62524 Federal Register / Vol. 71, No. 206 / Wednesday, October 25, 2006 / Notices 

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. 

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–35, 30% w/v (API p/n 90– 

0710–04); 

• Calcium Chloride CaCl2·2H2O (FW 

147.02) (API p/n 80–7040–72); 

• Citric Acid HOCCOOH 

(CH2COOH)2·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 prepackaged 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 μm 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, 80–7030– 

60.

1 vial or 30.0 g 

C7H8N2O2 (FW 

152.20) 

Citric Acid, 80–7035– 

61.

1 vial or 10.5 g 

HOCCOOH 

(CH2COOH)2·H2O 

(FW 210.14) 

Hydrochloric Acid Solution, 0.75N 

Dissolve one vial or 30.0 g of phydroxybenzoic 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 μm with a 

mstockstill on PROD1PC61 with NOTICES2

Federal Register / Vol. 71, No. 206 / Wednesday, October 25, 2006 / Notices 62525 

1Note: Some proprietary information of AstoriaPacific International was removed from this 

diagram. 

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 

CaCl2•2H2O (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 μm 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 μm 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. 

CV CV 11 2 2 =

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: 

V C V

C 2

1 1

2

=

 

For example, to prepare a 1.0 mg/L 

working calibrant from a 1000 mg/L 

stock calibrant, use 0.1 ml (100 μl) of the 

stock calibrant in 100 ml final volume. 

V

V

2

2

1 0

0 1

=

=

( .

.

 mg/L) (100 ml)

1000 mg/L

 

 ml

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. 

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 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 


mstockstill on PROD1PC61 with NOTICES2EN25OC06.002</MATH> EN25OC06.003</MATH> EN25OC06.004</MATH>
62526 Federal Register / Vol. 71, No. 206 / Wednesday, October 25, 2006 / Notices 

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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°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 and FASPacTM are 

trademarks of Astoria-Pacific, Inc., 

Clackamas, Oregon. 

• Brij-35 is a registered trademark of 

ICI Americas, Wilmington, Delaware. 

• Contrad 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°C, and 

• 1.0 g (one bottle) Glucose (API p/n 

80–7061–72), C6H12O6 (FW 180.16), 

dried at 110°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°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. 

Range Working standard concentration Standard to pipet 

(Stock, Intermediate or Working) 

Volume to pipet 

(μL)–(ml) 

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° 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° 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 

mstockstill on PROD1PC61 with NOTICES2

Federal Register / Vol. 71, No. 206 / Wednesday, October 25, 2006 / Notices 62527 

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 


mstockstill on PROD1PC61 with NOTICES2EN25OC06.001</GPH>