[Federal Register Volume 62, Number 85 (Friday, May 2, 1997)]
[Notices]
[Pages 24116-24119]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-11344]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention


Protocol to Measure the Quantity of Nicotine Contained in 
Smokeless Tobacco Products Manufactured, Imported, or Packaged in the 
United States

AGENCY: Centers for Disease Control and Prevention (CDC), Department of 
Health and Human Services.

ACTION: Request for comments.

-----------------------------------------------------------------------

SUMMARY: CDC's Office on Smoking and Health (OSH) is requesting 
comments from all interested parties on a standard methodology for 
measurement of quantity of nicotine in smokeless tobacco. The 
Comprehensive Smokeless Tobacco Health Education Act of 1986 (15 U.S.C. 
4401 et seq., Pub. L. 99-252) requires that each person who 
manufactures, packages, or imports smokeless tobacco provide the 
Secretary of HHS annually with a report on the quantity of nicotine 
contained in smokeless tobacco products; OSH has been delegated the 
authority to implement the nicotine reporting provisions of this law. 
The methodology (``Protocol for Analysis of Nicotine, Total Moisture, 
and pH in Smokeless Tobacco Products'') is the basis for such nicotine 
reporting and is intended to provide standardized measurement of 
nicotine, total moisture, and pH in smokeless tobacco products.

DATES: Written comments to this notice should be submitted to Patricia 
Richter, Centers for Disease Control and Prevention (CDC), Office on 
Smoking and Health, 4770 Buford Highway, NE., Mailstop K50, Atlanta, 
Georgia 30341-3724 on or before June 2, 1997. Comments may also be 
faxed to Patricia Richter at (770) 488-5848 or submitted by email to 
[email protected] as WordPerfect 5.0, 5.1/5.2, 6.0/6.1 or ASCII files.

FOR FURTHER INFORMATION CONTACT: Patricia Richter, Centers for Disease 
Control and Prevention (CDC), Office on Smoking and Health, 4770 Buford 
Highway NE., Mailstop K50, Atlanta, Georgia 30341-3724; telephone: 
(770) 488-5703.

SUPPLEMENTARY INFORMATION: In 1989, the smokeless tobacco industry 
submitted a business review letter to the Department of Justice (DOJ), 
in accordance with 28 CFR 50.6. This letter requested approval of a 
collaborative industry effort to determine standard nicotine reporting. 
Previous to this, each company employed different methods of nicotine 
and moisture analysis; however, HHS requested that a standard 
methodology be developed to ensure the accuracy and reliability of the 
information on nicotine and moisture, as well as to ensure 
comparability of the data. HHS did not have the resources to develop 
such a standardized methodology thus necessitating a collaborative 
industry process to develop the methodology.
    In January 1993, DOJ extended permission to the smokeless industry 
to begin the development of uniform methods for analyzing smokeless 
tobacco products for nicotine and moisture content. The smokeless 
tobacco industry formed a work group, which represented the ten major 
domestic manufacturers of smokeless tobacco. The first meeting of the 
work group was on July 7, 1993 and the group continued to meet 
throughout 1993 and 1994. After this series of meetings, a standard 
methodology was approved by the work group and submitted to OSH. The 
protocol was revised by OSH based on individual comments received from 
peer reviewers and the Division of Environmental Health Laboratory 
Sciences, National Center for Environmental Health, CDC. Once OSH has 
received comments, it will review the comments, make the necessary 
changes to the methodology, and publish the final methodology in the 
Federal Register. Once the final methodology has been published, OSH 
will implement the nicotine reporting requirements of the Act.

    Dated: April 24, 1997.
Joseph R. Carter,
Acting Associate Director for Management and Operations, Centers for 
Disease Control and Prevention (CDC).

Standardized methodology: Protocol for Analysis of Nicotine, Total 
Moisture, and pH in Smokeless Tobacco Products

I. Requirements 1, 2

A. Reagents \3\
    1. 2 N Sodium hydroxide (NaOH)
    2. Methyl t-butyl ether (MTBE)
    3. (-)-Nicotine (Fluka 72290) >99% purity \4\
    4. Quinoline (Aldrich)
    5. Standard pH buffers; 7.00 and 10.00
    6. Deionized distilled water
B. Glassware and supplies
    1. Volumetric flasks
    2. 25 mm x 200 mm Pyrex culture tubes with Teflon lined screw 
caps (Mfr #982625X)
    3. Pasteur pipettes
    4. Repipettors (10 mL and 50 mL)
    5. Linear shaker (configured to hold tubes in horizontal 
position) 5, 6
    6. Moisture dish--Al, diam. 45-65 mm, depth 20-45 mm, with tight 
fitting cover
    7. Teflon-coated magnetic stirring bar
    8. 50 mL polypropylene container
C. Instrumentation
    1. Robot Coupe Model RSI 6V Scientific Batch Processor or 
equivalent
    2. Capillary gas chromatograph with modified split capability 
(splitless/split), flame ionization detector, integrator, a 4 mm 
split/splitless glass liner and a 30 m  x  0.32 mm ID fused silica 
column crosslinked and coated with 5% phenyl and 95% methyl silicone 
at 1 m film thickness.
    3. Orion Model SA 720 pH meter equipped with Orion 8103 Ross 
Combination pH electrode.
D. Additional Equipment

    Forced-draft oven, regulated to 99.5  0.5  deg.C. 
Suggested dimensions: 19 x 19 x 19'' (48 cm). Approx. oven settings: 
fresh air intake vent \1/5\ open; air control damper \1/4\ open; air 
exhaust vent \1/3\ open.
E. Chromatographic Conditions 7, 8
    1. Detector temperature: 250  deg.C
    2. Injector temperature: 250  deg.C
    3. Flow rate at 100  deg.C--1.7 mL/min; with split ratio of 40:1 
\9\
    4. Injection volume: 2 l
    5. Column conditions: 110-185  deg.C at 10  deg.C min-1; 185-240 
 deg.C at 6  deg.C min -1, hold at final temperature for 
10 min. Equil. time: 5 min.
F. Sample Preparation \10\

    There exist six different categories of commercial smokeless 
tobacco products:

    1. Dry snuff;
    2. Wet snuff;
    3. Wet snuff portion packs;
    4. Plug;
    5. Twist; and
    6. Loose leaf.

    Because of their physical characteristics, samples of three of the 
six product categories must be ground before nicotine, total moisture, 
and pH analyses can be conducted. The objective of grinding the samples 
is to obtain a homogeneous sample with particles measuring 
approximately 4 mm. Grinding to achieve this particle size should take 
no more than 3 minutes. To ensure proper grinding and an adequate 
amount of the ground sample for analysis, the minimum sample size of 
all commercial products to be ground should not be less than 100 grams.
    To ensure precision of analyses for nicotine, total moisture, 
and pH, the samples

[[Page 24117]]

that require grinding should be ground using a Robot Coupe Model RSI 
6V Scientific Batch Processor or its equivalent. This is a variable 
speed (0 to 3000 RPM) processor. The variable speed motor is 
required to ensure proper grinding of the tobacco tissues (and in 
the case of pH determination, the wet snuff portion pack). Elevated 
temperatures can result in moisture loss and an underestimated value 
for moisture content. Hence, care must be taken during grinding to 
avoid elevated temperatures. The bowl should be cleaned after each 
grinding to obtain accurate results.
    1. Dry snuff. Dry snuff samples do not need to be ground since 
the product is a powder. The sample must be thoroughly mixed before 
weighing for nicotine, total moisture, and pH analysis.
    2. Wet snuff. Wet snuff samples do not need to be ground. The 
sample must be thoroughly mixed before weighing for nicotine, total 
moisture, and pH analysis.
    3. Wet snuff portion packs. The tobacco contents of the wet 
snuff portion packs do not need to be ground for nicotine, total 
moisture, or pH analysis. The tobacco packaging material (the 
``pouch'') should be separated from the tobacco and ground to obtain 
particles measuring approximately 4 mm for pH analysis. The tobacco 
of the wet snuff portion pack and the ground pouch are combined and 
thoroughly mixed before pH analysis.
    4. Plug tobacco. Break or cut apart plugs and add in portions to 
grinder at 2000 RPM. Reduce RPM or stop grinding if sample bowl 
becomes warm. Pulse the Robot Coupe, when needed, to complete 
grinding. Grind samples until approximately 4 mm in size. The total 
grinding time should be no more than 3 minutes.
    5. Twist tobacco. Separate twists, add to grinder and grind at 
2000 RPM. Reduce RPM or stop grinding if sample bowl becomes warm. 
Continue grinding until sample particles are approximately 4 mm in 
size. The total time for grinding should be no more than 3 minutes.
    6. Loose leaf. Grind in the same manner as described in 4 and 5 
to obtain product with particle size of approximately 4 mm.

II. Nicotine Analysis

A. Calibration Standards

1. Internal Standard (IS)

    Weigh 10.00 grams of quinoline, transfer to a 250 mL volumetric 
flask and dilute to volume with MTBE. This solution will be used for 
calibration of the instrument for the nicotine calibration curve 
(II.A.2), for the standards addition assay (II.B), and for 
preparation of the extracting solution (II.D).

2. Nicotine Calibration Curve

    a. Weigh 1.0000 gram of nicotine into a clean, dry 100 mL 
volumetric flask and dilute to volume with MTBE. This gives a 
nicotine concentration of 10 mg/mL for the stock solution.
    b. Accurately pipette 0.5 mL of IS from stock solution (II.A.1) 
to five clean, dry 50 mL volumetric flasks. To prepare a nicotine 
standard corresponding to a concentration of 0.8 mg/mL, pipette 
exactly 4.0 mL of the nicotine standard (II.A.2.a) to a 50 mL 
volumetric flask containing the internal standard and dilute to 
volume with MTBE. To obtain nicotine concentrations equivalent to 
0.6, 0.4, 0.2, and 0.1 mg/mL, pipette precisely 3.0, 2.0, 1.0, and 
0.5 mL, respectively, of the nicotine standard into the four 
remaining flasks and dilute to volume with MTBE.
    c. Transfer aliquots of the five standards to auto sampler vials 
and determine the detector response for each standard using gas 
chromatographic conditions described in I.E.
    d. Calculate least squares line for linear equation from these 
standards by obtaining the ratio of Areanicotine/
AreaIS. This ratio will be the Y value and the 
concentration of nicotine will be the X value for determining the 
linear equation of the line (Equation 1):

Equation 1:

Y=a+bX;

Where:

X=Concentration of nicotine in mg
Y=Areanicotine/AreaIS
a=intercept on the ordinate (y axis)
b=slope of the curve

    The final result will be reported in the following units:
    Concentration of nicotine=mg of nicotine/gram of tobacco sample.
    e. Determine the recovery of nicotine by pipetting 10 mL of the 
0.4 mg/mL nicotine standard to a screw capped tube containing 1.0 mL 
of 2 N NaOH. Cap the tube. Shake the contents vigorously and allow 
the phases to separate. Transfer an aliquot of the organic phase to 
an injection vial and inject. Calculate the concentration of 
nicotine using the equation of the line in II.A.2.d above. This 
should be repeated two more times to obtain an average of the three 
values. The recovery of nicotine can be obtained by using the 
following equation:

Equation 2:

Recovery=Nicotinecalculated/Nicotineactual

B. Standards Addition Assay

    Prior to analyzing a smokeless tobacco product for nicotine 
content, the testing facility must validate the system to verify 
that matrix bias is not occurring during nicotine extraction. This 
is done by analyzing the nicotine calibration standards in the same 
vegetable matrix as the smokeless tobacco. The standards addition 
assay should be performed with each smokeless tobacco product 
tested.
    1. Using an analytical balance, accurately weigh 1.000 
 0.020 gram of the homogeneous, prepared tobacco sample 
into a culture tube. Repeat this five times for a total of 6 culture 
tubes containing the smokeless tobacco product. Record the weight of 
each sample.
    2. To prepare a nicotine standard corresponding to a 
concentration of 0.8 mg/mL, pipette exactly 4.4 mL of the nicotine 
standard (II.A.2.a) to one of the culture tubes. To obtain nicotine 
concentrations equivalent to 0.6, 0.4, 0.2, and 0.1 mg/mL, pipette 
precisely 3.3, 2.2, 1.1, and 0.55 mL, respectively, of the nicotine 
standard into four of the remaining culture tubes. One of the 
culture tubes is not supplemented with nicotine and serves as an 
analytical blank. Allow the samples to equilibrate for 10 minutes.
    3. Pipette 5 mL of 2 N NaOH into each tube. Cap each tube. Swirl 
to wet sample and allow to stand 15 minutes.\11\
    4. Pipette 50 mL of extraction solution (II.D.1) into each tube. 
Cap each tube and tighten.\12\
    5. Place tubes in rack(s), place racks in linear shaker in 
horizontal position and shake for two hours.
    6. Remove rack(s) from shaker and place in vertical position to 
allow the phases to separate.
    7. Allow the solvent and nicotine supplemented samples and the 
blank to separate (maximum 2 hours).
    8. Transfer aliquots of the five standards and the blank from 
the extraction tubes to sample vials and determine the detector 
response for each using gas chromatographic conditions described in 
I.E.
    9. Subtract the Areanicotine/AreaIS of the 
blank from the Areanicotine/AreaIS of each of 
the standards.
    10. Calculate least squares line for linear equation from the 
corrected standards as described above (Equation 1) in II.A.2.d.
    The final corrected result will be reported in the following 
units:
    Concentration of nicotine = mg of nicotine/gram of tobacco 
sample.
    11. Determine the recovery of nicotine by pipetting 10 mL of the 
0.4 mg/mL nicotine standard to a screw capped tube containing 1.0 mL 
of 2 N NaOH and 10 mL of extraction solution (II.D.1). Cap the tube 
and tighten. Shake the contents vigorously and allow the phases to 
separate. Transfer an aliquot of the organic phase to an injection 
vial and inject. Calculate the concentration of nicotine using the 
equation of the line above in II.A.2.d. This should be repeated two 
more times to obtain an average of the three values. The recovery of 
nicotine can be obtained by using Equation 2:

Recovery = Nicotinecalculated/Nicotineactual

    12. Compare the results of steps II.A.2. and II.B. If they 
differ by a factor of 10% or more, the recovery of nicotine from the 
aqueous matrix is not equivalent to recovery from the vegetable 
matrix of the smokeless tobacco product. In this instance, the 
nicotine concentration of the smokeless tobacco product must be 
determined from a nicotine calibration curve prepared from nicotine 
standards in a vegetable-based matrix.

C. Quality Control Pool

    At least two quality control pools prepared in the smokeless 
tobacco product matrix are recommended to be included in each 
analytical run. The smokeless tobacco product should be enriched 
with nicotine at the high and low ends of expected values for the 
smokeless tobacco product. The pools must be analyzed in duplicate 
in every run. The quality control pool must be prepared in 
sufficient quantity to last for all analyses of a product lot.

D. Sample Extraction Procedure

    1. Extraction solution is prepared by pipetting 10 mL of the IS 
from the stock solution (II.A.1) to a 1000 mL volumetric flask and 
diluting to volume with MTBE.
    2. Using an analytical balance, accurately weigh 1.000 
 0.020 gram of prepared

[[Page 24118]]

tobacco sample into culture tube and record weight.13 The 
number of products sampled per lot should reflect an acceptable 
level of precision.14 The test material is to be 
representative of the product that is sold to the public and 
therefore should consist of sealed, packaged samples from each lot 
of finished product that is ready for commercial distribution.
    Triplicate determinations will provide precision data.
    3. Pipette 5 mL of 2 N NaOH into the tube. Cap the tube. Swirl 
to wet sample and allow to stand 15 minutes.11
    4. Pipette 50 mL of extraction solution into tube, cap tube and 
tighten.12
    5. Place tubes in rack(s), place racks in linear shaker in 
horizontal position and shake for two hours.
    6. Remove rack(s) from shaker and place in vertical position to 
allow the phases to separate.
    7. Allow the solvent and sample to separate (maximum 2 hours). 
Transfer an aliquot from the extraction tube to a sample vial and 
cap.
    8. Analyze the extract using GC conditions as described above 
(I.E) and calculate the concentration of nicotine using the linear 
calibration equation. Correct percent nicotine values for both 
recovery and weight of sample by using Equation 3.15
Equation 3:16
[GRAPHIC] [TIFF OMITTED] TN02MY97.049

    9. Report the final nicotine determination as mg of nicotine per 
gram of the tobacco product (mg nicotine/gram), to an accuracy level 
of two decimal places. All data should include the mean value with a 
95% confidence interval, the range of values, the number of samples 
tested per lot, and the estimated precision of the mean. Information 
will be reported for each manufacturer and variety (including brand 
families and brand variations) and brand name (e.g., Skoal Bandits, 
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).

III. Total Moisture Determination

    A. This procedure is referred to as ``Total Moisture 
Determination'' because AOAC Method 966.02 determines water and 
tobacco constituents that are volatile at temperatures of 
99.50.5 deg.C.
    B. Accurately weigh 5.00 grams of the sample (ground to pass 
 4 mm screen) 17 into a weighed moisture dish 
and place uncovered dish in oven.18 The number of 
products sampled per lot should reflect an acceptable level of 
precision.14 The test material is to be representative of 
the product that is sold to the public and therefore should consist 
of sealed, packaged samples from each lot of finished product that 
is ready for commercial distribution. Triplicate determinations will 
provide precision data.
    C. Do not exceed 1 sample/10 sq in. (650 sq cm) shelf space, and 
use only 1 shelf. Dry 3 hr at 99.5  0.5  deg.C. Remove 
from oven, cover, and cool in desiccator to room temp. (about 30 
min). Reweigh and calculate percent moisture.
    D. Report the final moisture determination as a percentage (%), 
to an accuracy level of one decimal place. All data should include 
the mean value with a 95% confidence interval, the range of values, 
the number of samples tested per lot, and the estimated precision of 
the mean. In addition, information for each manufacturer and variety 
(including brand families and brand variations) and brand name 
(e.g., Skoal Bandits, Skoal Long Cut Cherry, Skoal Long Cut 
Wintergreen, etc.) will be reported.

IV. pH Measurement

    A. Test samples as soon as possible after they are received. The 
number of products sampled per lot should reflect an acceptable 
level of precision.14 The test material is to be 
representative of the product that is sold to the public and 
therefore should consist of sealed, packaged samples from each lot 
of finished product that is ready for commercial distribution. 
Triplicate determinations will provide precision data.
    B. Accurately weigh 2.00 grams of the sample. Place in a 50 mL 
polypropylene container with 10 mL deionized distilled water.
    C. Place teflon-coated magnetic stirring bar in container and 
stir mixture continuously throughout testing.
    D. Measure pH of sample after two-point calibration with 
standard pH 7.00 and 10.00 buffers on a pH meter calibrated to an 
accuracy of two decimal places.
    E. Calculate the mean of pH values at 5, 15, 30, and 60 minutes.
    F. Report the final pH determination to an accuracy level of two 
decimal places. All data should include the mean value with a 95% 
confidence interval, the range of values, the number of samples 
tested per lot, and the estimated precision of the mean. Information 
will be reported for each manufacturer and variety (including brand 
families and brand variations) and brand name (e.g., Skoal Bandits, 
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).
    G. Estimate the ``free base nicotine'' content with the 
Henderson-Hasselbalch equation (Equation 4), based on measured pH 
and nicotine content.

Equation 4:
[GRAPHIC] [TIFF OMITTED] TN02MY97.050

pKa = 8.02 (CRC Handbook of Chemistry and Physics, 1989-1990)
[B] = amount of free base nicotine
[BH+] = amount of ionized nicotine

    H. Report the final estimated free base nicotine as a percentage 
(%) of the total nicotine content, to an accuracy level of two 
decimal places and as mg of free base nicotine per gram of the 
tobacco product (mg free base nicotine/gram), to an accuracy level 
of two decimal places. All data should include the mean value with a 
95% confidence interval, the range of values, the number of samples 
tested per lot, and the estimated precision of the mean. Information 
will be reported for each manufacturer and variety (including brand 
families and brand variations) and brand name (e.g., Skoal Bandits, 
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).

Sample calculation:

Mean total nicotine = 10.30 (mg/g)
Mean pH = 7.50
pKa = 8.02
[GRAPHIC] [TIFF OMITTED] TN02MY97.051

[GRAPHIC] [TIFF OMITTED] TN02MY97.052

V. Assay Criteria for Quality Assurance

A. Establishing limits for Quality Control Parameters

    All quality control parameters must be determined within the 
laboratory in which they are to be used. At least 10 within-
laboratory runs must be performed to establish temporary confidence 
intervals for the quality control parameters. Permanent limits 
should be established after 20 runs and should be reestablished 
after each additional 20 runs.

B. Exclusion of Outliers from the Calibration Curve\16\

    The coefficient of determination between 
Areanicotine/AreaIS and nicotine concentration 
should be equal to 0.99 or higher. Any calibration standard having 
an estimated

[[Page 24119]]

concentration computed from the regression equation (Equation 1) 
which is different from its actual concentration by a factor of 10% 
can be excluded from the calibration curve. Up to two concentrations 
may be excluded, but caution should be used in eliminating values, 
since bias may be increased in the calibration curve. If an outlier 
value is eliminated, its duplicate value must also be discarded to 
avoid producing a new bias. All unknowns must fall within the 
calibration curve; therefore, duplicate values excluded at either 
end of the calibration curve will restrict the useful range of the 
assay.

C. Quality Control Pools and Run Rejection Rules

    The mean estimated nicotine concentration in a pool should be 
compared with the established limits for that pool based on at least 
20 consecutive runs. An analytical run should be accepted or 
rejected based upon the following set of rules adapted from Westgard 
et al. (1981).
    1. When the mean of one QC pool exceeds the limit of x 
 3 standard deviations (SD), then the run is rejected as 
out of control. Here, x and SD represent the overall mean and 
standard deviation of all estimated nicotine concentrations for a 
particular pool in the runs which were used to establish the control 
limits.
    2. When the mean nicotine concentrations in two QC pools in the 
same run exceed the same direction, then the run must be rejected. 
The same direction is the condition in which both pools exceed 
either the x + 2 SD or the x-2 SD limits.
    3. When the mean nicotine concentrations in one or two QC pools 
exceed their x + 2 SD limits in the same direction in two 
consecutive runs, then both runs must be rejected.
    4. When the mean nicotine concentrations in two QC pools are 
different by more than a total of 4 SD, then the run must be 
rejected. This condition may occur, for example, when one QC pool is 
2 SD greater than the mean, and another is 2 SD less than the mean.

Endnotes

    The comments and notes listed below can be described as Good 
Laboratory Practice guidelines; they are described in detail in this 
protocol to ensure minimal interlaboratory variability in the 
determination of nicotine, total moisture, and pH in smokeless 
tobacco.
    \1\ This protocol assumes that the testing facility will 
implement and maintain a stringent Quality Assurance/Quality Control 
program to include, but not be limited to, regular interlaboratory 
comparisons, routine testing of random blank samples, determination 
of the quality and purity of purchased products, and proper storage 
and handling of all reagents and samples.
    \2\ When a specific product or instrument is listed, it is the 
product or instrument that was used in the development of this 
method. Equivalent products or instruments may also be used. The use 
of company or product name(s) is for identification only and does 
not imply endorsement by the Centers for Disease Control and 
Prevention.
    \3\ All chemicals, solvents, and gases are to be of the highest 
purity.
    \4\ Companies must ensure that the purity of the nicotine base 
is certified by the vendor and that the chemical is properly stored. 
However, nicotine base oxidizes with storage, as reflected by the 
liquid turning brown. If oxidation has occurred, the nicotine base 
should be distilled prior to use in making a standard solution.
    \5\ Horizontal shaking will allow more intimate contact of this 
three phase extraction. There is a minimal dead volume in the tube 
due to the large sample size and extraction volume. This 
necessitates horizontal shaking.
    \6\ If linear shaker is not available, a wrist action shaker 
using 250 mL stoppered Erlenmeyer flasks can be substituted. Values 
for nicotine are equivalent to those obtained from the linear 
shaker.
    \7\ After installing a new column, condition the column by 
injecting a tobacco sample extract on the column, using the 
described column conditions. Injections should be repeated until 
areas of IS and nicotine are reproducible. This will require 
approximately four injections. Recondition column when instrument 
has been used infrequently and after replacing glass liner.
    \8\ Glass liner and septum should be replaced after every 100 
injections.
    \9\ Most older instruments operate at constant pressure. To 
reduce confusion, it is suggested that the carrier gas flow through 
the column be measured at the initial column temperature.
    \10\ The testing facility must ensure that samples are obtained 
through the use of a survey design protocol for sampling ``at one 
point in time'' at the factory or warehouse. The survey design 
protocol must address short-, medium-and long-term product 
variability (e.g., variability over time and from contai ner to 
container of the tobacco product) as defined by ISO Protocol 8243, 
Annex C. Information accompanying results for each sample should 
include, but not be limited to:
    1. For each product--manufacturer and variety (including brand 
families and brand variations) and brand name (e.g., Skoal Bandits, 
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.) 
information.
    2. Product ``category,'' e.g., loose leaf, plug, twist, dry 
snuff, moist snuff, etc.
    3. Lot number.
    4. Lot size.
    5. Number of randomly sampled, sealed, packaged (so as to be 
representative of the product that is sold to the public) smokeless 
tobacco products selected per lot (sampling fraction) for nicotine, 
moisture, and pH determination.
    6. Documentation of method used for random sample selection.
    7. ``Age'' of product when received by testing facility and 
storage conditions prior to analysis.
    \11\ Use non-glass 10 mL repipette for transferring NaOH 
solution.
    \12\ Use 50 mL repipette for transferring MTBE.
    \13\ For dry snuff, use 0.500  0.010 gram sample.
    \14\ The testing facility is referred to ISO Procedure 8243 for 
a discussion of sample size and the effect of variability on the 
precision of the mean of the sample (ISO 8243, 1991).
    \15\ When analyzing new smokeless tobacco products, extract 
product without IS to determine if any components co-elute with the 
IS or impurities in the IS. This interference could artificially 
lower calculated values for nicotine.
    \16\ The calculated nicotine values for all samples must fall 
within the low and high nicotine values used for the calibration 
curve. If not, prepare a fresh nicotine standard solution and an 
appropriate series of standard nicotine dilutions. Determine the 
detector response for each standard using chromatographic conditions 
described in I.E.
    \17\ The method is a modification of AOAC Method 966.02 (1990) 
in that the ground tobacco passes through a 4 mm screen rather than 
a 1 mm screen.
    \18\ When drying samples, do not dry different products (e.g., 
wet snuff, dry snuff, loose leaf) in the oven at the same time since 
this will produce errors in the moisture determinations.

References

    AOAC (Association of Official Analytical Chemists). Official 
Methods of Analysis. 966.02: Moisture in Tobacco. (1990) Fifth 
Edition. K. Helrich (ed). Association of Official Analytical 
Chemists, Inc. Suite 400, 2200 Wilson Boulevard, Arlington, Virginia 
22201 USA.
    CRC handbook of chemistry and physics. RC Weast, DR Lide, MJ 
Astle, and WH Beyer (eds). 70th ed. Boca Raton, Florida: CRC Press 
(1989-1990) D-162.
    Henningfield, J.E., Radzius A., Cone E.J. (1995). Estimation of 
available nicotine content of six smokeless tobacco products. 
Tobacco Control 4:57-61.
    ISO (International Organization for Standardization). IOS 8243: 
Cigarettes--Sampling. (1991). Second Edition. Prepared by Technical 
Committee ISO/TC 126, Tobacco and tobacco products. International 
Organization for Standardization, Case Postale 56, CH-1211 Geneve 
20, Switzerland.
    Westgard JO, Barry P, Hunt M, and Groth T. (1981). A multi-rule 
Shewhart chart for quality control in clinical chemistry. Clinical 
Chemistry 27:493.

[FR Doc. 97-11344 Filed 5-1-97; 8:45 am]
BILLING CODE 4163-18-P