routine analysis of cannabis for pesticides and mycotoxins ... · dspe-2 mltube with 150 mg mgso4,...

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

http://www.waters.com/webassets/cms/library/docs/720006373en.pdf

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

http://www.waters.com/webassets/cms/library/docs/720006465en.pdf

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


10Nov2018_16

6.669e+005

Fenhexamid6.70

min9.25 9.50 9.75 10.00 10.25 10.50 10.75

%

0

100


10Nov2018_16

7.588e+005

Spinetoram10.02

9.76

min6.200 6.300 6.400 6.500

%

0

100


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


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


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


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


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



5.313e+004THPI5.95

min7.600 7.650 7.700 7.750 7.800 7.850 7.900

%

0

100



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



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!

routine analysis of cannabis for pesticides and mycotoxins ... · dspe-2 mltube with 150 mg mgso4,...

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