ab sciex presentation

DuoSpray™ IonizationA Novel Approach to Analyzing the California Mandated List of Pesticides in Cannabis

Robert Di Lorenzo, PhDApplication Support Scientist

Diana TranApplication Support Scientist NACRW 2018

RUO-MKT-11-8253-A

© 2018 AB Sciex

• Market Overview

‒ State Regulations

• SCIEX Technology

• Analytical Data

‒ Meeting California requirements for testing

‒ Value Added Analysis

• Q & A

Overview

© 2018 AB Sciex

Introduction

www.marijuanapacking.com

www.marijuanastocks.com

© 2018 AB Sciex

• State law determines which pesticides are to be tested and what the

appropriate action limits are in Cannabis

• Oregon has action limits for 59 pesticides and limits are the same for

flower and concentrates

• California’s new pesticide testing requirements contain 65 compounds

‒ 7 additional compounds compared to Oregon

‒ CA pesticide panel excludes MGK-264

‒ Category I (detect or non-detected)

‒ Category II (has specified action limits for different cannabis matrices)

Pesticide Testing Regulations

Introduction

© 2018 AB Sciex

Introduction

OR Pesticide Residue Panel

https://public.health.oregon.gov

Abamectin Dimethoate Naled

Acephate Ethoprophos Oxamyl

Acequinocyl Etofenoprox Paclobutrazol

Acetamiprid Etoxazole Permethrins

Aldicarb Fenozycarb Phosmet

Azoxystrobin Fenpyroximate Piperonyl Butoxide

Bifenazate Fipronil Prallethrin

Bifenthrin Flonicamid Propiconazole

Boscalid Fludioxonil Propoxur

Carbaryl Hexythiazox Pyrethrins

Carbofuran Imazalil Pyridaben

Chlorantraniliprole Imidacloprid Spinosad

Chlorfenapyr Kresoxim-methyl Spiromesifen

Chlorpyrifos Malathion Spirotetramat

Clofentezine Metalaxyl Spiroxamine

Cyfluthrin Methiocarb Tebuconazole

Cypermethrin Methomyl Thiacloprid

Daminozide Methyl Parathion Thiamethoxam

Dichlorvos MGK-264 Trifloxystrobin

Diazinon Myclobutanil

https://swamis420.wordpress.com/2012/06/26/tropicana-strain-review/

Additional CA PesticidesCaptan Mevinphos

Chlordane Pentachloronitrobenzene

Coumaphos Spinetoram

Dimethomorph

Historically analyzed by GC-MS

© 2018 AB Sciex

Introduction

Category I Pesticides


Category II Pesticides

Aldicarb Coumaphos Etofenoprox Mevinphos

Carbofuran Daminozide Fipronil Paclobutrazol

Chlordane Dichlorvos Imazalil Propoxur

Chlorfenapyr Dimethoate Methiocarb Spiroxamine

Chlorpyrifos Ethopro(phos) Methyl Parathion Thiacloprid

Abamectin Chlordantraniliprole Fludioxonil PCNB Spirotetramat

Acephate Clofentezine Hexythiazox Permethrin Tebuconazole

Acequinocyl Cyfluthrin Imidacloprid Phosmet Thiamethoxam

Acetamiprid Cypermethrin Kresoxim-methyl Piperonylbutoxide Trifloxystrobin

Azoxystrobin Diazinon Malathion Prallethrin

Bifenazate Dimethomorph Metalaxyl Propiconzaole

Bifenthrin Etoxazole Methomyl Pyrethrins

Boscalid Fenhexamid Myclobutanil Pyridaben

Captan Fenpyroximate Naled Spinosad

Carbaryl Flonicamid Oxamyl Spiromesifen

No CA defined limits

CA defined action limits

Analyzed via APCI

© 2018 AB Sciex

Canadian List – Lots of Pesticides, Low Levels

Introduction

• 95 Pesticides (32 new) on Health Canada draft list for cannabis and related products

• Generally 20-40x lower

MRLs than Oregon‒ Many at 10 ppb in matrix

• Regulate for fresh

flower and oil in

addition to dried

product

© 2018 AB Sciex

Introduction

Compound MRL (ppm)

Allethrin 0.1

Azadirachtin 0.5

Benzovindiflupyr† 0.01

Buprofezin 0.01

Clothianidin 0.025

Cyantranilipole 0.01

Cyprodinil* 0.01

Deltamethrin* 12.5

Dinotefuran 0.05

Dodemorph 0.05

Endosulfan sulfate 0.5

Endosulfan-alpha 0.1

Endosulfan-beta 0.5

Etridiazol 0.01

Fensulfothion 0.01

Fenthion 0.01

Compound MRL (ppm)

Fenvalerate* 3.75

Fluopyram 0.01

Iprodione 0.5

Kinoprene† 0.05

Methoprene 1

Novaluron 0.025

Phenothrin 0.025

Pirimicarb 0.01

Pyraclostrobin 0.01

Resmethrin 0.05

Spirodiclofen 0.01

Tebufenozide 0.01

Teflubenzuron 0.025

Tetrachlorvinphos 0.01

Tetramethrin 0.05

Thiophanate-methyl* 0.025

*- Minimum value in cases where MRL in flower is not yet determined

†- Few commercial sources for standard

© 2018 AB Sciex

• Pesticide residue testing

is required by the state -

variable

• Lack of affordable in-

house LC-MS expertise

• Method development

takes too long

Challenges for a new cannabis testing lab

© 2018 AB Sciex

Questions, we had to ask ourselves…

Motivation

Do you really need a GC and LC to run the entire panel?

Are all of those pesticides amenable to LC-MS analysis?

Can they all be done by ESI?

Problematic compounds on the California List of pesticides:

• Captan (low sensitivity)

• Chlordane (APCI Ionization)

• Pentachloronitrobenzene (PCNB) (APCI Ionization)

© 2018 AB Sciex

SCIEX Hardware

LC

FLOW

ESI

APCI

Corona Pin

ESI Spray

Flow diversion to either ionization

mode for APCI/ESI analysis

IonDrive™

QJet Guide

© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY

Pesticides

© 2018 AB Sciex

• SOP

‒ Sample Preparation

‒ System Performance Testing

• Acquisition Method

‒ Optimized Parameters

‒ Plug and play

‒ LC Separation of interferences

‒ Compatibility on two instruments

• Quantitation Method

‒ Internal STD designations

‒ Calibrators and quality controls

• Data Analysis

‒ Verified LOQ analysis for pesticides and cannabinoids

• Report Template

vMethod™ Application for Quantitation of Pesticide Residues and

Cannabinoids in Cannabis Matrices v1.0

Detailed SOP

© 2018 AB Sciex

• 1 gram of homogenized flower is vortexed in

10 mL of Acetonitrile

• Sonicated for 15 minutes

• Centrifuged at 4000 rpm

• Extracts frozen, supernatant removed

• Injection volume of 1 of µL for LC-MS/MS

analysis

‒ The majority of the pesticides will have to

be detuned

‒ Although the additional sensitivity is

needed for Captan and PCNB to meet the

CA Action Limits.

Cannabis Flower Extraction

Pesticide MRM Analysis

Simple Solvent Extraction

© 2018 AB Sciex

Pesticide Chromatography – Oregon List

Kinetex Biphenyl, 4.6x150mm (16 minute gradient)

Acephate

Avermectin B1aAvermectin B1b

Acequinocyl

© 2018 AB Sciex

Using TCLP Chlordane and APCI Negative Ionization…

Chlordane

• Chlordane has 8 chlorine atoms

• Typical chlorinated pattern

• Chlordane underwent in source

fragmentation with APCI

M –HCl-

M -2HCl-

M -3HCl-

M -4HCl-

Cis/trans Chlordane are the

most abundant in mix

M

Q1 Scan

© 2018 AB Sciex

Using RESTEK PCNB Standard with APCI Negative Ionization

PCNB

Q1 Scan

• PCNB has 5 chlorine atoms

• Typical chlorinated pattern

• PCNB also underwent in source

fragmentation with APCI

• Loss of HCl-

• Forms an ammonium adduct

Product Ion Scan

(MS2)

© 2018 AB Sciex

Luna Omega Polar C18, 4.6x150mm (30 minute gradient)

Pesticide Chromatography – California List

APCI APCI

Analytical flow is diverted to APCI Analytical flow is diverted to APCI

© 2018 AB Sciex

Analyzed in ESI

Parathion Methyl

Unspiked Flower

Extract

0.05 ppm in flower

1 ppb in vial

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

1 ppm in flower

20 ppb in vial

0.05 – 1 ppm in flower

1-20 ppb in vial

R2 = 0.9990

Solvent Standard

20 ppb in vial

CA- Schedule 1

© 2018 AB Sciex

Analyzed in ESI

Cyfluthrin

Unspiked Flower

Extract

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

Solvent Standard

20 ppb in vial

1 ppm in flower

20 ppb in vial

0.05 ppm in flower

1 ppb in vial 0.05– 1 ppm in flower

1-20 ppb in vial

CA- 2 ppm

© 2018 AB Sciex

Analyzed in ESI Ionization

Coumaphos

Unspiked Flower

Extract

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

Solvent Standard

20 ppb in vial

0.05 ppm in flower

1 ppb in vial

1 ppm in flower

20 ppb in vial

0.05 – 1 ppm in flower

1-20 ppb in vial

R2 = 0.9989

CA- Schedule 1

© 2018 AB Sciex

Analyzed in APCI

Pentachloronitrobenzene

Unspiked Flower

Extract

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

Solvent Standard

20 ppb in vial

1 ppm in flower

20 ppb in vial

0.05 ppm in flower

1 ppb in vial 0.05 – 1 ppm in flower

1-20 ppb in vial

R2 = 0.9755 with first pointFirst STD can be

excluded as LOD

First STD can be

excludedCA- 100 ppb

© 2018 AB Sciex

Analyzed in ESI

Captan

Unspiked flower

extract

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

1 ppm in flower

20 ppb in vial

5 ppm in flower

100 ppb in vial

Unspiked flower

extract

0.1 ppm in flower

2 ppb in vial

0.5 ppm in flower

10 ppb in vial

1 ppm in flower

20 ppb in vial

5 ppm in flower

100 ppb in vial

MRM 1

MRM 2

CA Action Limit is 0.7 ppm for

inhalables

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Analyzed in APCI

Chlordane

CA – Schedule 1

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Mycotoxins – Linearity and Reproducibility

Aflatoxin B1: r > 0.994, CV(n=4) = 5.76% at 12.5 ppt

Aflatoxin B2: r > 0.994, CV(n=4) = 8.71% at 12.5 ppt

Aflatoxin G1: r > 0.994, CV(n=4) = 8.12% at 12.5 ppt

Aflatoxin G2: r > 0.996, CV(n=4) = 7.59% at 12.5 ppt

Replicates at 1.9 ppb equivalent

Current Mycotoxins MRL = 20 ppb


For when you need a little bit more…

© 2018 AB Sciex

What is MS3?

QTRAP Analysis

© 2018 AB Sciex

Hardware

QTRAP Analysis

2. Q2

fragments

precursor ion

with no low

mass cut-off

© 2018 AB Sciex

MS3 for Quantitation

QTRAP Analysis

© 2018 AB Sciex

What can MS3 do for Cannabis matrices? Using Pyrethrin Pyrethrin I as an example…

QTRAP Analysis

MRM of Pyrethrin Pyrethrin I

MS3 of Pyrethrin Pyrethrin I

Distinguishes real Pyrethrin Pyrethrin from isobaric interferences

Flower Blank

Flower Blank

© 2018 AB Sciex

Cyfluthrin MS3 in Cannabis Flower Extract

QTRAP Analysis

Flower Blank

Flower Blank

2 ppb in vial

2 ppb in vial

10 ppb in vial

10 ppb in vial

20 ppb in vial

20 ppb in vial

MRM of Cyflutrhin

MS3 of Cyfluthrin

11 ppb

in matrix

© 2018 AB Sciex

Changing Landscape – 100 ppb MRL for Category I

Category I Pesticides


Category II Pesticides

Aldicarb Coumaphos Etofenoprox Mevinphos

Carbofuran Daminozide Fipronil Paclobutrazol

Chlordane Dichlorvos Imazalil Propoxur

Chlorfenapyr Dimethoate Methiocarb Spiroxamine

Chlorpyrifos Ethopro(phos) Methyl Parathion Thiacloprid

Abamectin Chlordantraniliprole Fludioxonil PCNB Spirotetramat

Acephate Clofentezine Hexythiazox Permethrin Tebuconazole

Acequinocyl Cyfluthrin Imidacloprid Phosmet Thiamethoxam

Acetamiprid Cypermethrin Kresoxim-methyl Piperonylbutoxide Trifloxystrobin

Azoxystrobin Diazinon Malathion Prallethrin

Bifenazate Dimethomorph Metalaxyl Propiconzaole

Bifenthrin Etoxazole Methomyl Pyrethrins

Boscalid Fenhexamid Myclobutanil Pyridaben

Captan Fenpyroximate Naled Spinosad

Carbaryl Flonicamid Oxamyl Spiromesifen

All at 100 ppb MRL

CA defined action limits

Analyzed through APCI

© 2018 AB Sciex

IonDrive™ Technology Key Innovations

IonDrive™ Turbo V Source IonDrive™ QJet Guide IonDrive™ HE Detector

Driving Performance by Driving More Ions While Maintaining Ruggedness

Make More Ions Collect More Ions Detect More Ions

© 2018 AB Sciex

• Larger diameter (11 mm) heaters

• Optimized geometry

• More efficient heat transfer

• Covers a larger cross-section of the

spray cone

• Wider “sweet spot” when optimizing

probe position

• More robust against fluctuations in

gas flow dynamics, and source to

source differences

IonDrive™ Turbo V Source – Make More Ions

© 2018 AB Sciex


Original Turbo V™ IonDrive™ Turbo V

© 2018 AB Sciex

Cannabis Matrix

Mycotoxins

TerpenesCannabinoids

PesticidesTotal

Cannabis

Solution

How many instruments/extracts/analyses are necessary?

Extract + Cleanup 1 & 2

LC-MS/MS

GC-MS(/MS)Extract + Cleanup 3

LC-MS/MS

Extract + Cleanup 4

LC-UV

2-injectionsExtract + Cleanup 5

GC-FID (or MS)

1 Extract

1 Column

1 Solvent System

1 Instrument


Potency


• Current regulations require the quantitative

reporting of cannabinoid compounds

‒ Concentrations differ greatly between strains

and analytes

‒ High abundance

‒ THC, THCA, CBD, CBDA

‒ Low abundance

‒ CBG, CBN, CBC, THCV, CBDV…

‒ Concentrations > 90% by weight to < 0.01%,

depending on analyte/strain

• Commonly performed by LC-UV

‒ Two injections needed

‒ One for high abundance compounds

‒ One for low abundance compounds

‒ Concentration range: 0.05 – 30% w/w

Potency Analysis


• Dilute extract 1:6

‒ Use for Pesticides, Aflatoxins

• Dilute serially further 2000x

‒ Use for Potency

Sample Dilution

Current vMethod Sample Prep Protocol

Calibration standards

Concentration in Extract Equivalent Concentration in Sample

10 ppb 0.3%

50 ppb 1.5%

200 ppb 6%

500 ppb 15%

800 ppb 24%

1000 ppb 30%

Need to go lower (< 0.05%)

Need to go higher (~100%)


• Need to increase linear dynamic range (LDR) to

the low end and to the high end

• Instead of diluting 1:6 extract 2000x, only dilute

200x

‒ Large dilution will still eliminate all matrix effects

• 10 ppb standard now becomes equivalent to

0.03% in sample

‒ Low end reached

• Extend standard concentrations to ~33 ppm

which would be equivalent to 99% in sample

‒ High end reached

• Problem: ESI is prone to saturation at high end

Sample Dilution

Modifications to extend potency range (0.03%-100%)

New Calibration Standards

Concentration in Extract Equivalent Concentration in Sample

10 ppb 0.03%

50 ppb 0.15%

200 ppb 0.6%

500 ppb 1.5%

800 ppb 2.4%

1200 ppb 3.0%

3000 ppb 9.0%

6000 ppb 18%

10 000 ppb 30%

20 000 ppb 60%

30 000 ppb 90%

Solution: Use APCI instead of ESI

Bonus: May be able to simultaneously

analyze other compound classes

(Hint: It’s terpenes)


CASE 2

CASE 3

MRM Monitoring

Modifications to extend potency range (0.03%-100%)

• Many possible MRM transitions can be monitored for each analyte

‒ Varying degree of sensitivity seen for each transition

‒ Currently only 2 transitions are used per analyte

‒ 1 for quantitation, 1 for confirmation

• May saturate detector at high end of calibration range

THCA

9 Transitions

THC

7 Transitions

CBD

6 Transitions

CBDA

8 Transitions

Analyze entire

calibration range with

one MRM per analyte

Full LDR?

Stop

Yes

Use one MRM for high

end and another for

low end of calibration No

Full LDR?

Stop

Yes

Detune DP and CE to

eliminate saturation at

high end of calibration

Full LDR?

Stop

Yes

No

Solution Flowchart

CBDV

CBD

CBDACBGA

CBG

THCV

CBN

CBC

THC

THCA

CASE 1


THC – Case 1 - Full range in one transition

Results – Extended Linear Dynamic Range

Linear Range

10-30 000 ppb in vial

0.03-90% in sample

R2 = 0.999


THCA – Case 2 – Two transitions required


Linear Range


0.03-30% in sample

R2 = 0.995

Linear Range

1200-30 000 ppb in vial

3.6-90% in sample

R2 = 0.995


CBDV – Case 3 – Detuning required


Linear Range


0.03-30% in sample

R2 = 0.999

• All transitions monitored for CBDV saturate at the high end of the curve

• Need to further detune DP and CE to extend range to 100%

• Sample concentration is expected to be below 1%


• Calibration extended to effective concentrations

of 0.03%-90% by weight in sample

‒ All but CBDV and THCV, which are linear

between 0.03-30%

‒ These are not expected to be above 1%

• APCI less prone to saturation than ESI

‒ Some analytes only need one transition monitored

• Excellent reproducibility and accuracy

‒ All calibrations R2 ≥ 0.995

‒ CV < 10%

‒ Most < 5%

‒ Accuracy

‒ Within 20% for 0.03% calibration standard

‒ Within 10% for all other standards

‒ Most within 5%

CannabinoidCalibration

Range 1R2 Calibration

Range 2R2

THC 0.03-90% 0.999

THCA 0.03-30% 0.995 3.6-90% 0.995

CBD 0.03-90% 0.999

CBDA 0.03-3.6% 0.999 3.6-90% 0.998

CBG 0.03-90% 0.999

CBGA 0.03-9% 0.999 9-90% 0.998

CBN 0.03-90% 0.999

CBC 0.03-30% 0.998 0.15-90% 0.999

CBDV 0.03-30% 0.999

THCV 0.03-30% 0.999

Summary

• Potency analysis can be performed in

a single injection


Terpenes


• 200+ terpenes have been identified in

Cannabis

‒ Unique strains presenting varying terpene

profiles

‒ Contribute primarily to flavor and aroma

‒ Potentially have beneficial health outcomes

‒ ‘Entourage’ effect

• Only Nevada requires terpene analysis

‒ Consumers and aficionados are highly

interested in terpene profiles

‒ Growers interested in profiling terpenes as a

metric for product quality and batch

reproducibility

Terpene Profiling

© 2018 AB Sciex

• Six commonly reported terpenes

• Not amenable to ESI

‒ Usually analyzed by GC

• Quite responsive to APCI

• Challenging separation

‒ Series of structural isomers with identical MRM

transitions

‒ All relevant compounds must be separated

chromatographically

• Previously shown to be possible by LC-

MS/MS, but using a different column and

solvent system than used in vMethod

Terpene Profiling

Linalool β-Myrcene α-Pinene Limonene α-Humuleneβ-Caryophyllene

© 2018 AB Sciex

Chromatography in solvent

Linalool

b-Myrcene

a-Pinene

Limonene a-Humulene

b-Caryophyllene

CBDCBDA

CBGA

CBG

CBDV

THCV

CBC

THCA

THC

© 2018 AB Sciex

• Extending the terpene profiling from 6 to 20+

terpenes

• Still using same column, solvent system and

extract as existing vMethod™

• Mix of monoterpenes, sesquiterpenes, their

hydroxylated analogues and some miscellaneous

aroma compounds

• Challenging chromatography achieved with

baseline separation of nearly all isomers

‒ Most abundant and most commonly observed

terpenes included in method

Extended ProfilingLinalool

Geraniol

Eucalyptol

Isop

ule

gol

Terp

ineol

R,S

-Born

eo

l

R,S-Pulegone

Ocim

ene

α,β-Pinene

Limonene (137/95)

Eudesmol

α-Bisabolol

Guaiol (205/149)

Myrc

ene

β-Caryophyllene

β-Humulene

Menth

ol (1

39

/97)

Citro

nello

l(1

57/8

3)

R,S-Pulegone (153/93)

Caryophyllene oxide (221/203)


• Leveraging the power of APCI to

simultaneously analyze for

compounds natively produced in

Cannabis‒ Namely terpenes and cannabinoids

‒ Phenotype strains using multivariate

regression tools

• Potential to qualitatively and

quantitatively assess similarities and

differences between strains

‒ Consumer standpoint

‒ Health benefit standpoint

Cannabis Phenotyping

SCIEX Users Meeting – KC Hyland


• ESI and APCI used effectively on diluted

extracts to meet MRLs for California

mandated list of pesticides

• Mycotoxins seamlessly added to verified

analysis method

Pesticides and Mycotoxins

• Exploiting the power of APCI to ionize non-

polar compounds with minimal matrix effects

and large LDR

• Linear dynamic range extended for potency

analysis

‒ Simultaneous analysis of low and high level

cannabinoids in one injection

‒ Concentration range between 0.03-90% w/w

‒ Potential to extend LDR further

• Quantitative analysis of most abundant

terpenes

• Challenging separation achieved for extended

list of terpene isomers

Cannabinoids and Terpenes

Summary

All analyses performed using a

single extract

All analyses able to be performed on

a single LC-MS/MS system


Questions?

© 2018 AB Sciex

For Research Use Only. Not for use in diagnostic procedures.

AB Sciex is doing business as SCIEX.

© 2018 AB Sciex. The trademarks mentioned herein are

the property of AB Sciex Pte. Ltd. or their respective owners.

AB SCIEX™ is being used under license.

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