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Appendix 11 .
R .J . Reynolds Sidestream Smoke Methods Applied in
the 1999 Massachusetts Benchmark Study
R . J . Reynolds Sidestream Methods
1 . Determination of "Tar", Nicotine, Water and Carbon Monoxide
in Sidestream Cigarette Smoke------------------------------------------------------------2
2 . Analysis of Benzo[a]pyrene in Sidestream Cigarette Smoke---------------------43
3 . Analysis of 1-Aminonaphthalene, 2-Aminonaphthalene, 3-Aminobiphenyl
and 4-Aminobiphenyl in Sidestream Cigarette Smoke-----------------------------59
4 . Analysis of 1,3-Butadiene, Isoprene, Acrylonitrile, Benzene, Toluene and
Styrene in Sidestream Cigarette Smoke----------------------------------------------77
No interlaboratory validation process has been conducted using this analytical method .
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Determination of "Tar," Nicotine, Water and Carbon
Monoxide in Sidestream Smoke
Research & Development
R .J. Reynolds Tobacco Company
Winston-Salem, NC
No interlaboratory validation process has been conducted using this analytical method .
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Table of Contents
I . PR I N C I PLE OF M EAS U REM E NT------------------------------------------------- 4
11 . DE FI N ITI ONS-------------------------------------------------------------------------- 4
I I I . SAM PLE REQU I REM E NTS--------------------------------------------------------- 5
IV. APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION-- 5
A . Apparatus and Instruments--------------------------------------------------------- 5
B . Chemicals and Reagents------------------------------------------------------------ 9
C . Preparation of Standards------------------------------------------------------------ 9
D . I n stru ment Cond itions------------------------------------------------------------- 16
V . PROCEDURE---------------------------------------------------------------------------- 20
A . Preparation of Calibration Curves for Sidestream Pad Nicotine and Water-
----------------------------------------------------------------------------------------------- 2 0
B . Preparation of Pad Blanks----------------- -------------------------------------- 20
C . Analysis of Checks and Pad Blanks----------------------------------------------- 21
D . Preparation of Calibration Curves for Chimney and Impinger Nicotine-----21
E . Preparation of Monitors and Test Cigarettes for Smokin ---------------------22
F . Preparation of Smoking Machine--------------------------------------------------- 22
G . Smoke/Analyze Monitors and Test Cigarettes------ -------------------------- 24
H . Analysis of Filter Pads for Sidestream Pad Nicotine and Water--------------27
I . Analysis of Solutions for Chimney and Impinger Nicotine---------------------28
J . Data Review--------------------------------------------------------------------------- 28
K . Calculations---------------------------------------------------------------------------- 29
VI . REFERENCES------------------------------------------------------------------------- 31
Figures
1. Sidestream Flow - Command Panel--------------------------------------------------32
2. Gas Analyzer Panel ---------------------------------------------------------------------- 32
3. CO/CO2 Analyzers - Typical Display------------------------------------------------- 33
4. Sidestream Smoke Collection-----------------------------------------------------------34
5. BAT Fishtail Chimney Dimensions---------------------------------------------------- 35
6. Chromatogram of Monitor Extract for Sidestream Pad Nicotine Analysis
(Packed Column, Isothermal)----------------------------------------------------------- 36
7. Chromatogram of Calibration Standard for Sidestream Pad Nicotine Analysis
(Packed Column, I sothermal )----------------------------------------------------------- 36
8. Chromatogram of Monitor Extract for Sidestream Pad Water Analysis
(Packed Column, Isothermal)----------------------------------------------------------- 37
No interlaboratory validation process has been conducted using this analytical method .
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9 . Chromatogram of Calibration Standard for Sidestream Pad Water Analysis
(Packed Column, Isothermal)-------_--------------- _------------------------------- 37
10 . Chromatogram of Calibration Standard for Sidestream Pad Nicotine
Analysis (Megabore Column, Temperature Programmed)-----------------------38
11 . Chromatogram of Sample Extract for Sidestream Pad Nicotine Analysis
(Megabore Column, Temperature Programmed)---------------------------------- 38
12 . Chromatogram of Calibration Standard for Sidestream Pad Water Analysis
(Packed Column, Temperature Programmed)--------------------------------------39
13. Chromatogram of Sample Extract for Sidestream Pad Water Analysis
(Packed Column, Temperature Programmed)--------------------------------------39
14.Chromatogram of Test Extract for Chimney Nicotine------------------------------40
15 .Chromatogram of Test Extract for Impinger Nicotine------------------------------40
Tables
1. Oven Program for a Megabore Nicotine Column & Water Column-------------17
2. Column Parameters for a Megabore Nicotine Column & Water Column------17
3. Column Parameters for a Packed Nicotine Column & Water Column----------18
4. Oven Program for the Chimney & Impinger Nicotine Analysis on a Megabore
C o I u m n------------------------------------------------------------------------------------- 19
5 . Column Parameters for the Chimney & Impinger Nicotine Analysis on a
M e g a b o r e C o I u m n---------------------------------------------------------------------------19
6. Dilutions of the Chimney and Impinger Solutions Prior to UV Analysis--------26
No interlaboratory validation process has been conducted using this analytical method .
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Safety Note :
This procedure may involve the use of hazardous materials and/or
equipment. Prior to the use of this procedure, the user is responsible for
establishing appropriate safety and health practices
PRINCIPLE OF MEASUREMENT
Sidestream smoke from ISO-conditioned cigarettes is generated as specified
in the study protocol' . Cigarettes are smoked with 50% ventilation blocking
under a puffing regimen of a 45 mL puff volume of 2 second duration, once
every 30 seconds . Sidestream smoke is all smoke emitted from the lit end of
a burning cigarette during the smolder process . During smoking a glass
fishtail chimney is positioned over the burning cigarette and allows smoke to
be directed in a controlled manner. Sidestream smoke exiting the fishtail
chimney is drawn through a Cambridge filter pad and finally into an impinger
containing isopropanol with octadecane as an internal standard . After
smoking is complete, the Cambridge filter pad is extracted in an
isopropanol/octadecane/ethyl alcohol solution and the extract analyzed for
nicotine and water by gas chromatography . The chimney is rinsed with the
extraction solution (isopropanol/octadecane/ethyl alcohol solution) and the
wash collected . Impinger contents and washes are also collected . Aliquots
of both chimney and impinger extracts are then analyzed for nicotine by gas
chromatography .
II . DEFINITIONS
1 . SS: Sidestream smoke
2 . TPM : Total Particulate Matter
3 . Cigt : Cigarettes
4 . LIMS : Laboratory Information Management System
5. EZChrome EliteT"" : Chromatography Data Acquisition Software
6. GC : Gas Chromatography
7 . Clearing Puff : Any puff taken after the cigarette has been extinguished or
removed from the cigarette holder .
8 . Nicotine Check Solutions : Standard solutions prepared independently of
the nicotine calibration standards . Nicotine check solutions are analyzed
to check instrument stability after the calibration and during sample
analysis .
No interlaboratory validation process has been conducted using this analytical method .
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9 . Monitor : A product specifically designed for use as a test piece to ensure
that the cigarette test procedures have been correctly applied .
Ill . SAMPLE REQUIREMENTS
Sample required per determination : 3 cigarettes per determination
IV . APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION
A . Apparatus and Instruments
1 . 20-Port Linear Smoking Machine, RJR Modified Phipps & Bird smoking
machine equipped with 100 mL syringes, modified to accept fishtail
chimneys .
2 . 100 mL Syringes, Model #5177, Popper & Sons Inc . New Hyde Park,
NY .
3 . Analytical Balance, Mettler Model AE 163, Mettler-Toledo, Inc .
Columbus, OH .
4 . Soap Bubble Flow Meter,Heinreich Borgwaldt Model R24 .01, 80 mL,
KC Automation, Richmond, VA .
5. Mainstream 44 mm Cambridge Filter Holder, Filtrona stock code
66300, Filtrona Instruments & Automation, Richmond VA modified in-
house to fit RJR smoking machine .
6 . Labyrinth Seals Filtrona no . 66332 (yellow) ; Filtrona no . 66333
(orange) ; Filtrona no . 66334 (green) ; Filtrona no . 66316 (blue), Filtrona
Instruments & Automation, Richmond VA .
7 . Neoprene Washers, Diameter 4 .50 - 5 .49 mm, Filtrona no . 66335 ;
diameter 5 .50 - 6 .49 mm, Filtrona no . 66336 ; diameter 6 .50 - 7 .49
mm, Filtrona no . 66337 ; diameter 7 .50 - 9 .00 mm, Filtrona no . 66317,
Filtrona Instruments & Automation, Richmond VA .
8 . Sidestream 44 mm Cambridge Filter Holder - RJR in-house
manufacture .
9 . Glass fiber filter pads (locally known as Cambridge filter pads) 44 mm
in diameter, catalog no . 559715, Performance Systematix Inc .,
Caledonia, MI .
10 . Saran sample gas bags, 5-liter, 12" x 12", catalog no . A4353, Anspec
Anaytical Specialties Inc ., Geneva, IL .
No interlaboratory validation process has been conducted using this analytical method .
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11 . Infrared Carbon Monoxide Analyzer - Beckman Model 880, equipped
with dual ranges : 0-10% mainstream, 0-2% sidestream ; linear output,
Rosemount Analytical Inc ., Orrville, OH .
12 .Vacuum Pumps - Piab Models M-16 and M-32, Piab Vacuum
Products, Rockland, MA .
13 . Barometer - Fortin type mercurial, Princo, Southampton, PA .
14 . Flow meters - constructed with multitube frames, catalog no . E-03215-
46, 150 mm flow tubes, catalog no . E-03217-19, and high-resolution
valves, catalog no . E-03218-64, Cole-Parmer Instrument Co ., Vernon
Hills, IL .
15 . BAT Fishtail Chimney, hand-crafted by Public Scientific Glass,
Pfafftown, NC .
16 . Pressure and Vacuum Tubing - red, thick wall, Fisherbrand*, catalog
no . 14-167F, Fischer Scientific, Atlanta, GA .
17 . Poly-flow Tubing -'/<" O .D ., Imperial Eastman Brand, PN 466167, RJR
Central Supply .
18 . Silicone Medial Grade Tubing - Helix Medical Inc ., 250 I .D . x .376"
O .D ., catalog no . 32828-018, VRW, Scientific Products, Westchester,
PA . Or equivalent .
19 . Keck® Quick Disconnect Adapters - yellow, catalog no . E-06841-51,
dark blue, catalog no . E-06841-53, light blue, catalog no . E-06841-60,
Cole-Parmer Instrument Co ., 625 E . Bunker Ct., Vernon Hills, III .
20 . Keck° Original Taper Clips - catalog no . E-06840-15, Cole-Parmer
Instrument Co ., 625 E . Bunker Ct., Vernon Hills, III .
21 . Impinger - Dreschel gas washing bottle, 125 mL, catalog no . 5516-08,
Ace Glass Inc ., Vineland, NJ .
22 . Device for measuring temperature and humidity - Rotronic Hygroskop
GT, Rotronic Instrument Corp ., Huntington, NY .
23.100% Mercerized Cotton Thread size 50 (black) - Brewer Sewing
Supplies, Chicago IL .
24 . Tweezers - Fisher Scientific, Atlanta GA .
No interlaboratory validation process has been conducted using this analytical method .
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25 . Metal Millimeter Rulers - General Tools, MFG . Co ., catalog no .
C636EM - 6" Rule, New York, NY .
26 . Mechanical Markers - RJR in-house manufacture .
27 . Metal Rods for adjustment of mechanical markers - RJR in-house
manufacture .
28 . Digital Flow Meter - CES Model 209 SE, Custom Electronics Systems,
Inc . Winston-Salem, NC .
29 . UV Spectrophotometer - Beckman Model DU-70, with batch sampler
and 1 cm flow cell, Beckman Instruments, Inc ., Irvine, CA .
30 . Digital Diluter - Hamilton Model 100004, Hamilton Co ., Reno NV .
31 . Reciprocating Shaker - New Brunswick Scientific Model R-2, New
Brunswick Scientific, Edison, NJ .
32 . PC/Software - 386 or higher processor, lab-PC interface board,
LabWindows Version 2 .0, National Instruments Corporation, Austin,
TX .
33 . Gas Chromatograph - Hewlett Packard model 5880A, flame ionization
detector (nicotine) and thermal conductivity detector (water), with a
HP7673A automatic sampler, Hewlett Packard, Co ., Wilmington DE .
34 . Nicotine Columns
a . Column - DB-WAX fused silica megabore, 1 micron film thickness,
0 .53 mm ID, 15 m length, catalog no . 125-7012, J & W Scientific
Co ., Folsom, CA .
b. Column - RJR packed 6-foot, 4 mm I .D . Partisorb PEG Basic LL
media, glass column . Empty column : Hewlett Packard, catalog no .
19094A-A46, Hewlett Packard Co ., Wilmington, DE . Packing
material: catalog no . 4520-999, Whatman Inc ., Clifton, NJ .
35. Water Column - 1/8" I .D . stainless steel 6-foot, Porapak Q 801100,
catalog no . 1-2792, Supelco Inc ., Bellefonte, PA .
36. Convection oven - Precision mechanical convection oven, Fischer
Scientific, Atlanta, GA .
37 . Air generator - Aadco 737 pure air generator, model no . 737-11 B,
Aadco Co ., Clearwater, FL .
No interlaboratory validation process has been conducted using this analytical method .
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38 . Flow check - Alltech Digital Flow Check, catalog no . 4700, Alltech
Associates, Inc., Deerfield, IL .
39 . Flowmeter - Humonics Optiflow 520 Digital Flowmeter, Catalog no .
HFM-520, J&W Scientific Co ., Folsom, CA .
40 .Gyrotory Shaker-model G2, New Brunswick Scientific, Edison, NJ .
41 . Automatic dispenser - Oxford automatic dispenser, 10 mL, Oxford
Laboratories, Foster City, CA .
42 . Class A Volumetric Flasks - Kimax Brand* or equivalent, 50 mL,
catalog no. 10-209C, 100 mL, catalog no . 10-209D, 200 mL, catalog
no . 10-209E, 250 mL, catalog no . 10-209F, Fisher Scientific, Atlanta,
GA .
43 . Class A pipettes - Fisherbrand* or equivalent, sizes 0 .5 mL, catalog no .
13-650-2A, 3 mL, catalog no . 13-650-2D, 6 mL, catalog no . 13-650-2G,
8 mL, catalog no . 13-650-2J, 13 mL, special order, 15 mL, catalog no .
13-650-2M, 20 mL, catalog no . 13-650-2N, Fisher Scientific, Atlanta,
GA .
44 . Syringes - Hamilton Series 1700 Gastight Syringes, 10 µL, catalog no .
14-815-1, 25 NL, catalog no . 14-815-29, 250 µL, catalog no . 13-684-
102, 500µL, catalog no . 13-684-106, Fisher Scientific, Atlanta, GA .
45 .125 mL Erlenmeyer flasks with glass stoppers- Pyrex brand or
equivalent, catalog no . 10-098D, Fisher Scientific, Atlanta, GA .
46 . GC vials - Pre-capped nitrogen purged GC Vials, catalog no . 200-117,
Sun International, Wilmington, NC .
47 . Syringes - Hamilton Series 1700 Gastight Syringes, 10 µL, catalog no .
14-815-1, 25 pL, catalog no . 14-815-29, 250 µL, catalog no . 13-684-
102, 500µL, catalog no . 13-684-106, Fisher Scientific, Atlanta, GA .
48 . GC vials with crimp top caps - vials, catalog no . 200-000, crimp tops,
catalog no ., 200-100, Sun International, Wilmington, NC .
49 . EZChrom EliteT"" Chromatography Data System, Scientific Software,
Inc ., San Ramon, CA .
No interlaboratory validation process has been conducted using this analytical method .
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B . Chemicals 8 Reacaents
1 . Isopropyl alcohol, Low Water Content 4-liter bottles, catalog no . 324-4,
CAS# 67-63-0, Burdick and Jackson, Muskegon, IL .
2 . Ethyl Alcohol, 200 proof denatured alcohol, CAS# 64-17-5, 1 pint
bottles, Aaper Alcohol & Chemical Co ., Shelbyville, KY
3 . Carbon Monoxide- certified gases to verify the large cylinders used
for CO analyzer calibration, Scott Specialty Gases, Durham, NC :
Nominally : 0 .25% CO
Nominally : 0 .75% CO
4. Carbon Monoxide - large cylinder gases for calibration of CO analyzer,
Machine & Welding Supply Co ., Winston-Salem, NC :
Nominally : 0 .25% CO
Nominally : 0 .75% CO
5 . Octadecane - Aldrich Chemical Company, Inc . catalog # 0-65-2, CAS#
593-45-3, Aldrich Chemical Co ., Milwaukee, WI .
6 . L-nicotine - 99% purity, Acros, catalog no . AC 18142-0250, CAS# 34-
11-5, Fisher Scientific, Atlanta GA .
7 . Drierite - W . A . Hammond Drierite Company, Xenia, OH .
C . Preparation of Standards
Safety Note:
Nicotine is a toxic chemical and readily absorbed through the
skin. It may be fatal if inhaled, swallowed, or absorbed through
the skin. Always wear nitrile gloves and protective
eyewear/clothing when handling.
1 . Extraction Solution : Dissolve 1 .2 g of octadecane and 15 mL of 200
proof ethyl alcohol in each 4 Liter bottle of low water content isopropyl
alcohol and mix by hand shaking . Allow the solution to equilibrate
overnight before use .
2 . Standards for Nicotine & Water in the Sidestream Cambridge filter
pads :
2.1 . Nicotine Standard Stock Solution (typically prepared monthly)
• Put a small amount of extraction solution in a 250 mL
volumetric flask .
No interlaboratory validation process has been conducted using this analytical method .
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• Weigh 2 .5 g (to the nearest 0 .1 mg) of nicotine (99% purity)
into the 250 mL volumetric flask and dilute to volume with
extraction solution . Stopper and shake well .
• Label the flask with the nicotine concentration, lot number of
nicotine used to prepare the stock solution, date, and analyst's
initials .
• Calculation for Nicotine
.0 mg _ 9
2 .5000 g x 0 .99 purity x 1000 mg - 2475 .9 mg
g 250 mL mL
2 .2 . Nicotine Calibration Standards (prepare fresh with each new
batch of extraction solution)
• Prime the Oxford dispenser .
• Into six volumetric flasks, pipette the following amounts of the
specified solution, dilute to volume with extraction solution, and
determine the amount of nicotine in 10 mL of solution . Stopper
and shake well . Label each flask by standard level and
nicotine concentration, date and analyst's initials . Nicotine
Stock and Standards are kept in a walk-in freezer at
approximately 0 ° F .
LO=BLANK: 100 mL extraction solution in a 100 mL volumetric
flask
L1=STD1 : 20 mL of L2 Standard in a 50 mL volumetric flask
(2OmLxO .0495___ mg I-
NIC . 50 mL mL) -0 .0198 mL mg x10mL=0 .198mg
L2=STD2 : 0 .5 mL of Standard Stock Solution in a 100 mL
volumetric flask
1O .5mLx9 .9 mg
NIC : .0495mL mgx10mL=0 .495mg
100mL mi =0
L3=STD3 : 3 mL of Standard Stock Solution in a 100 mL
volumetric flask
No interlaboratory validation process has been conducted using this analytical method .
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. (3mLx9
.9 z) m HIL
NIC .2970x10mL=2 .97mg
100mL =0
L4=STD4 : 6 mL of Standard Stock Solution in a 100 mL
volumetric flask
16mLx9.9--')
: mL" -mgNIC - 0 .5940 mg x10mL=5 .94mg
100 mL mL
L5=STD5 : 8 mL of Standard Stock Solution in a 100 mL
volumetric flask
8mLx9 .9 mg
NIC = 0 .7920 mg mL x 10 mL= 7 .92 mg
: 100mL mL)
L6=STD6 : 13 mL of Standard Stock Solution in a 100 mL
volumetric flask
mgl
(13 mL x 9 .9
mL J
NIC : ` =1 .2870 mg x10mL=12 .87mg
100 mL mL
2 .3 . Nicotine Check Stock Solution (typically prepared monthly)
• Put a small amount of extraction solution in a 250 mL volumetric
flask.
• Weigh 2 .5 g (to the nearest 0 .1 mg) of nicotine (99% purity)
into the 250 mL volumetric flask and dilute to volume with
extraction solution . Stopper and shake well .
• Label the flask with the nicotine concentration and lot number
of nicotine used to prepare stock solution, date, and analyst's
initials .
• Calculation for Nicotine
.0 mg = 9
2 .5000 g x 0 .99 purity x 1000 mg _ 2475 .9a9
g 250 mL mL
2.4. Preparation of Nicotine Checks Standards (prepare fresh with
each new batch of extraction solution)
• Into four 100 mL volumetric flasks, pipette the following
amounts of the Nicotine Check Stock Solution, dilute to volume
No interlaboratory validation process has been conducted using this analytical method .
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with extraction solution, and determine the amount of nicotine
in 10 mL of solution . Stopper and shake well .
• Label each flask by check level, nicotine concentration, date
and analyst's initials.
• Nicotine Check Stock and Check Solutions are stored in a
walk-in freezer .
CHK1 : 0 .5 mL of Check Stock Solution in a 100 mL volumetric
flask
.5 mL x 9 .9 mg 0
mL) mg
NIC : 0 -x
= .0495 lOmL=0 .495 mg
100 mL mL
CHK2 : Prepare CHK2 . FTC Smoke Lab smoke 20 ports (5
cigarettes per port) of monitors according to STD0001 STM9 .
Extract monitor pads according to STD0006STM3 and combine
the solution into a 250 mL volumetric flask . Mix thoroughly .
Use this smoke extract check as a comparison of each GC for
the day as well as to previous days performance, to ensure
that all GC's are yielding similar results .
CHK3 : 3 mL of Check Stock Solution in a 100 mL volumetric
flask
( (3mLx9 .9-J mg
NIC : mL =0 .2970mgx10mL=2 .97mg
100 mL mL
CHK4 : 6 mL of Check Stock Solution in a 100 mL volumetric
flask
(6mLx9 .9J
mgNIC .5940mL mx10mL=5 .94mg
: 100mL mL =0
CHK5 : 13 mL of Check Stock Solution in a 100 mL volumetric
flask
NIC :
C
13mLx9 .9 mg
mL -1 J
- .2870mgx10mL=12 .87mg
100 mL mL
2.5 . Water Calibration Standards (prepare fresh with each new batch
of extraction solution)
No interlaboratory validation process has been conducted using this analytical method .
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• Add the following amounts of distilled to seven oven dried 50
mL volumetric flasks and dilute to volume with extraction
solution Determine the amount of water in 10 mL of solution .
• Label each flask by standard level, water concentration, date,
and analyst's initials .
• Stopper and shake well .
LO : Extraction Solution
L1 : 10 µL of distilled water into 50 mL of extraction solution .
Water :
0 .0100 g x 1000 mg
g ) mg
.200 mL x 10 mL = 2 .000 mg
SO mL =0
L2 : 50 µL of distilled water into 50 mL of extraction solution .
Water:
C
0 .0500 g x 1000 mg I
50 g J=1 .O00 mL mg
mL xlOmL=10 .000mg
L3 : 100 µL of distilled water into 50 mL of extraction solution .
Water:
0 . 1000 g x (1000 mgg ~
=2 .000mg x10mL=20.000mg
50 mL mL
L4 : 150 µL of distilled water into 50 mL of extraction solution .
Water :
0 .1500 g x 1000 mg
.000 mL x 10 mL = 30 .000 mg
SO mL g = 3
L5 : 250 µL of distilled water into 50 mL of extraction solution .
Water :
0 .2500 g x 1000 mg
g ) mg
.000ML x 10 mL = 50 .000 mg
50mL =5
L6 : 400 µL of distilled water into 50 mL of extraction solution .
No interlaboratory validation process has been conducted using this analytical method .
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Water:
0.4000 g x [moo mg 1
.000mLx10mL=80 .000mg
SOmL g J=8
L7 : 500 µL of distilled water into 50 mL of extraction solution .
Water :
C
0 .5000 g x 1000 mg J I
50 mL g =10
.000 mL x l O mL =100 .000 mg
3. Standards for Chimney & Impinger Nicotine
3 .1 . Nicotine Chimney & Impinger Standard Stock Solution
(prepare fresh with each batch of low water isopropyl alcohol,
octadecane, and ethyl alcohol extraction solution)
• Put a small amount of extraction solution in a 100 mL
volumetric flask .
• Weigh 0 .1 g (to the nearest 0 .1 mg) of nicotine (99% purity)
into the 100 mL volumetric flask and dilute to volume with
extraction solution . Stopper and shake well .
• Label the flask with nicotine concentration, lot number of
nicotine used to prepare the stock solution, date, and analyst's
initials .
• Calculation for Nicotine
.00 mg = 0
0 . 1000 g x 0 .99 purity x 1000 mg _ 99 .99 mg
g 100 mL mL
3 .2 . Nicotine Chimney & Impinger Standards (prepare fresh with
each batch of extraction solution)
• Prime the Oxford dispenser .
• Into six 100 mL volumetric flasks, pipette the following
amounts of the nicotine stock solution, dilute to volume with
extraction solution . Stopper and mix well . Label each flask by
standard level, nicotine concentration, date and analyst's
initials . The calibration is based on the nicotine concentration
per 50 mL .
L0= Extraction solution
No interlaboratory validation process has been conducted using this analytical method .
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L1=STD1 : 0 .25 mL of Standard Stock Solution
10.25 mL x 0 .99 mg J -
NIC mL .002475mL x m 50 mL= 0 .12375 mg
. 100mL - 0
L2=STD2 : 2 mL of Standard Stock Solution
NIC .
C 2rnLxO .99 mg
J
mi = 0.0198'ng x 50 mL = 0.9900 mg
100 mL mL
L3=STD3 : 5 mL of Standard Stock Solution
i
5mLx0 .99mg
NIC : ~ mI'~ =0 .0495 m x 50 mL = 2 .475 mg
100 mL mL
L4=STD4 : 10 mL of Standard Stock Solution
NIC .
C
10 mL x 0 .99 mg -
mL J
-0 .0990mgx50mL=4 .950mg
100 mL mL
L5=STD5 : 15 mL of Standard Stock Solution
115 mL x 0 .99 mg ~
NIC : m =0 .1485 mgx50mL=7 .425mg
100 mL mL
3 .3 . Nicotine Chimney & Impinger Check Stock Solution (prepare
fresh with each batch of extraction solution)
• Put a small amount of extraction solution in a 100 mL
volumetric flask .
• Weigh 0 .2 g (to the nearest 0 .1 mg) of nicotine (99% purity)
into the 100 mL volumetric flask and dilute to volume with
extraction solution . Stopper and shake well .
• Label the flask with nicotine concentration, lot number of
nicotine used to prepare the stock solution, date, and analyst's
initials .
• Calculation for Nicotine
.00 mg _ 1
0 .2000 g x 0 .99 purity x 1000 mg _ 198 .98 mg
g 100 mL mL
No interlaboratory validation process has been conducted using this analytical method .
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3 .4 . Nicotine Chimney & Impinger Checks (prepare fresh with each
batch of extraction solution)
Low check:
Into a 100 mL volumetric flask, pipette 2 mL of the nicotine check
stock solution, dilute to volume with the extraction solution, and
mix well . Determine the nicotine concentration per 50 mL, as in
the example below :
2 x 1 .98 mgjmL _ 3 .96 mg _ 0 .036 mg x 50 mL 1 .98 mg
=
100 mL 100 m L mL 54 mL
High check:
Pipette 5 mL of nicotine check stock solution into a 100 mL volumetric
flask, dilute to volume with extraction solution, and mix well .
Determine the amount of nicotine per 50 mL as follows :
/
5x1 .98mg mL=9 .90mg _ 0 .099mgx50mL= 4 .950mg
100 mL 100 m L mL 50 mL
D . Instrument Conditions
1 . Sidestream Pad Nicotine and Water
A Hewlett Packard Gas Chromatograph model 5880A, equipped with a
flame ionization detector and a thermal conductivity detector, and a
HP7673A automatic sampler or equivalents .
For the nicotine analysis, one of the following GC columns is required :
• J & W Scientific 15m, 0 .53mm I .D ., 1µm film thickness, DBWAX,
catalog no . 125-7012 .
• RJR packed 6-foot, 4mm I .D . Partisorb PEG Basic LL media, glass
column . Empty column from Hewlett Packard catalog no . 19094A-
A46 . Packing material from Whatman Inc ., catalog no . 4520-999 .
For the water analysis, the following GC column is required :
• Supelco Inc ., 1/8" I .D . stainless steel 6-foot, Porapak Q 80/100,
catalog no . 1-2792 .
For a GC containing the megabore nicotine column and a water
column, the following conditions apply :
No interlaboratory validation process has been conducted using this analytical method .
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Table 1 : Oven Program for a Megabore Nicotine Column & a Water
Column :
Column oven initial temperature (°C) 125
Column oven initial time (min) 1 .0
Column oven program rate (°C/min) 15
Column oven final temperature (°C) 180
Column oven final time (min) 2 .8
Total Run Time (min) 7 .5
Water injection takes place 2 .4 minutes into temperature program run .
Table 2 : Column Parameters for a Megabore Nicotine Column & a Water
Column
Nicotine Column Parameters Water Column Parameters
Detector FID Detector TCD
Detector temperature (°C) 250 Detector temperature (°C) 250
Detector H2 flow (mL/min) at 50 psi >_ 30 Detector makeup gas Helium =35
(mUmin)
Detector makeup gas Nitrogen _25
(mL/min)
Detector air flow (mL/min) at 35 psi -300
Injection mode Split Injection mode Packed
Injector temperature ()C) 250 Injector temperature (°C) 220
Column flow Helium (mL/min) =10 Column flow rate Helium -30
(mL/min)
Split Vent Flow Helium (mL/min) =100 Reference Column flow Helium =30
(mL/min)
Septum Purge Flow Helium 3-5
(mLlmin)
Injection Volume (pL) 1 .0 Injection Volume (NL) 1 .0
Autoinjector mode Normal Autoinjector mode Normal
Autoinjector sample pumps 6 Autoinjector sample pumps 6
Autoinjector viscosity delay 0 Autoinjector viscosity delay 0
Autoinjector sample washes 2 Autoinjector sample washes 2
Autoinjector solvent A washes 2 Autoinjector solvent A washes 2
Autoinjector solvent B washes 2 Autoinjector solvent B washes 2
No interlaboratory validation process has been conducted using this analytical method .
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For a GC containing the packed nicotine column and a water column,
the following conditions apply :
Oven Temperature : 160 °C isothermal
Run time 6 .5 min
To maximize peak resolution the oven temperature can be altered on
packed instruments depending on the packing of the column installed .
The temperature shall be between 150°C and 160°C . This
temperature will be optimized after column installation based on the
resolution of standards and the comparison sample .
Table 3 : Column Parameters for a Packed Nicotine Column & a Water
Column
Nicotine Column Parameters Water Column Parameters
Detector FID Detector TCD
Detector temperature (°C) 300 Detector temperature (°C) 250
Detector H2 flow (mL/min) at 50 psi _ 30 Detector makeup gas Helium -35
(mL/min)
Detector air flow (mL/min) at 35 psi _300
Injection mode Packe Injection mode Packed
d
Injector temperature ()C) 200 Injector temperature (°C) 220
Column flow Helium (mUmin) =50 Column flow rate Helium _30
(mUmin)
Reference Column flow Helium _30
(mL/min)
Injection Volume (NL) 1 .0 Injection Volume (NL) 1 .0
Autoinjector mode Normal Autoinjector mode Normal
Autoinjector sample pumps 6 Autoinjector sample pumps 6
Autoinjector viscosity delay 0 Autoinjector viscosity delay 0
Autoinjector sample washes 2 Autoinjector sample washes 2
Autoinjector solvent A washes 2 Autoinjector solvent A washes 2
Autoinjector solvent B washes 2 Autoinjector solvent B washes 2
Run Time (min) 6 .5 Run Time (min) 6 .5
No interlaboratory validation process has been conducted using this analytical method .
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2 . Chimney and Impinger Nicotine :
Suitable chromatographic conditions for a HP 5880A GC equipped with a
flame ionization detector and a J&W Scientific Co ., DB-Wax fused silica
megabore, 1 µm film thickness, 0 .53 mm I .D ., 15 m length include :
Table 4 : Oven Program for the Chimney & Impinger Nicotine
Analysis on a Megabore Column
Column oven initial temperature (°C) 125
Column oven initial time (min) 1 .0
Column oven program rate (°C/min) 12
Column oven final temperature (°C) 190
Column oven final time (min) 3 .6
Total Run Time (min) 10
Table 5: Column Parameters for the Chimney & Impinger Nicotine
Analysis on a Megabore Column
Detector FID
Detector temperature (°C) 300
Detector H2 flow (mL/min) at 50 psi _ 30
Detector makeup gas Nitrogen (mL/min) = 43
Detector air flow (mL/min) at 35 psi = 300
Injection mode Split
Injector temperature (°C) 250
Column flow Helium (mUmin) = 10
Split vent flow Helium (mL/min) = 66
Septum purge flow Helium (mUmin) = 3
Injection Volume (µL) 1 .0
Autoinjector mode : Normal
Autoinjector sample pumps 6
Autoinjector viscosity delay 0
Auotinjector sample washes 2
Autoinjector solvent A washes 2
Autoinjector solvent B washes 2
No interlaboratory validation process has been conducted using this analytical method .
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V . PROCEDURE
A. Preparation of Calibration Curve for Sidestream Pad Nicotine and
Water
1 . Perform GC calibration each time a new batch of extraction solution is
prepared .
2 . Prime each GC by making two injections of a standard or sample
solution per channel before actual sample analysis begins .
3 . Start EZChrom data acquisition on the appropriate channels using the
Elite Calibration Scheduler Program .
4 . Transfer nicotine standards (L1-L6) and water standards (L0-L7) to GC
vials .
5. Place the nicotine standards in positions 21-26 and water standards in
positions 1-8 on each GC .
6 . "Start Program" on the GC terminal for the nicotine channel and then
on the GC terminal for the water channel . "Start Program" on the
nicotine channel injects samples in positions 21-26 in duplicate . "Start
Program" on the water channel injects samples in positions 1-8 in
duplicate .
7 . After running this program, EZChrom performs a linear least squares
regression . The calibration curve is reviewed to insure that all
injections are identified, the correlation coefficient is greater than
0 .9989, and that response factors, slope and intercept values are
acceptable based on past performance . Continue with analysis if the
calibration curve is acceptable . Take corrective action if the calibration
curve is not acceptable .
B . Preparation of Pad Blanks
A . Analyze pad blanks and nicotine checks each day prior to analysis of
samples .
2 . Prepare a pad blank flask by placing a clean Cambridge filter pad into
a 125 mL Erlenmeyer flask containing 40 mL of extraction solution and
submit with the sidestream samples .
3 . Place the flask on the table top shaker and shake for 45 minutes at
200 rpm . After shaking, transfer approximately 1 mL aliquots of the
No interlaboratory validation process has been conducted using this analytical method .
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pad blank into two GC vials . Each vial is analyzed, in duplicate, on the
GC .
C . Analysis of Checks and Pad Blanks
1 . Prime the GC by making two injections of a standard or sample
solution per channel .
2. Place the pad blank vials in positions 1 & 2 on the GC .
3 . Transfer check solutions into GC vials and place on the GC in
positions 96-100 .
4. Start EZChrom data acquisition on appropriate channels using the
following batch names :
PAD BLANK BATCH FOR WATER INSTRUMENTS : SSPAD .SEQ
NICOTINE CHECK BATCH FOR NICOTINE INSTRUMENTS :
SSCHECK .SEQ
5 . Start program 300 on the GC terminal for the nicotine channel and
then on the GC terminal for the water channel .
Program 300 on the nicotine channel injects samples in position 96-
100 one time each .
Program 300 on the water channel injects samples in position 1 & 2 in
duplicate and then the CHECK 2 (comparison sample) in position 97
once.
6. Obtain nicotine checks and comparisons from the Elite Nicotine
Checks Program, and the pad blanks and comparisons by running the
Elite Review Pad Blanks Program .
7 . Continue with analysis if the obtained check values are within ± 5% of
the calculated values . Take corrective action if the check values are
not acceptable .
8. Record the following values on the GC Verification form :
(1) the comparison and Check 4 for the nicotine channels .
(2) the comparison and average of the four pad blank injections for the
water channel .
9. Enter Pad Blank Value
Enter the obtained average of the four pad blank injections for the water
channel into the Elite Pad Blank Entry Program .
No interlaboratory validation process has been conducted using this analytical method .
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D . Preparation of Calibration Curve for Chimney and Impinger Nicotine
1 . Perform calibration the week in which samples are smoked by
analyzing the standards in duplicate after priming the system with a
single injection of the highest standard . Standards are followed by low
and high check standard solutions (in triplicate) to ensure that the
calibration standards have been prepared correctly . EZChrom
performs a linear least squares regression .
2 . Calibration Acceptance Criteria
Review the calibration curve to ensure that all injections are identified,
the correlation coefficient is greater than 0 .9957, and that response
factors, slope and intercept values are acceptable based on past
performance .
3 . Continue with sample analysis if the calibration curve and check solutions
are acceptable . If results are not acceptable, take the necessary
corrective action before continuing .
E . Preparation of Monitors and Test Cigarettes for Smoking
1 . Obtain a LIMS run set up according to established LIMS procedures .
Ensure monitors and test cigarettes have been conditioned, marked,
and loaded as specified in the study protocol1 .
F. Preparation of Smoking Machine
1 . Set up the 20-port sidestream smoking machine with the puffing
parameters' of 45 ± 0 .5 mL puff volume, 2 ± 0 .1 seconds puff duration
and 30 ± 1 seconds puff interval .
2 . Set Up Glassware/Equipment
2 .1 . Flasks for sidestream nicotine analysis on filter pads :
Remove twenty-one 125 mL Erlenmeyer flasks from the oven,
stopper and allow to cool completely . Fill each flask with 40 mL
extraction solution .
2 .2 . Impingers for sidestream nicotine collection :
Set up and number 16 impinger bottles and fill each with 40 mL
extraction solution from the GC Nicotine and Water Analysis
Laboratory . Place a glass stopper in each bottle immediately
after it is filled . Upon return to the laboratory where the actual
smoking of samples occurs, replace each glass stopper with an
impinger stem and secure with plastic ring clips . Set impingers
No interlaboratory validation process has been conducted using this analytical method .
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on the backside of the smoking machine but do not connect to
the vacuum source . Make connections later after flow rates are
checked .
Note : Four ports (3, 8, 13 and 18) are designated for sidestream
vapor phase collection and impingers are not connected to
these ports on the backside of the smoke machine . Thus,
only 16 impingers are required .
2 .3 . Volumetric flasks for chimney and impinger nicotine analysis :
Set up twenty 50 mL volumetric flasks on a shaker tray labeled
"Chimneys ." Set up an additional 16 volumetric flasks on the
tray labeled "Impingers ."
3 . Weigh both mainstream and sidestream filter holders and place in
position on the smoking machine . Attach sidestream holders to
smoking machine by attaching red vacuum tubing (- 1 3/4" section) to
the top of the sidestream holders with the end marked "H" next to the
holder and the end marked "C" next to the chimney . Leak check each
mainstream holder and ensure that sidestream holders are screwed on
securely .
4 . Check the smoking machine hood air flow and adjust if necessary .
See specifications listed on the smoking machine hood .
5. Calibrate CO Analyzers
Note : Sidestream CO analyzers must be calibrated each week prior to
sample analysis .
5 .1 Without connecting impingers, turn on sidestream air flow
(figure 1) and both high and low calibration gas cylinders .
Sampling of sidestream smoke for CO concentration is
performed during the smoking process through the use of a
continuous flow system . Perform calibration by analyzing
certified gas standards at the following nominal concentrations :
Sidestream : 0 .20% CO
0 .75% CO
5 .2 At the LIMS workstation, under the Data Acquisition menu,
select Sidestream Smoke Machine . Under File, select Collect
Calibration Data .
No interlaboratory validation process has been conducted using this analytical method .
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5 .3 Check the flow rates of the sidestream CO/CO2 ports (3, 8, 13, &
18) with a digital flow meter and adjust if needed to equal 50
mL/sec ± 0 .5 mUsec .
5 .4 Zero each sidestream CO and CO2 analyzer by pressing Zero,
then Enter button on each analyzer (figure 3) . When each
analyzer reads zero, return to the PC and click on OK to collect
data on "zero" gas . When data have been collected, ensure that
the two-way valve is set on "High" to acquire the high standard
gas and flip the four toggle switches above the analyzers (figure
2) to allow the gas to flow into the analyzers .
5 .5 Allow the readings on the digital display to settle for a few
seconds, then press Span and Enter. At the PC, click on OK to
collect data for the high gas . After data have been collected, flip
the two-way valve to the "Low" position to allow the low calibration
gas to enter the analyzers . Allow the readings to settle and then
click on OK at the PC to begin collection of data .
5 .6 When data have been collected, turn the two-way valve back to
the "High" position and flip the four toggle switches to the off
position . Turn off flow at the gas cylinders and turn off sidestream
air flows .
5 .7 At the PC click on File, then calibrate .
5 .8 Calibration Acceptance Criteria
The LabWindows software performs a linear regression of
instrument response and the resulting curve is used for data
quantitation . Check the index to ensure goodness of fit (0 .999, or
greater) . Continue with analyses of test samples/monitors if
calibration is acceptable . If not acceptable, recalibrate analyzers
before continuing with analyses of test samples and monitors .
G . Smoke/Analyze Monitors and Test Samples
Note : The atmosphere in the laboratory where the smoking is performed
is maintained at 75 ± 2°F and 60 ± 2 % relative humidity .
1 . Place assembled impingers onto rear section of the smoking machine .
Using the Keck disconnect and tubing assemblies attach tubing from
impinger front (internal stem connection to the sidestream filter pad
holder and from impinger rear (bulb) to vacuum source of system .
Take care not to reverse these connections . Turn on air flow and
ensure flow rates of 50 .0 mL/sec. ± 0 .5 mL/sec (3 L/min .) through
each sidestream port using a digital flow meter . These flow rates are
No interlaboratory validation process has been conducted using this analytical method .
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established and monitored at the front of the sidestream pad holder .
Note : Efficiency at this procedure is important to minimize solvent
loss in the impinger!
2 . After flowrates have been adjusted and verified, turn off the flow of air
and attach a numbered fishtail chimney (figure 4) to each sidestream
holder via the vacuum tubing and anchor each chimney into the
smoking machine chimney support . Raise chimneys to the "loading"
position (highest position) and load cigarettes into respective holders .
String down each microswitch and adjust each cigarette so that the
string rests atop the butt length mark on the cigarette .
3 . Place the aluminum smoke plate into the support brackets attached to
the Plexiglas side panels of the smoke machine . Lower the chimneys
to their lowest position and adjust bottom of chimney to 6 millimeters
above aluminum plate or until chimney rear slit is 1 - 2 mm from the
cigarette, whichever comes first . Use spacers provided for this task .
4 . Raise the chimneys to the intermediate position (lighting position) and
arrange each lighter so that it touches the end of the cigarette . Press
the red Start button on the sidestream command panel (figure 1) to
activate sidestream flows and ensure all 5 green lights for ports 1 - 20
are on .
5 . At the PC workstation, click on File and select Start Acquisition .
Check that all sidestream ports have adequate flows by observing the
flow meters at the back of the smoke machine and on the front
analyzer cabinet . Vigorous bubbling should also be evident in the
impingers and the sidestream analyzer front panels should indicate
good zero baselines .
6 . Press the Clock button on the smoke processor unit to begin the
smoke cycle . This button also initiates the real time start of the
analyzer software program . When the Clock button is pressed, a 55-
second countdown is started, the first 15-20 seconds of which is used
to calculate the baseline for the sidestream CO analyzers . When the
countdown has reached approximately 25 seconds (lighting time
depends on the condition of the lighters), light the cigarettes for
approximately 20-25 seconds . Immediately after lighting, lower the
chimneys to their lowest position, all the way down to completely
cover each cigarette . Ensure the cigarette rods are centered in the
chimneys and the fire cones are not touching the chimney sides .
7 . When the first cigarette has finished burning and the microswitches
are tripped, the chimneys automatically rise to their highest position .
At this point, extinguish the fire cone from each cigarette, wait until all
No interlaboratory validation process has been conducted using this analytical method .
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flows have shut off (green lights on sidestream control panel will be
off) and then turn off the Clock .
8 . At the PC workstation, under File, select Analyze Data to obtain
sidestream CO values . Click on Send to LIMS then Exit .
9 . Load the next cigarette, string down microswitches and repeat steps 1
- 6 above . Repeat process until all cigarettes have been smoked .
When the last cigarette has finished burning, leave the extinguished
butts in the holders until the first clearing puff has been taken .
Disconnect the impingers from the machine, remove the mainstream
holders and take the remaining 5 clearing puffs . Puff counts will then
be transferred automatically to LIMS .
10 . Collect sidestream CO raw data with LabWindows (a PC based
software system for data acquisition) and transfer to LIMS for final
calculation
11 . Process Filter Pads
11 .1 Re-weigh the sidestream pad holders, remove the pads and
place them into the 125 mL Erlenmeyer flasks containing the
extraction solution . Prepare a pad blank by placing a clean pad
into another flask with extraction solution . Transfer these flasks
to the GC Nicotine & Water Analysis Lab . Shake these flasks
for 45 minutes at 200 rpm and analy~ze for nicotine and water
content according to STD0023STM . After GC has removed the
necessary aliquots for analysis, transfer these extracts back to
the sidestream smoke collection room for UV
spectrophotometric analysis .
12 . Process Chimneys and Impingers
12 .1 Wash down each chimney with extraction solution and collect
each wash in labeled 50 mL volumetric flasks for UV analysis at
310 nm . After spectrophotometric analysis transfer these
extracts to the GC Lab for subsequent nicotine analysis
according to STD0004STM8 .
12 .2 Transfer impinger contents to 50 mL volumetric flask . Rinse the
impingers and tubing with extraction solution, add the washings
to the appropriate volumetric flask and dilute to volume with
extraction solution . Transfer these extracts to the GC Lab for
subsequent nicotine analysis according to STD0004STM8 .
No interlaboratory validation process has been conducted using this analytical method .
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13 . Dilute Sidestream Filter Pads/Chimney Extracts for Spectrophotometric
Analysis
13 .1 Both the pad and chimney extracts must be diluted prior to UV
analysis . Dilution is based upon the average sidestream WTPM
observed for each sample . This dilution is achieved through the
use of a Hamilton diluter according to the table below . Refer to
ACD0011 SOP4 for general operation and maintenance
guidelines on Automatic Dilution Systems . Refer to
STD0010SOP5 for general operation and maintenance
guidelines on a Beckman DU-70 Spectrophotometer with Batch
Sampler.
Table 6 : Dilutions of the Chimney and Impinger Solutions prior to UV
Analysis
Chimneys Pads
WTPM, Factor Dilution % Dilution %
mg/cigt
0 .0 - 1 .0 1 .25 no N/A no N/A
dilution dilution
1 .1 - 10 .0 1 .0938 6 .1 /0 .9 61/90 7 .1 /0 .9 71 /90
10 .1 - 25 .0 0 .5556 7 .1 /0 .9 71/90 7 .6/0 .4 76/40
25 .1 - 35 .0 0 .4688 7 .2/0 .8 72/80 7 .7/0 .3 77/30
> 35 .0 0 .5000 7 .5/0 .5 75/50 7 .8/0 .2 78/20
Note : Chimney and sidestream pad extracts must be analyzed
within 36 hours of smoking .
13 .2 Obtain duplicate readings of each extract (absorbance units)
and print a hard copy report . Transfer readings to the LIMS
system for calculations and storage .
H . Analysis of Filter Pads for Sidestream Pad Nicotine & Water
1 . Filter pads containing sidestream smoke condensate collected by
machine smoking are placed into 125 mL Erlenmeyer flasks containing
40 mL of extraction solution, stoppered immediately . Transfer flasks to
the GC Nicotine and Water Analysis Laboratory for extraction and
subsequent analysis of nicotine and water .
2 . Position the tray of filled flasks on a table top shaker and shake at 200
rpm for 45 minutes .
3 . Transfer approximately 1 mL of the test sample extract into sealed
autoinjector GC vials by using a new 3 mL sterile syringe for each
No interlaboratory validation process has been conducted using this analytical method .
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sample . Used syringes shall be disposed of in appropriate waste
containers .
4 . Use the Elite Sequence Builder Program (Refer to the RJR
Chromatography manual6) to schedule the samples on the GC
channels .
5 . Verify that the "sidestream" template is selected before building the
sequence .
6. Transfer the sample vials to the GC autosampler and start the GC .
7 . The amount of nicotine and water present in the test sample extracts
are interpolated from the appropriate EZChrom calibration curve and
transferred to LIMS for data evaluation and approval . Results are
expressed as mg/cigt .
8 . Use the Elite Real Time Display Program from the DATAACQ Menu in
LIMS to verify that all chromatography data are present . When all data
are present, notify the sidestream smoke analyst . The sidestream
smoke analyst shall compare the monitor results with the control limits
to ensure the monitor is within acceptable limits .
9 . If the sidestream smoke analyst identifies data that are unacceptable
the GC analyst shall re-analyze the suspect sample(s) .
9.1 Transfer approximately 1 mL aliquot(s) of the test sample extract
to be reanalyzed into GC vial(s) .
9 .2 Use the Elite Sequence Builder Program (Refer to the RJR
Chromatography manual6) to schedule the sample(s) on the GC
channels .
9.3 Reanalyze and notify the sidestream smoke analyst when
complete .
I . Analysis of Solutions for Chimney and Impinger Nicotine
1 . Upon receipt of the test extracts from the smoke lab analyst, transfer a
portion of each sample/monitor solution into vials and cap for
subsequent GC analysis . In addition, samples for chimney nicotine
analysis have a low check solution placed before the first sample and a
high check solution after the last sample . Samples for impinger
nicotine analysis should have check solutions in positions 3, 8, 13, and
18 .
No interlaboratory validation process has been conducted using this analytical method .
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2 . After GC analysis is complete, examine the chromatography
(EZChrom) data for consistency and integrity . Analysis of the low and
high check solutions must yield values within ± 5% of their actual
concentrations .
3 . Once chromatography data have been reviewed, smoke analyst review
the monitor values to ensure they are within the established control
limits before approving the data . If the monitors are not within range,
re-smoke or reanalyze as the situation requires .
4 . Perform calculations automatically via the chromatography and LIMS
data systems . Results are expressed in mg/cigt according to the
following equations :
J . Data Review
1 . After all requested analyses have been completed, at the PC
workstation (under the LIMS "ReportlApprove" option) print out a hard
copy of all raw and final (calculated) data .
2 . Examine all data for consistency and integrity . Compare monitor
results against control values to determine validity of data . If monitor
results are not within acceptable control limits or if samples are outside
of calibration ranges, correct the problem and re-smoke or re-analyze
the samples as the situation requires before approving the data .
K . Calculations
1 . Puffs per cigarette = Total # puffs per port/# cigarettessmoked
. 2 (g) - Wt.1(g) x 1000 mg
2 . Sidestream Pad TPM (mg/cigt) = Wt
#cigts g
Wt . 2 = weight of sidestream filter holder after smoking
Wt . 1= weight of sidestream filter holder prior to smoking
3 . Sidestream Pad Water (mg/cigt)
• Pad blank water result (mg) = aY + b
a= slope of calibration curve
water peak area
Y=
internal standard area
b= Y - axis intercept of the calibration line
No interlaboratory validation process has been conducted using this analytical method .
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• Sample water result (mg) = aY + b
a= slope of calibration curve
water peak area
Y=
internal standard area
b= Y - axis intercept of the calibration line
• Final water result (mg/cigt) = [water sample (mg) - pad blank (mg)]
# cigts
t) = aY + b
4. Sidestream Pad Nicotine km g /ci g
# cigts
a= slope of calibration curve
water peak area
Y=
internal standard area
b= Y - axis intercept of the calibration line
5. Sidestream Pad Tar (mg/cigt) =[SS Pad TPM(mg/cigt)]- [Nicotine(mg /cigt ) + H20(mg/cigt)
6 . Chimney Tar (mg/cigt) =
[SS Pad Tar (mg/cigt)]x (Ave . Chimney Absorbance reading) x Dilution factor u sed
# cigts x Ave . Pad Absorbance reading
7. Total Sidestream Tar (mg/cigt) = SS Pad Tar (mg/cigt)+ Chimney Tar( mg/cigt ) )
Sidestream CO (mg/cigt) = Data value(from LabWindows program) x P ressure(mm Hg)
8.
Temperature(Kelvin)
9. Chimney Nicotine (mg/cigt) = C(mg/mL) x 50
# cigt
Where C(mg/mL) = aY + b
a= slope of calibration curve
Y nicotine peak area
=
internal standard area
b= Y - axis intercept of the calibration line
and 50 = volume (mL)
and # cigt = 3
No interlaboratory validation process has been conducted using this analytical method .
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10 . Impinger Nicotine (mg/mL)= Same as for "Chimney Nicotine" above (#10)
11 . Total Sidestream Nicotine (mg/cigt) =
SS Pad Nicotine (mg/cigt) + Impinger Nicotine (mg/cigt) + Chimney Nicotine (mg/cigt)
12 . Impinger Nicotine/Total SS Nicotine, % =
Impinger Nicotine (mg/cigt) x 100
SS Pad Nicotine (mg/cigt) + Impinger Nicotine (mg/cigt) + Chimney Nicotine (mg/cigt)
. 2(g) - Wt .1(g) X 1000 mg
13 . Mainstream Pad TPM (mg/cigt) _ Wt
#cigts g
Wt . 2 = weight of mainstream filter holder after smoking
Wt . 1= weight of mainstream filter holder prior to smoking
No interlaboratory validation process has been conducted using this analytical method .
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VI . REFERENCES
1 . Massachusetts Constituents Testing Study Protocol, Brown & Williamson
Tobacco Corporation, Lorillard Tobacco Company, Philip Morris Incorporated
and R .J . Reynolds Tobacco Company, April 20, 1999 .
2 . RJR Standard Test Method, STD0013STM, Determination of Carbon
monoxide and Carbon dioxide in the Vapor Phase of Smoke by NDIR,
Rev .4, May 1999 .
3 . RJR Standard Test Method, STD0006STM, Gas Chromatographic
Determination of Nicotine and Water in Smoke, Rev . 5, May 1999 .
4 . ACD0011 SOP, Standard Operating Procedure for Automatic Dilution
Systems, Rev. 1, November 1997 .
5 . ACD0010SOP, Standard Operating Procedure for the Use of Beckman
DU-70 Spectrophotometer With Batch Sampler, Rev . 1, November 1997 .
6 . RJR Chromatography Manual, EZCHROM ELITET" Chromatography Group
Users Manual, October 1996 .
7 . RJR Standard Test Method, STD0023STM, Determination of Sidestream
Nicotine and Water by Gas Chromatography, Rev . 1, February 1998 .
8 . RJR Standard Test Method, STD0004STM, Determination of Sidestream
Chimney and Impinger Nicotine by Gas Chromato raphy, Rev . 3, February
1999 .
9 . RJR Standard Test Method, STD0001 STM, Determination of TPM and "Tar"
in Mainstream Smoke by FTC Method, Rev . 2, April 1999 .
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 1 : SIDESTREAM FLOW - COMMAND PANEL
5
C
FLOW
1-5 0"-0 ~
~
6-10 0 'e-0
11-15 0 '~ ~' ,1-0 ~N uP
O 0 Q ~'Q
OFF DOWN
16-2QS0 10 STRRT STOP/
RESET FLOW CHIMNEYS
1 = Green indicator lights
2 = Pushbutton switch
3 = Flow on/off toggle switch
4 = Toggle switch for chimney positions (up/down, center = off)
5 = 16 pin connector
FIGURE 2 : GAS ANALYZER PANEL
Flow knob 0 0 0 0
High
SS port # 3 8 13 10
I
Indicator lamp ®
Low
(Calibrate)
Toggle switch
$ 1 1 1 1
(Sample)
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 3 : CO/COZ ANALYZERS - TYPICAL DISPLAY
CO -1 CO2 - 1
Diait~l display of CO % Diaite al diszof CD/o
F1 m Zer F1 m m
~n [ IF 8 8 an F2 8 8
Ente tat S hift Enter 5tatd S Pod h ift
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 4 : SIDESTREAM SMOKE COLLECTION
6mm
PLATE POSITIONED 6 mm
BELO W CHIMNEY
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 5 : BAT FISHTAIL CHIMNEY DIMENSIONS
BAT Fishtail Chimney
Rear View All measurements in millimeters
27 1
--24
Front View
470
Side View
18 Bottom View
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 6 : CHROMATOGRAM OF MONITOR EXTRACT FOR SIDESTREAM
PAD NICOTINE ANALYSIS (PACKED COLUMN, ISOTHERMAL)
1 .375 Mi.ac Aap 0 .T42805VeNz
p< 1 Naw<
Rviv .~ianT~<
isro co.cnna'
v
o. ws
o. w.o
0 0.5 1.0 55 2.0 2.5 9 .o zs 4 .0 4.s s .0
.
Mircrte-
FIGURE 7 : CHROMATOGRAM OF CALIBRATION STANDARD FOR
SIDESTREAM PAD NICOTINE ANALYSIS (PACKED COLUMN,
ISOTHERMAL)
--~~
Na~e '
w.vevvmYure '.
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 8 : CHROMATOGRAM OF MONITOR EXTRACT FOR SIDESTREAM
PAD WATER ANALYSIS (PACKED COLUMN, ISOTHERMAL)
.
Timc: 0.000 Minutc . Amp: 0.688677 VoIN
1 .6
1 .. . ----i
1.M 0 .4
4 .3
42
0 .1
tAl
0 .81 W .3
0 .8 i0 .8
0.7 W .7
0 .0 0.5 1 .0 1 .5 2 .0 2:5 3.0 3 .5
Minutc:
FIGURE 9 : CHROMATOGRAM OF CALIBRATION STANDARD FOR
SIDESTREAM PAD WATER ANALYSIS (PACKED COLUMN, ISOTHERMAL)
.6 Timc: 0.000 Minutcs Amp : 0 .683734 Volts
1 ,
1 .5
H .4
1 .1
0 .9a W .9
0 .8 r0 .8
0 .7 4---J 1
0.0 .
05 .
10 1 .5 2 .0 25
. 30
. 3S
Minutec
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FIGURE 10 : CHROMATOGRAM OF CALIBRATION STANDARD FOR
SIDESTREAM PAD NICOTINE ANALYSIS (MEGABORE COLUMN,
TEMPERATURE PROGRAMMED)
FIGURE 11 : CHROMATOGRAM OF SAMPLE EXTRACT FOR SIDESTREAM
PAD NICOTINE ANALYSIS (MEGABORE COLUMN, TEMPERATURE
PROGRAMMED)
+
_ .__~ a eo
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FIGURE 12 : CHROMATOGRAM OF CALIBRATION STANDARD FOR
SIDESTREAM PAD WATER ANALYSIS (PACKED COLUMN, TEMPERATURE
PROGRAMMED)
FIGURE 13 : CHROMATOGRAM OF SAMPLE EXTRACT FOR SIDESTREAM
PAD WATER ANALYSIS (PACKED COLUMN, TEMPERATURE
PROGRAMMED)
.~,, .. ooye
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 14 : CHROMATOGRAM OF TEST EXTRACT - CHIMNEY NICOTINE
.
Timc: 0.133 Minute : Amp: 0.712338 Valt-
1 .021 1 .02
Pc k Npmc
0 .981
1
Rc rnt~ Timc
W .98
0 .9M W .94
0 .901
0.861
0.sz
0.781
E ro
co
~
r
~
a a cm
~
78
0.741 r N It Ir) ~' .74
N
m
0.701 40.70
0.661 10.66
0 1 2 '4 7
~7 9 10
FIGURE 15 : CHROMATOGRAM OF TEST EXTRACT - IMPINGER NICOTINE
Amp : 0 .81158 Volts
kN
entl
V
0
t
0 2 4 5 6 s 0
Mintc:
No interlaboratory validation process has been conducted using this analytical method .
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Analysis of Benzo[a]pyrene in Sidestream Cigarette
Smoke
Research & Development
R. J. Reynolds Tobacco Company
Winston-Salem, NC
No interlaboratory validation process has been conducted using this analytical method .
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Table of Contents
I . PRI NC I PLE OF M EASU REM ENT ---------------------------------------------------45
11 . DEFI N I TI ONS-------------------------------------------------------------------------------45
I I I . SAM PLE REQU I RE M E NTS-------------------------------------------------------------45
IV. APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION---45
A . Apparatus and Instrument--------------------------------------------------------45
B . Chemicals and Reagents--------------------------------------------------------47
C . Preparation of Standards---------------------------------------------------------48
D . I n s t r u m e n t C o n d i t i o n s------------------------------------------------------------- 4 9
V . PROCEDURES-------------------------------------------------------------------------49
A. Smoking of Cigarettes -------------------------------------------------------------49
B. Sample Preparation ----------------------------------------------------------------50
C. Sample Analysis --------------------------------------------------------------------51
D. Calculations --------------------------------------------------------------------------52
VI . REFERENCES --------------------------------------------------------------------------54
Figures
1. A typical chromatogram of the calibration standard ------------------------------55
2. A typical chromatogram of SS for I R4F---------------------------------------------56
3. B[a]Psidestream smoke collection apparatus using the fishtail chimney----57
4. A typical system blank chromatogram obtained by simulating the smoking of
two cigarettes (15 puffs per cigarette)---------------------------------------------------58
Tables
1 . SS B[a]P calibration standards------------------------- -------- ---------------------- 48
2 . Critical integration parameters ---------------------------------------------------------49
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Safety Note :
This procedure may involve the use of hazardous materials and/or
equipment. Prior to the use of this procedure, the user is responsible for
establishing appropriate safety and health practices .
I. PRINCIPLE OF MEASUREMENT
Sidestream smoke from the ISO-conditioned cigarettes, with 50% ventilation
blocking, is generated under a puffing parameter of 45 mL puff volume every
30 seconds with a two second duration as specified in the study protocol' .
The wet total particulate matter from the smoke is collected on a Cambridge
filter attached to the top of a fishtail chimney2 . After smoking, the chimney is
rinsed with acetone, the rinse is concentrated and the residue dissolved in
cyclohexane . This cyclohexane and a cyclohexane extract of the Cambridge
filter are combined3, 4 . A portion of the combined cyclohexane extract is
subjected to normal phase solid phase extraction to remove chromatographic
inten`erences5 . The eluent from the solid phase extraction is taken to dryness
and the residue dissolved in acetonitrile, which is subsequently analyzed by
high pressure liquid chromatography3,4 . The benzo[a]pyrene concentration is
determined by an external standard calibration using a fluorescence
detector3•4 .
II . DEFINITIONS
1 . HPLC : High Pressure Liquid Chromatography
2. B[a]P : Benzo[a]pyrene
3. SS : Sidestream Smoke
4. MS : Mainstream Smoke
5 . WTPM : Wet Total Particulate Matter
6 . SPE : Solid Phase Extraction
7 . CF : Cambridge Filter
8 . ACN : Acetonitrile
III . SAMPLE REQUIREMENTS
Sample required per determination : Two cigarettes
IV. APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION
A. Apparatus and Instruments
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1 . Balance : Model AE163 dual range analytical balance, Mettler
Instrument Corp ., Columbus, OH .
2 . Twenty port smoking machine : Model SM350, modified to accept eight
fishtail chimneys, Filtrona Instruments and Automation, Ltd ., Bletchley,
Milton Keynes, England .
3 . Volume checker : Heinrich Borgwaldt Model R24 .01 soap bubble flow
meter, 150-mL, KC Automation, Inc ., Richmond, VA .
4. Leak checker: Heinrich Borgwaldt Model R24 .0639 leakage tester, KC
Automation, Inc ., Richmond, VA .
5 . Flow meter : Flow Indicator and Totalizer, Model 209SE, Custom
Electronics Systems, Inc ., Winston-Salem, NC .
6 . Fishtail chimney : hand crafted, Public Scientific Glass Co ., Pfafftown,
NC .
7. Filter pad and holder : Model CM113A pad and 44 mm filter pad holder,
Cambridge filter Corporation, East Syracuse, NY .
8 . Electric lighter : Borgwaldt Electric Lighter, Model R29 .01, Borgwaldt
Technik GmbH, Hamburg, Germany.
9 . HPLC system : Model 510 pump, Model 680 gradient controller, Model
717 plus autosampler and Model 474 fluorescence detector, Waters
Corporation, Milford, MA.
10 .HPLC analytical column : Vydac 201TP54 C18 column, 4 .6 x 250 mm,
5pm particle size, The Separations Group, Hesperia, CA .
11 . HPLC guard column : Brownlee RP-18 Spheri-5MPLC guard refill in a
Brownlee 3-cm MPLC holder, Brownlee Labs, Inc ., Santa Clara, CA .
12 . Pipette and tips : Model EDP-Plus pipette and RC-1000 tips, Rainin
Instrument Co ., Emeryville, CA .
13 .Autosampler vials : #200 492 vials, #200 606 caps with septa and #
200 784 0 .25-mL insert, Sun International, Wilmington, NC .
14 . SPE extraction device : Supelco Visiprep 24 DL, Supelco, Bellefonte,
PA .
15 . SPE cartridges : Sep-Pak Vac RC (500 mg) NH2, Waters Corporation,
Milford, MA .
16 .Concentrator and tubes : TurboVap II Concentration Workstation Model
46367 and #45817 200-mL tubes with 1 .0-mL stems, Zymark
Corporation, Hopkinton, MA .
No interlaboratory validation process has been conducted using this analytical method .
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17 . Evaporator : Model 111 Analytical Evaporator, Organomation
Associates, Inc ., South Berlin, MA .
18 . Vortexer : Model M37615 Thermolyne Maxi Mix I I type 37600 Mixer,
Barnstead/Thermolyne, Dubuque, IA .
19 . Shaker : Burrell Wrist Action Shaker Model 45, Burrell Corp ., Pittsburg,
PA
20 . Extract filtering device : #AV525UORGCC Whatman Autovial 0 .45 Nm
PTFE, Whatman, Inc ., Clifton, NJ .
21 . Collection tubes : #73500-16100 Kimble 16 x 100 mm culture tubes,
Fisher Scientific, Atlanta, GA.
22 .Transfer pipettes : #313-678-6A Pasteur pipettes, 5 3/4 inch, Fisher
Scientific, Atlanta, GA .
23 . Pipettes : various volumes, class A, Fisher Scientific, Atlanta, GA .
24 . Erlenmeyer flasks : #10-047B, Pyrex 125-mL, 24/40 standard taper,
Fisher Scientific, Atlanta, GA .
25 . Magnetic stirrer : Thermolyne nuova II, Model SP18425,
Barn stead/Th ermolyne, Dubuque, IA .
26 . Graduated cylinder : #08-550J, Fisherbrand 2000-mL, Fisher Scientific,
Atlanta, GA .
27 . Data acquisition software : EZChrom Chromatography Data System
Version 6 .6, Scientific Software, Inc ., San Ramon, CA .
28 . Data reduction software : Microsoft Excel 97, Microsoft Corp .,
Redmond, WA .
B . Chemicals and Reagents
1 . B[a]P (CAS# 50-32-8), Catalog No . B1,008-0, 97% purity, Aldrich
Chemical Co ., Milwaukee, WI .
2 . ACN, HPLC grade, Catalog No . 015-4, Burdick & Jackson, Muskegon,
MI
3 . Cyclohexane, HPLC grade, Catalog No . 053-4, Burdick & Jackson,
Muskegon, MI .
4 . Hexane, HPLC grade, Catalog No . 216-5, Burdick & Jackson,
Muskegon, MI .
No interlaboratory validation process has been conducted using this analytical method .
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5. Acetone, GC grade, Catalog No . A928-4, Fisher Scientific, Atlanta, GA .
6 . Water, >17 .5 megohm-cm, 0 .2 pm filtered, inhouse supply passed
through a Barnsread Nanopure II system consisting of one Catalog No .
D0836, two Catalog No . D0809 and one Catalog No . D0820 cartridges,
Barnsread Co ., Boston, MA .
7 . Nitrogen, 5 .9+ grade, BOC Gases, Murray Hill, NJ .
8 . Molecular sieves, Catalog No . M514-500, 4 angstrom, Fisher
Scientific, Atlanta, GA .
Note : Approximately 20 mL of activated molecular sieves are added to four
liter bottles of hexane and cyclohexane .
C . Preparation of Standards
1 . To prepare primary stock standard, accurately weigh 5 .0 mg of B[a]P
into a 1000-mL volumetric flask, dilute to volume with ACN, yielding a
5000 ng mL-1 B[a]P solution .
2 . To prepare secondary stock standard, dilute 10 mL of the primary
stock standard to 100 mL volumetrically with ACN, yielding a 500 ng
mL-1 B[a]P solution .
3 . To prepare tertiary stock standard, dilute 10 ml of secondary stock
standard to 100 mL volumetrically with ACN, yielding a 50 ng mL-1
B[a]P solution .
4 . Prepare working standards by transferring the amounts in Table 1 into
separate 100-mL volumetric flasks and diluting to volume with ACN .
Table 1 : SS B[a]P Calibration Standards
Standard Transfer Solution Transfer Final Concentration
Level Amount (ng mL"')
(mL)
1 Standard Level 4 10 0 .5
2 tertiary stock 2 1 .0
standard
3 tertiary stock 5 2 .5
standard
4 tertiary stock 10 5 .0
standard
No interlaboratory validation process has been conducted using this analytical method .
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Standards should be prepared every three months and freezer-stored (~ -
10°C) when not in use .
D. Instrument Conditions
1 . HPLC Analytical Column : Vydac 201TP54, 4 .6 X 250 mm, 5pm
particle size
2 . HPLC Guard Column : Brownlee (OD-GU) RP-18 SPHERI-5, 30 X 4 .6
mm, 5pm particle size in a Brownlee 3-cm MPLC holder
3. Flow rate: 1 .3 mL min"'
4 . Mobile Phase : ACN/Water (90/10% by volume)
5 . Column temperature : Ambient
6. Runtime : 40 min (autosampler) and 38 min (data acquisition system)
7. Injection volume : 50 µL
8 . Detector settings : excitation wavelength of 378 nm, emission
wavelength of 405 nm, gain setting at X1000, both excitation and
emission slits set at 18 nm
9 . Retention time & integration : Typical retention time of the analyte is
shown in the chromatogram of a standard (Figure 1) and a SS
sample extract of 1 R4F reference cigarette (Figure 2) . Typical
integration parameters used to generate peak height data for
quantitation are tabulated in Table 2 .
Table 2 : Critical Integration Parameters
Events Start Time Stop Time (min) Value
(min)
Integrator Off 0 .000 5 .000 0
Threshold 0 .000 40 .000 1500
Width 0 .000 40 .000 2 .931
Valley to Valley 5 .000 20 .000 0
Integrator Off 20.000 40.000 0
V . PROCEDURES
A . Smoking of Cigarettes
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1 . Follow the random run order of the samples as specified in the study
protocol' .
2 . Prior to smoking, cigarettes are marked for insertion length into
Cambridge holder and for butt (smoking) length . Cigarettes must be
conditioned under 72 ± 2°F and 60 ± 2% relative humidity for a
minimum of 48 hours, but not to exceed 10 days as specified in ISO
3402 . The cigarettes are taped to block fifty percent of their
ventilation .
3 . Set up the eight-port linear smoking machine using the fishtail
chimney apparatus to collect the particulate phase of SS smoke (see
Figure 3) . Set the puffing regimen to deliver a 45 ± 0 .5 mL puff
volume every 30 ± 1 seconds with a two ± 0 .1 second duration as
specified in the study protocol' .
4 . Connect vacuum tubing to SS Cambridge pad holder from a vacuum
pump to draw 3 .0 L min"' (verified with flow meter) through the fishtail
chimney .
5 . Preweigh the MS and SS filter holders and position on the machine .
Check for leaks using a water column while drawing the 45 mL puff
volume .
6 . Insert cigarette into the MS holder to depth mark, install chimneys and
adjust to proper height (6 mm above plate) .
7 . Activate SS flow rate (3 .0 L min-') and light cigarettes using an electric
lighter . Terminate smoking when butt length line on cigarette is
reached based on visual observation .
8 . Smoke two cigarettes per port . Clear fishtail chimney of remaining
smoke by allowing to draw 3 .0 L min-' for about 60 seconds . After
smoking, reweigh both MS and SS holders and record total puff count .
Subtract "before weight" from "after weight" and express results as mg
WTPM per cigarette for both MS and SS smoke . Express puff count
on a per cigarette basis . Place SS pad in 125-mL Erlenmeyer flash
and glass stopper .
9 . Analyze blank samples (five) intermittently through the random order
run by simulated smoking of two unlit cigarettes, applying 15 puffs per
cigarette (see Figure 4) .
B . Sample Preparation
1 . Sample Extraction
a . After smoking, immediately rinse fishtail chimney with
approximately 20 mL acetone into 200-mL concentrator tube .
Place tubes in concentrator which is set for 38°C and 12 psi
No interlaboratory validation process has been conducted using this analytical method .
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nitrogen pressure . Place concentrator on endpoint mode and
evaporate acetone rinse of chimney to sensor mark as indicated by
response from evaporator. Add 10 mL cyclohexane volumetrically
to concentrate and vortex 30 seconds . Add this 10 mL of the
cyclohexane extract of the concentrate to the 125-mL Erlenmeyer
flask containing the SS WTPM pad from smoking .
b . Extract SS Cambridge pad with equivalent amount of cyclohexane
which corresponds to the total WTPM in mg ±5 mL, e .g ., if total
WTPM is 53 mg, use 55 mL cyclohexane added volumetrically .
Use a minimum of 20 mL cyclohexane (10 mL from hood rinse plus
10 mL to extract pad) . Shake for one hour using wrist action
shaker and filter an aliquot of the extract into 4-mL autosampler
vial .
2 . Solid Phase Extraction
a . Precondition SPE cartridges with 10 mL hexane via gravity flow (do
no let SPE cartridges become dry). Place collection tubes into SPE
extraction device . Pipette 500 pL of the cyclohexane extract onto
the SPE cartridge and allow to penetrate via gravity into the SPE
cartridge . When extract has penetrated SPE cartridge, add 10 mL
hexane . Allow hexane to elute through cartridge using gravity .
When all the hexane has eluted from the cartridge and no flow is
observed, apply one inch Hg vacuum until cartridge has dry
appearance .
b . Transfer collection tubes containing hexane eluent to evaporator
preheated to 40°C . Apply gentle flow of nitrogen until eluent is
completely evaporated . Pipette 1 mL ACN into dry collection tube
containing residue and vortex for 30 seconds . Place a portion of
ACN extract of the residue into autosampler vials containing 250
NL insert and subject to HPLC analysis in duplicate .
C . Sample Analyses
1 . Create a peak table in the EZChrom method file by indicating B[a]P
retention time (13 .2 min), window (10%), units (ng mL-1), quantitate
(height), fit type (linear), force zero (no), calibration flag (replace) and
concentration levels of standards (0 .5, 1 .0, 2 .5, and 5 .0 ng mL-') . Enter
runtime and integration parameters (Table 2) into acquisition setup of the
method file .
2 . Prepare run sequence file on EZChrom under method used for the
analysis of B[a]P . Arrange sequence such that there are duplicate
No interlaboratory validation process has been conducted using this analytical method .
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injections of both standards and samples . Inject standards before and
after samples (bracket calibration) by indicating standards as calibration
run types at the beginning and end of the sequence file . Enter
concentration levels as 1-4, four being the highest concentration standard .
Calibration is performed with each set of sample analyses . Enter samples
as unknowns and enter as the unknown multiplier the total volume of the
cyclohexane extract :
Multiplier = volume of cyclohexane extract (mL) X final volume (mL)
number of cigarettes smoked X aliquot volume (mL)
Multiplier - volume cyclohexane extract (mL) X 1 .0 mL
2X0 .5mL
Multiplier = volume cyclohexane extract (mL)
Where final volume = mL ACN to extract residue from collection
tube and aliquot volume = mL cyclohexane extract transferred to
SPE cartridge
3 . Turn on pump and detector and allow system to equilibrate as indicated by
a steady, flat baseline . Purge autosampler with ACN . Inject a standard
and verify retention time of B[a]P (Figure 1) .
4 . Load all standard and sample vials into autosampler . Program
autosampler for duplicate injections, 50 NL injection volume and a 40 min
runtime . Start analyses by beginning run through autosampler .
5 . Accept or reject analyses based on the correlation coefficient of the
calibration curve (>0 .98) and visual inspection of all chromatograms for
retention time, resolution and peak shape .
D . Calculations
1 . Determine the B[a]P (ng cigt-1) by an external standard method using the
regression equation derived from the calibration curve . B[a]P
concentration is obtained using the following equation :
B[a]P (ng cigt"1)- sample peak height X Multplier
responce factor
standard peak height
where response factor =
standard concentration
No interlaboratory validation process has been conducted using this analytical method .
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The limit of quantitation (LOQ) is based on the response of the lowest
concentration standard (0 .5 ng mL-') using the minimum amount of
cyclohexane extract (20 mL) .
LOQ (ng cigt-')-_ lowest concentration standard peak height X Multiplier
responce factor
50,0000 µV X 20
102957
= 9 .7 ng cigf'
2. Transfer data into a spreadsheet format and calculate the averages of
duplicate injections . Determine the averages, standard deviations and
percent relative standard deviations for each cigarette brand using
Microsoft Excel 97 software .
No interlaboratory validation process has been conducted using this analytical method .
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VI . REFERENCES
1 . Massachusetts Constituents Testing Study Protocol, Brown & Williamson
Tobacco Corporation, Lorillard Tobacco Company, Philip Morris
Incorporated and R . J . Reynolds Tobacco Company, April 20, 1999 .
2 . Protor, C . J ., C . Martin, J . L . Beven and H . F . Dymond, Evaluation of an
Apparatus Designed for the Collection of Sidestream Tobacco Smoke,
Analyst, 113, 1509-1513, 1988 .
3 . Risner, C . H ., The Determination of Benzo[a]pyrene in the Total Particulate
Matter of Cigarette Smoke, J . Chromatogr. Sci., 26, 113-120, 1988 .
4 . Risner, C . H ., The Determination of Benzo[a]Pyrene and Benz[a]Anthracene
in Mainstream and Sidestream Smoke of Kentucky Reference Cigarette
I R4F and a Cigarette Which Heats but Does Not Burn Tobacco : A
Comparison, Beitr. Tabak. lnt., 15, 11-17, 1991 .
5 . Dumont, J ., F . Larocque-Lazure and C . lorio, An Alternate Isolation
Procedure for the Subsequent Determination of Benzo[a]Pyrene in Total
Particulate Matter of Cigarette Smoke, J. Chromatogr. Sci., 31, 371-374,
1993 .
No interlaboratory validation process has been conducted using this analytical method .
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Figure 1 : A typical chromatogram of a calibration standard .
rK ss.oo4 fttts r mp, ovoiL,
11 o
a
A
m
5 10 1S 20 25 3S
~
No interlaboratory validation process has been conducted using this analytical method .
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Figure 2 : A typical chromatogram of SS for 1R4F .
j Tnc SBAOO Miautt; 1up: O Voks
, 1A
4
a
0•
0 S 10 15~29 35
Miaots
No interlaboratory validation process has been conducted using this analytical method .
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Figure 3 : B[a]P in sidestream smoke collection apparatus using the fishtail
chimney .
FISHTAIL
CHIMNEY
MAINSTREAM
CAMBRIDGE PAD
6mm ?
PLATE POSITIONED 6 mm
BELOW CHIMNEY
No interlaboratory validation process has been conducted using this analytical method .
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Figure 4 : A typical system blank chromatogram obtained by simulating the
smoking of two cigarettes (15 puffs per cigarette) .
rre 37.94TftC trp, 003795Yok,
1 n,
0
m
~
r
m
a.a .0
T
9 10 15 20 35
No interlaboratory validation process has been conducted using this analytical method .
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Analysis of 1-Aminonaphthalene, 2-Aminonaphthalene,
3-Aminobiphenyl and 4-Aminobiphenyl in Sidestream
Cigarette Smoke
Research and Development
R. J . Reynolds Tobacco Company
Winston-Salem, NC
No interlaboratory validation process has been conducted using this analytical method .
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Table of Contents
I . PRI NCI PLE O F M EASU REM ENT -------------------------------------------------61
11 . DEFI N ITI ONS ---------------------------------------------------------------------------61
111 . SAMPL E REQU I REMENTS ---------------------------------------------------------62
IV . APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION --62
A. Apparatus and Instrument--------------------------------------------------------62
B. Chemicals and Reagents---------------------------------------------------------63
C. Preparation of Standards---------------------------------------------------------64
D. I nstru ment Cond itions------------------------------------------------------------65
V . PROCEDURE ---------------------------------------------------------------------------66
A . Smoking of Cigarettes------------------------------------------------------------- 67
B . S a m p l e P r e p a r a t i o n---------------------------------------------------------------- 6 8
C . S a m p l e An a I ys i s--------------------------------------------------------------------6 9
D . C a I c u I a t i o n--------------------------------------------------------------------------6 9
E . S y s t e m S u i t a b i I i t y------------------------------------------------------------------- 6 9
V I . R E F E R E N C E S--------------------------------------------------------------------------6 9
Figures
1A . Total ion chromatogram of Standard #4 ------------------------------------70
1 B . Selected Ion Chromatogram of Standard #4 ------------------------------71
2A . Total ion chromatogram of the 1 R4F Reference Cigarette smoke
sample---14
2B . Selected Ion Chromatogram of the 1 R4F
Reference Cigarette smoke sample ------------------------------------------72
3A . Total ion chromatogram of the Blank sample -----------------------------73
3B . Selected Ion Chromatogram of the Blank sample --------------------- 74
4 . Sidestream smoke collection apparatus using fishtail chimney----------75
Tables
1 . PAA Stock Solutions --------------------------------------------------------------64
2 . SSPP PAA Calibration Concentrations----------------------------------------65
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Safety Note :
This procedure may involve the use of hazardous materials and/or
equipment. Prior to the use of this procedure, the user is responsible for
establishing appropriate safety and health practices
I . PRINCIPLE OF MEASUREMENT
Sidestream smoke from ISO-conditioned cigarettes, with 50% ventilation
blocking, is generated under puffing parameters of 45-mL puff volume with 2
second duration once every 30 seconds as specified in the study protocol' .
The wet total particulate matter from the smoke of one cigarette is collected on
a Cambridge pad . After smoking, the Cambridge pad is placed in a flask
containing extraction solution . The Cambridge pad is extracted for one hour
on a wrist action shaker . A portion of this extract is subjected to solid phase
extraction to remove chromatographic interferences . The eluent from the solid
phase extraction is acylated with pentafloropropionic acid anhydride to form
stable amides . This derivatized sample is then further purified using solid
phase extraction . The primary aromatic amine (PAA) concentration is
determined by internal standard calibration using gas chromatography/mass
spectrometry/selected ion monitoring (GC/MS/SIM) .
II . DEFINITIONS
1 . GC : Gas chromatography
2 . PAA : Primary aromatic amines
3 . SSPP : Sidestream particulate phase
4 . WTPM : Wet total particulate matter
5 . CF : Cambridge filter pad
6 . RSD : Relative standard deviation
7 . MW : Molecular weight
8 . MS : Mass spectrometer
9 . PFPA : Pentafluoropropionic anhydride
10 . TIC : Total ion chromatogram
11 . SIM : Selected ion monitoring
No interlaboratory validation process has been conducted using this analytical method .
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12. SPE : Solid phase extraction
13.TMA : Trimethylamine
Ill . SAMPLE REQUIREMENTS
One cigarette is required per sample determination . Six replicate determinations
are performed for each brand .
IV. APPARATUS, CHEMICALS, REAGENTS AND INSTRUMENTATION
A. Apparatus and Instrumentation
1 . 20 port Linear Smoke Machine for sidestream smoking, Winston-
Salem .
2 . Flow meter : Flow Indicator and Totalizer, Model 209SE, Custom
Electronics System, Inc ., Winston-Salem, NC .
3 . Fishtail chimney : Handcrafted, Public Scientific Glass Co ., Pfafflown,
NC .
4 . Coresta Holder with Single Cambridge Pad, Coresta Part No . 66300 .
5. Balance : Model AE163 dual range analytical balance, Mettler
Instrument Corp ., Hightstown, NJ
6 . Erlenmeyer flasks : #10-047B, Pyrex 125-mL, 24140 standard taper,
Fisher Scientific, Atlanta, GA .
7 . Magnetic stirrer: Thermolyne Nuova II, model SP18425,
Barnstead/Thermolyne, Dubuque, IA .
8 . Graduated cylinder : #08-550J, Fisherbrand 2000-mL, Fisher Scientific,
Atlanta, GA .
9 . HP 68901MS-1 equipped with autosampler and HP5973 Mass
Selective Detector and a computerized data station (HP ChemStation )
for data acquisition and reduction, Hewlett-Packard Co ., Wilmington,
DE .
10 . GC column : DB5ms 0 .33 micron film thickness, 25 m x 0 .2 mm, Cat .
No . 1285522, J&W Scientific Inc ., Folsom, CA .
No interlaboratory validation process has been conducted using this analytical method .
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11 . Turbovap I I Concentration Workstation, Zymark Corporation,
Hopkinton, MA .
12 . Rainin Pipetters : Electronic digital pipettes inculding, 0-100 µL, 0-250
µL, & 0-1000 µL, Rainin Instrument Co ., Woburn, MA .
13 . Vac Elut SPS 24 for Sep-Pak Elution, Varian, Harbor City, CA
14 .4420 Eppendon` Easypet, Brinkman Instruments, Westbury, NY .
15 .100 µL limited volume vials for GC, Product No . 9480, Alltech
Associates,Inc ., Deerfield, IL .
16 . Pasteur Pipettes, Cat . No. 13-678-6B, Fisher Scientific, Atlanta, GA.
17 . Waters Sep-Pak Vac 12 cc (2G) NH2 Cartridges, W9004Q1, Waters
Corporation, Milford, MA .
18 . Waters Sep-Pak Vac 6 cc (1 G) Accell Plus QMA Cartridges,
T7329B1,Waters Corporation, Milford, MA .
B . Chemicals and Reagents
1 . Trimethylamine hydrochloride, 06325LN, 98% Purity, Aldrich Chemical,
MILWAUKEE, WI .
2 . Sodium hydroxide pellets, SX0590-1, 97% Purity, MCB Reagents,
Cincinnati, OH .
3 . Sodium sulfate (Anhydrous), 986530, Fisher Scientific, Fair Lawn, NJ .
4 . Molecular sieves, 944297070062, Advanced Specialty Gas Equipment,
South Plainfield, NJ .
5 . Pentafluoropropionic anhydride, 65193, Pierce, Rockford, IL .
6 . 1-Aminonaphthalene, 80151ET, 98% Purity, Aldrich Chemical Co .,
Milwaukee, WI .
7 . 2-Aminonaphthalene, 80033HT, 95% Purity, Aldrich Chemical Co .,
Milwaukee, WI .
8 . 3-Aminobiphenyl, FIE01, 98 % Purity, TCI, Tokyo, Japan .
9 . 4-Aminobiphenyl, 03107AT, 98% Purity, Aldrich Chemical, Milwaukee,
WI .
No interlaboratory validation process has been conducted using this analytical method .
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10. [zH7]-2-Aminonaphthalene, B447P1, 98% Purity, CDN Isotopes,
Vandreuil Quebec.
11 . [zH9]-4-Aminodiphenyl, B448P1, 99% Purity, CDN Isotopes, Vandreuil,
Quebec .
12. Hexane UV, BT841, 99 .9% Purity, Burdick and Jackson, Muskegon,
MI .
13 . Dichloromethane, BS365, 99 .9% Purity, Burdick & Jackson,
Muskegon, MI.
14 . (±)-a-Tocopherol, 10209MQ, 97% Purity, Aldrich Chemical,
Milwaukee, WI .
C. Preparation of Standards
NOTE : All of the primary standards and solutions, stored at
approximately -20 °C, must be allowed to warm up to ambient
temperature prior to use .
1 . Primary stock solutions are prepared by accurately weighing
approximately the specified PAA amounts in Table 1 into a 10 mL
volumetric flasks . Add approximately 5 mL hexane and swirl the
flasks gently till all the crystals dissolve . Fill to the mark with hexane
and mix well . These stock solutions are stored in amber volumetric
flasks in the freezer at -20°C (expires after 6 months) .
Table I PAA Stock Solution
Compounds Weight Purity Volume Stock
(mg) (%) (mL) Solution
/mL
H, -2-aminona hthalene 1 .00 98 10.0 100
1-aminona hthalene 1 .00 98 10.0 100
2-aminona hthalene 1 .50 95 10 .0 150
H9 -4-aminobi hen I 2 .50 99 10 .0 250
3-aminobi hen I 1 .50 98 10 .0 150
4-aminobi hen I 1 .00 98 10.0 100
2 . To prepare secondary stock standard solution, dilute 100 µL of the
primary stock standard to 100 mL volumetrically with hexane .
3 . Prepare calibration standards by manual dilution of the secondary
stock solution followed by derivatization .
No interlaboratory validation process has been conducted using this analytical method .
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a . Allow the stock solution to warm up to room temperature before
dispensing . Using a Rainin Pipetter, pipette the necessary amount
of each solution into a Zymark tube containing approximately 15 mL
of dried hexane .
b . Add 50 µL of TMA and 20 µL of PFPA into the Zymark tube, swirl
and let sit at room temperature for at least one hour .
c. Take solution to dryness in the TurboVap concentrator set at 38°C
and 12 psi nitrogen pressure .
d . Reconstitute in 250 µL of dried hexane .
Calibration standards were made from the stock solution at concentrations
found in Table 2 .
Table 2 : MSPP PAA Calibration Concentration
Compounds Stdl Std2 Std3 . Std4 Std5
ng cigt" ng cigt"' ng cigt"' ng cigt"' ng cigt"'
125 .0 125 .0 125 .0 125 .0 125 .0
aminonaphthalene
1-aminonaphthalene 50 .0 100 .0 150 .0 200 .0 250 .0
2-aminonaphthalene 50 .0 100 .0 150 .0 200 .0 250 .0
[ H9]-4-aminobiphenyl 25 .0 25.0 25 .0 25.0 25.0
3-aminobiphenyl 7 .5 15 .0 30 .0 60 .0 80 .0
4-aminobiphenyl 7 .5 15 .0 30 .0 60 .0 80 .0
D. Instrument Conditions
1 . GC column : DB5ms 30 m x 0 .20 mm, 0 .33 µm film thickness .
2 . Injection mode : splitless, 30 sec . purge . One µL injection . Injection
port temperature 240°C .
3. Constant flow : average linear velocity 49 cm/sec .
4. Gas type : Helium
5 . GC oven conditions : set initial temperature 60°C, hold 1 .0 minute ;
program rate 20°C min-'to 120°C no hold, Second level : program rate
of 2 .0°C min'to 200°C no hold ; third level : 20 °C to 280°C, hold 15
minutes .
No interlaboratory validation process has been conducted using this analytical method .
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6 . Run time : 63 minutes .
7 . Operate mass spectrometer in the SIM acquisition mode . For the
aminonaphthalenes, monitor ions 289 .1, 296 .1 for quantitation and
monitor ions 142 .1,and 122 .1 as qualifier ions . For the aminobiphenyls,
monitor ions 315 .1, 324 .1 for quantitation and monitor ions 153 .1, and
168 .1 as the qualifier ions .
8 . Mass spectrometer temperature zones : MS quads 106°C, MS source
230°C, interface temperature 240°C .
9 . Retention time and integration parameters : Typical retention times of
the analytes are shown in the TIC of a standard (Figure 1), a SS
sample extract of 1 R4F reference cigarette (Figure 2 .) and a Blank
sample (Figure 3) . Integration parameters : peak width 0 .04 sec . and
threshold 10 .0 .
V .PR0CEDURES
A. Smoking of Cigarettes
1 . Follow the random run order of the samples as specified in the study
protocol' .
2 . Prior to smoking, cigarettes are marked for insertion length into the
Cambridge holder and for butt (smoking) length . Cigarettes must be
conditioned under 72 ± 2°F and 60 ± 2% relative humidity for a
minimum of 48 hours, but not to exceed 10 days as specified in ISO
3402 . The cigarettes are taped to block fifty percent of their ventilation .
3 . Set up the 20-port linear smoking machine using the fishtail chimney
apparatus to collect the particulate phase of SS smoke (see Figure 4) .
Set the puffing regimen to deliver a 45-mL puff volume every 30
seconds with two-second duration as specified in the study protocol' .
4 . Connect vacuum tubing to SS Cambridge pad holder from a vacuum
pump to draw 3 .0 L min"' (verified with flow meter) through the fishtail
chimney .
5 . Preweigh the SS filter holders and position on the machine . Check for
leaks using a water column while drawing the 45 mL puff volume .
6 . Insert cigarette into the SS holder to depth mark, install chimney and
adjust to proper height (6 mm above plate) .
No interlaboratory validation process has been conducted using this analytical method .
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7. Activate SS flow rate (3 .0 L min -') and light cigarettes using an electric
lighter. Terminate smoking when butt length line on cigarette is
reached .
8 . Smoke one cigarette per port . Afterwards, reweigh both MS holders
and record total puff count . Subtract "before weight" from "after
weight" and express results as mg WTPM per cigarette for MS smoke .
Express puff count on a per cigarette basis .
9 . Analyze blank samples (five) intermittently through the random order
run by simulated smoking of one cigarette, applying 15 puffs per
cigarette.
10 .After smoking, Cambridge pads are immediately placed into a 125 mL
flask containing the extraction solution and the flask is stoppered (see
Sample Preparation) .
B . Sample Preparation
1 . Solvents : Add a volume of 25 mL of dried molecular sieves, which have been
activated at 170°C, for four liters of solvent of hexane and methylene chloride .
2 . Preparation of Trimethylamine : Prepare two molar TMA by adding three
grams of TMA-HCI to six mL water and neutralizing to a pH of approximately
7 with 50% sodium hydroxide solution . The TMA is back extracted into 15 mL
of hexane . To eliminate moisture contamination, the TMA is stored over one
mL of molecular sieves per 15 mL of TMA in hexane .
3. Preparation of (±)-a-tocopherol solution : The 0.2mM (±)-a-tocopherol solution
is prepared by adding 0 .02g of liquid (±)-a-tocopherol in 100 mL of hexane .
4 . Prepare the extraction solution for each sample in 125-mL Erlenmeyer flasks
by adding 100 mL of dried hexane, 25 g of sodium sulfate, 300 µL of TMA, 50
µL of 0 .2M (±)-a-tocopherol and 100 µL of internal standard solution . These
flasks can be made up the night before adding all of the above constituents
except for the sodium sulfate and the internal standard solution .
5 . Sample extraction : Place Erlenmeyer flasks containing the Chambridge pads
on a wrist action shaker and shake at a speed of 5 for one hour .
6 . Solid phase extraction : Place a 10 mL, 20mL, 50mL or 100 mL aliquot
(depending on the mg of `tar) of the hexane extract on an Amino (NH2) Sep-
Pak cartridge which has been preconditioned with 50 mL of hexane . After
elution of the extract, the Sep-Pak is washed with 125 mL of hexane to
remove nonpolar components . The PAA are then eluted from the Sep-Pak
with 100 mL of 8 :2, hexane :methylene chloride . Derivatize this fraction with
No interlaboratory validation process has been conducted using this analytical method .
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50 µL TMA in hexane followed by 20 µL PFPA and react at room temperature
for one hour . Purify the derivatized sample using a 1 g quaternary amine
(OMA) anion exchange Sep-Pak column . The column is preconditioned with
20 mL of 8 :2 hexane :methylene chloride . The derivatized sample is taken
through the column at a flow rate of 2 mL min'' . The column is then washed
with an additional 10 mL of 8 :2 hexane :methylene chloride . The combined
effluent is put into a Zymark tube and taken to dryness in a Turbo-Vap
concentrator. The sample is reconstituted in 250 pL of 8 :2 hexane :methylene
chloride for GC/SIM analysis .
C. Sample Analysis
1 . Preparation of Calibration Curve : Place five derivatized calibration
standards in the HP 7673A sample tray . Fill wash vials with hexane .
At the ChemStation, perform an auto tune for the mass spectrometer
and check ratios of the 69, 219, and 502 ions . Set up a sequence
table for each vial repeating this sequence so there are at least two
injections for each standard . Set up calibration table using the
ChemStation software package for quantitation . Establish a standard
curve of concentration ratio versus relative response ratio for each
compound using the HP Chem station software package provided with
the instrument . Base the response ratio on the molecular ions
corresponding to the PFPA derivatives of 1-aminonaphthalene, 2-
aminonaphthalene, [~2H7]-2-aminonaphthalene, 3- aminobiphenyl, 4-
aminobiphenyl and [ H9]-4-aminobiphenyl (m/z 289 .05, 296 .09, 315 .06
and 324 .12, respectively) . The acceptance or rejection of the
calibration is determined by examining the correlation coefficient and
visual inspection of the calibration curves . The correlation coefficient
shall be no less than 0 .950 . Calibration standards that do not meet
this requirement shall be remade and re-analyzed . The instrument is
autotuned every other week and calibrated every day . Reference
cigarette (1R4F) smoke samples are analyzed with each group of
samples and must be within 15% of historically observed MS PAA
concentrations .
2 . Sample Analysis: Set up sequence table with one injection per sample
and Run sequence .
D . Calculations
PAA Concentration (ng cigt-') = C (ng cigf')l # of cigt .
Where C (ng cigt-') = aY + b
a = slope of calibration curve
No interlaboratory validation process has been conducted using this analytical method .
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Y = peak area of PAA
Int. std . peak area
b = Y axis intercept of calibration line
# of cigt = 1
E . System Suitability
For each group of samples, monitor instrument precision by injecting a
1 R4F SS WTPM sample six times . The concentrations of 1-
aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl and 4-
aminobiphenyl should have a coefficient of variance not greater than 5
% . The minimum detectable quantity for 1-aminonaphthalene and 2-
aminonaphthalene is 0 .05 ng/cigt . The minimum detectable quantity of
3-aminobiphenyl and 4-aminobiphenyl is 0 .100 ng/cigt .
VI . REFERENCES
1 . Massachusetts Constituents Testing Study Protocol, Brown & Williamson
Tobacco Corporation, Lorillard Tobacco Company, Philip Morris Incorporated
and R . J . Reynolds Tobacco Company, April 20, 1999 .
No interlaboratory validation process has been conducted using this analytical method .
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Figure 1A . Total Ion Chromatogram (TIC) of Standard #4 . Peak
identifications are as shown .
Abundance
2200W
2000001
180000
1600001
14000W
1200001
1000W
800001
6/0001
'fiJ0W
200W
_F__ r ^r7r ~
Time- 17 .0 19 .0 21 .0 23 .0 25 .0 27 .0 29.0
No interlaboratory validation process has been conducted using this analytical method .
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FIGURE 1 B . SELECTED ION CHROMATOGRAMS (SIM) OF STANDARD #4 .
PEAK IDENTIFICATIONS AND IONS MONITORED ARE AS SHOWN .
Abunclance Ion 289.10 (288 .80to 289.80) :
100000 u
c ~
v a
80000 ~ A
t
60000 a
a
~
40000 e al
20000
0
Tirr~e--> 17 .0 19 .0 21 .0 23.0 25_0 27 .0 29.0
Abundance ~ lon 296_ 10 (295 .80 to 296_80)
100000 R
~
~
80000 R 6
G ~
0
60000
40000
200001
O
Time-> 17.0 19.0 21 .0 23.0 25 .0 27 .0 29.0
Abundance Ion 315.1(l (314_330 to 315 .Sq .
100000 '' T
C v
N L
80000 L
~.
G
L
Z
60000 O C
.~ E
40000 v
200001
0 ~-rrrr~ r-rr-
Tirne- 17.0 19.0 21 .0 23 .0 25 .0 27 .0 29 .0
;obuncfance Ion 324.10 (323 .80 to 324 .80) ~
~
100000 d
~
0
80000 c
E
60000 ~
40000 x
20000
0
17 .0 19.0 21 .0 23.0 25 .0 27 .0 29.0
L
Tarie--7
No interlaboratory validation process has been conducted using this analytical method .
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Figure 2A . Total Ion Chromatogram (TIC) of the I R4F Reference Cigarette
smoke sample . Peak identifications are as shown
TIC 1401014_D
x.rndance
170000
160000
150000
140000
130000
120000
110000
100000
90000
80000
70000
50000{ i
400001
30000
20000
10000~
0
Tirw~__} 17_O 19_0 21 .0 23.0 25_0 27 .0 29 .00
No interlaboratory validation process has been conducted using this analytical method .
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Figure 2B . Selected Ion Chromatograms (SIM) of the 1 R4F Reference
Cigarette smoke sample . . Peak identifications and ions monitored are as
shown .
lon 289 .10 (28a .80 to 289-60) :
A .Gundenoe
80000
U
~ ~
C
~
00000 L
..
a'~
~
C ~
0 a
40000 a m
£ a
m .a
~
20000
N
O - 10, T T T
Tlrna--~ 16 001 7 .001 B .0019 .0020 .002'i .0022 .0023 .0024 .0025 .0028-0027 .0026 .0029T
T T T T 1
.0030 .00
Abundanee ~on 298 .10 !•265 .80 ta 296 8O) :
C
~
80000 ~
•~-
r
a
m
0000 C
G
G
.~
40000
N
20000 x
0 ~
18 .001 7 .001 a.0019 .0020 .0021 .0022 .0023 .0024 .0025 .0026 .0027 .0028 .0029 .0030 .00
an . .
._?'_5 .'If+ ' 14 .8i.' to :31ti 8 ... .
4.bundenca
80000
60000
L
C t
L d
0000
~
20000 ~ E
~
O r. rrTr r r r-~-r r T- T T T T T 4T `Tr 4' r`T~'T"~"1
1 B .001 7 .001 8 .0019 .0020 .0021 .0022 .0023 .0024 .0025 .0028 .0027 .0028 .0029 .0030 .00
P.buntl3nca lon 324 .10 (323 .80 to 324 .50) :
80000
60000
40000
20000
_ . . . . . . . .r . . . .~ . . .r . .~ . . .~ . .,1 . . . .1 .
Tima--> 16 .001 7 .009 8 .0019 .0020 .0021 .0022 .0023 .0024 .0025 .0026 .0027 .0028 .002G .0030 .00
No interlaboratory validation process has been conducted using this analytical method .
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Figure 3A . Total Ion Chromatogram of the Blank sample .
AGUnaYan ..!
LO000
ea000
~
CO000
6
E
s
I
S J -
u,~ II D
6
O Y 1 E
5
I
r
aaoo
juj Li l .^-,N
r ay
~
V
rU
AA
TIIl1R--- IT .O 19 .0 .0 23 .0 2T .n 29 .0
No interlaboratory validation process has been conducted using this analytical method .
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Figure 3B . Selected Ion Chromatogram of the Blank sample
Abundance G
I pn 269 . 10
600004
N
~
50000 ~
~ ~
n.
4D000 ~
O
~ cOC"
30000 C C
O
20000 E
cd .~
~
10000 cd
D N r,
Tlme--- 17 .0 19 .0 21 .0 23 .0 25 .0 27 .0 29 .0
Abundance
lon 296 .10
600004
50000
40000
30000
20000 ~
10000 ~ j
{O
TnMe--- 17 .0 19 .0 21 .0 23 .0 25 .0 27 .0 29 .0
P.bundance
Ion 315 .10
600001
50000
40000
30000
20000
1OOO0 ~
D Lr°T'TTrI"TTYrTYTY"'~"YT"'Y
1
Time--=- 17 .0 19 .0 21 .0 23 .0 25 .0 27 .0 29 .0
~
c
AbunS.4ance
lon 324 .10 ~
600001 a.
L
50000 O
C
.~
40000
~
30000
~
20000
10000 N
W
Tlme--- 17 .00 19 .0 21 .0 23 .0 25 .0 27 .0 29 .0
No interlaboratory validation process has been conducted using this analytical method .
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Figure 4 . Sidestream smoke collection apparatus using fishtail chimney .
FISHTAIL
CHIMNEY
MAINSTREAM
CAMBRIDGE PAD
6mm ?
PLATE POSITIONED 6 mm
BELOW CHIMNEY
No interlaboratory validation process has been conducted using this analytical method .
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Analysis of 1,3-Butadiene, Isoprene, Acrylonitrile,
Benzene, Toluene and Styrene in Sidestream Cigarette
Smoke
Research & Development
R. J . Reynolds Tobacco Company
Winston-Salem, NC
No interlaboratory validation process has been conducted using this analytical method .
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Table of Contents
I . PRI NCI PLE OF M EASU REM ENT--------------------------------------------------79
I I . DEFI N ITI ONS----------------------------------------------------------------------------79
111 . SAMPLE REQUI REMENTS------------------------------------------------------79
IV . APPARATUS, INSTRUMENTATION, CHEMICALS AND REAGENTS---80
A . Apparatus and Instrumentation-------- ---__--------- _----------------------- 80
B . Chemicals and Reagents----------------------------------------------------------82
C . Preparation of Standards-----------------------------------------------------------82
D . I nstru ment Conditions--------------------------------------------------------------85
V . P R O C E D U R E---------------------------------------------------------------------------8 6
A . Smoking of Cigarettes------------------------------------------------------------ 86
B . Preparation of Calibration Curve-----------------------------------------------87
C . Sample Analysis -------------------------------------------------------------------88
D . Calculations-------------------------------------------------------------------------- ----------88
V I . R E F E R E N C E S--------------------------------------------------------------------------8 9
Figures
1 . Total ion chromatogram for standard 3------------------------------------------------ 90
2 . Total ion chromatogram for Kentucky Reference 1 R4F----------------------------90
3 . Total ion chromatogram for a blank smoke sample---------------------------------91
4 . Extracted ion chromatogram for 1,3-Butadiene in 1 R4F---------------------------92
5 . Extracted ion chromatogram for Acrylonitrile and Acrylonitrile-d3 in 1 R4F----92
6 . Extracted ion chromatogram for Isoprene in 1 R4F----------------- -------------- 93
7 . Extracted ion chromatogram for Benzene and Benzene-d6 in 1 R4F-----------93
8 . Extracted ion chromatogram for Toluene in 1 R4F----------------------------------94
9 . Extracted ion chromatogram for Styrene in 1 R4F---------------------------------94
10 . Modified Filtrona sidestream smoking machine-------------------------------------95
11 . Sidestream traps on Modified Filtrona smoking machine------------------------ 96
12 . Fishtail chimney used for sidestream smoking--------------------------------------97
13 . Schematic of fishtail chimney -----_------------------------------------------------- 98
Tables
1 . Preparation of Standards----------------------------------------------- ------------------ 83
2 . Selected Ions Monitored-------------------------------------------------------------------86
3 . Integration Parameters----- ------- ------------------------------------------------------ 86
No interlaboratory validation process has been conducted using this analytical method .
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Safety Note :
This procedure may involve the use of hazardous materials and/or
equipment . Prior to the use of this procedure, the user is responsible for
establishing appropriate safety and health practices .
PRINCIPLE OF MEASUREMENT
Sidestream smoke from 13 commercial brand style cigarettes, ISO
conditioned, is generated using a 45 mL puff volume taken every 30
seconds with a two second puff duration . The cigarette filters are 50%
ventilation blocked as specified in the study protocol' . The sidestream
vapor phase cigarette smoke (SSVP) is collected using a series of four
impingers each containing methanol . The first impinger is maintained
at 0°C and the remaining three at -78°C . The combined impinger
solutions are analyzed by gas chromatography (GC) with selected ion
monitoring mass spectrometry (SIM)2-5 . Quantitation is performed
using internal standard calibration .
II . DEFINITIONS
1 . GC : Gas chromatography/ chromatograph
2 . MS : Mass spectrometry/ spectrometer
3. SIM : Selected ion monitoring mass spectrometry
4 . MSPP : Mainstream particulate phase cigarette smoke
5 . WTPM : Wet total particulate matter
6 . SSVP : Sidestream vapor phase cigarette smoke
III . SAMPLE REQUIREMENTS
Sample required per determination : Three cigarettes
No interlaboratory validation process has been conducted using this analytical method .
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IV . APPARATUS, INSTRUMENTATION, CHEMICALS AND REAGENTS
A. Apparatus and Instrumentation
1 . GC : Model 5890 equipped with a Model 7673 autosampler,
Hewlett-Packard, Palo Alto, CA .
2 . GC column : 60 m, 0 .32 mm id, 5 µm film thickness DB-1 fused
silica capillary column, J&W Scientific, Folsom, CA .
3. MS : Model 5972, Hewlett-Packard, Palto Alto, CA .
4. Balance : Model AE240 and AE163 dual range analytical balances,
Mettler Instrument Corporation, Columbus, OH .
5 . Smoking machine : modified Model SM350, fitted with RJR
sidestream portable flow system, Filtrona Instruments, Richmond
VA .
6 . Volume checker : 150 mL soap bubble flow meter, Heinrich
Borgwaldt Model R24 .01, KC Automation Inc, Richmond, VA.
7 . Leak checker : 250 mL leveling bulb, ring stand, in-house, RJR,
Winston-Salem, NC .
8. Vacuum tubing : red thick wall, general purpose, 3/8 inch id and 1/8
inch wall, Fisher Scientific, Springfield, IL .
9. Tubing Adapter : blue, 8-10 mm id tubing, catalog no . E-06841-50,
Cole-Parmer Instrument Co ., Vernon Hills, IL .
10 . Tubing Coupler Adapter: white, catalog no . E-06841-54, Cole-
Parmer Instrument Co ., Vernon Hills, IL .
11 . Port Adapter : catalog no . E-06841-53, Cole-Parmer Instrument Co .,
Vernon Hills, IL .
12 . Cambridge filter pad holder:45mm pad holder, catalog no . 9840-
000, Phipps & Bird, Richmond, VA .
13 . GC autosampler vials : 12x32 mm clear borosilicate crimp-top vials
with caps, catalog no . 500-584 Sun Brokers, Wilmington, NC .
14 . Sleeve serum stoppers : 14 .5 mm sleeve serum stoppers, catalog
no . 14-126BB, Fisher Scientific, Springfield, NJ .
No interlaboratory validation process has been conducted using this analytical method .
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15 . Gastight syringes : 25-µL, 50-µL, 100-µL, 250-µL and 2000-µL
Pressure-Lok syringes, Precision Sampling Corporation, Baton
Rouge, LA.
16 . Disposable syringes : 3-mL B-D 3CC23G1 syringes and needles,
catalog no . 14-826-11, Fisher Scientific, Springfield, NJ .
17. Filter pad : CM113A, fiberglass, 44-mm diameter, Cambridge Filter
Corporation, East Syracuse, NY .
18 . Filter pad holder : 44-mm, cat . No . 66300, Filtrona Instruments,
Richmond VA .
19 . Impingers : 70-mL glass impinger with extra course frit, custom
made, Scientific Silicates, Waterloo, Ontario, Canada .
20. Dewar flasks : 1 000-mL and 4300-mL volumes, catalog nos 10-
197B and 10-197D, Fisher Scientific, Springfield, NJ .
21 . Pipettes : 5 and 20-mL Glass, class A, catalog nos . 13-650-2F and
13-650-2N, Fisher Scientific, Springfield, NJ .
22 . Nalgene tubing : 5/16 inch inside diameter and 7/16 inch outside
diameter, catalog no . 14-176-30, Fisher Scientific, Springfield, NJ .
23 . Sealing film : Parafilm "M", 4 inch, catalog no . 13-374-12, Fisher
Scientific, Springfield, NJ .
24 . Disposable Pasteur pipettes : 5'/4 inch, glass, catalog no . 13-678-
20A, Fisher Scientific, Springfield, NJ .
25 . Volumetric flasks : 10 and 25-mL Pyrex volumetric flasks, catalog
nos . 20-812D and 10-210A, Fisher Scientific, Springfield, NJ .
26 . Data acquisition software : ChemStation-IBM compatible computer
with Microsoft Windows and DOS software G1143C Version
C .02 .00, Microsoft Corp ., Redmond, WA .
27 . Data reduction software : Microsoft Excel 97, Microsoft Corp .,
Redmond, WA.
28 . Fishtail chimney : made to specifications, Public Scientific Glass
Co ., Pfafftown, NC .
No interlaboratory validation process has been conducted using this analytical method .
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B. Chemicals and Reagents
1 . Methanol, HPLC grade, catalog no . 230-4, Burdick & Jackson,
Muskegon, MI .
2 . Perdeuterated acrylonitrile, catalog no . DLM-820, Cambridge
Isotope Laboratories, Woburn, MA .
3. Perdeuterated benzene, catalog no . DLM-1, Cambridge Isotope
Laboratories, Woburn, MA .
4. Isopropanol, catalog no . 324-4, Burdick & Jackson, Muskegon, MI .
5. Dry ice, technical grade, Sunnyside Ice, Winston-Salem, NC .
6. Benzene, CAS# 71-43-2, catalog no . 31,995-3, 99% purity, Aldrich
Chemical Co ., Milwaukee, WI .
7 . Isoprene, CAS # 78-79-5, catalog no . I-1, 955-1, 99% purity, Aldrich
Chemical Co ., Milwaukee, WI .
8 . 1,3-Butadiene, CAS # 106-99-0, catalog no . 29,503-5, 99% purity,
Aldrich Chemical Co ., Milwaukee, WI .
9. Acrylonitrile, CAS # 107-13-1, catalog no . 11,021-3, 99% purity,
Aldrich Chemical Co ., Milwaukee, WI .
10 . Styrene, CAS # 100-42-5, catalog no . 24,086-9, 99% purity,
Aldrich Chemical Co ., Milwaukee, WI .
11 . Toluene, CAS # 108-88-3, catalog no . 17,941-B, 99% purity,
Aldrich Chemical Co ., Milwaukee, WI .
12 . Helium, grade 5 BOC, Machine & Welding Co ., Winston-Salem,
NC.
C . Preparation of Standards
NOTE : All of the primary standards are prepared weekly and are stored at
approximately -78°C, then allowed to warm to ambient temperature
prior to use .
No interlaboratory validation process has been conducted using this analytical method .
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1 . Prepare the internal standard stock solution by adding 142 ± 5
mg (weighed accurately to nearest 0 .01 mg) of perdeuterated
benzene and 85 ± 5 mg (weighed accurately to nearest 0 .01
mg) of perdeuterated acrylonitrile by syringe to methanol in a
clean, dry, rubber sleeve stoppered 25-mL volumetric flask .
Dilute to the mark by the syringe addition of methanol . Shake
vigorously to ensure complete mixing . Transfer via syringe to
evacuated capped GC vials . Label and store the internal
standard stock solution at -78°C until use . Internal standard
should be prepared weekly .
2 . Prepare a stock solution by addition of acrylonitrile (65 mg),
benzene (210 mg), toluene (340 mg) and styrene (65 mg),
weighed to the nearest 0 .1 mg, to a 10-mL volumetric flask and
dilute to volume with methanol (Stock 1) . Prepare a second
stock solution containing 1,3-butadiene (5 mg) and isoprene (45
mg), weighed to the nearest 0 .1 mg, in a 10-mL volumetric flask
similarly (Stock 2) . Prepare five calibration standards by the
addition of internal standard stock solution (100 µL) to each of
five clean, dry, rubber sleeve stoppered 25-mL volumetric flasks
containing 20-mL of methanol . Add µL amounts of Stock 1 and
Stock 2 to the five volumetric flasks as indicated below (Table 1)
and dilute to volume with methanol via syringe . Final standard
concentrations are calculated as µg/cigt by multiplying the
standard weight by the dilution volume and dividing by the
number of cigarettes smoked .
Table 1 . Preparation of Standards
Calibration Standard 11'
(2 µL Stock I and 40 µL of Stock 2)
Analyte Final Conc . ()ig/cigt)
1,3-butadiene 12 .31
acrylonitrile 4 .15
isoprene 57 .75
benzene 14 .02
toluene 22 .38
styrene 4 .52
a AII calibration standards contain 189 .3 µg/cigt perdeuterated benzene
and 113 .3 µg/cigt perdeuterated acrylonitrile .
No interlaboratory validation process has been conducted using this analytical method .
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Calibration Standard 2a
(5 µL Stock I and 100 µL of Stock 2)
Analyte Final Conc . (µglcigt)
1,3-butadiene 30 .77
acrylonitrile 10 .38
isoprene 144 .37
benzene 35.05
toluene 55.95 77
styrene 11 .31 ~
a AII calibration standards contain 189 .3 µg/cigt perdeuterated benzene
and 113 .3 µglcigt perdeuterated acrylonitrile .
Calibration Standard 3a
(10 µL Stock I and 400 µL of Stock 2)
Analyte Final Conc . (µg/cigt)
1,3-butadiene 123 .07
acrylonitrile 20 .76
isoprene 577 .47
benzene 70.11
toluene 111 .89
styrene 22 .62
a AII calibration standards contain 189 .3 µglcigt perdeuterated benzene
and 113 .3 µg/cigt perdeuterated acrylonitrile .
Calibration Standard 4'
(20 µL Stock 1 and 800 µL of Stock 2)
Analyte Final Conc . (µglcigt)
1,3-butadiene 246 .13
acrylonitrile 41 .52
isoprene 1154 .93
benzene 140 .21
toluene 223 .79
styrene 45 .23
a All calibration standards contain 189 .3 µg/cigt perdeuterated benzene
and 113 .3 µg/cigt perdeuterated acrylonitrile .
No interlaboratory validation process has been conducted using this analytical method .
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Calibration Standard 5a
(40 µL Stock 1 and 1600 µL of Stock 2)
Analyte Final Conc . (µg/cigt)
1,3-butadiene 492 .27
acrylonitrile 83.04
isoprene 2309 .87
benzene 280 .43
toluene 447 .57
styrene 90 .47
a AII calibration standards contain 189 .3 µg/cigt perdeuterated benzene
and 113 .3 µglcigt perdeuterated acrylonitrile .
3 . Prepare all calibration standards and the internal standard on a
weekly basis . Use a new set of standards for quantitative
analysis of each of the three subgroups of cigarettes as
described in the protocol' . Store all calibration standards at -
78°C until use . Allow to warm to ambient temperature prior to
GC/MS analysis .
D . Instrument Conditions
1 . Set the following GC conditions :
Column : DB-1 column (60 m x 0 .32 mm x 5 µm film)
Head pressure : 12 psi, helium carrier gas
Injection volume: I µL
Split vent flow : 55 mUmin at 250°C
Inlet Liner: Jennings cup
Initial oven temperature : 0°C
Initial oven temperature rate : 2 .5°C/min
Second oven temperature : 90°C .
Second temperature rate : 20°C/min
Final oven temperature : 275°C
Final oven time : 5 minutes
Runtime : 50 minutes 15 seconds
Retention times : see Table 2
2 . Set the following mass spectrometer conditions :
Detector mode : SIM
Monitor ions : see Table 2, underlined ions are used for
quantitation, other ions are qualifier ions
Multiplier voltage : 200 EMV offset
No interlaboratory validation process has been conducted using this analytical method .
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Tuning program: maximum sensitivity autotune
A typical standard chromatogram is shown in Figure 1 . A total ion
chromatogram of Kentucky Reference 1 R4F is shown in Figure 2 . Figure
3 shows a total ion chromatogram of a blank smoke sample . Extracted
ion chromatograms of the analytes are shown in Figures 4-9 . The
integration parameters used to generate peak areas are shown in Table 3 .
Table 2 . Selected Ions Monitored
Component Ions Resolution DwellTime RT(min)
Monitored (msec)
1,3-Butadiene 53,54 low 100 9 .4
Acrylonitrile 52,53 high 75 17 .6
Acrylonitrile-d3 54,56 high 75 17 .5
Isoprene 67,68 high 75 18 .4
Benzene 77,78 low 100 33 .6
Benzene-d6 82,84 low 100 33 .4
Toluene 91,92 low 100 40.8
Styrene 104 low 100 44 .7
Table 3 . Integration Parameters
Events Value Time
Initial Area Reject 0 Initial
Initial Peak Width 0.07 Initial
Shoulder Detection Off Initial
Initial Threshold 12 .0 Initial
Force through zero No
V . PROCEDURE
A . Smoking of Cigarettes
1 . Generate SSVP from three cigarettes using a modified Filtrona
SM350 linear twenty-port smoking machine fitted with an RJR
sidestream portable flow system (Figure 10) . Block 50 % of the
cigarette ventilation by taping . Mark cigarettes for butt lengths
(tipping plus 3 mm) and insertion depths into holders . Trap
SSVP using a series of four impingers (70 ml volume, with
coarse fritted stem extended and containing 15 ml of methanol) .
Connect the four impingers in series via nalgene tubing
(dedicated tubing for each cigarette type - 5/16 in ID ) and wrap
all seals with sealing film . Cool the first trap to 0 ± 2°C and the
remaining three to -78± 2°C (dry ice and isopropanol) (Figure
No interlaboratory validation process has been conducted using this analytical method .
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11) . Trap MSPP onto a 44-mm Cambridge pad . Collect
sidestream smoke using the fishtail chimney (Figures 12 & 13) .
Equip the top of the fishtail chimney with a 44-mm Cambridge
pad assembly via nalgene tubing . Pull a flow rate of 3L/min
through the entire sidestream system . Maintain the hood flow at
800 ftlmin . Configure the smoking machine to take one 45 mL
puff every 30 seconds of two second puff duration . Determine
the mass of the 44-mm Cambridge pads used for collection of
WTPM prior to and after smoke generation . Smoke cigarettes
to the marked butt length . After smoke collection, maintain all
four impingers at -78 ± 2°C . Transfer the contents of each into
impinger #2 . Add internal standard stock solution (100 µL) and
mix combined impinger contents . Transfer the solution into GC
vials, ensuring that the temperature remains near -78 ± 2°C
during all sample transfers. Weigh and record the 44-mm
Cambridge pads and holders . Determine the WTPM per
cigarette by subtracting the after smoking pad and holder
weights from the before smoking pad and holder weights and
dividing by the number of cigarettes smoked . Record puff count
for each smoking and express on a per cigarette basis .
2 . For each analysis, generate blank samples by taking 13 puffs of
air through the system per cigarette for a total of 39 puffs per
blank smoke sample .
3 . Analyze Kentucky Reference 1 R4F cigarettes with each
analysis in order to ensure consistency of the data .
B . Preparation of Calibration Curve
1 . Transfer the five calibration standards to the autosampler tray of
the HP 5890 GC . Construct a sequence beginning with injection
of a methanol blank followed by the calibration standards . Add
samples for analysis to the sequence table . A new series of
calibration standards should be analyzed every 2-3 days .
2 . Prepare calibration curves by regression analysis using the
internal standard method . Utilize acrylonitrile-d3 as internal
standard for 1,3-butadiene, acrylonitrile and isoprene . Utilize
benzene-d6 as internal standard for benzene, toluene and
styrene quantitation . Perform all quantitation using the HP
Chemstation software . Manually inspect each peak and
manually reintegrate improperly integrated peaks . Integration
parameters used to qualify peaks are listed in Table 3 .
No interlaboratory validation process has been conducted using this analytical method .
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3. Tune the mass spectrometer using "maximum sensitivity
autotune" conditions with perFlurotributylamine as calibration
standard .
4 . Load the GC-MS method . Start the sequence . Correlation
coefficients for the calibration curves should be no less than
0 .98 .
C . Sample Analysis
1 . Place samples in the GC autosampler tray following the
calibration standards . Analyze samples in the same order as
described in the study protocol' .
2 . Quantitate samples using the HP Chemstation quantitation
software by the internal standard method . Utilize acrylonitrile-d3
as internal standard for 1,3-butadiene, acrylonitrile and
isoprene . Utilize benzene-d6 as internal standard for benzene,
toluene and styrene quantitation . Perform all quantitation using
the HP Chemstation software . Manually inspect each peak and
manually reintegrate improperly integrated peaks .
3 . Accept the results from test cigarettes relying on the criteria
documented in the study protocol' .
D . Calculations
Utilize internal standard regression analysis of calibration
standards for analyte concentration determination . Calculate
standards in terms of pg/cigt by multiplying the weight of the
standard by the volume of the dilution and dividing the result by
the number of cigarettes smoked . Perform quantitation using the
HP Chemstation programming . The software interpolates the
concentration of the analyte according to the formula C=(y-b)/m,
where :
C= Analyte concentration in sample (pg/cigt)
y= ratio of target compound area to ISTD area
b = calibration curve intercept
m= slope of calibration curve
No interlaboratory validation process has been conducted using this analytical method .
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VI . REFERENCES
1 . Massachusetts Constituents Testing Study Protocol, Brown & Williamson
Tobacco Corporation, Lorillard Tobacco Company, Philip Morris Incorporated
and R . J . Reynolds Tobacco Company, April 20, 1999 .
2 . Byrd, G .D ., K .W . Fowler, R . D . Hicks, M .E. Lovette and M . F . Borgerding,
Isotope Dilution Gas Chromatography-Mass Spectrometry in the
Determination of Benzene, Toluene, Styrene and Acrylonitrile in Mainstream
Cigarette Smoke, Journal of Chromatography, 503, 359-368, 1990.
3 . Darrall, K .G ., J .A Figgins, R .D . Brown and G .F . Phillips, Determination of
Benzene and Associated Volatile Compounds in Mainstream Cigarette
Smoke, Analyst, 123, 1095-1101, 1998 .
4 . Brunnemann, K .D ., M .R . Kagan, J .E . Cox and D . Hoffmann, Analysis of 1,3-
Butadiene and Other Selected Gas-Phase Components in Cigarette
Mainstream and Sidestream Smoke by Gas Chromatography-Mass Selective
Detection, Carcinogenisis, 11, 1863-1868, 1990 .
5. Brunnemann, K .D ., M .R . Kagan, , J .E.Cox and D . Hoffmann, Determination of
Benzene, Toluene and 1,3-Butadiene in Cigarette Smoke by GC-MSD, Exp .
Pathol., 37, 108-113, 1989 .
No interlaboratory validation process has been conducted using this analytical method .
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Fi ure 1
Total Ion Chromatogram for Standard 3
18000
1eoao
17000
18000
1 sooo
14000
13000
12000
11000
10000
9000
8000
7000
9000
5000
4000
3000
2000
1000
a
Fi ure 2
Total Ion Chromatogram for Kentucky Reference I R4F
85000
8000.0
75000
70D00
65W
60000
55000
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
Tim->
No interlaboratory validation process has been conducted using this analytical method .
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Figure 3
Total Ion Chromatogram for a Blank Smoke Sample
Abundarica
TIC : '150'1 OlS .O
22 000 1
20000 '
18000
1 .3000i
i
14000 +
~
12000 i
1007
BO00 I il
I
BO00 •'
4000 I
20001
5 .00 '10_00 15 .00 20 .00 25 .00 30 .00 35 .00 40 .00 45 .00
No interlaboratory validation process has been conducted using this analytical method .
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Fi ure 4
Extracted Ion Chromatogram for 1,3-Butadiene in 1R4F
Abundance
lon 53 .00 (52 .70 to 53 .70) : 1401006 .~
i lon 54 .00 (53 .70 to 54 .70) : 140";1 006 . E:)
3500'
3000f1j
2500
1
2000I
1500'
1000
5001
O~
8 .20 8 .40 8 .60 8 .80 9 .00 9 .20 9 .40 9 .60 9 .8010 .0010 .2010 .4010 .601 O_80
Tima-->
Figure 5
Extracted Ion Chromatogram for Acrylonitrile and Acrylonitrile-d3 in 1 R4F
~ non 53.00(52.70to53.70)_1401006.D
,
;
1400 I Ilan 54.00 (53.70 to 54.~ 0): 140,1006.D
1200 lion 56.00 (55 .70 to 470) : 1401 b06 . D
i
1000 ;
i
800 ,
600
200 1 i-
-~_. _,_ ------- ~~
0 i
18.10 18.20 18 .30 18.40 18.50 18 .60 18. 70 18.80 18.90 19.00 19.10
No interlaboratory validation process has been conducted using this analytical method .
92 of 98
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Figure 6
Extracted Ion Chromatogram for Isoprene in 1R4F
eaooo Ion 67.00 (66 .70 to 67 .70) : 1501008 .9
55000 Ion 68 .00 (67 .70 to 68 .70) : 1501008 .b
50000 i
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
1B .o0
Figure 7
Extracted Ion Chromatogram for Benzene and Benzene-d6 in 1 R4F
Ion 84 .00 (83 .70 to 84.70) : 1501008 .D
Ion 82 .00 I,(~8,1 .70 to 82.70): 1501008 .D
~1
Ion 78 .Oq (7~j .70 to 78.70): 1501008 .D
lon 77 .0?(76I70 to 77 .70) : 1501008 .D
a
No interlaboratory validation process has been conducted using this analytical method .
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Fi ure8
Extracted Ion Chromatogram for Toluene in 1 R4F
Ion 91,40 (90 .70 to 91 .70) : 1401006 .D
50000
45000 Ion 9$ .0~ (91 .70 to 92.70) : 1401006 .D
40" I 1i
35000
i
30000
25000
20000
15000 j I ~ II
10000
5000
0 '
4^`^ Trat~- - d1 7H -4YM --41 33- .---
TIm6->
Figure 9
Extracted Ion Chromatogram for Styrene in 1R4F
8000 lon 104 :100 (103 .70 to 104 .70) : 1501008 .D
i
7000
I
6000
5000
~
4000 ~
3000
~
2000
1000 i
.___-------__
0 __-_ _- ~ - - --__
~aa nn aa 7n da 00, aa 90 . 45 on d5 10 aS Jn 45'tn 450 45 So . , 4`y.60- 45, 4,0~ 4' pn~_5 ~n
No interlaboratory validation process has been conducted using this analytical method .
94 of 98
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Figure 10
Modified Filtrona Sidestream Smoking Machine
No interlaboratory validation process has been conducted using this analytical method .
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Figure 11
Sidestream Traps on Modified Filtrona Smoking Machine
No interlaboratory validation process has been conducted using this analytical method .
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Figure 12
Fishtail Chimney Used for Sidestream Smoking
No interlaboratory validation process has been conducted using this analytical method .
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Figure 13
Schematic of Fishtail Chimney
BAT Fishtail Chimney
Rear V iew All measurements in millimeters
27
-24
I
Front View
25
Side View
90
23 Bottom View
No interlaboratory validation process has been conducted using this analytical method .
98 of 98
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