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

1

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

2

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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http://www.gerstel.com/en/mps-gc-ms-autosampler.htm

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

4

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

5

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


6 Test Sample 1 Brand 1 TS1-2 mL KCl-10 µL ISTD/PFCS 0.5006





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




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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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


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

10

1

10

1

00

1

00

0

10

00

0 n

g

Typical Chromatograms from LOD Analysis 2014

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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


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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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

14

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 ?


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


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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


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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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Documents

Determination of Potential Flavor Additives in Tobacco