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Summary of Regulatory Methods and Procedures for Determination of Harmful and Potentially Harmful Components in Tobacco Smoke

Margarita H. Docheva
Margarita H. Docheva
, 
Desislava M. Kirkova
Desislava M. Kirkova
 y 
Liliya S. Stoyanova
Liliya S. Stoyanova
   | 09 jun 2024
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Publicado en línea: 09 jun 2024
Páginas: 112 - 135
Recibido: 04 jul 2023
Aceptado: 21 feb 2024
DOI: https://doi.org/10.2478/cttr-2024-0002
Palabras clave
© 2024 Margarita H. Docheva et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Chemical components in tobacco smoke and evaluation of their harmful effects according to IARC (5), FDA (6), WHO Study Group (3), and Fowles and Dybing (4).

Group Compound Chemical structure IARCa (group, year) FDAb WHO study groupc Carcinogenicity or toxicity data Fowles and Dybingd
TACI TNCRI CRI
Alkaloids Nicotine CAS 54-11-5 – – – – –
Aldehydes Acetaldehyde CAS 75-07-0 Group 2B, 1999 CA, RT, AD 6.1 67.1 9.18×10−5
Acrolein CAS 107-02-8 Group 2A, 2021 RT, CT – 1099 –
Formaldehyde CAS 50-000-0 Group 1, 2012 CA, RT – 19.8 9.90×10−6
Crotonaldehyde CAS 4170-30-3 Group 2B, 2021 CA – – –
Propionaldehyde CAS 123-38-6 – CT, RT – – –
n-Butyraldehyde CAS 123-72-8 – – – – –
Aromatic amines 3-Aminobiphenyl CAS 2243-47-2 – – – – –
4-Aminobiphenyl CAS 92-67-1 Group 1, 2012 CA – – 3.60×10−7
1-Aminonaphthalene (1-Naphthylamine) CAS 134-32-7 Group 3, 1987 CA – – –
2-Aminonaphthalene (2-Naphthylamine) CAS 91-59-8 Group 1, 2012 CA – – 1.80×10−7
Hydrocarbons Benzene CAS 71-43-2 Group 1, 2018 CA, CT, RTD 2.6 0.64 6.71×10−5
1,3–Butadiene CAS 106-99-0 Group 1, 2012 CA, CT, RTD 9.9 2.4 3.02×10−4
Isoprene CAS 78-79-5 Group 2B, 1999 CA – – –
Styrene CAS 100-42-5 Group 2A, 2019 CA, RT – 0.01 –
Toluene CAS 108-88-3 Group 3, 1999 RT, RTD – 0.22 –
Polycyclic aromatic hydrocarbons Benzo[a]pyrene CAS 50-32-8 Group 1, 2012 CA 0.0086 – 1.93×10−6
Tobacco-specific nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) CAS 64091-91-4 Group 1, 2012 CA 3.4 – 7.80×10−6
N′-Nitrosonornicotine (NNN) CAS 16543-55-8 Group 1, 2012 CA 0.29 – 3.80×10−5
N′-Nitrosoanabasine (NAB) CAS 37620-20-5 Group 3, 2007 – – –
N′-Nitrosoanatabine (NAT) CAS 71267-22-6 Group 3, 2007 – – –
Phenols Catechol CAS 120-80-9 Group 2B, 1999 CA 0.58 1.2 –
m-Cresol CAS 108-39-4 – CA, RT – 0.01 –
p-Cresol CAS 106-44-5 – CA, RT – 0.01 –
o-Cresol CAS 95-48-7 – CA, RT – 0.01 –
Phenol CAS 108-95-2 Group 3, 1999 CA, RT – 0.07 –
Hydroquinone CAS 123-31-9 Group 3, 1999 – 1.2 – –
Resorcinol CAS 108-46-3 Group 3, 1999 – – – –
Other organic compounds Acetone CAS 67-64-1 – RT – – –
Acrylonitrile CAS 107-13-1 Group 2B, 1999 CA, RT 1.4 2.1 1.29×10−4
Quinoline CAS 91-22-5 Group 2B, 1999 CA – – –
Pyridine CAS 110-86-1 Group 2B, 2019 – – – –
Metal and metalloids Arsenic CAS 740-38-2 As Group 1, 2012 CA, CT, RT – 0.16 1.16×10−4
Cadmium CAS 7440-34-9 Cd Group 1, 2012 CA, RT, RDT 1.7 2.6 2.16×10−5
Chromium CAS 7440-47-3 Cr Group 3, 1990 CA, RT, RDT – – 3.15×10−5
Lead CAS 7439-92-1 Pb Group 2B, 1987 CA, CT, RDT 0.00 – 7.68×10−9
Mercury CAS 7439-97-6 Hg Group 3, 1993 CA, RDT – 0.02 –
Nickel CAS 7440-02-0 Ni Group 2B, 1990 CA, RT – – 1.43×10−7
Selenium CAS 7782-49-2 Se Group 3, 1987 RT – – –
Other constituents Ammonia CAS 7664-41-7 NH3 – RT – 0.07 –
Carbon monoxide CAS 630-08-0 CO – RDT – 1.3 –
Hydrogen cyanide CAS 74-90-8 HCN – RT, CT – 17.2 –
Other constituents Nitrogen oxides CAS 10102-43-9 NOn – – – 3.1 –

Determination of selected carbonyls in mainstream cigarette smoke.

SOP 08. Standard Operating Procedure for Determination of Aldehydes in Mainstream Cigarette Smoke Under ISO and Intense Smoking Conditions. 31 August 2018 (16) CRM No. 74. Determination of Selected Carbonyls in Mainstream Cigarette Smoke by HPLC (August 2019) (17)
Compounds
Formaldehyde, acetaldehyde, acrolein (acrylaldehyde) Formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and n-butyraldehyde as their 2,4-dinitrophenylhydrazones
Method summary
Cigarettes are smoked on a smoking machine according to the ISO or intense regime. One cigarette is smoked per smoke trap, containing 300 mg CX-572 particles and a glass fibre filter pad. This standard operating procedure is suitable for the quantitative determination of the following three aldehydes in mainstream (MS) cigarette smoke: formaldehyde, acetaldehyde, and acrolein to be analyzed by liquid chromatography. The aldehydes in mainstream tobacco smoke are extracted by adding a solution containing a mixture of carbon disulphide and methanol and are derivatized with a solution of 2,4-dinitrophenylhydrazine (DNPH). The samples are subjected to analysis using high-performance liquid chromatography (HPLC), equipped with ultraviolet (UV) or diode array detector (DAD). Cigarettes are smoked on a smoking machine that has been fitted with impingers, but without the specified filter pad holder containing the glass fiber filter. The carbonyls in mainstream tobacco smoke are trapped by passing each puff through an impinger device containing an acidified solution of 2,4-dinitrophenylhydrazine (DNPH) in acetonitrile. An aliquot of the smoke extract is then syringe-filtered and diluted with 1% Trizma™ base in aqueous acetonitrile. The samples are subjected to analysis using reverse phase HPLC/UV or HPLC/DAD.
Apparatus and equipment
Linear smoking machine Linear smoking machine
Rotary smoking machine Rotary smoking machine
HPLC system with a UV and/or DAD detector HPLC system with a UV and/or DAD detector
Carboxen 572 particles, 20/45 mesh size
Derivatization solution
DNPH solution: DNPH solution:
Approximately 1 g DNPH-HCl is added to a 50-mL volumetric flask, then 10 mL of 85% phosphoric acid is carefully added. Approximately 150 mL deionized water is added to a 200-mL volumetric flask, then 28 mL of 85% phosphoric acid is carefully added. The solution is made up to a volume of 200 mL with deionized water. 6.8 g (0.024 mole) of DNPH (approximately 30% water) is weighed into a 2-L amber volumetric flask and 1 L of acetonitrile is added. 58 mL of the diluted phosphoric acid solution is added and gently mixed. The volume is diluted with deionized water.
Trizma™ base dilution solution:
MeCN : 1% aqueous Trizma™ (80:20 v/v) - 2.00 g of Trizma™ base is dissolved in 200 mL of deionized water in a 1-L volumetric flask. The volume is diluted with acetonitrile.
Linear range
The calibration should cover the concentration range of interest. The linear range is approximate to the aldehyde content in tobacco smoke. The calibration should cover the concentration range of interest. The linear range is approximate to the carbonyl content in tobacco smoke.
Number of cigarettes to be smoked
ISO / Intense smoking regime ISO / Intense smoking regime
Linear smoking machine:  1 Linear smoking machine:  2
Rotary smoking machine:  1 Rotary smoking machine:  5
Sample preparation
The aldehydes mainstream apparatus is assembled on the smoking machine using Carboxen 572 and glass fibre filter pad. The method is used for simultaneous determination of aldehydes (SOP 08) and volatile organics (SOP 09) in tobacco smoke. Transfer 0.5 mL to 5 mL flask and add 200 μL of DNPH solution and diluted to 5 mL. An aliquot of solution is analyzed by HPLC/UV or HPLC/DAD. The carbonyl mainstream apparatus is assembled on the smoking machine without using the filter pads and filter holders and cigarettes are smoked. The DNPH smoke extract solution is allowed to sit for five to thirty minutes. After that 6 mL of 1% Trizma™ base solution is pipetted into a 10 mL volumetric flask. An aliquot of solution is analyzed by reverse-phase HPLC/UV or HPLC/DAD.
Sample analysis
High performance liquid chromatography with UV or DAD Detector High performance liquid chromatography with UV or DAD Detector (RP HPLC - UV or RP HPLC - DAD).
HPLC equipment and its operating conditions
LC column: Ascentis RP-Amide (150 mm × 4.6 mm, 3 μm) or equivalent LC column: 250 mm x 4 mm, Reversed Phase (RP) C18 (5 μm), or equivalent
Column Temperature: 30 ± 5 °C Disposable Guard Column: 4 mm x 4 mm, RP C18 (5 μm), or equivalent
Injection volume: 20 μL
UV detection: 360 nm or maximum wavelength at 300–400 nm Column Temperature: 30 °C
Injection volume: 20 μL
Degasser: On UV or DAD detection: 365 nm
Binary pomp flow: 1.0 mL/min Flow rate: 1.5 mL/min
Mobile Phase A: water Mobile Phase:
Mobile Phase B: acetonitrile

Solvent A: Prepare 2 L of 30% Acetonitrile, 10% THF, 1% IPA in Type I water, filter and degas (UHP Helium sparged)

Solvent B: Prepare 2 L of 65% Acetonitrile, 1% THF, 1% IPA in Type I water, filter and degas (UHP Helium sparged)

Solvent C: Acetonitrile (UHP Helium sparged)
Total analysis time: 45 min
Gradient conditions:
0.0 100% A 0% B
10.0 55% A 45% B
25.0 55% A 45% B Gradient conditions:
30.0 0% A 100% B 0.0 100% A 0% B 0% C
30.0 0% A 100% B 8.0 70% A 30% B 0% C
31.0 55% A 45% B 20.0 47% A 53% B 0% C
40.0 55% A 45% B 27.0 0% A 100% B 0% C
30.0 0% A 0% B 100% C
32.0 0% A 0% B 100% C
34.0 95% A 5% B 0% C
Equilibration 10.0 100% A 0% B 0% C

CRM No. 78 – Determination of Selected Phenolic Compounds in Mainstream Cigarette Smoke by High Performance Liquid Chromatography with Fluorescence Detector.

Method description
Compounds
Hydroquinone, resorcinol, catechol, phenol, p-cresol, m-cresol, and o-cresol
Method summary
Cigarettes are smoked on a routine smoking machine and mainstream smoke is trapped on a glass-fibre filter pad (GFP). The GFP is extracted with aqueous acetic acid. An aliquot of the extract is syringe filtered and analysed by HPLC/FLD.
Apparatus and equipment
Linear smoking machine or Rotary smoking machine HPLC/FLD
Extraction solution
1% acetic acid solution
Linear range
The calibration should cover the concentration range of interest. The linear range is approximate to the phenolic compounds in tobacco smoke.
Number of cigarettes to be smoked
ISO smoking regime
Linear smoking machine: 5
Rotary smoking machine: 10
Sample preparation
Extraction of filter pads:
The pads from the holders are removed, transferred into an Erlenmeyer flask and 40 mL 1% acetic acid solution (5 cigarettes) and 80 mL 1% acetic acid solution (10 cigarettes) is added. The extraction of phenolic compounds from the pad is until the CFP has disintegrated. The CFP extracts may need to be diluted.
Sample analysis
HPLC/FLD
HPLC equipment and its operating conditions
HPLC column with pentafluoro phenylpropyl (PFP) stationary phase; an example of the column dimensions are 3-μm, a 150-mm column and 4.6 mm internal diameter or equivalent. Wavelength programmable fluorescence detector settings (example):
Time (min) Excitation (nm) Emission (nm)
Column Temperature: Ambient 0.0 280 310
Auto-sampler Tray Temperature: 4 °C 12.4 280 310
Injection volume: 10 μL or 20 μL 12.5 274 298
23.0 274 298
Mobile Phases: 24.0 280 310
Mobile phase A: Prepare 2 L of 1% acetic acid in deionized water and degas 28.0 280 310
Mobile phase B: Prepare 2 L of 1% acetic acid in methanol and degas
Flow rate: 0.8 mL/min
The following is an example of gradient conditions:
Time (min) % A % B
0 78 22
8 78 22
8.5 55 45
21 55 45
22 0 100
28 0 100

CRM No. 95 – Determination of aromatic amines in mainstream cigarette smoke by gas chromatography mass spectrometry with negative chemical ionization (22).

Method description
Compounds
1-Aminonaphthalene, 2,6-dimethylaniline, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, o-anisidine, o-toluidine
Internal standard
2,6-Dimethylaniline-d11, 2-aminonaphthalene-d7, 4-aminobiphenyl-d9, o-toluidine-d9
Method summary
Cigarettes are smoked on a standard smoking machine. The mainstream smoke is collected on a glass-fibre filter pad (GFP). After addition of internal standards, the GFP is extracted with dichloromethane using a laboratory shaker for 20 min. The extract is filtered, derivatized with HFBA, purified on a Florisil SPE and analysed by GC/MS-NCI.
Apparatus and equipment
Linear smoking machine or Rotary smoking machine GC/MS-NCI
Extraction solution
Dichloromethane
Linear range
1-Aminonaphthalene: 0.4 – 5.0 ng/mL
3-Aminobiphenyl: 0.07 – 0.9 ng/mL
2,6-Dimethylaniline: 0.1 – 3.1 ng/mL
4-Aminobiphenyl: 0.07 – 0.9 ng/mL
2-Aminonaphthalene 0.4 – 5.0 ng/mL
o-Anisidine 0.1 – 1.1 ng/mL
o-Toluidine 1 – 16 ng/mL
Number of cigarettes to be smoked
ISO smoking regime Intense smoking regime
• Linear smoking machine: 5 • Linear smoking machine: 2–3
• Rotary smoking machine: 10 • Rotary smoking machine: 5
Sample preparation
Extraction of filter pads: Sample derivatization: Sample clean-up:
The pads are removed from the holders, transferred into the Erlenmeyer flask and 50 mL dichloromethane + 1 mL internal standard intermediate solution is added. The extraction of aromatic amines from the pad is for 40 min. 5 mL aliquot is derivatized with HFBA (25 μL for ISO regime and 50 μL for intense regime) for 40 min. SPS cartridge (Florisil) is pre-conditioned with 12 mL of dichloromethane (discard). Whole sample extract is transferred into the cartridge and is allowed to pass through the cartridge (collect). The cartridge is eluted with 8.5 mL dichloromethane (collect). The both eluents are combined, mixed well and an aliquot is transferred to a GC vial for GC/MS-NCI analysis.
Sample analysis by GC/MS-NCI
GC equipment and its operating conditions: Mass detection conditions:
GC column: low/mid-polarity, with a (14%-cyanopropylphenyl)-methylpolysiloxane stationary phase, a 30-m column with a 0.32-mm internal diameter and 1-μm film thickness or equivalent. Interface/transfer line temperature: 240 °C (ion trap 200 °C)
MS source temperature: 150 °C
MS quadrupole temperature: 106 °C
MS mode: NCI
Data acquisition mode: Selected Ion Monitoring (SIM)
Injector temperature: 250 °C Reagent gas: Methane at 40% flow
Injection mode: Splitless Ion peak for identification (m/z):
Injection volume: 3 μL o-Toluidine: 283
Mode: Constant flow 2,6-Dimethylaniline: 297
Flow rate: 1.5 mL/min o-Anisidine: 299
Column temperature: 40 °C (0.5 min), 15 °C/min to 240 °C (hold for 5 min), 50 °C/min to 270 °C (hold for 10 min) 1-Aminonaphthalene: 319
2-Aminonaphthalene: 319
3-Aminobiphenyl: 345
4-Aminobiphenyl: 345
o-Toluidine-d9: 290
2,6-Dimethylaniline-d11: 306
2-Aminonaphthalene-d7: 326
4-Aminobiphenyl-d9: 354

Comparison between SOP 05 and CRM No. 58 for the determination of benzo[a]pyrene in mainstream cigarette smoke.

SOP 05. Standard Operating Procedure for Determination of Benzo[a]pyrene in Mainstream Cigarette Smoke under ISO and Intense Smoking Conditions, February 2015 (40) CRM No. 58. Determination of Benzo[a]pyrene in Mainstream Cigarette Smoke by GC-MS, November 2019 (41)
Compounds
Benzo[a]pyrene Benzo[a]pyrene
Internal standard
B[a]P-d12 B[a]P-d12
Method summary
Mainstream smoke total particulate matter from the cigarette sample is trapped onto a GFP. Internal standard is spiked onto the GFP, which is extracted with cyclohexane. The cyclohexane extract is purified using SPE by passing through a pure silica unbound phase SPE cartridge, and the eluent is collected. The sample is analyzed by GC/MS with electron ionization detection. Mainstream smoke total particulate matter from the cigarette sample is trapped onto a GFP. Internal standard is spiked onto the GFP, which is extracted with methanol, and the methanol extract is diluted with water. Crude water/methanol smoke extract is purified using SPE by passing through a cyclohexyl bonded silica (CH) SPE cartridge, followed by the elution of B[a]P with cyclohexane. The sample is analyzed by GC/MS operated in the SIM data acquisition mode for identification, confirmation and quantification of B[a]P.
Apparatus and equipment
Linear smoking machine or Rotary smoking machine GC/MS Linear smoking machine or Rotary smoking machine GC/MS
Extraction solution
Cyclohexane Methanol
Linear range
2 – 60 ng/mL 1 – 200 ng/mL
Number of cigarettes to be smoked
ISO smoking regime ISO smoking regime
• Linear smoking machine: 5 • Linear smoking machine: 5 – 10
• Rotary smoking machine: 20 • Rotary smoking machine: 10 – 20
Intense smoking regime Intense smoking regime
• Linear smoking machine: 3 • Linear smoking machine: 5 – 10
• Rotary smoking machine: 10 • Rotary smoking machine: 10 – 20
Sample preparation
Extraction of filter pads: The pads from the holders are removed, transferred into Erlenmeyer flask and cyclohexane and internal standard (40 mL cyclohexane + 40 μL of the B[a]P-d12, for 44-mm pads, or 100 mL cyclohexane+ 100 μL of the B[a]P-d12, for 92-mm pads) are added to the flask. The extraction of B[a]P from the pad is for 60–80 min. Extraction of filter pads: The pads from the holders are removed, transferred into Erlenmeyer flask and methanol and internal standard (20 mL + 200 μL of the B[a]P-d12 for 44-mm pads, or 50 mL + 200 μL of the B[a]P-d12 for 92-mm pads) are added to the flask. The flask is shaken vigorously until the filter pad disintegrates. The solution is filtered through a glass suction filter or using paper filtration and the volume is supplemented to at least 40 mL for a 44-mm filter pad, or to at least 80 mL for a 92-mm pad. Distilled water is added into the flask in order to obtain a solution containing 60% water and 40% methanol.
Sample clean-up: SPE cartridge is pre-conditioned with 10 mL cyclohexane (discard). 10 mL of sample extract is pipetted onto the cartridge and is allowed to pass through the cartridge (collect). The cartridge is eluted with two further 15-mL aliquots of cyclohexane, allowing the cartridge to run dry after the last aliquot is passed through. The cyclohexane solution is evaporated almost to dryness and is dissolved with 1 mL cyclohexane. Sample clean-up: SPE cartridge is pre-conditioned with 10 mL methanol and 10 mL of a mixture of water and methanol (60:40, v/v). The extraction solution is passed through the SPE cartridge and the cartridge is dried (discarded). The cartridge is eluted with 15 mL cyclohexane. The volume of the cyclohexane solution is reduced to 1 mL. If necessary, the cyclohexane solution can be dried from the water. The obtained solution is transferred into an amber autosampler vial and capped with a PTFE lined septum cap.
Sample analysis
GC/MS GC/MS
GC equipment and its operating conditions
GC Column - Fused silica capillary column with a methylphenyl (5%) polysiloxane stationary phase, a 30-m column with a 0.25-mm internal diameter and 0.25-μm film thickness is suitable GC Column - Fused silica capillary column with a methylphenyl (5%) polysiloxane stationary phase, 30-m column with a 0.25 mm internal diameter and 0.25 μm film thickness is suitable.
Injector temperature: 280 °C (ion trap: 200 °C) Injector temperature: 290 °C
Injection mode: Splitless Injection mode: Splitless
Injection volume : 1 μL or 2 μL Injection volume: 1 μL
Mode: Constant flow Mode: Constant flow
Flow rate: 1.2 mL/min (ion trap 1.0 mL/min) Flow rate: 0.9 mL/min
Column temperature: 150 °C for 0 min; 6 °C/min to 260 °C, hold at 260 °C for 7 min 50 °C/min to 290 °C, hold at 290 °C for 20 min Column temperature: 80 °C (hold for 3 min); 5 °C/min to 290 °C (hold for 20 min)
Mass detection conditions
Interface/transfer line temperature: 280 °C (ion trap 200 °C) Interface/transfer line temperature: 270 °C
MS source temperature: 230 °C MS source temperature: 230 °C
Dwell time: 50 ms
Ionization mode: Electron ionization
Detection: Full-scan m/z 30-300 Ion peak for identification (m/z):
Ion peak for identification(m/z): Quantification Confirmation
B[a]P 252 B[a]P 252 126
B[a]P-d12 264 B[a]P-d12 264 132

Comparison between SOP 09 and CRM No. 70 for the determination of volatile organics in mainstream cigarette smoke.

SOP 09. Standard Operating Procedure for Determination of Volatile Organics in Mainstream Cigarette Smoke under ISO and Intense Smoking Conditions 31.08.2018 (29) CRM No. 70. Determination of Selected Volatile Organic Compounds in Mainstream Cigarette Smoke by GC-MS - September 2019 (30)
Compounds
1,3-Butadiene, benzene 1,3-Butadiene, isoprene, acrylonitrile, benzene, toluene
Internal standard
Benzene-d6 Benzene-d6
Method summary
Mainstream cigarette smoke according to the Health Canada Intense (HCI) or the ISO regime is performed. One cigarette per smoke trap is smoked, containing 300 mg CX-572 particles and a glass fibre filter pad. The volatile organic components are extracted by adding a solution containing a mixture of carbon disulfide and methanol onto the Carboxen particles and glass fibre filter pad. The analytical determination of VOCs (1,3-butadiene and benzene) is performed by GC/MS with electron ionization mode. VOCs are collected by passing the mainstream smoke of cigarettes through a GFP as specified in ISO 3308 or in ISO 20778 into cryogenic traps (impingers) containing methanol. The impinger solutions are fortified with benzene-d6 and analysed by GC/MS.
Apparatus and equipment
Linear smoking machine or Rotary smoking machine GC/MS Linear smoking machine or Rotary smoking machine, Dewar flasks, Two impingers connected in series, GC/MS
Extraction solution
Option A: Carbon disulfide and methanol 10 mL of methanol is added to each impinger and placed into the Dewar flasks containing the dry ice / isopropanol solution (the temperature must be at or below −70 °C).
Option B: Carbon disulfide and methanol (1:4) mixed solution, containing internal standard solution
Linear range
Option A – Linear smoking machine 1,3-Butadiene: 5 – 50 μg/mL
• 1,3-Butadiene: 20 – 160 μg/mL Isoprene: 12 – 600 μg/mL
• Benzene: 8 – 64 μg/mL Acrylonitrile: 4 – 200 μg/mL
Option A – Rotary smoking machine Benzene: 4 – 200 μg/mL
• 1,3-Butadiene: 10 – 80 μg/mL Toluene: 4 – 200 μg/mL
• Benzene: 4 – 32 μg/mL
Option B – Linear smoking machine
• 1,3-Butadiene: 20 – 160 μg/mL
• Benzene: 8 – 64 μg/mL
Option B – Rotary smoking machine
• 1,3-Butadiene: 10 – 100 μg/mL
• Benzene: 4 – 40 μg/mL
Number of cigarettes to be smoked
ISO smoking regime ISO smoking regime
• Linear smoking machine: 3 • Linear smoking machine: 5 or 10
• Rotary smoking machine: 3 • Rotary smoking machine: 10
Intense smoking regime Intense smoking regime
• Linear smoking machine: 3 • Linear smoking machine: 3 or 6
• Rotary smoking machine: 3 • Rotary smoking machine: 5
Sample preparation
Option A: 2 mL (for linear) or 4 mL (for rotary) of carbon disulfide is added into the vial or flask. 8 mL (for linear) or 16 mL (for rotary) methanol is added slowly. Carboxen CX-572 is added quickly into the two-phase solution. After the bubbling stops, the glass fibre filter pad is placed into the vial or flask. The mixture is shaken gently on a rotary shaker for 30 min at 120 cycles per min. 5 mL of the supernatant solution is transferred into a vial and 100 μL (for linear) or 50 μL (for rotary) of internal standard solution is added. 1 mL portion of the eluate solution is transferred to a 1.5 mL autosampler vial and analyzed by GC/MS. After all samples are smoked following ISO regime, each impinger is spiked with 100 μL of benzene-d6 spiking solution. For the intense regime, each impinger is spiked with 200 μL of benzene-d6 spiking solution. The impingers are stoppered and gently mixed. An aliquot of the combined impinger solutions is transferred into an amber GC vial and analyzed for volatiles using GC/MS.
Option B: The Carboxen 572 particles and the glass fibre filter pad are transferred into the vial or flask. 10 mL (for linear) or 20 mL (for rotary) of solvent mixture is added slowly into the vial or flask. The mixture is shaken gently on a rotary shaker for 30 min at 120 cycles or rounds per min. 1 mL portion of the eluate solution to a 1.5 mL autosampler vial is transferred and analyzed by GC/MS.
Sample analysis
GC/MS GC/MS
GC equipment and its operating conditions
GC Column: InertCap AQUATIC-2 with a 60-m column with 0.25-mm internal diameter and 1.4-μm film thickness is suitable. GC Column: Fused silica capillary column with a 60-m column with 0.25-mm internal diameter and 1.4-μm film thickness is suitable.
Injector temperature: 240 °C (ion trap: 200 °C) Injector temperature: 150 °C
Injection mode: Split Injection mode: Split
Injection split ratio: 1:10 Injection split ratio: 1:30
Injection volume: 1 μL or 2 μL (Ion trap 1:5; after 1.5 min 1:120) Injection volume: 3 μL
Mode: Constant flow Injection split flow: 30 mL/min
Flow rate: 1.0 mL/min (ion trap 1.0 mL/min) Column temperature: 40 °C (6 min); 20 °C / min to 225 °C (hold for 6 min)
Column temperature: 40 °C (6 min); 6 °C/min to 250 °C
Mass detection conditions
Interface/transfer line temperature: 180 °C (ion trap 200 °C) Interface/transfer line temperature: 240 °C
MS source temperature: 200 °C (ion trap 180 °C) MS source temperature: 240 °C
Dwell time: 50 ms Acquisition mode: SIM (or scan)
Ionization mode: Electron ionization (ionizing voltage at 70 eV) Solvent delay: Column dependent
Detection: Full-scan m/z 30 – 300 Detection: Full-scan m/z 40 – 200
Ion peak for identification (m/z): Ion peak for identification (m/z):
Benzene: 78 Quantification Confirmation
Benzene-d6: 84 1,3-butadiene 54 53
1,3-Butadiene: 54 Isoprene 67 68
Acrylonitrile 52 53
Benzene 78 77
Benzene-d6 84 83
Toluene 91 92

Parameters of different smoking regimes (8).

Parameters Smoking regime
Standard Intensive
ISO Massachusetts Canada
Puff volume (mL) 35 45 55
Puff duration (s) 2 2 2
Puff frequency (/min) 1 2 2

Determination of tobacco-specific nitrosamines in cigarette mainstream smoke by SOP 03 and CRM No. 63.

SOP 03. Standard Operating Procedure for Determination of Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke Under ISO and Intense Smoking Conditions – June 2014 (44) CRM No. 63. Determination of tobacco specific nitrosamines in cigarette mainstream smoke – GC-TEA method, January 2019 (45)
Compounds
NNN, NNK, NAT, and NAB NNN, NAB, NAT, and NNK
Internal standard
Deuterium-labelled 3-(1-nitrosopyrrolidin-2-yl)pyridine(NNN-d4) N-Nitrosopentyl-(3-picolyl)-amine (NNPA)
Deuterium-labelled 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK-d4)
Deuterium-labelled N-nitrosoanatabine (NAT-d4)
Deuterium-labelled N-nitrosoanabasine (NAB-d4)
Method summary
Conditioned cigarettes are smoked using standard procedures. Mainstream smoke is trapped on a glass fiber filter pad. After addition of an internal standard, the filter pad is extracted with ammonium acetate. The extract is filtered and analysed by high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS-MS) with electrospray ionization. Analyte ions are detected in the MS-MS mode. Conditioned cigarettes are smoked using standard procedures. Mainstream smoke is trapped on a glass fiber filter pad. After addition of an internal standard, the filter pad is extracted with dichloromethane. Sample clean-up of the extraction solution is accomplished with one of the following methods:

Elution of the extract through an alumina column, followed by the elution of the TSNAs with an acetone/dichloromethane (50:50 v/v) mixture;

Elution of the extract through a combined silica-alumina column, followed by the elution of the TSNAs with an 8% methanol in dichloromethane solution.
The extract is concentrated followed by quantitative analysis using gas chromatography with a thermal energy analyser for detection (GC-TEA).
Apparatus and equipment
Linear smoking machine or Rotary smoking machine Linear smoking machine or Rotary smoking machine
• HPLC system interfaced to MS-MS (triple quadrupole mass spectrometer) • Gas chromatography with a thermal energy analyser
Extraction solution
Ammonium acetate Dichloromethane
Linear range
NNN: 0.5 – 200 ng/mL NNN: 50 – 2000 ng/mL
NAB: 0.5 – 200 ng/mL NAB: 50 – 2000 ng/mL
NAT: 0.5 – 200 ng/mL NAT: 50 – 2000 ng/mL
NNK: 0.5 – 200 ng/mL NNK: 50 – 2000 ng/mL
Number of cigarettes to be smoked
ISO smoking regime ISO smoking regime
• Linear smoking machine: 5 • Linear smoking machine: 10
• Rotary smoking machine: 20 • Rotary smoking machine: 20
Intense smoking regime
• Linear smoking machine: 3
• Rotary smoking machine: 10
Sample preparation
Extraction of filter pads Extraction of filter pads
The pads from the holders are removed, transferred into the 60-mL amber bottle and added 100 mmol/L aqueous ammonium acetate extraction solution (20 mL for 44-mm pads, 50 mL for 93-mm pads) + internal standard (200 μL for a 44-mm pad, 500 μL for a 92-mm pad). The extraction of nitrosamines from the pad is for 30 min. Remove aliquots (e.g. 1–2 mL) of the extract, filter the aliquots with at most 0.45 μm membrane filter, and place in an autosampler vial. The pads from the holders are removed, transferred into the Erlenmeyer flask and added 100 mL dichloromethane + 400 μL internal standard (10 cigarettes) or 200 mL dichloromethane + 800 μL internal standard (20 cigarettes). The extraction of nitrosamines from the pad is for 30 min. The volume is completed with dichloromethane to 150 mL (10 cigarettes) and 300 mL (20 cigarettes). An aliquot of the solution is concentrated to approximately 5 mL. The elution solution is concentrated to a volume 2 mL.
Sample clean-up - 1: In the liquid chromatographic column alumina slurry (50 mL of dichloromethane and 10 g of basic alumina) is placed. The extract is added. 30 mL of dichloromethane is added and is passed through the column (waste). The TSNAs are eluted with 100 mL of a solution of acetone – dichloromethane (50 : 50 v/v). The elution solution evaporated and dissolved to a volume of 2 mL.
Sample clean-up - 2 : In the liquid chromatographic column is placed approximately 2 g of anhydrous sodium sulphate, 15 g of alumina, 15 g of silica and 2 g of anhydrous sodium sulphate. The extract is added. 100 mL of dichloromethane is added and passed through the column (waste). The TSNAs are eluted with 160 mL of a solution of 8 % methanol in dichloromethane. The elution solution evaporated and dissolved to a volume of 2 mL.
Sample analysis
HPLC system interfaced to MS-MS Gas chromatograph – Thermal Energy Analyser (GC-TEA)
Chromatography equipment and its operating conditions
HPLC column capable of distinct separation of TSNA and isotope labelled TSNA peaks from those of other cigarette emission components, e.g. Agilent Zorbax Eclipse XDB-C18 (2.1 x 150 mm, 3.5-μm particle size) GC Column: Fused silica capillary column with a 50 % methyl/50 % phenyl polysiloxane stationary phase, a 30-m column with a 0.53mm internal diameter and 1-μm film thickness is suitable for this analysis.
Injection volume: 5 μL Injector temperature: 230 °C
Column oven temperature: 40 ± 1 °C (Alter as appropriate for the column used.) Injection mode: Splitless
Injection volume: 2 μL
Degasser: On Mode: Constant flow
Binary pump flow: 0.2 mL/min Column temperature: 150 °C for 2 min, 3 °C/min to 230 °C, 20 °C/min to 250 °C, hold at 250 °C for 3 min
Mobile phase A: 0.1 % acetic acid in water (v/v)
Mobile phase B: 0.1 % acetic acid in methanol (v/v)
Gradient: Shown in Table 3
Total analysis time: 12 min TEA conditions
Mobile Phase A: 0.1% acetic acid in water (v/v) TEA interface temperature: 240 °C
Mobile Phase B: 0.1% acetic acid in methanol (v/v) TEA pyrolysis temperature: 500 °C
Gradient conditions:
0.0 50 % A 50 % B
3.0 10 % A 90 % B
4.0 0 % A 100 % B
5.0 0 % A 100 % B
5.5 50 % A 50 % B
12.0 50 % A 50 % B
eISSN:
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General Interest, Life Sciences, other, Physics
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