CORESTA Recommended Method No. 58 DETERMINATION OF … · CRM No. 58 – November 2019 Page 4/12 2....
Transcript of 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 Page 3/12
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.
CRM No. 58 – November 2019 Page 4/12
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.
CRM No. 58 – November 2019 Page 5/12
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.
CRM No. 58 – November 2019 Page 6/12
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.
CRM No. 58 – November 2019 Page 7/12
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.
CRM No. 58 – November 2019 Page 8/12
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.
CRM No. 58 – November 2019 Page 9/12
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).
CRM No. 58 – November 2019 Page 10/12
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 5 Virginia blended
Sample 6 Virginia blended
Sample 7 Charcoal filtered
CRM No. 58 – November 2019 Page 11/12
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
CRM No. 58 – November 2019 Page 12/12
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