©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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©2016 Waters Corporation 2
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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©2016 Waters Corporation 3
QQQ vs HRMS Where’s Waldo?
Targeted Analysis (QQQ)
Non-Targeted Analysis (HRMS)
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©2016 Waters Corporation 4
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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©2016 Waters Corporation 5
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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©2016 Waters Corporation 6
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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©2016 Waters Corporation 7
Why move from QQQ to HRMS Screening?
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©2016 Waters Corporation 8
Speed/Flexibility
Sensitivity
Selectivity
Informatics
Significant Developments in HRMS Screening over the last 20 years....
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©2016 Waters Corporation 9
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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©2016 Waters Corporation 10
Why ? Scope of HRMS Screening Experiment
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©2016 Waters Corporation 11
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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©2016 Waters Corporation 12
Atrazine - HRMS
Low Energy
High Energy
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©2016 Waters Corporation 13
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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©2016 Waters Corporation 14
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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©2016 Waters Corporation 15
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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©2016 Waters Corporation 16
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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©2016 Waters Corporation 17
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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©2016 Waters Corporation 18
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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©2016 Waters Corporation 19
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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©2016 Waters Corporation 20
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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©2016 Waters Corporation 21
Tobacco Extract Pesticide Screening using UPLC-HRMS
R.T.
MSE
IMS
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©2016 Waters Corporation 22
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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©2016 Waters Corporation 23
Benefits of Ion Mobility: Resolution in Three Dimensions
Peak capacity
Mobility separation is orthogonal
Resolution
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©2016 Waters Corporation 24
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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©2016 Waters Corporation 25
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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©2016 Waters Corporation 26
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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©2016 Waters Corporation 27
Binary Compare Reference vs. Unknown Sample
IPA Solvent Blank
1:10 Diluted E-liquid In IPA
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©2016 Waters Corporation 28
Compound Identification – Trend Plots
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©2016 Waters Corporation 29
Chemical elucidation process – Unknown Characterization
LE
HE
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©2016 Waters Corporation 30
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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