Total Reducing Sugars Analytical Method, 62524-62527 [06-8836]

Agencies

• Alcohol and Tobacco Tax and Trade Bureau
• DEPARTMENT OF THE TREASURY

[Federal Register Volume 71, Number 206 (Wednesday, October 25, 2006)]
[Notices]
[Pages 62524-62527]
From the Federal Register Online via the Government Printing Office [www.gpo.gov]
[FR Doc No: 06-8836]



Federal Register / Vol. 71, No. 206 / Wednesday, October 25, 2006 / 
Notices

[[Page 62524]]


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DEPARTMENT OF THE TREASURY

Alcohol and Tobacco Tax and Trade Bureau

[Notice No. 66]


Total Reducing Sugars Analytical Method

AGENCY: Alcohol and Tobacco Tax and Trade Bureau, Treasury.

ACTION: General notice.

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SUMMARY: The Alcohol and Tobacco Tax and Trade Bureau issues this 
notice to describe the analytical method that it uses to determine the 
total reducing sugars content in the cigars and cigarettes that it 
analyzes. We reference this method in our notice of proposed rulemaking 
entitled ``Tax Classification of Cigars and Cigarettes,'' which is 
published elsewhere in this issue of the Federal Register. We are 
giving notice of this method so that interested members of the public 
may use the same method to evaluate the total reducing sugars of 
tobacco products for the purpose of commenting on the proposed rule.

FOR FURTHER INFORMATION CONTACT: Linda Wade Chapman, Regulations and 
Rulings Division, Alcohol and Tobacco Tax and Trade Bureau, 1310 G 
Street NW., Suite 200-E, Washington, DC 20220; telephone 202-927-8210.

SUPPLEMENTARY INFORMATION:

Background

    The Alcohol and Tobacco Tax and Trade Bureau (TTB) is responsible 
for the administration of the Federal excise tax and related provisions 
that apply to tobacco products under Chapter 52 of the Internal Revenue 
Code of 1986, as amended. Cigars and cigarettes are among the tobacco 
products covered by those provisions.
    Elsewhere in this issue of the Federal Register, we are publishing 
a notice of proposed rulemaking, ``Tax Classification of Cigars and 
Cigarettes,'' which sets forth proposed new regulatory standards for 
the classification of cigars and cigarettes for tax purposes. The 
proposed regulatory standards include a specification regarding the 
total reducing sugar content of cigar and cigarette filler tobacco. 
This specification is based on the results of a study TTB performed on 
cigars and cigarettes using the Astoria 2 + 2 Flow Analyzer, which is 
the apparatus that TTB currently uses for tobacco product analysis. The 
purpose of this notice is to advise the public of the apparatus and 
method we employ to determine the total reducing sugars found in 
tobacco products, thereby allowing interested industry members and 
others to evaluate the total reducing sugar content of tobacco products 
in the same way for the purpose of commenting on the proposed new 
regulatory standards.
    TTB uses the Astoria 2 + 2 Flow Analyzer and the Astoria-Pacific 
International Total Reducing Sugars A250 method with one modification--
we prepare the extraction solution without methanol, thereby increasing 
the extraction time to 30 minutes, instead of 15 minutes, per operating 
note 10 in method A250.
    The method is set forth below.

Laboratory Method

Total Reducing Sugars--A250

    (Astoria-Pacific International, Rev. D 8/2003)

A. Scope and Application

    This method is used for the determination of total reducing sugar 
in acetic acid extracts of tobacco. The applicable range of this method 
is 20 to 400 mg/L. The range can be extended by changing the detector 
sensitivity or by sample dilution.

B. Summary of Method

    Samples (containing sucrose) are first hydrolyzed by invertase to 
form reducing monosaccharides.\1\ Reducing sugars react with p-
hydroxybenzoic acid hydrazide (PAHBAH) in an alkaline media to form a 
yellow color measured at 410 nm.\2\ Calcium is used to enhance the 
color development.\3\

C. Interferences

    No interferences are known for this methodology.

D. Sample Handling and Preservation

    Casing and flavoring products should be refrigerated until 
analyzed. Samples should be stored in airtight containers prior to 
analysis. Extracted samples are stable for 48 hours when refrigerated.

E. Raw Materials Required

    Note: Chemicals should be of ACS grade or equivalent.

     Acetic Acid, Glacial CH3COOH (FW 60.05);
     Brij[supreg]-35, 30% w/v (API p/n 90-0710-04);
     Calcium Chloride CaCl2[middot]2H2O 
(FW 147.02) (API p/n 80-7040-72);
     Citric Acid HOCCOOH 
(CH2COOH)2[middot]H2O (FW 210.14) (API 
p/n 80-7035-61);
     Deionized Water (ASTM Type I or II);
     Fructose C6H12O6 (FW 
180.16) (API p/n 80-7062-72);
     Glucose C6Hl2O6 (FW 
180.16) (API p/n 80-7061-72);
     Hydrochloric Acid HCl (FW 36.46);
     Invertase (See Operating Note 2) (API p/n 80-
7050-61K);
     p-Hydroxybenzoic Acid Hydrazide (PAHBAH) 
C7H8N2O2 (FW 152.20) (API 
p/n 80-7030-60);
     Methanol CH3OH (FW 32.04);
     Sodium Hydroxide NaOH (FW 40.00); and
     Sucrose C12H22O11 (FW 
342.30) (API p/n 80-7063-72).
    (Reagents may be purchased in a pre-packaged kit, API p/n 80-7000-
13K).

F. Reagent Preparation

1. Sodium Hydroxide Solution, 1 N (1 L)
    Caution: The dissolution of sodium hydroxide in water releases a 
great amount of heat.

Sodium Hydroxide...........................  40.0 g
    NaOH (FW 40.00)
Deionized Water
 

    Dissolve 40.0 g of sodium hydroxide in 800 ml of deionized water 
contained in a 1 L volumetric flask. Dilute to the mark. Mix well. 
Filter to 0.45 [mu]m with a membrane filter. Keep tightly closed; 
stable for one month at room temperature.
2. Hydrochloric Acid Solution, 0.75 N (1 L)

Hydrochloric Acid, Concentrated (36-37%)...  63 ml
    HCl (FW 36.46)
Deionized Water
 

    Cautiously add 63 ml of hydrochloric acid to 800 ml of deionized 
water contained in a 1 L volumetric flask. Dilute to 1 L with deionized 
water and mix well. Filtering is not necessary. Keep tightly closed; 
stable for 60 days at room temperature.
3. p-Hydroxybenzoic Acid Solution (PAHBAH) (500 ml)

p-Hydroxybenzoic Acid Hydrazide, 80-    1 vial or 30.0 g
 7030-60.
    C7H8N2O2 (FW 152.20)
Citric Acid, 80-7035-61...............  1 vial or 10.5 g
    HOCCOOH
     (CH2COOH)2[middot]H2O (FW
     210.14)
Hydrochloric Acid Solution, 0.75N
 

    Dissolve one vial or 30.0 g of p-hydroxybenzoic acid hydrazide and 
one vial or 10.5 g of citric acid in 400 ml of 0.75 N hydrochloric acid 
solution contained in a 500 ml volumetric flask. Dilute to volume with 
hydrochloric acid solution and mix well using a magnetic stirring bar. 
Filter to 0.45 [mu]m with a

[[Page 62525]]

membrane filter. Store at room temperature; stable for 60 days.
4. Calcium Chloride Solution (500 ml)

Calcium Chloride, 80-7040-72..........  1 vial or 0.34 g
    CaCl22H2O (FW 147.02)
Deionized Water
 

    Dissolve one vial or 0.34 g of calcium chloride in 400 ml of 
deionized water contained in a 500 ml volumetric flask. Dilute to 
volume with deionized water and mix well. Filter to 0.45 [mu]m with a 
membrane filter. Store at room temperature; stable for 60 days.
5. Extraction Solution, 1% acetic acid, 2% methanol (v/v) (20 L)

    Note: If analyzing Nitrate/Nitrite simultaneously, see Operating 
Note 10.


Acetic Acid, Glacial.......................  170 ml
    CH3COOH (FW 60.05)
Methanol...................................  340 ml
    CH3OH (FW 32.04)
Deionized Water
 

    Add 170 ml of glacial acetic acid and 340 ml of methanol to 16.49 L 
of deionized water contained in a 20 L polyethylene carboy. Do not 
dilute to volume. Mix well. Store at room temperature. Stable for 30 
days.

    Note: Extraction Solution can be prepared in smaller quantities. 
This solution will be used for extraction, calibrants and sampler 
wash.

6. Invertase Solution (500 ml)

Invertase, 80-7050-61K................  1 vial or 0.20 g
    (See operating note 2)
Extraction Solution
 

    Dissolve one vial or 0.20 g invertase into 300 ml extraction 
solution contained in a 500 ml volumetric flask. Dilute the solution to 
volume with Extraction Solution and mix well. Filter to 0.45 [mu]m with 
a membrane filter. Store refrigerated; stable for 10 days.
7. Sampler Wash Solution (1 L)

Extraction Solution........................  1 L
Brij-35, 30% solution......................  1 ml
 

    Add 1 ml of Brij-35, 30% solution to 1 L Extraction Solution and 
mix well.
8. Startup and Shutdown Solution (1 L)

Deionized Water............................  1 L
Brij-35, 30% solution......................  1 to 2 ml
 

    Add 1 to 2 ml of Brij-35, 30% solution to 1 L deionized water and 
mix well.

G. Calibrants

    Specific Stock and Working Calibrant preparation instructions can 
be found on the back of the flow diagram. Be sure to use the flow 
diagram which covers the concentration range you wish to analyze.
    Working calibrants may be prepared to cover alternate ranges by 
adding the appropriate volumes of stock or intermediate calibrant to 
100 ml volumetric flasks that contain approximately 80 ml of extraction 
solution. Dilute the solution to 100 ml with extraction solution and 
mix well.
    The following formula can be used to calculate the amount of stock 
(or intermediate) calibrant to be used.

[GRAPHIC] [TIFF OMITTED] TN25OC06.002

Where:

C1 = desired concentration (in mg/L) of working calibrant 
to be prepared;
V1 = final volume (in ml) of working calibrant to be 
prepared (generally 100 ml);
C2 = concentration (in mg/L) of stock (or intermediate) 
calibrant; and
V2 = volume (in ml) of stock (or intermediate) calibrant 
to be used.

    Rearranging the equation to solve for V2 yields:
    [GRAPHIC] [TIFF OMITTED] TN25OC06.003
    
    For example, to prepare a 1.0 mg/L working calibrant from a 1000 
mg/L stock calibrant, use 0.1 ml (100 [mu]l) of the stock calibrant in 
100 ml final volume.
[GRAPHIC] [TIFF OMITTED] TN25OC06.004

    Add this amount of stock calibrant to the volumetric flask and then 
dilute to volume with the extraction solution.

H. Operation Procedure

    1. Set up the cartridge as shown in the flow diagram [The flow 
diagram is shown at the end of this document.\1\ Check all tubing and 
connections. Replace if necessary.
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    \1\ Note: Some proprietary information of Astoria-Pacific 
International was removed from this diagram.
---------------------------------------------------------------------------

    2. Place reagent lines in startup solution.
    3. Turn on the power to all units including heat bath and latch 
pump platens to begin liquid flow.
    4. Verify that the bubble sizes and spacing are consistent 
throughout the cartridge. If bubbles are splitting up as they enter or 
exit a coil or heat bath, check and replace fittings if necessary. The 
bubbles should flow smoothly without dragging. If dragging occurs, add 
more Brij-35 to the startup and sampler wash solutions.
    5. Check all reagent containers on the instrument for particulate 
matter. Reagents should be filtered prior to use. Be sure all 
containers are properly labeled and filled before pumping reagents.
    6. After the heat bath has reached the desired temperature and a 
stable baseline has been verified on the startup solution, place 
reagent lines in reagent bottles.
    7. If using data collection software, set up the appropriate sample 
table.
    8. Allow reagents to run for 5 to 10 minutes and verify a stable 
baseline.
    9. Load the sampler tray with calibrants, blanks, samples, and QC 
or monitor samples.
    10. Select the appropriate parameters for the detector and sampler. 
(See Flow Diagram at the end of methodology.)
    11. Begin analysis.
    12. At the end of analysis place all reagent lines in startup/
shutdown solution and turn off the heat bath. Pump for 20 to 30 minutes 
to flush all of the reagents out of the cartridge and to allow the heat 
bath to cool.
    13. Turn off the power to all units and release pump platens.

I. Operating Notes

    1. If this cartridge is run as a single cartridge on the system, a 
sample ``helper'' line may be necessary. Problems such as poor 
precision, erratic washout between peaks, irregular peak shape, or 
inconsistent ISAC artifacts could be an indication that a ``helper'' 
line is needed. (See Section 9 of the Astoria[supreg] Analyzer 
Operation Manual.) Pecking may also be turned ON.
    2. Invertase, grade VII from Bakers Yeast, available from Sigma 
Chemical Company, St. Louis, MO is recommended.
    3. To check hydrolysis efficiency, prepare a check calibrant of the 
same nominal concentration from sucrose. If hydrolysis efficiency is 
poor, replace the invertase reagent.
    4. This cartridge has a 0.015'' ID pulse suppressor installed at 
the NaOH reagent addition tee. Insure that no air bubbles are present 
in this suppressor before analyzing samples. Air bubbles or debris in 
the suppressor will produce an unstable baseline and loss of 
sensitivity.
    5. Another common cause of low sensitivity and noise in the 
baseline is debris in the flowcell. Particulate matter from the 
reagents and samples become lodged in the flowcell restricting the 
amount of light that is passed through the flowcell. Flushing the 
flowcell with approximately 10 ml of sampler wash solution with a 
syringe will dislodge

[[Page 62526]]

any debris. Following proper filtration procedures for the reagents and 
samples will reduce the frequency of clogging.
    6. Avoid using carbon in the filtration of samples. It will effect 
the color development.
    7. Adding 1 ml/L of Brij-35 to the reagents just before use rather 
than when they are prepared will decrease the possibility of bacteria 
or algae contamination.
    8. If bubbles are sticking in a debubbler, cleaning the debubbler 
will allow bubbles to escape smoothly out the debubble line. Bubbles 
sticking in the debubbler can cause a loss in the overall precision of 
the peak height. To clean, soak the debubbler for 2-3 hours in a 
mixture of 20-30% Contrad[supreg]NF (API p/n 80-0007-04) and hot tap 
water. Rinse thoroughly.
    9. Cover all reagents and other solutions to avoid interference due 
to dust and other particulates.
    10. Methanol in the extraction buffer has been found to cause a 
0.2-0.3% increase in Nitrate results. If running nitrate simultaneously 
with total reducing sugars, removing the methanol from the extraction 
solution will take care of the bias. However, you will need to increase 
the extraction time to 30 minutes.

J. Sample Preparation Procedure

    Note:
    This procedure is applicable for all tobacco products including 
leaf, stem, scrap, and cut filler. The total sugar results achieved 
from this procedure will be on a dry weight basis.

    1. Place approximately 3 to 5 grams of product into a screen wire 
basket.
    2. Place baskets into a 90[deg]C convection oven for one hour.
    3. Remove baskets from oven.
    4. Grind samples to 20 mesh using a Wiley Mill Grinder or 
equivalent.
    5. Place ground samples into air tight containers.
    6. Using an analytical balance, accurately weigh approximately 100 
mg of the dried and ground tobacco sample.
    7. Transfer the weighed sample to a 125 ml Erlenmeyer flask.
    8. Accurately dispense 100 ml of Extraction Solution into the 125 
ml flasks.
    9. Stopper the flasks and place them on a wrist action or orbital 
shaker for 15 minutes.
    10. Filter the samples through Schleicher and Schuell 560 
pleated filter paper or equivalent.
    11. Contain the filtered solution in plastic vials with hinged caps 
until the appropriate time for analysis.
    12. If refrigerated, samples will be stable for 48 hours.

K. References

    1. Official Methods of Analysis of the Association of Official 
Analytical Chemists (AOAC), Thirteenth Edition 31.134, 1980, 
Washington, DC.
    2. Ferraro, J.J., Caccavo, F.A., Saifer, A., p-Hydroxybenzoic Acid 
Hydrazide Procedure for Serum Glucose Adapted to the Technicon ``SMA 
12/60'', and Compared with Other Glucose Methods. Clin Chem 22/2, 263--
266 (1976).
    3. Davis, R. E., A Combined Automated Procedure for the 
Determination of Reducing Sugars and Nicotine Alkaloids in Tobacco 
Products Using a New Reducing Sugar Method. Tobacco Science, XX: pp. 
146-151 (1976).

Acknowledgements

     Astoria[supreg] and FASPacTM are trademarks of 
Astoria-Pacific, Inc., Clackamas, Oregon.
     Brij[supreg]-35 is a registered trademark of ICI Americas, 
Wilmington, Delaware.
     Contrad[supreg] is a registered trademark of Decon 
Laboratories, Bryn Mawr, PA.

Calibrant Preparation

Total Reducing Sugars in Extracts of Tobacco

    200-A250-A00
    20-400 mg/L Sugars
    (Astoria-Pacific International, Rev. A 11/2003)
4,000 mg/L Sugar Stock Standard (500 ml)
    (1) Dissolve into 300 ml extraction solution:
     1.0 g (one bottle) Fructose (API p/n 80-7062-72), 
C6H12O6 (FW 180.16), dried at 
110[deg]C, and
     1.0 g (one bottle) Glucose (API p/n 80-7061-72), 
C6H12O6 (FW 180.16), dried at 
110[deg]C.
    (2) Dilute to 500 ml with extraction solution.
    (3) Add 2 drops Chloroform, CHCl3 (FW 119.38) and mix 
well.
    (4) Store @ 2-8[deg]C. Stable for 60 days.
Working Standard (100 ml)
    Use adjustable, microliter pipettes to add the designated volumes 
of stock, intermediate or working standard to 100 ml volumetric flasks 
containing approximately 80 ml of extraction solution. Dilute each 
solution to the mark with the extraction solution and mix well. Make 
standards covering the range being run.

----------------------------------------------------------------------------------------------------------------
                                                                        Standard to pipet
                 Range                        Working standard       (Stock, Intermediate or    Volume to pipet
                                               concentration                 Working)             ([mu]L)-(ml)
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20-400 mg/L............................  400 mg/L.................  4,000 mg/L...............     10,000 C6 10.0
20-400 mg/L............................  300 mg/L.................  4,000 mg/L...............       7,500 C5 7.5
20-400 mg/L............................  200 mg/L.................  4,000 mg/L...............       5,000 C4 5.0
20-400 mg/L............................  100 mg/L.................  4,000 mg/L...............       2,500 C3 2.5
20-400 mg/L............................  40 mg/L..................  4,000 mg/L...............       1,000 C2 1.0
20-400 mg/L............................  20 mg/L..................  4,000 mg/L...............         500 C1 0.5
----------------------------------------------------------------------------------------------------------------


    Note: To prepare alternate calibrant concentrations consult the 
methodology.

2,000 mg/L Sucrose Stock for Liquid Control (500 ml)
    (1) Dissolve into 300 ml extraction solution:
     1.0 g (one bottle) Sucrose (API p/n 80-7063-72), 
C12H22O11 (FW 180.16), dried at 
110[deg] C.
    (2) Dilute to 500 ml with extraction solution.
    (3) Add 2 drops Chloroform, CHCl3 (FW 119.38) and mix 
well.
    (4) Store @ 2-8[deg] C. Stable for 60 days.
150 mg/L Sucrose Liquid Control (100 ml)
    (1) 7.5 ml of 2,000 mg/L Sucrose Stock.
    (2) Dilute with Extraction Solution to 100 ml.
    (3) Mix well.

BILLING CODE 4810-31-P

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[GRAPHIC] [TIFF OMITTED] TN25OC06.001

BILLING CODE 4810-31-C

    Signed: October 17, 2006.
John J. Manfreda,
Administrator.
[FR Doc. 06-8836 Filed 10-24-06; 8:45 am]
BILLING CODE 4810-31-P