[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.
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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
pir1@cdc.gov 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
