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LABSTAT INTERNATIONAL ULC. 262 Manitou Drive
Kitchener, Ontario, Canada N2C 1L3 Phone: (519) 748-5409 Fax: (519) 748-1654 Web: www.labstat.com
Peter JOZA , David LI, Andrew MASTERS, Bill RICKERT
1
Determination of Pyrazine Flavourants in Electronic
Cigarette Cartridges and Liquids by Headspace
Solid Phase Microextraction - Gas Chromatography-Mass Spectrometry (HS-SPME-GCMS)
2014 CORESTA CONGRESS October 12-16, 2014 Québec City, Canada
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Objectives
2
Apply the HS-SPME-GCMS used for profiling volatile and semi-volatile compounds in tobacco products to e-liquids and e-cigarette aerosol
Quantify four targeted compounds:
2,3,5-Trimethylpyrazine (TriMPZ), 2,3,5,6-Tetramethylpyrazine (TetraMPZ), 2-Acetylpyrrole (APL) 2-Acetylpyrazine (APZ)
Provide some measure of the changes in the delivery of flavour components (targeted and non-targeted) during the “life” of an e-cigarette
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Background
67th TSRC: A rapid profiling method which can be applied to the detection of flavorings in various tobacco products
HS-SPME-GCMS Profiles Flavoring ID
Cigarettes, Cigar/Cigarillos
RYO, Pipe, Chew, Snus,
Snuff, Shisha, Dissovable Wiley, NIST/EPA/NIH, FFNSC
3
Can the same process be used for the analysis of e-liquids and e-cig aerosols?
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Considerations
4
The impact of the e-liquid matrix on the quantification and detection limits of flavour components using HS-SPME?
The type(s) and amount of flavour components
• Are these reflective of tobacco products?
The transfer of flavour components of an e-liquid
• Are these transferred to the e-cigarette aerosol?
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Sample Preparation for HS-SPME-GCMS Analysis
Base Method vs. Emissions / e-Liquids
ISTD/FPCS:
d6-Benzene
d5-Acetophenone
d5-Benzophenone
Add 20 μL ISTD/FPCS
HS-SPME-GCMS
Add 2 mL 3.0 M KClaq
0.5 g of tobacco filler in 10 mL HS vial
Cap the vial and keep at room temp for 2 hrs
Spike FLs std solution
5
Collection Pad
(up to 250mg)
Add 10 μL ISTD
ISTD/FPCS:
d5-2-ethylpyrazine
d7-quinoline
Chromatographic
Differences
Standard
Preparation
Only
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Instrument Parameters
Instrument Parameters
SPME Fiber 65 µm PDMS/DVB
Sample Ads Temp/Time 50 C/20 min
Sample Des Temp/Time 260 C/5 min
Fiber Condition Temp/Time 260 C/26 min
GC Column DB-WAXETR (30 m x 0.25 mm x 0.25 µm)
GC Injector Splitless mode, inlet temp 260 C.
MS Mode Full Scan (35 - 400 m/z)
GC Run Time 50 min (program 40 to 260°C)
6
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Instrument Parameters
Compound RT (min) Quantifier Qualifier
d5-2-ethylpyrazine 15.6 112 113
2,3,5-Trimethylpyrazine
(TriMPZ) 17.5 122 42
2,3,5,6-Tetramethylpyrazine
(TetraMPZ) 19.4 136 54
d7-quinoline 30.1 136 108
2-Acetylpyrrole (APL) 23.4 122 80
2-Acetylpyrazine (APZ) 30.9 94 109
7
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Instrument Parameters
8
Nicotine
Propylene Glycol
Nicotyrine
Fibre Bleed Residual Solvent
~ 4ng TriMPZ
and TetraMPZ
~ 20ng APL
and APZ
Calibration Standard
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Instrument Parameters
9
d5-2-ethylpyrazine
TriMPZ TetraMPZ
d7-quinoline
APL APZ
Nicotine
Propylene Glycol
Nicotyrine
Fibre Bleed Residual Solvent
~ 4ng TriMPZ
and TetraMPZ
~ 20ng APL
and APZ
Calibration Standard
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Determination of Matrix Effect
10
Impact of Propylene Glycol and Glycerin
0
200000
400000
600000
800000
1000000
1200000
0 20 40 60 80 100
Res
po
nse
(A
rea)
Propylene Glycol (%)
TriMPZ
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
0 20 40 60 80 100
Res
po
nse
(A
rea)
Propylene Glycol (%)
APL
0.05
0.06
0.07
0.08
0.09
0.1
0.11
0 20 40 60 80 100
Res
po
nse
Rat
io
Propylene Glycol (%)
TetraMPZ/d5-2-EthylPZ
Calibration Standards Matrix
50:50 (v/v) Propylene Glycol to Glycerin solution
8 mg/mL Nicotine 250mg matrix at each level
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r2=0.997
Quantification of Target Analytes
LOD Determination - Root Mean Square Error (RMSE) *
Generation of a 4 or 5 point calibration curve
Instrument response (area counts) vs. standard concentration
Standards of concentration within an order of magnitude of estimated LOD (e.g. TetraMPZ - 1.10ng to 10.6ng)
* Handbook of Residue Analytical Methods for Agrochemicals, p68-70, Editor-in-Chief Dr Philip W Lee, DuPont Crop Protection,
USA; Published by John Wiley & Sons Ltd in 2003. 11
RMSE
LOD
LOQ
Compound LOQ (ng)
TriMPZ 4.27
TetraMPZ 2.75
APL 107
APZ 26.1
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Quantification of Target Analytes
12
Compound Range
(ng)
TriMPZ 1 - 750
TetraMPZ 1 - 850
APL 10 - 8000
APZ 10 - 8200
d5-2-ethylpyrazine - 194ng d7-quinoline - 112ng
(ISTD for APZ)
All r2 > 0.99
TIC Response (semi-quantitative)
TriMPZ / TetraMPZ: 1 to 4µg
APL and APZ: 10 to 40µg
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Analysis of E-liquids – Experimental
13
18 different formulations tested
• 13 e-liquids (1 advertized as “tobacco flavour)
• 1 e-liquid originating from a tobacco extract • 4 e-cigarette cartomizers (fresh)
• 4 variants of same product line (1 mentholated) • Centrifuge cartomizer at 1500 rpm for 5 min.
• 1 e-cigarette cartomizer (after use – first 20 puffs)
Single replicate for each formulation
• 0.25g sample size
Opportunity to identify other flavour components
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Analysis of E-liquids - Chromatography
14
Sample 1
Tobacco Flavour
Sample 2
No Description
Sample 5
Americano
Sample 1
TriMPZ
Saturated Signal
Sample 2
Requires a more dilute sample for analysis
Sample 5
APZ
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Analysis of E-liquids - Results
15
Sample No Matrix InfoTriMPZ
(ng/g)
TetraMPZ
(ng/g)
APL
(ng/g)
APZ
(ng/g)
1 E-Liquid Tobacco Flavour > UCL 197 - NQ
2 E-Liquid - 5.33 > UCL > UCL
3 E-Liquid Forest Fruit Mix 61.4 NQ - -
4 E-Liquid 7 Leaves 2601 > UCL - -
5 E-Liquid Americano 290 - NQ 8.75
6 E-Liquid Strawberry 5.13 NQ - -
7 Cubana 544 NQ - 195
8 E-Liquid 6.02 4.08 8767 1166
9 E-Liquid 6.21 4.94 - 31166
10 E-Liquid 45.9 7.07 NQ 402
11 E-Liquid 5.06 4.04 - NQ
12 E-Liquid 4.37 4.44 - 57.3
13 Tobacco Extract E-Liquid 52.7 NQ 129 -
14 Cartomizer Liquid 1 > UCL 3.7 NQ -
15 Cartomizer Liquid 2 > UCL NQ 106 -
16 Cartomizer Liquid 3 > UCL - - -
17 Cartomizer Liquid 4M > UCL NQ 117 -
18 Cartomizer Liquid (Used) > UCL - - -
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Analysis of E-liquids - Results
16
Sample No Matrix InfoTriMPZ
(ng/g)
TetraMPZ
(ng/g)
APL
(ng/g)
APZ
(ng/g)
1 E-Liquid Tobacco Flavour > 16 µg 197 - NQ
2 E-Liquid - 5.33 68795 * 47656 *
3 E-Liquid Forest Fruit Mix 61.4 NQ - -
4 E-Liquid 7 Leaves 2601 3638 * - -
5 E-Liquid Americano 290 - NQ 8.75
6 E-Liquid Strawberry 5.13 NQ - -
7 Cubana 544 NQ - 195
8 E-Liquid 6.02 4.08 8767 1166
9 E-Liquid 6.21 4.94 - 31166
10 E-Liquid 45.9 7.07 NQ 402
11 E-Liquid 5.06 4.04 - NQ
12 E-Liquid 4.37 4.44 - 57.3
13 Tobacco Extract E-Liquid 52.7 NQ 129 -
14 Cartomizer Liquid 1 4625 * 3.7 NQ -
15 Cartomizer Liquid 2 4089 * NQ 106 -
16 Cartomizer Liquid 3 5605 * - - -
17 Cartomizer Liquid 4M 10716 * NQ 117 -
18 Cartomizer Liquid (Used) 3842 * - - -
* Estimated using TIC area count
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E – Cig Aerosol – Experimental
17
1 brand (cartomizer 1 from E-liquids) • 4 replicates • 5 cartridges per replicate • Collection in 25 puff increments (5 increments) • Same cartomizers used for all 5 increments (20 total) • Freshly charged battery every 50 puffs • 44mm pad collection (½ pad SPME, ½ pad liquid extract)
Puffing Regimen • 70mL puff volume • 3.5 second puff duration (square wave profile) • 30 second puff frequency
Identify changes in delivery of flavour components (targeted and non-targeted)
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E – Cig Aerosol – Results
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Puff Segment TPM
(mg/cart)
Cartridge Wt. Loss (mg/cart)
Propylene Glycol
(mg/cart)
Nicotine (mg/cart)
Glycerin (mg/cart)
1 – 25
69.6
± 2.0
(2.9%)
67.5
± 1.8
(2.7%)
4.50
± 0.15
(3.4%)
3.02
± 0.10
(3.3%)
51.6
± 1.3
(2.5%)
26 – 50
65.1
± 2.0
(3.1%)
63.3
± 2.2
(3.5%)
4.27
± 0.10
(2.3%)
2.84
± 0.07
(2.5%)
48.5
± 1.6
(3.4%)
51 – 75
61.7
± 1.0
(1.6%)
60.2
± 1.1
(1.8%)
4.13
± 0.18
(4.4%)
2.72
± 0.11
(4.1%)
46.1
± 1.4
(3.0%)
76 – 100
58.2
± 1.2
(2.1%)
56.8
± 1.2
(2.1%)
4.06
± 0.06
(1.5%)
2.65
± 0.03
(1.2%)
44.3
± 0.7
(1.6%)
101 - 125
54.9
± 1.9
(3.4%)
54.1
± 1.9
(3.4%)
3.70
± 0.14
(3.7%)
2.42
± 0.09
(3.8%)
40.5
± 1.8
(4.4%)
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E – Cig Aerosol – Results
19
Consistent loss in delivery of major aerosol components
Delivery of 5th segment is 80% that of the 1st segment (average)
75.0
80.0
85.0
90.0
95.0
100.0
2 3 4 5
Per
cen
tage
(%
) o
f Fi
rst
Pu
ff S
egm
ent
Puff Segment
Changes in Aerosol Delivery
MS TPM Cartridge Wt. Loss Propylene Glycol Nicotine Glycerol
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E – Cig Aerosol – Chromatography
20
E-liquid
Collection Segment 1
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E – Cig Aerosol – Chromatography
21
Only 2,3,5-Trimethylpyrazine (TriMPZ) found in the emmission
E-liquid
Collection Segment 1
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HS-SPME Results – Targeted
22
Puff Segment Nicotine
(mg/cart) TriMPZ * (ng/cart)
1 – 25
3.02
± 0.10
(3.3%)
472
± 19
(4.0%)
26 – 50
2.84
± 0.07
(2.5%)
435
± 14
(3.3%)
51 – 75
2.72
± 0.11
(4.1%)
427
± 16
(3.7%)
76 – 100
2.65
± 0.03
(1.2%)
412
± 18
(4.0%)
101 - 125
2.42
± 0.09
(3.8%)
386
± 7
(1.8%)
* TriMPZ results extrapolated using TIC response
Delivery of TriMPZ in the 5th segment is 82% that of the 1st segment
Results are consistent with major components of e-cigarette aerosol
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E – Cig Aerosol – Chromatography
23
No observed changes in TIC ratios amongst non-targeted compounds
Collection Segment 5
Collection Segment 4
Collection Segment 3
Collection Segment 2
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HS-SPME Aerosol – Non-Targeted
24
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
ETHYL ACETATE
ETHANOL
2-METHOXY-3-METHYLPYRAZINE
2,3,5-TRIMETHYLPYRAZINE
PROPYLENE GLYCOL
OXOPHORONE
BETA-DEMASCONEISOMER
2-HYDROXY-3-METHYL-2-CYCLOPENTEN-1-ONE
NICOTYRINE
ETHYLVANILLIN
Segment 2 Segment 5
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Summary
25
HS-SPME-GCMS can be used for the determination of flavour components for both e-liquids and e-cigarette aerosol
• Optimization/Compromises dependent on levels
Matrix matching (glycerin / propylene glycol) need be considered for trace level quantitation
Screening of e-liquid is useful for identification of targeted compounds
This method can be used to assess the transfer of flavour components of an e-liquid to the E-cig aerosol.
• Targeted and non-targeted compounds
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Acknowledgements
26
Mr. MingZhong Cui
Mrs. Helena Coetzer
Thank You !
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