ASMS 2019 Breakfast sessionI. Tackle Emerging Cannabis

Regulations with ConfidenceII. Intelligent GC Systems

Dr. Anthony Macherone

Agilent & Johns Hopkins SOM

Dr. Anastasia Andrianova

Application Scientist, Agilent

Agenda

• Part I

• Brief Overview

– Sample Preparation, Potency, Metals, Terpenes, Residual Solvents and, Microbial Screening

• Pesticides

– Why LC-MS/MS and GC-MS/MS are required

– Sample Preparation

– Method & Performance

– Data Processing & Reporting

Part II

• Intelligent GC Systems

Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories

where such use is permitted under state/country law.

Tackle Emerging Cannabis Regulations with Confidence

Regulatory Testing Overview

Agilent Provides a ComprehensiveWorkflow

Field Deliverable Potency Testing

App Note: 5991-9285

1220VL-VWD with OpenLab software

• 11 cannabinoids including ∆9-THC and ∆8-THC

• Sample report including total potency & total CBD calculations

Method translates to 1260 systems

On-site consulting

Real-Time Potency

App Note: 5991-8677

Platform: Agilent Cary 630 FT-IR

• Fitted with a diamond absolute total reflectance (ATR) accessory

Quick & Real-Time Determination of Cannabinoid Potency

• Cannabis concentrates (shatter, butter, waxes) and distillates

• Sample measurement in seconds

• Non-destructive sample analysis

• No consumables required

Detailed Sample Preparation

Figure 1: Schematic representation of sample preparation procedure for LC-MS/MS analysis

Figure 1: Schematic representation of sample preparation procedure for GC-MS/MS analysis

Multi-Element Analysis of Cannabis

App note: 5991-8482

• 25 elements in cannabis and related products

Recommended configuration:

• 7800 ICP-MS + High Matrix Introduction system

Terpenes

App note: 5991-8499

• 22 common target terpenes in 6 minutes (10-min cycle time)

Recommended configuration:

• 7697A / 7890B or Intuvo / 5977B Inert HSS-GC-FID/MS system

Residual Solvents

App note: 5991-7625 (not cannabinoid specific)

Recommended configuration:

• 7697A /7890B or Intuvo / 5977B Inert HSS-GC-FID/MS system

Microbial Screening

Quantitative PCR for Cannabis industry

• Co-marketed with Medicinal Genomics

Agilent & Medicinal Genomics app note 5994-0430EN

Platform: Aria MX Real-Time PCR System

• The reagent kits offered by Medicinal Genomics allows the customer to test the following:

– Microbial testing on the cannabis flower

– Gender Detection

– Powdery mildew

– CBDA Synthase gene

– THCA Synthase gene

Tackle Emerging Cannabis Regulations with Confidence

Why LC-MS/MS & GC-MS/MS are Required Platforms

Pentachloronitrobenzene, PCNB

Poorly ionizes in electrospray (ESI)

Application literature space: APCI in negative ionization mode (Ni-APCI)

Publically described ionization mechanism*

1. Loss of HCl

2. Formation of ion adduct

*Tran, D.; et al. Document number: RUO-MKT-02-7607-A. 2018, AB Sciex Framingham, MA

Primary ions: C6Cl4NO3- & C6Cl5O

-

LC-QTOF Ni-APCI HRAM MS Spectrum

Red Gaussian = Theoretical; Blue centroid = Actual Data

LC-QTOF Ni-APCI C6Cl4NO3- Isotopic Fidelity

Red Gaussian = Theoretical; Blue centroid = Actual Data

LC-QTOF Ni-APCI C6Cl5O- Isotopic Fidelity

LC-QTOF Ni-APCI Ni-APCI HRAM MS/MS Product Ion Spectra

Summary

Ni-APCI LC-MS/MS uses non-specific, non-linear precursor ion / product ion pairs

• Even quadratic curves are shown with r2 of 0.97* – not acceptable for CA

EI GC-MS/MS highly-specific precursor ion / product ion pairs

• > 3.5 times more selective

• > Linear r2 > 0.999

• > 10 times more sensitive

State regulatory entities that do not allow monitoring acequinocyl hydroxy should not allow the monitoring of phenoxides of PCNB via Ni-APCI LC-MS/MS

• Tetrachloronitrophenoxide

• Pentachlorophenoxide

*Tran, D.; et al. Document number: RUO-MKT-02-7607-A. 2018, AB Sciex Framingham, MA

Tackle Emerging Cannabis Regulations with Confidence

Residual Pesticides Analysis

This Photo by Unknown Author is licensed under CC BY-NC

No QuEChERS

• Adding water increases pH – affects base labile pesticides

• Reaction exotherm – affects thermally labile pesticides

No dispersive SPE

• d-SPE does not offer enough capacity

• PSA co-scavenges acidic pesticides

• GCB co-scavenges planar pesticides

No “dilute & shoot”

• Poor analytical performance

• Increased maintenance, decreased throughput (Billable $$)

Sample Preparation for Dry Cannabis Flower

Initial Comments

SPE Cleanup and Dilution (California)

LC/MS/MS Sample Preparation - Cannabis Flower (Steps 1-7)

2/ Add 15 mL acetonitrile &shake a further 5 minutes @ 3000/min

4/ Decant supernatant solvent into unconditioned SampliQ

C18 EC SPE Cartridge (PN 5982-1365)Gravity elution.

5/ Add a 5 mL aliquot of acetonitrile to the original tube, stirring up the pellet

of plant material and shake for 3 minutes at high speed. Transfer all

solids and ACN to the SPE cartridge. Finally, wash tube with a further 5 mL

ACN and transfer to SPE cartridge

6/ Bring the collected eluent (extract) up to

25mL with acetonitrile (25 fold dilution)

1/ Weigh 1.0 g of chopped cannabis flower material into 45mL tube.

Add 2 ceramic homogenizer pellets and

shake for 5min at high speed.

3/ Centrifuge 5000 rpm, 5 min

7/ Take 50 μL of extract and mix with 450 μL 25/75%

water/methanol (v/v) in a sample vial. (250-fold dilution)

Cap & vortex for 30 sec.

Samples are ready for LC/MS/MS analysis

The sample preparation steps outlined above constitute a

resultant 1/250 total dilution.

SPE Cleanup and Dilution (California)

GC/MS/MS Sample Preparation - Cannabis Flower (Steps 1-7)

2/ Add 15 mL acetonitrile &shake a further 5 minutes @ 3000/min

4/ Decant supernatant solvent into unconditioned SampliQ

C18 EC SPE Cartridge (PN 5982-1365)Gravity elution.

5/ Add a 5 mL aliquot of acetonitrile to the original tube, stirring up the pellet

of plant material and shake for 3 minutes at high speed. Transfer all

solids and ACN to the SPE cartridge. Finally, wash tube with a further 5 mL

ACN and transfer to SPE cartridge

6/ Bring the collected eluent (extract) up to

25mL with acetonitrile (25 fold dilution)

1/ Weigh 1.0 g of chopped cannabis flower material into 45mL tube.

Add 2 ceramic homogenizer pellets and

shake for 5min at high speed.

3/ Centrifuge 5000 rpm, 5 min

7/ Take 200 μL of extract and mix with 800 μL acetonitrile in a sample vial. (125-fold dilution)

Cap & vortex for 30 sec.

Samples are ready for GC/MS/MS analysis

The sample preparation steps outlined above constitute a

resultant 1/125 total dilution.

Agilent LC/MS/MS Instruments for Pesticide & Mycotoxin Analysis

Ultivo LC/MS/MS 1260 Infinity II Multisampler /

6470 LC-MS/MS

LC/MS/MS

LC-MS/MS Analysis“California method”

Agilent 1260 or 1290 Infinity II / 6470 or Ultivo LC-MS/MS

Chromatographic Column

• Poroshell 120 Phenyl-Hexyl 2.7 µm, 3.0mm x 100mm

Guard column

• Poroshell 120 Phenyl-Hexyl 2.7 µm, 3.0mm x 5mm

Mobile Phase A

• 5mM ammonium formate with 0.1% formic acid in water

Mobile Phase B• 0.1 % Formic acid in methanol

Cycle time: 11 min

Overlaid chromatogram of California pesticides list and mycotoxins in extracted flower matrix, actual concentration 500ppt (pre-extraction concentration=125ppb.)

Mycotoxins in Cannabis Matrix – California@ 4ppb in Cannabis Matrix 1260/6470

Aflatoxins

B1

B2

G1

G2

Ochratoxin A

Avermectin B1ar2=0.9996

Spynosyn Ar2=0.9999

Daminozider2=0.9997

Example Calibration Curves, California List.

(a)

(c)

(b)

Examples of chromatography near LLOQ

(a) Avermectin B1a, 0.1ppb (ng/mL)

(b) Daminozide, 0.1ppb (ng/mL)

Examples of chromatography near LLOQ

(c) Spinosyn A, 0.1ppb (ng/mL)

Examples of chromatography near LLOQ

Agilent GC/MS/MS Instrument for Pesticide Analysis

7010 GC/MS/MS

GC/MS/MS

GC-MS/MS Analysis“California method”

Agilent 7890 GC, 7693 AS, 7010 GC-MS/MS

Chromatographic Columns

• 2 X HP-5MS UI column (P/N: 19091S-431)

Multi-Mode Inlet (cooled with air)

• Ultra-Inert 2-mm dimple split-less liner (P/N: 5190-4006)

• Ultra-Inert Non-Stick Advanced Green 11-mm septa (5190-3158)

Purged Ultimate Union configured for Mid-Column backflush

Cycle time: 15 min

Post batch, run JetClean 2 min

Chromatograms of Quintozine (PCNB) and Methyl Parathion

GC-MS/MS MRM chromatogram for the PCNB

quantifier and qualifying ions at 8 ppb in 20% - 30%

cannabis flower extract matrix, and the linear

calibration curve over the range of 8 ppb through

250 ppb in matrix.

GC-MS/MS MRM chromatogram for the methyl

parathion quantifier and qualifying ions at 8 ppb in 20%

- 30% cannabis flower extract matrix, and the linear

calibration curve over the range of 8 ppb through 250

ppb in matrix.

Chromatograms of Captan and cis-Chlordane

SRM chromatograms for captan and deuterated captan-d6

internal standard at 8 ppb in 20% - 30% cannabis flower

extract matrix, and the linear calibration curve over the

range of 8 ppb through 250 ppb in matrix.

GC-MS/MS method at 8 ppb in 20% - 30% cannabis

flower extract matrix, and the linear calibration curve over

the range of 8 ppb through 250 ppb in matrix

California, Nevada, Oregon & Canada Optimal Methods

LC-MS/MS & GC-MS/MS• Same instruments - 1260/6470 LC-QQQ and Ultivo LC-QQQ

• Ultivo is a smaller footprint option

• 7010 GC-QQQ

Increased lab productivity• Optimized methods for each region

– Slight Chromatography differences per region

– Sample preparation routines can differ per region

– Minimized LC-QQQ Analysis times

• Incorporate mycotoxin analysis

– Mycotoxins included in LC-MS/MS pesticide method

Coming soon• New matrix specific methods

– Cannabis Concentrate safety testing for pesticides & mycotoxins

MassHunter Quantitative Analysis GUI Choices for Cannabis Data

Unified data analysis and reporting tools for both platforms

• Same software data analysis package for LC/MS/MS & GC/MS/MS

Graphical user interface (GUI)

• Options address an instrument operator’s method development or compliance permissions and experience

• Scientist “flavor”

• Complete method setup, batch review and reporting capabilities

• Analyst “flavor”

• Simplified and uncluttered GUI for use in the daily production environment

• Batch review and report generation only allowed from pre-defined data review criteria, methods and templates

• Laboratory can more easily control how data is processed and reported.

Custom report templates

• Specifically designed for the cannabis analysis requirements of each geographic region

• Available as an integral element of the MassHunter Quantitation software

Cannabis Sample Report US States1

Cannabis Sample Report Canada

Intelligent GC Systems

Dr. Anastasia Andrianova

GC/MS Applications Scientist, Agilent

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• Pete Stone

• Jennifer Hitchcock

• Jean-Francois Roy

• Christophe Deckers

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Acknowledgements

Questions?