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

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]

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