Routine Analysis of Cannabis for Pesticides and Mycotoxins
using UPLC-MS/MS and GC-MS/MSMarian Twohig, Ph.D.
Principal Scientist, Waters Corporation
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
The Analytical Journey
Which direction to go in?
The true method of knowledge is
experiment
-William Blake
The Analytical Journey
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
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
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.
Challenges
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
Matrix Suppression and ComplexityESI+ Red Trace
ESI- Green Trace
Pesticide MRM’s at ppb levels in extract
Cannabis flower extract
100 ppb Standard 100 ppb Pre-spike
Not Detected
388 > 273
371 > 273
Matrix Suppression: Spiromesifen 100 ppb ESI
388 > 273
371 > 273
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
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
Sample Extraction, Clean Up and Analysis
Some Natural Cannabis Constituents
THCATHC
CBDA
CBD
Myrcene
Cannabinoids – Potential Interferences
TerpenesGC Compounds
Other Cannabis ConstituentsCommon to Most Plants
Chlorophyll
Contaminates GC Injectors
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
• 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
¡ 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
Acetonitrile Extraction Only Versus dSPE
Shaded area indicates compounds that co-elute with high abundance cannabis resin constituents
0
20
40
60
80
100
120
Dami
nozid
eAc
epha
teOx
amyl
Metho
myl
Flomi
cami
dTh
iameth
oxam
Imida
clopri
dDim
ethoa
teAc
etami
prid
Thiac
loprid
Aldica
rbDic
hlorvo
sPro
poxu
rCa
rbofur
anCa
rbaryl
Metal
axyl
Imaza
lilNa
led (D
ibrom
)Ch
loran
tranil
prole
Phos
met
Parat
hion m
ethyl
Spiro
mesif
enAz
oxyst
robin
Methi
ocarb
Spiro
xami
neBo
scalid
Paclo
butra
zol
Malat
hion
Myclo
butan
ilBif
enaza
teSp
irotet
ramat
Ethop
ropFe
noxyc
arbFip
ronil
Kreso
xim-m
ethyl
Tebu
cona
zole
Diazin
onPro
picon
azole
Clofen
tezine
Spino
sad A
MGK-2
64Pra
llethr
inTri
floxy
strob
inCh
lorfen
pyr
Pyret
hrin I
ISp
inosad
DPip
erony
l buto
xide
Chlor
pyrifo
sHe
xythi
azox
Etoxa
zole
Pyret
hrin I
Fenp
yroxim
atCy
fluthr
inCy
perm
ethrin
Pyrid
aben
Abam
ectin
B1a
Perm
ethrin
Etofen
prox
Bifen
thrin
Aceq
uinoc
yl
% 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
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
Abam
ectin
B1a
Acep
hate
Aceq
uino
cyl
Acet
amip
ridAf
lato
xin
B2Af
lato
xin
G1
Afla
toxi
n G
2Af
lato
xinB
1Al
dica
rbAz
oxys
trob
inBi
fena
zate
Bife
nthr
in**
Bosc
alid
Carb
aryl
Carb
ofur
anCh
lora
ntra
nilip
role
Chlo
rdan
e ci
s**
Chlo
rdan
e tr
ans*
*Ch
lorf
enap
yrCh
lorp
yrifo
sCl
ofen
tezin
eCo
umap
hos
Cyflu
thrin
Cype
rmet
hrin
Dam
inoz
ide
DDVP
(Dic
hlor
vos)
Diaz
inon
Dim
etho
ate
Dim
etho
mor
phEt
hopr
op(h
os)
Etof
enpr
oxEt
oxaz
ole
Fenh
exam
idFe
noxy
carb
Fenp
yrox
imat
eFi
pron
ilFl
onic
amid
100 ug/g
500 ug/g
0
20
40
60
80
100
120
140
Flud
ioxo
nil
Hexy
thia
zox
Imaz
alil
Imid
aclo
prid
Kres
oxim
-met
hyl
Mal
athi
onM
etal
axyl
Met
hioc
arb
Met
hom
ylM
ethy
l par
athi
onM
evin
phos
Myc
lobu
tani
lN
aled
Orc
hato
xin
AO
xam
ylPa
clob
utra
zol
PCN
B**
Perm
ethr
inPh
osm
etPi
pero
nylb
utox
ide
Pral
leth
rinPr
opic
onaz
ole
Prop
oxur
Pyre
thrin
IPy
reth
rin II
Pyrid
aben
Spin
etor
amSp
inos
ad A
Spin
osad
DSp
irom
esife
nSp
irote
tram
atSp
iroxa
min
eTe
buco
nazo
leTh
iacl
oprid
Thia
met
hoxa
mTH
PI *
*Tr
iflox
ystr
obin
100 ug/g
500 ug/g
0.1 µg/g0.5 µg/g
0.1 µg/g0.5 µg/g
Reproducibility
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Mevinp
hos
Methyl p
arathion
Fenhexam
id
Chlorpyrifos
Coumaphos
Dimeth
omorph
Azoxystro
bin
Cyperm
ethrin
Spinetor
am
Spinosad
A
Fipronil
%RSD (n=5) Pre-spike 0.10 µg/g
LC Column Protection Products/Guard Columns
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
Workflow for Multi-Residue Pesticide AnalysisUPLC-MS/MS and GC-MS/MS
LCAmenablePesticides
GCAmenablePesticides
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
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
Method Generation: QuanpediaThree Clicks to an Analysis Method – 1, 2, ....
QuanpediaThree Clicks to an Analysis Method – 3
Quanpedia Generated Acquisition MethodMS acquisition method
MS processing method
• 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?
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
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
Quantitation of Fenhexamid
Residuals < 2%
R2 = 0.99925-500 ppb in sample2.5-50 ppb in vial
25 ppb2.5 ppb in vial
Quantitation of FenhexamidStandards (n=4)
Pre-spikes (n=6)
Matrix Matched Curve
Note in-sample concentrations listed in the Std. Conc. column
Precision and Accuracy
• 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
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
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
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
• 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
To strive, to seek, to find, and not to yield Alfred Lord Tennyson
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
Thank you for your attention!