100 5
5.779e+003Chlordane 1
9.50
9.45
Chlordane 29.66
9.56
Captanas THPI
Bifenthrin
Cis/trans-chlordane PCNB
0
100
200
300
400
500
600
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49Pe
ak R
espo
nse
Injection Number
PCNB
Bifenthrin
12% RSD
6% RSD
GC-MS/MS Reproducibility
0.7 µg/g
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• Quantitative and confirmatory pesticides detection in cannabisBroader analyte coverage for both LC and GC pesticides, Mycotoxins analysed as a subset of the LC-MS/MS assay.
• Simple and generic sample extractionIncreased ease, decreased training needs, fewer steps where losses/variability can occur
Summary• Reduced matrix suppression using
dSPEImproved robustness, higher uptime, longer column life, less frequent source cleaning
• Fast ImplementationQuanpedia database and quantitation workflow ensures rapid implementation
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To strive, to seek, to find, and not to yield Alfred Lord Tennyson
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ProVerde Laboratories• Chris Hudalla• Rebecca Stevens• James Roush
Acknowledgements
Waters Corp• Kim Tran• Kari Organtini• Gordon Fujimoto• Michael S.Young• Doug Stevens• Narendra Meruva• Andy Aubin
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Thank you for your attention!