coresta recommended method no. 58 determination of … · crm no. 58 – november 2019 page 4/12 2....

Cooperation Centre for Scientific Research

Relative to Tobacco

Smoke Analytes Sub-Group

CORESTA Recommended Method

No. 58

DETERMINATION OF BENZO[a]PYRENE

IN MAINSTREAM CIGARETTE SMOKE BY GC-MS

November 2019

CRM No. 58 – November 2019 2/12

CORESTA RECOMMENDED METHOD Nº 58

Title:

DETERMINATION OF BENZO[a]PYRENE IN MAINSTREAM CIGARETTE

SMOKE BY GC-MS

Status: Valid

Note: This document will be periodically reviewed by CORESTA

Document history:

Date of review Information

February 2004 Version 1

March 2013 Version 2

July 2014 Version 3

January 2018 Version 4 (formatting changes only)

November 2019 Version 5 (editorial changes for ISO 20778 Intense Smoke Regime

and provide citation for supporting collaborative study)

CRM No. 58 – November 2019 /12

CORESTA RECOMMENDED METHOD N° 58

DETERMINATION OF BENZO[a]PYRENE

IN MAINSTREAM CIGARETTE SMOKE BY GC-MS

(November 2019)

0. INTRODUCTION

Between 1999 and 2003, a Special Analytes Task Force reviewed the existing methodologies

for the determination of Benzo[a]pyrene (B[a]P) in the mainstream smoke of cigarettes. Several

methods were proposed for this determination, which mainly were based on two types of

analytical methodologies: High Performance Liquid Chromatography with Fluorescence

Detection (HPLC-FLD) and Gas Chromatography with Mass Spectrometric detection (GC-

MS). In both cases, it was necessary to purify the smoke condensate extract before the

chromatographic analysis to obtain a correct separation of the B[a]P peak from the matrix co-

extracts.

The Task Force decided in the first instance to develop a method using HPLC-FLD. However,

after several Joint Experiments it appeared that achieving a significant reduction of the initially

observed variability would be technically difficult. The Task Force then decided to investigate

a GC-MS method as an alternative and was able to demonstrate through Joint Experiments that

a lower variability can be obtained with this methodology.

This CORESTA Recommended Method (CRM), produced through collaboration involving 13

laboratories from 8 countries, provides an optimized procedure for the determination of B[a]P

in cigarette mainstream smoke. The repeatability and reproducibility (r & R) of this method,

evaluated in a collaborative study in 2003 involving 13 laboratories, seven cigarette samples

using the ISO 3308 smoking regime, were assessed according to ISO 5725 Parts 1 and 2.

Additional (r & R) data are provided from 10 samples with different tar yields from a

collaborative study in 2012 using both ISO 3308 and Health Canada T-115 (HCI) smoking

regimes, which involved 12 laboratories from 11 countries [1,2].

At the time the second collaborative study was conducted, the study protocol stipulated the use

of Health Canada Official Method (T-115) for Intense smoking conditions as there was not an

ISO standard that defined Intense smoking conditions. ISO 20778, Routine analytical cigarette-

smoking machine — Definitions and standard conditions was published in 2018 and is

equivalent to Health Canada Intense conditions and is referred to hereafter.

1. FIELD OF APPLICATION

This method is applicable to the determination of B[a]P in the Total Particulate Matter (TPM)

of cigarette mainstream smoke with TPM yields between 1,5 mg/cigarette and 42 mg/cigarette

where the cigarettes are smoked following either ISO 3308 or ISO 20778.


2. NORMATIVE REFERENCES

ISO 3308, Routine analytical smoking machine – Definition and standard conditions

ISO 3402, Tobacco and tobacco products – Atmosphere for conditioning and testing

ISO 4387/Amd 1, Cigarettes - Determination of total and nicotine-free dry particulate matter

using a routine analytical smoking machine

ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results – Part 1:

General principles and definitions

ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results – Part 2:

Basic method for the determination of repeatability (r) and reproducibility (R) of a standard

measurement method

ISO 8243, Cigarettes – Sampling

Health Canada, Official Method T-115, Determination of "Tar", Nicotine and Carbon

Monoxide in Mainstream Tobacco Smoke

ISO 20778, Cigarettes – Routine analytical cigarette-smoking — Definitions and standard

conditions with an intense smoking regime

ISO 20779, Cigarettes – Generation and collection of total particulate matter using a routine

analytical smoking machine with an intense regime

3. METHOD SUMMARY

3.1 Cigarette mainstream smoke is trapped on a glass fiber filter pad (Cambridge Filter Pad

or CFP as specified in ISO 3308 or in ISO 20778)

3.2 Internal standard (Benzo[a]pyrene-D12, B[a]P-D12) is spiked onto the CFP, the TPM

collected on the CFP is extracted with methanol and the methanol extract is diluted with

water.

3.3 Crude water/methanol smoke extracts are purified using Solid Phase Extraction (SPE)

by passing through a cyclohexyl bonded silica (CH) SPE cartridge, followed by the

elution of B[a]P with cyclohexane.

3.4 The samples are analyzed by GC-MS operated in the Single Ion Monitoring (SIM) data

acquisition mode for identification, confirmation and quantification of B[a]P.

4. APPARATUS AND EQUIPMENT

4.1 Laboratory equipment for the preparation of samples, standards, and reagents – for

example:

4.1.1 Analytical balance, capable of measuring to 0,01 mg

4.1.2 Volumetric flasks covering a range of volumes (10 ml and 100 ml)

4.1.3 Gas tight syringes covering a range of volumes (25 µl, 100 µl, 250 µl and 1000 µl)

4.1.4 GC-MS system equipped with a computerized control and data acquisition and

processing system. The system must be able to pilot the mass spectrometer in order to

obtain chromatographic data under Single Ion Monitoring (SIM) data acquisition mode

or equivalent on a capillary column.


4.1.5 Fused silica capillary GC column with a methylphenyl (5 %) polysiloxane stationary

The GC must be configured to perform splitless injections phase, 30 m long with

0,25 mm internal diameter and 0,25 µm film thickness is suitable for analysis.

4.1.6 Rotary evaporator or equivalent

4.1.7 Vacuum sample preparation unit

4.1.8 SPE cartridges with cyclohexyl bonded silica phase; a 6 ml volume and 1g packing per

cartridge.

5. REAGENTS AND SUPPLIES

5.1 Methanol

5.2 Distilled water

5.3 Cyclohexane

5.4 Toluene

5.5 Benzo[a]pyrene (known purity)

5.6 Benzo[a]pyrene-D12 (known purity)

Note: All reagents shall be at least analytical grade quality.

Warning notice: The solvents and chemicals to be used for this method are classified as toxic,

highly toxic, harmful, carcinogenic, mutagenic, sensitizing, teratogenic, irritant, corrosive,

easily flammable and dangerous for the environment. The instructions specified in the

individual material safety data sheets concerning safe handling; storage and waste disposal as

well as protective equipment must be followed.

6. PREPARATION OF GLASSWARE

For the preparation of samples, standards and reagents, all glassware shall be thoroughly

cleaned before use to avoid any contamination.

7. PREPARATION OF SOLUTIONS

Not applicable.

8. PREPARATION OF STANDARDS

8.1 Primary B[a]P stock solution

Dissolve approximately 10 mg B[a]P, weighed to the nearest 0,01 mg, in 10 ml of

toluene.

8.2 Secondary B[a]P stock solution

Dilute 1 ml of the primary B[a]P stock solution to 100 ml with methanol.

8.3 B[a]P-D12 stock solution

Dissolve approximately 10 mg B[a]P-D12, weighed to the nearest 0,01 mg, in 10 ml of

toluene.


8.4 B[a]P-D12 spiking solution

Using a gas syringe, transfer 100 µl of the B[a]P-D12 stock solution into a 100 ml

volumetric flask and bring to volume with methanol. This solution has a concentration

of approximately 1 µg/ml.

8.5 Working standard solutions

Prepare 6 working standard solutions that cover the concentration range of interest.

For example, transfer 20 µl of the B[a]P-D12 stock solution (section 8.3) and 10 to 2000

µl of the secondary B[a]P stock solution (section 8.2) into 100 ml volumetric flasks and

dilute to the mark with cyclohexane. These solutions have a concentration of

approximately 0,2 µg/ml of B[a]P-D12 and concentrations from 1 ng/ml to 200 ng/ml of

B[a]P.

8.6 Storage

The above standard solutions have been shown to be stable for up to six months if stored

at ≤ 20° C.

9. SAMPLING

Take cigarette samples in accordance with ISO 8243.

10. CIGARETTE PREPARATION

Condition the cigarette samples in accordance with ISO 3402.

11. SAMPLE GENERATION – SMOKING OF CIGARETTES

The smoking parameters for which the method has been studied are defined in ISO 3308 and

in ISO 20778 (Table 1).

Table 1. Smoking parameters for ISO 3308 and ISO 20778 smoking regimes

Smoking regime Puff volume

(ml) Puff frequency

(seconds) Puff duration

(seconds) Ventilation

Blocking (%)

ISO 3308 35 60 2 0

ISO 20778 55 30 2 100

For the ISO 3308 smoking regime, five (5) to 10 cigarettes are smoked onto a 44 mm CFP, and

10 to 20 cigarettes are smoked onto a 92 mm CFP.

If the maximum amount of TPM that a CFP is able to retain is exceeded, the number of

cigarettes shall be reduced.

For low tar products smoked under the ISO 3308 smoking regime a greater number of cigarettes

may be smoked to achieve a minimum TPM of 10 mg for a 44 mm pad an 20 mg for a 92 mm

pad.

Note: CFPs of 44 mm diameter are capable of retaining up to 150 mg of Total Particulate

Matter (TPM) and pads of 92 mm diameter up to 600 mg TPM.


12. SAMPLE ANALYSIS

12.1 Sample (CFP) extraction

12.1.1 Remove the filter pad from its holder, fold the filter two times (with the smoke

condensate inside) and wipe the inside of the holder with the folded CFP.

12.1.2 Transfer the filter pad into an Erlenmeyer flask (100 ml for a 44 mm pad; 200 ml for a

92 mm pad).

12.1.3 CFP Extraction:

• For a 44 mm pad, add 20 ml of methanol into the flask, then add 200 µl of the

B[a]P- D12 spiking solution (section 8.4) with a suitable syringe.

• For a 92 mm pad, add 50 ml of methanol into the flask, then add 400 µl of the B[a]P-

D12 spiking solution (section 8.4) with a suitable syringe.

12.1.4 Shake the flask vigorously until the filter pad has disintegrated and filter the solution

through a glass suction filter or using paper filtration.

12.1.5 Wash the filter remainder with approximately 15 ml of methanol for a 44 mm pad or

25 ml of methanol for a 92 mm filter pad. Add this washing solution to the filter

extract and complete to a volume with methanol which is at least 40 ml for a 44 mm

filter pad, or at least 80 ml for a 92 mm pad. For convenience bigger final volumes

can be used, but without unnecessarily diluting the solution.

12.1.6 Transfer an aliquot of the obtained solution into a flask. The volume of this aliquot

shall not exceed 40 ml which is convenient for this procedure. However, a smaller

aliquot can be used in order to shorten the elution time during the SEP clean-up step

(section 12.2).

12.1.7 Add distilled water into the flask in order to obtain a solution containing 60 % of water

and 40 % of methanol, and shake. For example, if an aliquot of 40 ml is used, add 60

ml of distilled water.

12.2 Sample clean-up

12.2.1 The CH SPE cartridge is pre-conditioned with 10 ml of methanol and 10 ml of a

mixture of water and methanol (60:40, v/v).

12.2.2 In the vacuum sample preparation unit, let the extraction solution pass through the CH

SPE cartridge under vacuum at a flow rate of approximately 2 ml/min (1 drop per

second).

12.2.3 Rinse the flask with 10 ml of a mixture of water and methanol (60:40, v/v). Dry the

cartridge with a stream of air for at least 30 minutes.

12.2.4 Elute the cartridge with 15 ml of cyclohexane.

12.2.5 Reduce the volume of the cyclohexane solution to about 0,5 ml. Then add cyclohexane

in order to obtain a volume of 1 ml in a volumetric flask. In spite of the drying

procedure described in section 12.2, the cyclohexane solution may still contain a

significant amount of water and a two-phase solution can be obtained after the volume

reduction. In this case, the cyclohexane phase shall be separated from the water phase

before adjusting the final volume to 1 ml. Alternatively, the cyclohexane solution may

be dried on a water adsorbent before volume reduction.

12.2.6 Transfer the obtained solution into an amber autosampler vial and cap with a PTFE

lined septum cap.


12.3 GC-MS determination

12.3.1 GC-MS operating conditions

Set up and operate the GC-MS system in accordance with the manufacturer’s instructions.

The following conditions are suitable for analysis:

• Injector temperature: 290 °C

• Mode: constant flow

• Flow: 0,9 ml/min

• Injection: 1 µL splitless

• Column temperature: 80 °C (3 min) 5 °C/min to 290 °C (20 min)

• Transfer line temperature: 270 °C

• MS Source: 230 °C

• Ion traces: B[a]P: m/z 252 (quantification) and 126 (confirmation)

• B[a]P-D12: m/z 264 (quantification) and 132 (confirmation)

These chromatographic conditions shall be adapted in order to obtain sufficient

resolution of the B[a]P and B[a]P-D12 peaks. A typical chromatogram of cigarette

smoke extract is given in Appendix 1.

12.3.2 Calibration

Inject successively each working standard solution (section 8.5) into the GC-MS

system. Record the area of the B[a]P and the B[a]P-D12 peaks. Generate a calibration

curve for B[a]P by calculating a linear equation regression of the area ratios of B[a]P

to B[a]P-D12 peaks as a function of the B[a]P concentrations. The intercept of this

regression line should be close to zero.

Inject one working standard solution (section 8.5) after approximately every 10

samples and if the measured concentration for this solution is different by more than

15 % of the nominal value, investigate and then repeat the calibration procedure.

12.3.3 Determination of B[a]P

12.3.4 Inject the sample, calculate the area ratio of B[a]P to B[a]P-D12 peaks and obtain the

concentration of B[a]P in the solution by comparing this ratio with the B[a]P

calibration line.

Note: It was observed by several laboratories that the B[a]P-D12 peak abundance may show

significant variations [1, 2]. The reasons for this variability of the GC-MS response

have not been fully investigated. However, this phenomenon has no effect on the final

result because the internal standard compensates for these variations.


12.3.5 Calculation

The concentration of B[a]P in cigarette smoke is calculated as follows:

𝑀 =𝐶 × 𝑉 × 𝑉𝑒

𝑛 × 𝑉𝐶

Where

M is the mass of B[a]P in cigarette smoke expressed in ng/cigarette

C is the concentration of B[a]P in the sample solution expressed in ng/ml

V is the volume of the sample solution expressed in ml (V = 1 ml)

n is the number of cigarettes smoked

Ve is the volume of the extraction solution (section 12.2)

Vc is the volume of the aliquot of the extraction solution taken for the clean-up

(section 12.2).


13. REPEATABILITY AND REPRODUCIBILITY

The first collaborative study supporting this Recommended Method was conducted in 2003 and

involved 13 laboratories and five replicate analyses of seven cigarette samples including the

University of Kentucky reference cigarette 2R4F. The study cigarettes covered a wide range

of blends and cigarette design constructions. Repeatability (r) and reproducibility (R) were

calculated following the ISO 5725 Part 1 and 2 statistical procedures (Table 2).

Table 2. Results from the 2003 Collaborative Study

Sample description B[a]P (ng/cigarette)

Mean r R

2R4F 7,28 1,27 2,52

A 1,81 0,49 1,01

B 5,27 1,06 2,52

C 6,54 1,11 2,21

D 7,76 1,47 2,88

E 8,71 1,39 2,72

F 14,07 2,26 5,94

In 2012, mean yield, r and R data were obtained from a second collaborative study involving

12 laboratories. This study provided data on the measurement of B[a]P in 10 cigarette samples

(seven commercial products and 3R4F, 1R5F, and the CORESTA Monitor CM6) smoked under

both the ISO 3308 and ISO 20778 regimes [1,2]. The samples are shown in Table 3. After

outlier removal, the r and R values were calculated according to ISO 5725 Part 1 and 2 statistical

procedures and are presented in Table 4.

Table 3. 2012 Collaborative study sample identification [1,2]

Sample ID Product/ Blend Type

CM6 CORESTA Monitor 6 Test Piece

1R5F Kentucky Reference 1R5F

3R4F Kentucky Reference 3R4F

Sample 1 Dark air-cured

Sample 2 American blended

Sample 3 American blended

Sample 4 Virginia blended



Sample 7 Charcoal filtered


Table 4. Results from the 2012 Collaborative Study

ISO 3308 smoking regime ISO 20778 smoking regime

Sample description

TPM yield (mg/cigarette)

B[a]P (ng/cigarette)

TPM yield (mg/cigarette)

B[a]P (ng/cigarette)

Mean N* Mean r R Mean N*

Mean r R

CM6 17,55 10 15,48 2,48 5,19 41,99 11 27,86 4,65 8,45

1R5F 2,14 10 1,64 0,41 0,8 26,72 12 7,13 1,25 2,28

3R4F 9,88 11 6,66 0,85 2,00 39,94 12 15,51 1,68 4,93

1 11,81 10 8,49 2,00 3,11 37,10 9 17,81 3,06 6,79

2 9,82 10 8,00 1,26 2,15 35,30 9 18,37 1,88 4,85

3 7,41 9 8,22 1,24 2,57 30,67 10 20,69 4,88 7,85

4 4,21 8 3,84 0,69 1,74 24,72 9 10,63 1,97 3,67

5 2,15 8 2,14 0,55 1,32 17,02 10 7,52 1,67 2,93

6 11,98 9 7,66 0,99 2,69 32,82 9 14,24 2,28 4,85

7 1,46 8 1,85 0,58 1,14 21,25 10 9,12 1,83 3,98

*N - number of data sets taken for statistical analysis after removal of outliers

14. REPORT

14.1 Test Results

14.1.1 The expression of the laboratory data depends on the purpose for which the data are

required, and the level of laboratory precision. Any further statistical analyses should

be calculated and expressed on the basis of the laboratory data before any rounding

has taken place.

14.1.2 The amount of B[a]P in the mainstream smoke of cigarette should be reported in ng/cig

and results should be rounded to the nearest 0,1 ng. However, mean yields and r and

R values reported in Tables 2 and 4 are reported to two decimal places.

15. REFERENCES

[1] CORESTA Smoke Analytes Sub-Group Technical Report – “2012 Collaborative Study

on B[a]P, VOCs, and Carbonyls in Mainstream Cigarette Smoke”, August 2019.

[2] Intorp, M., Purkis S.W., and Hauleithner A.: Updates of CORESTA Recommended

Methods after Further Collaborative Studies Carried Out under Both ISO and Health Canada

Intense Smoking Regimes, Beiträge zur Tabakforschung International/Contributions to

Tobacco Research, 25 (2013), pp. 700-707


APPENDIX 1

Example of a chromatogram of a cigarette smoke extract

Using the capillary GC column and the chromatographic conditions described in sections 4

and 12.3.1, respectively, the retention times of the B[a]P and B[a]P-D12 peaks are between 40

and 45 minutes. The sum of ion traces 252 and 264 are displayed. The upper chromatogram

shows the part of chromatogram located between 40 and 65 min and the lower one is a zoom

around the B[a]P and B[a]P- D12 peaks.

B[a]P d12

B[a]P

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