non-targeted high resolution screening of tobacco products for … · 2016-11-03 · ©2016 waters...

©2016 Waters Corporation 1

Non-Targeted High Resolution Screening of

Tobacco Products for Potential Contaminants

using UPLC/APGC/QTOF-MS

Naren Meruva1, PhD, MBA

Principal Scientist

Eimear McCall2, Gareth Cleland1 and Jennifer Burgess1

1Waters Corporation, Milford, MA, USA

2Waters Corporation, Wilmslow, SK9 4AX, UK

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Overview

Tobacco regulatory compliance testing requirements

Targeted (QQQ) vs. Non-targeted (HRMS) analysis

Challenges in contaminant screening

HRMS acquisition types – MSE and HDMSE

Performance criteria for HRMS methods

Tobacco Applications

– Pesticide screening, Extractables and Leachables testing

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QQQ vs HRMS Where’s Waldo?

Targeted Analysis (QQQ)

Non-Targeted Analysis (HRMS)

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HPHC Testing – Targeted Analysis

Chemical Class Methodology No. of Constituents

Nicotine & Minor Alkaloids GC-FID/MS or

LC-UV 3

Aldehydes/Ketones LC-UV or LC-MS/MS

7

Volatiles/Semi-volatiles GC-MS 23

Tobacco Specific Nitrosamines LC-MS/MS 2

Polyaromatic Hydrocarbons GC-MS or GC-MS/MS

17

Phenols LC-FLD 5

Primary Aromatic Amines GC-MS or LC-MS/MS

6

Heterocyclic Aromatic Amines LC-MS/MS 8

Volatile Nitrosoamines GC-MS or LC-MS/MS

8

Metals ICP-MS 8

Others – Aflatoxin B1, Dioxins & Furans

Varies 6

Science Based Tobacco Regulation

FDA harmful and potentially harmful constituents – 93 HPHCs

Development of Standardized Methods

20 priority constituents established

Chromatography-Mass Spectrometry Methods Desirable

High selectivity, sensitivity and resolving power for analysis of trace constituents in complex matrices

Source: FDA Guidance for Industry - Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act, March 2012.

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Targeted vs. Non-Targeted Analysis

Targeted Analysis – Well defined target list of analytes

– Selective acquisition &/or processing modes

– Analytical standards available for every compound

Non-targeted (suspect) screening – Usually in combination with targeted screening

– Screen against a comprehensive library of known compounds

– Analytical standards available for most compounds

Unknown screening – No defined target list

– Compound not present in the library, maybe a new chemical structure

– Structural elucidation required

QQQ

QTOF-MS

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MRM #1

MRM #2

MRM #3

MRM #4

MRM #5

MRM #6

MRM #7

Why move from QQQ to HRMS Screening?

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Why move from QQQ to HRMS Screening?

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Speed/Flexibility

Sensitivity

Selectivity

Informatics

Significant Developments in HRMS Screening over the last 20 years....

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Precursor (low energy) and fragment (high energy)

Increased confidence in elucidation and confirmation

UPLC-APGC/QTOF-MS Single Platform Qualitative and Quantitative Screening

Full scan data

MSE MS/MS

GC/QTOF-MS

UPLC/QTOF-MS

UPLC+APGC/QTOF-MS

Accurate mass Scientific Libraries Resolving power

Pesticide/Contaminant Screening Natural Product Characterization Leachables and Extractables Omics Workflow

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Why ? Scope of HRMS Screening Experiment

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MSE – a GOLD standard in Data Independent Analysis (DIA)

Cycle between low energy and high energy on collision cell

Accurate mass precursor and fragment ions in a single injection

Unbiased, comprehensive data acquisition- it’s not just targets that are of importance

MSE Low energy - precursors

High energy - fragments

301.07556

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Atrazine - HRMS

Low Energy

High Energy

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SANTE 11945/2015 – Performance Criteria for Qualitative Screening

Screening:

Applicability of the screening method is defined by the false non-compliant (positive) and false compliant (negative) rates – Tolerance ≤ 5% false negative rate

– Desirable ≤ 5% false positive rate

Mass accuracy tolerance = ≤5 ppm

Mass resolution tolerance = ≥20k (FWHM)

Identification:

2 ions with mass accuracy ≤5 ppm

S/N >3

XIC must overlap

Ion ratios ± 30 % (relative)

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Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75

%

0

100

15Sept2014_XevoG2XS_sens_Verapamil_TofMRM_Fragments_SD033_MDW_Plasma_P10 Sm (Mn, 2x2) 1: TOF MSMS ES+ 165.091 0.0050Da

2.26e3Area

Area% 100.00

Area 68.83

Height 2229

Time 3.26

S/N:PtP=70.88

11Sept2014_XevoG2XS_sens_SD033_MDW_Plasma_P10 Sm (Mn, 2x2) 1: TOF MS ES+ 455.292 0.0050Da

726Area

Area% 100.00

Area 22.74

Height 654

Time 3.38

S/N:PtP=5.88

Xevo G2-XS TofMRM + Target Enhancement

90 pg/mL S/N 5.9

Tof MRM 455.3>165.091 90 pg/mL S/N 71

Full scan 455.292

Screening approaches have evolved to meet sensitivity &

specificity requirements

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Xevo G2-XS Linearity 5.6 to 11600 pg/mL

Compound name: Verapamil Tof MRM Xevo G2-XS

Correlation coefficient: r = 0.999041, r^2 = 0.998083

Calibration curve: 2.01888 * x + -5.65928

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 2000 4000 6000 8000 10000

Res

pons

e

-0

5000

10000

15000

20000

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Multi Residue Analysis of Tobacco: Typical Challenges

Sample throughput: >1000 pesticides commercially available, applied across variety of foods and feeds

Regulatory requirements: Approx 740 compounds regulated in EU at low ppb concentrations

CORESTA has issued GRLs for about 120 pesticides in tobacco

Accessibility of analysis: LC and GC amenable pesticides

Non-targeted analysis: Metabolites or unknown contaminants

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Experimental: LC Conditions

LC System: Waters ACQUITY I-Class

Column: ACQUITY UPLC BEH C18 2.1 x 100 mm, 1.7 µm

Column Temp: 45 ˚C

Sample Temp: 4 ˚C

Flow Rate: 0.450 mL/min.

Mobile Phase A: 10mM ammonium acetate (pH5.0) in Water

Mobile Phase B: 10mM ammonium acetate (pH 5.0) in MeOH

Flow rate: 0.45 ml/min

Needle wash: 50:50 water: methanol (v/v)

Syringe purge: 10:90 Methanol: water (v/v)

Total run time: 17 min

Injection volume: 10 µL

Gradient: UPLC

Time % A % B

0.00 98 2

0.25 98 2

12.25 1 99

13.00 1 99

13.01 98 2

17.00 98 2

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Experimental: GC Conditions

GC System: A7890 (with APGC Interface)

Column: DB-5MS 30m

Carrier Gas: Nitrogen

Flow Rate: 1.2 mL/min

Initial Temperature: 50 degrees C (1 min. hold)

Gradient: 29.5 ˚C/min to 320 ˚C; hold for 2.41 min

Runtime: 12.83 minutes

Inlet Mode: Splitless

Inlet Type: Multimode

Temperature: 180 ˚C

Injection volume: 1 µL

Make-up Gas: Nitrogen

Make-up Gas Flow: 250 mL/min

Transfer Line Temperature: 310 ˚C

APGC

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Experimental: Xevo G2 XS QToF Parameters

LC-MS Ionisation

Electrospray (ESI)

Capillary (kV) 0.8 Sampling Cone 20.0 Source Temperature 120°C Source Offset 80 Desolvation Temperature 550°C Cone Gas Flow 50 L/Hr Desolvation Gas Flow 1000 L/Hr Acquisition range 50-1200 m/z Scan time 0.25 sec Lockmass LeuEnk

(556.2771m/z)

GC-MS Ionisation

Atmospheric pressure (API)

Corona current (µA) 3.0 Sampling Cone 20.0 Source Temperature 120°C Source Offset 80 Cone Gas Flow 175 L/Hr Auxiliary Gas Flow 50 L/Hr Acquisition range 50-1200 m/z Scan time 0.25 sec Lockmass Siloxane Bleed

(281.0517m/z)

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Comprehensive Scientific Libraries

Waters screening library (pesticides, vet drugs, toxins) – Over 2200 entries of which over 700 contain method related information (RT and

fragment ion accurate masses)

Toxicology – 1000+ entries that include detection results

– Includes designer and synthetic drugs

Traditional Chinese Medicine – Contains about 6000 compounds from 600 herbs

– Latin name, Chinese name, chemical classification

Synthetic Adulterants Library - Collaborative project in development

Leachables and Extractables

Chemspider – Over 34,000,000 compounds from 490+ data sources

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Tobacco Extract Pesticide Screening using UPLC-HRMS

R.T.

MSE

IMS

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Pesticide Screening in Tobacco using HRMS

Carbendazim at 0.1 ppm in tobacco

20X lower than GRL in tobacco

Low Energy Precursor Ion

High Energy Fragment Ions

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Benefits of Ion Mobility: Resolution in Three Dimensions

Peak capacity

Mobility separation is orthogonal

Resolution

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Precursor Ion Low Energy Spectrum

Fragment Ions High energy spectrum

Before Cleanup - Conventional LC-MS Spectrum (DIA)

Mass Spectral Clean up Using Ion Mobility-MS

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Mass Spectral Clean up Using Ion Mobility-MS

Precursor Ion Low energy spectrum

Fragment Ions High energy spectrum

After Cleanup – Drift time aligned

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Extractables and Leachables in E-cigarette Products

+ Paper Wrap w/Binders

Mouth Piece

Outer End Caps End caps

Analyzed IPA solvent blank and extracts of e-cigarette refill cartridge components using UPLC/QTOF-MS and APGC/QTOF-MS:

– Inner end caps

– Outer end caps

– Mouth piece

– Gauze with flavor formulation

– Metal shell

– Paper wrap with binders

Characterization of extractables and leachables is important for ensuring the safety, quality and efficacy of inhalation tobacco products

(Inner Gauze)

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Binary Compare Reference vs. Unknown Sample

IPA Solvent Blank

1:10 Diluted E-liquid In IPA

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Compound Identification – Trend Plots

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Chemical elucidation process – Unknown Characterization

LE

HE

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Summary

Robust, reliable workflows for improved efficiencies

Accurate mass high resolution MS provides targeted, suspect and non-

targeted screening on a single instrument

Flexible hardware and software to meet your screening requirements

Pesticide/Contaminant Screening Natural Product Characterization Leachables and Extractables Omics Workflow

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non-targeted high resolution screening of tobacco products for … · 2016-11-03 · ©2016 waters...

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