Determination of Potential Flavor Additives in Tobacco ...
Transcript of Determination of Potential Flavor Additives in Tobacco ...
LABSTAT INTERNATIONAL ULC.
262 Manitou Drive
Kitchener, Ontario, Canada N2C 1L3
Phone: (519) 748-5409 Fax: (519) 748-1654 Web: www.labstat.com
David LI, Peter JOZA, Andrew MASTERS, Bill RICKERT
68th Tobacco Science Research Conference
Sept. 28 – Oct.1, 2014
Charlottesville, VA, USA
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Determination of Potential Flavor Additives in
Tobacco Products by Headspace - Solid Phase
Microextraction - Gas Chromatography Mass
Spectrometry (HS-SPME-GCMS)
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Background 67th TSRC: A rapid profiling method which can be applied to the detection
of flavorings in various tobacco products
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67th TSRC: the chemical composition of the added flavorings can be
identified unambiguously.
HS-SPME-GCMS Profiles Flavoring ID
Cigarettes, Cigar/Cigarillos
RYO, Pipe, Chew, Snus,
Snuff, Shisha, Dissovable Wiley, NIST/EPA/NIH, FFNSC
Flavorings, FL – Intrinsic Tobacco Flavors & Extrinsic Flavorants
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Objectives
To establish the analytical sequence for a
successful analysis
To semi-quantify frequently used flavorings without
multiple instrument runs
To distinguish
• Extrinsic flavorings from Intrinsic flavorings
• Flavorings from extraneous constituents
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Sample Preparation
for HS-SPME-GCMS Analysis
ISTD/FPCS:
d6-Benzene
d5-Acetophenone
d5-Benzophenone
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Spike 20 μL ISTD/FPCS (Fiber Performance Check Std)
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 Only for QC and LOD
3R4F for QC and LOD
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Instrument Parameters
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Instrument Parameters
SPME Fiber 65 µm PDMS/DVB
Sample Ads Temp/Time 50 C/20 min
Sample Des Temp/Time 250 C/5 min
Fiber Condition Temp/Time 250 C/40 min
GC Column DB-5MS (60 m x 0.25 mm x 0.25 µm)
GC Injector Splitless mode, inlet temp 250 C.
MS Mode Full Scan (35 - 500 m/z)
GC Run Time 60 min
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Mentholated filler
Menthol
Non-Flavored filler
ISTD-1 ISTD-2
Nicotine Neophytadiene
Analytical Sequence
Flavored filler
• To design the chemical composition of spiking solution for QC samples
• To understand sample flavoring characteristics and identify the major flavorings
• To arrange sample run order to avoid carryover for analytical run
Prescreen Run
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Analytical Sequence
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Sample # Sample Type Tobacco Information Sample Composition Tobacco Weight/g
1 Fiber Bank Empty 10 mL vial
2 Ref tobacco 3R4F RT-2 mL KCl-10 µL FPCS 0.5029
3 Ref tobacco CM7 RT-2 ml KCl-10 µL FPCS 0.5075
4 LFCS 3R4F RT-2 mL KCl-10 µL ISTD/PFCS 0.5021
5 Fiber Bank Empty 10 mL vial
6 Test Sample 1 Brand 1 TS1-2 mL KCl-10 µL ISTD/PFCS 0.5006
7 Test Sample 2 Brand 2 TS2-2 mL KCl-10 µL ISTD/PFCS 0.5054
8 Test Sample 3 Brand 3 TS3-2 mL KCl-10 µL ISTD/PFCS 0.5041
9 Test Sample 4 Brand 4 TS4-2 mL KCl-10 µL ISTD/PFCS 0.5028
10 Ref tobacco 3R4F RT-2 mL KCl-10 µL FPCS 0.5027
Example of Instrument Run List
• Avoidance of carryover: Prescreen run
• Fiber & lab environment backgrounds: Fiber blank
• Instrument stability: Opening & closing verifications
• Instrument sensitivity: Lab Fortified Control Sample, LFCS
• Fiber performance: FPCS added to all test samples
1 Fiber Bank Empty 10 mL vial
2 Ref tobacco 3R4F RT-2 mL KCl-10 µL FPCS 0.5029
10 Ref tobacco 3R4F RT-2 mL KCl-10 µL FPCS 0.5027
4 LFCS 3R4F RT-2 mL KCl-10 µL ISTD/PFCS 0.5021
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Semi-Quantitative Approach
LOD Determination - Root Mean Square Error (RMSE)
• Generation of a 4/5-points calibration curve
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Instrument response (area count)
vs. std concentration
Standards of concentration within an
order of magnitude of estimated LOD
• Calculation of RMSE
“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.
Std Concentration
Are
a C
ou
nt
20 40 60 80 100
Calculated LOD
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Semi-Quantitative Approach
Experiment Setup for LOD Analysis
• Divide 33 of frequently used flavorings into three group
• Spike 3R4F with flavorings standards (1, 2, 4, 8, 10, 20, 40, • • •, ng)
Group 1 Group 2 Group 3
Benzaldehyde (-)-α-Pinene Amyl Alcohol
Benzyl alcohol (R)-(+)-Limonene Camphene
Acetophenone (±)-Citronellal Citronellol
Methyl benzoate L-Menthone Eucalyptol
Methyl salicylate Menthol Isoamylisovalerate
Cinnamaldehyde (R)-(-)-Carvone Linalool
Piparonal Geraniol Linalyl acetate
Eugenol Anethole γ-Nonalactone
Vanillin Geranyl acetate Pentyl acetate
Ethyl vanillin β-Caryophyllene α-Phellandrene
Benzyl benzoate β-Ionone δ-Undecalactone
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ISTD-1 ISTD-2
Nicotine
Semi-Quantitative Approach
3R4F filler spiked with different amount of flavoring standards
1 2 3 4
5
6
7
8 9
10 11
FLs in Group 2
1. α-Pinene
2. Limonene
3. Citronellol
4. L-menthone
5. Menthol
6. Geraniol
7. Carvone
8. Anethole
9. Geranyl Acetate
10. Caryophylene
11. β-Ionone
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1
10
1
00
1
00
0
10
00
0 n
g
Typical Chromatograms from LOD Analysis 2014
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Semi-Quantitative Approach
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Sample Selection for LOD Analysis
Same Order: LOD vs. Std Concs
Yes No
Re-pick samples
Re-calculate LOD Accept LOD
y = 0.3965x + 0.02573
r2 = 0.993
Based on RMSE requirements:
• Pick five of the lowest spiked
samples
• Two limitations:
Flavoring peak S/N > 10
r2 > 0.90
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y = 51140x + 183100
r2 = 0.982
LOD = 13.7 ng
Semi-Quantitative Approach
Calculated LODs are within same order of magnitude with std concentrations 12
Typical Calibration Curves Used for LOD Calculation
y = 106600x + 106300
r2 = 0.995
LOD = 0.70 ng
LOQ = 2.40 ng
LOD = 45.5 ng • 3R4F matrix
• Two limitations: Peak S/N >10; r2 > 0.90
LOQ = 3.3 × LOD
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Semi-Quantitative Approach
Flavoring LODs: Aliphatic ( 1.4 ~ 131 ng/g) << Aromatic (11.7 ~ 13223 ng/g)
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Group 1 LOD, ng/g Group 2 LOD, ng/g Group 2 LOD, ng/g
Benzaldehyde 29.5 (-)-α-Pinene 4.9 Amyl Alcohol 11094.9
Benzyl alcohol 272.7 (R)-(+)-Limonene 6.1 Camphene 3.92
Acetophenone 27.3 (±)-Citronellal 3.1 Citronellol 31.1
Methyl benzoate 47.1 L-Menthone 2.5 Eucalyptol 1.41
Methyl salicylate 43.1 Menthol 29.8 Isoamylisovalerate 5.03
Cinnamaldehyde 903.1 (R)-(-)-Carvone 101 Linalool 4.47
Piperonal 573 Geraniol 558.3 Linalyl acetate 5.89
Eugenol 993.9 Anethole 11.7 γ-Nonalactone 1.69
Vanillin 9968.8 Geranyl acetate 49.3 Pentyl acetate 7.17
Ethyl vanillin 13223 β-Caryophyllene 20.5 α-Phellandrene 5.3
Benzyl benzoate 35.7 β-Ionone 14.6 δ-Undecalactone 131
LODs for 33 Flavorings
Eucalyptol 1.41
γ-Nonalactone 1.69
Vanillin 9968.8
Cinnamaldehyde 903.1
Benzyl alcohol 272.7
(±)-Citronellal 3.1
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Low-Level Calibration Curve
(LLCC, External method)
Semi-Quantitative Criteria
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Directly
Used as Criteria for Low-Level
Flavoring Semi-Quantification
(Non-Flavored Tobacco Products)
Eucalyptol Calibration Curve: External Method
Low-Level Calibration Curves Derived From LOD Analysis
y = 106600x + 106300
r2 = 0.995
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Semi-Quantitative Criteria
High-Level Calibration Curves
Boundary Response
(LLCC or HLCC) 15 Eucalyptol Calibration Curve: d5-Acetaphenone as ISTD
High-Level Calibration Curve
(HLCC, ISTD Method)
Directly
Used as Criteria for High-Level
Flavoring Semi-Quantification
(Flavored Tobacco products)
High-Level Calibration Curves Derived From LOD Analysis
y = 0.3941x + 0.03498
r2 = 0.993
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Semi-Quantitative Results
Unknown cig filler
3R4F cig filler
Example of Flavoring Quantification in Test Sample
ND results are omitted for clarity; Total 14 flavorings are detected in this case
Menthone 24.92 2.5 13 990202 1010020 18 18
β-Ionone 33.82 15 25 1216650 2531571 111 111
Linalyl acetate 27.36 5.9 9 476348 663865 13 13
33 established
HLCCs
Response
Comparison
Re-Q
ua
nt
Final Results
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1. Presence of Non-Naturally-Occurring Flavorings in Samples
e.g., Ethylvanillin, Ethylmaltol and Cinnamyl Anthranilate
2. Typical Use Level in Cigarettes
“An Overview of the Effects of Tobacco Ingredients on Smoke Chemistry and
Toxicity” R.R. Baker et al, Food and Chemical Toxicology, 42S (2004) S53-83.
“Permitted additives to Tobacco products in the UK”, Department of Health,
London, March 2000.
3. Amount Ratio Test Samples vs. Ref. 3R4F
4. Odor Threshold of Flavorings in Tobacco Matrix Tobacco Flavor Seminar Sept. 11-12, 1991
Published data from Leffingwell & Associates
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Identification of Flavoring Origins
Extrinsic Flavorings vs. Intrinsic Flavorings 2014
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Group 1 RT, min LOD, ng/g FL in cig, ng/g FL in 3R4F, ng/g Use Level, ng/g Ratio Threshold, ng/g Flavorings
Benzaldehyde 18.23 30 1229 44.7 60000 27.5 100 ?
Benzyl alcohol 20.82 273 63076 423.2 1700000 149.0 750 ?
Methyl salicylate 26.06 43 84 32.9 2500 2.6 ?
Piperonal 30.36 573 72023 ND 200000 4 ?
Vanillin 31.89 9969 320077 ND 864000 100 ?
Ethylvanillin 33.38 13223 279161 ND 250000 200 ?
Group 2
Limonene 20.51 6.1 248 9.5 1000 (Limon oil) 26.2 ?
L-Menthone 24.92 2.5 18 15.9 2500 1.1 ?
Menthol 25.45 30 1925 104.6 20000000 18.4 300 ?
β-Ionone 33.82 15 111 54.3 7500 2.0 0.007 ?
Group 3
Linalool 22.81 4.5 212 8.7 10000 24.4 6 ?
Linalyl acetate 27.36 5.9 13 ND 7500 600 ?
γ-Nonalactone 30.71 1.7 1982 ND 10000 65 ?
δ-Undecalactone 36.79 131 2087 ND 7500 950 ?
Identification of Flavoring Origins
Ethylvanillin 33.38 13223 279161 ND 250000 200 Extrinsic
Piperonal 30.36 573 72023 ND 200000 4 Extrinsic
Vanillin 31.89 9969 320077 ND 864000 100 Extrinsic
γ-Nonalactone 30.71 1.7 1982 ND 10000 65 Extrinsic
δ-Undecalactone 36.79 131 2087 ND 7500 950 Extrinsic
Menthone 24.92 2.5 18 15.9 2500 1.1 Intrinsic
Methyl salicylate 26.06 43 84 32.9 2500 2.6 Intrinsic
β-Ionone 33.82 15 111 54.3 7500 2.0 0.007 Intrinsic
Linalyl acetate 27.36 5.9 13 ND 7500 600 Intrinsic
Extrinsic
Extrinsic
Intrinsic
Extrinsic
Extrinsic
Extrinsic
Extrinsic
Intrinsic
Extrinsic
Intrinsic
Extrinsic
Intrinsic
Extrinsic
Extrinsic
Extrinsic Flavorings vs. Intrinsic Flavorings
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Test Cig Filler
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Benzaldehyde
Benzyl alcohol
Linalool
Menthol Piperonal
γ-Nonalactone
Ethylvanillin
δ-Undecalactone Vanillin Limonene
Identification of Flavorings Origins
Profile of the test sample labeled with ten detected characterizing flavorings
3R4F Cig Filler
Extrinsic Flavorings vs. Intrinsic Flavorings
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Interpretations on Extraneous Constituents
Identification of Flavoring Origins
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3R4F
CM7
Test Sample-Prescreen Run
Test Sample-Analytical Run
Longifolene:
• Flue-Cured Tobacco Note
• Pine Smoke Flavoring
It is highly possible that the
detected longifolene is from
the tobacco leaf cure process
rather than an intentionally
added flavoring.
X
X Characteristic masses of Longifolene are
not identified in reference samples
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Extraneous Constituents in Tobacco
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Extraneous Constituents
Detected in Cig Fillers
Ret Time
min Origin Interpretation
1-Propyl Acetate 8.62 Solvents used in Tobacco manufacturing
2-Ethylhexyl Acetate 24.22
MethylEthylbenzenes 18.04 Ink and adhesive components
Trimethylbenzene 19.27
1,1,3-Trimethyl-3-phenylindane 39.74 Polymer unit from packaging materials
Isopulegol 24.58
Menthol impurities from mentholated cigarettes
Isomenthone 25.11
Neomenthol 25.25
Menthyl Acetate 28.70
Menthyl isovalerate 35.64
Longicyclene 31.36 Flue-cured tobacco note from Pine smoke
Longifolene 32.32
Methyl Laurate 34.67 Inhibitor of tobacco maxillary bud growth
Identification of Flavoring Origins
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Summary
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The method developed can be used to semi-quantify
a number of frequently used flavorings in tobacco
products without multiple instrument runs
The methodology proposed can be used to distinguish
extrinsic from intrinsic flavorings as well as flavorings
from extraneous constituents
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Thank You!
LABSTAT INTERNATIONAL ULC.
262 Manitou Drive, Kitchener, Ontario, Canada N2C 1L3
Phone: (519) 748-5409 Fax: (519) 748-1654 Web: www.labstat.com
Thank You for Your Attention
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