一、烟草中糖的测定
1.1 总糖的测定——CORESTA Recommended Method N°89
Tobacco-determination of the content of total sugars-continuous-flow analy-sis method using hydrochloric acid/p-hydroxy benzoic acid hydrazide （PAHBAH） （April 2019）
0. INTRODUCTION
In 2015 the CORESTA Routine Analytical Chemistry Sub-Group （RAC）undertook a collaborative study for the determination of “Total Sugars”in tobacco by segmented continuous-flow analysis （CFA） . “Total sugars”is defined as the combined amount of reducing sugars and non-reducing sugars present in a sample. The predominant sugars found in tobacco are the monosaccharides fructose and glucose，which are both reducing sugars. The most common non-reducing sugar found in tobacco is the disaccharide sucrose. The CORESTA Recommended Method for reducing sugars （CRM N°38）was used as a basis for the development of this CRM to maintain compatibility and efficiency as reducing and non-reducing sugars are often analysed in parallel. Non-reducing sugars can be converted to reducing sugars by hydrolysis with either an acid or an enzyme. Once reduced they can react with one of several colour-forming compounds （e.g. PAHBAH）[1]，[2]. Additionally，the extraction solution （water or acetic acid）was examined，because of a note given in CRM N° 38 regarding that hydrolysis of sucrose may occur for some tobaccos if extracted with distilled water.
1. FIELD OF APPLICATION
This CRM specifies a method for the determination of the content of“Total Sugars”as glucose in tobacco by CFA using hydrochloric acid （HCl）for hydrolysis and p-hydroxybenzoic acid hydrazide （PAHBAH）for colour formation.
This method is applicable to unprocessed tobacco lamina and processed tobacco such as cigarette blend tobacco and roll-your-own （RYO）tobacco.
2. NORMATIVE REFERENCES
ISO 3696 Water for analytical laboratory use-Specification and test methods.
3. TERMS AND DEFINITIONS
No terms and definitions are listed in this document.
4. PRINCIPLE
An aqueous extract of the tobacco is prepared and the total sugar content （as glucose）of the extract is analysed by CFA. The extract is heated in the presence of HCl at 90℃，which hydrolyses any sucrose present to glucose and fructose. The reduced sample extract is passed through a dialyser to eliminate interference from coloured compounds in the sample and then reacts with PAHBAH in an alkaline medium at 85℃ to produce a yellow osazone complex.
Quantitation is by external standard using a series of glucose calibration standards （0.05 mg/mL～2.5 mg/mL） prepared with the same extraction solution. All measurements are performed at 420nm.
A collaborative study[3] has shown that the method gives comparable results for water and 5% acetic acid extracts. It is recommended that 5% acetic acid extracts should be used if analysis of reducing carbohydrates （CRM N°38）is to be carried out in parallel.
5. APPARATUS
Usual laboratory apparatus and，in particular，the following items：
5.1 Continuous-flow analyser，consisting of
-Autosampler
-Peristaltic pump
-Chemistry manifold with dialyser，heating bath and delay coils
-Photometric detector equipped with a 420nm filter
-Data acquisition system or recorder
See Annex A for examples of suitable flow diagrams.
6. REAGENTS
Use only reagents of recognized analytical grade. All reagents shall be used according to good laboratory practice and existing national regulations. Water must be high quality distilled or deionized （DI）water （according to ISO 3696）.
6.1 Polyoxyethylene lauryl ether （Brij-35Ⓡ，30% w/w solution），CAS # 9002-92-0
6.2 Acetic acid，glacial，CAS #64-19-7
6.3 Hydrochloric acid （HCl），37%，CAS #7647-01-0
6.4 Sodium hydroxide （NaOH），CAS #1310-73-2
6.5 Calcium chloride hexahydrate，CaCl2*6H2O，CAS #7774-34-7
6.6 p-Hydroxy benzoic acid hydrazide （PAHBAH），CAS #5351-23-5
6.7 Citric acid monohydrate，CAS #5949-29-1
6.8 Benzoic acid，CAS #65-85-0
6.9 D-glucose，CAS #50-99-7
7. PREPARATION OF SOLUTIONS
All reagents shall be of analytical grade quality. For best results，all solutions that include the dissolution of a solid should be filtered prior to use.
Appropriate safety and health practices shall be established according to national regulations.
7.1 System wash solution
Add 1mL of Brij-35Ⓡ，30% solution to about 800mL DI water and mix care fully. Then dilute to 1000mL with DI water. Replace every week in a clean bot tle. Depending on the system the amount of 0.5mL 30% solution of Brij-35Ⓡ per liter reagent might also be suitable.
7.2 Sampler wash solution
Use the extraction solution，DI water or acetic acid （5%）（7.3），as sampler wash solution.
7.3 Acetic acid solution 5% （v/v）
Add 50mL of acetic acid （glacial） to about 500mL of DI water. Dilute to1000mL with DI water and mix thoroughly.
7.4 Hydrochloric acid solution “A”，0.5mol/L （hydrolysis reagent）
Slowly add 42mL of hydrochloric acid （37%） to about 500mL of DI wa ter. Dilute to 1000mL with DI water，add 0.5mL of Brij-35Ⓡ，30% solution and mix thoroughly. Stable for as long as the solution remains clear.
7.5 Hydrochloric acid solution “B”，0.5mol/L
Slowly add 42mL of hydrochloric acid （37%） to about 500mL of DI water. Dilute to 1000mL with DI water and mix thoroughly. Stable for as long as the solution remains clear.
7.6 Sodium hydroxide solution，0.5mol/L
Dissolve 20g of sodium hydroxide in about 700mL of DI water. Dilute to1000mL，add 0.5mL Brij-35Ⓡ solution and mix thoroughly. Stable for as long as the solution remains clear.
7.7 Calcium chloride solution，0.008mol/L
Dissolve 1.75g of calcium chloride hexahydrate in about 700mL of DI wa ter. Dilute to 1000mL，add 0.5mL Brij-35Ⓡ solution and mix thoroughly. If a pre cipitate occurs when dissolving the calcium chloride hexahydrate，then filter the solution. Stable for as long as the solution remains clear.
7.8 p-Hydroxy benzoic acid hydrazide （PAHBAH）solution
Place 400mL of HCl solution （7.5）in a beaker，warm it to 45℃ and under constant stirring add 25g PAHBAH and 10.5g citric acid monohydrate to the HCl solution. Let the solution cool down，transfer it to a volumetric flask and dilute to volume with the HCl solution （7.5）.
7.9 Benzoic acid solution 0.1%（w/v）（stabilizing agent for the standard solutions）
Dissolve 2.0g of benzoic acid in 2 liters of DI water.
8. STANDARDS
8.1 D-glucose stock solution
Weigh，to the nearest 0.0001g，10.0g of glucose，dissolve in about 800mL of 0.1%benzoic acid （7.9，stabilizing agent if water extraction is used）respectively 5% acetic acid （7.3，if acetic acid extraction is used）and dilute to volume. This solution contains 10mg of glucose per liter. Store in a refrigerator.
8.2 D-glucose working standards
From the stock glucose solution，prepare a series of at least six calibration solu-tions according to the “Total Sugars”concentration which is expected to be found in the test samples （e.g.0.05 mg/mL～2.5 mg/mL）. Store in a refrigerator.
Table 1 D-glucose calibration standards-Nominal Concentrations
9. SAMPLE PROCEDURE
9.1 Preparation of samples for analysis
Prepare the tobacco for analysis by grinding （the sample should totally pass a 1 mm sieve）and analyse. If the tobacco is too wet for grinding it can be dried at a temperature not exceeding 40℃. For result calculation on a dry weight basis determine the moisture content.
9.2 Test portion
Weigh to the nearest 0.1 mg，approximately 250mg，of the ground tobacco into a 50mL conical flask. Add 25mL of the extraction solution （water or 5%acetic acid solution）. Stopper and shake for 30 minutes at a suitable mixing speed.
9.3 Preparation of test extract
Filter the extract through a quantitative filter paper such as Whatman No 401 （or equivalent ashless，quantitative filter paper）filter paper，rejecting the first fewmL of the filtrate，then collect the filtrate. Run the sample and standards through the system in the normal manner （e.g. priming with a high-level standard，calibration standards and samples with an intermediate calibration solution after every 25 sam-ples）. If sample concentration lies outside the range of the standards，the sample shall be diluted and run again.
When using 5%acetic acid extracts，the wash solution shall be 5%acetic acid.
Note：If this method is performed simultaneously with the other CFA methods， combined standards may be possible.
1 Whatman No.40 is an example of a suitable product available commercially. This information is given for the convenience of the users of this recommended method and does not constitute an endorsement by CORESTA of this product.
10. CALCULATION
10.1 Prepare a calibration curve by plotting Total Sugars （as d-glucose）instrument response against standard concentration. Compute sample concentration by comparing sample response with the standard curve.
10.2 Calculate the percentage of “Total Sugars”，w，in the tobacco using the formula A：
where
c is the “Total sugars”concentration，expressed in milligrams per millilitre， obtained from the calibration curve （10.1）；
V is the volume，in millilitres，of the sample （see 9.2），normally 25mL；
m is the mass，in milligrams，of the sample （see 9.2）；
Calculate，if applicable，the percentage of“Total sugars”on a dry weight basis，wd，in the tobacco using the formula B：
where
c is the “Total sugars”concentration，expressed in milligrams per millilitre， obtained from the calibration curve （10.1）；
V is the volume，in millilitres，of the sample （see 9.2），normally 25mL；
m is the mass，in milligrams，of the sample （see 9.2）；
M the moisture content，expressed as percentage by mass，of the tobacco （see 9.1）
The test result shall be expressed to one decimal place.
11. REPEATABILITY AND REPRODUCIBILITY
In 2015 an international collaborative study involving eight laboratories and five samples （three straight grade tobaccos，one cigarette blend and one RYO tobacco） was conducted.
The repeatability limit （r）and reproducibility limit （R）were calculated for this “Total Sugars”（HCl/PAHBAH）method using both water and a 5%acetic acid extraction （see Tables 2 and 3）.
The difference between three single results，found on different extractions by one operator using the same apparatus within a short time interval （the time it takes to analyse～50 sample cups）and without recalibration of the equipment during the time of analysis，will exceed the repeatability limit （r）on average not more than once in 20 cases in the normal and correct operation of the method.
Single results reported by two laboratories will differ no more than the reproduc-ibility limit （R）on average not more than once in 20 cases in the normal and correct operation of the method.
Table 2 Extraction with Water
Table 3 Extraction with 5% Acetic Acid
12. TEST REPORT
The test report shall provide the Total Sugars results to precision of one decimal place. It shall also provide all details necessary for the identification of the sample.
ANNEX A-SUITABLE FLOW DIAGRAMS
（Informative）
Note：For both Macro and Micro Flow systems，the position of the de-bubbler should be as near as possible to the pump. For good reproducible results a well-shaped flow bubble pattern is necessary.
In Table 4 the flow rates of the tubing are given in （mL/min）.
Figure 1 Suggested Flowchart for Macro Flow （mL/min）Systems
Figure 2 Suggested Flowchart for Micro Flow （μL/min）Systems
Table 4 Flow rates of the tubing
BIBLIOGRAPHY
[1 ]Clarke，M. B. ；Bezabeh，D. Z. ；Howard，C. T. Determination of Carbo-hydrates in tobacco Products by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry：A Comparison with Ion Chromatography and Application to Product Discrimination. J. Agric. Food Chem.2006，54，1975-1981
[2 ] Shifflett， J. R.； Jones， L. A.； Limowski， E. R.； Bezabeh， D. Z. Comparison of Segmented Flow Analysis and Ion Chromatography for the Quantitative Characterization of Carbohydrates in Tobacco Products. J. Agric. Food Chem.2012，60，11714-11722
[3]Routine Analytical Chemistry Sub-Group Technical Report.2015 Collaborative Study Developing a CRM for the Determination of Total Sugars in Tobacco by Continuous Flow Analysis.
1.2 还原糖的测定——CORESTA Recommended Method N°38
Detemination of reducing carbohydrates in tobacco by continuous flow analysis
（August 2010)
0. INTRODUCTION
A CORESTA Task Force studied the various widely-used procedures for the determination of reducing sugars in tobacco in order to adopt one of them as the CORESTA Recommended Method. Two procedures were adopted as CORESTA Recommended Methods N°37 and N°38. Studies carried out by the CORESTA Task Force between 1989 and 1993 have shown that the two methods may not produce identical results. For some tobaccos the results obtained with Method N°37 are higher than those of Method N°38 because Method N°37 is sensitive to interferences from reducing substances，other than sugars，present in tobacco. This is reflected in the titles of the two methods. CORESTA has decided to publish both methods since Method N°37 is easier to implement，while Method N°38 is more specific. When reporting the results indicate the method used.
1. FIELD OF APPLICATION
This method is applicable to leaf samples and tobacco blends.
2. PRINCIPLE
A tobacco extract in 5% acetic acid solution （see note 1）is prepared and the reducing carbohydrates content of the extract is determined by reaction with p-hydroxybenzoic acid hydrazide. In alkaline medium at 85℃， a yellow osazone is formed having an absorption maximum at 410nm.
Note 1：Collaborative studies have shown that when extracting with distilled water，hydrolysis of sucrose occurs for some tobaccos.
3. REAGENTS
All reagents shall be used according to good laboratory practice and existing national regulations.
3.1 Brij-35 Solution （Polyoxyethylene Lauryl Ether）
Add 1dm3 distilled water to 250g Brij-35，warm and stir until dissolved.
3.2 Sodium Hydroxide Solution （NaOH，0.5mol/L）
Prepare 1dm3 of 0.5mol/L sodium hydroxide from ampoules or dissolve 20.0g sodium hydroxide in 800cm3 distilled water. Mix and allow to cool. After total dissolution，add 0.5 cm3 Brij-35 solution （3.1）and dilute to 1 dm3 with distilled water.
3.3 Calcium Chloride Solution （CaCl2·6H2O，0.008mol/L）
Dissolve 1.75g calcium chloride hexahydrate in distilled water （see note 2）add 0.5 cm3 Brij-35 solution （3.1） and dilute to 1 dm3 with distilled water.
Note 2：If a precipitate occurs，filter the solution through a Whatman N°1 （or equivalent）filter paper.
3.4 Acetic Acid Solution （CH3 COOH，5% V/V）
Prepare a 5% （V/V）solution of acetic acid from “glacial”acetic acid （used in preparation of standards and samples and for wash solution on continuous flow analyzer）.
3.5 Hydrochloric Acid Solution （HCl，0.5mol/L）
Place 500cm3 distilled water in a 1 dm3 volumetric flask. Slowly add 42cm3 fuming hydrochloric acid （37% m/m）. Dilute to volume with distilled water.
3.6 p-Hydroxybenzoic Acid Hydrazide Solution （PAHBAH），（HOC6 H4 C-ONHNH2）
Place 250cm3 0.5mol/L hydrochloric acid （3.5）into a 500cm3 volumetric flask. Add 25g p-hydroxybenzoic acid hydrazide and allow to dissolve. Add 10.5g citric acid monohydrate [HOC（CH2 COOH）2 COOH·H2O]. Dilute to volume with 0.5mol/L hydrochloric acid solution. Store at 5℃，and take out of the refrigerator only enough volume to cover the daily needs.
Note 3：The purity of PAHBAH （＞97%m/m）is very important since a precipitate may be formed in the analytical stream if impurities are present. The PAHBAH can be recrystallised from distilled water （see Beilstein 10，174）. The PAHBAH is not pure when the following is observed：
a）dark particles present with white PAHBAH crystals；
b）yellow colour in 5% PAHBAH prepared in 0.5mol/L HCl；
c）difficulty in dissolving PAHBAH crystals in 0.5mol/L NaOH；
d）foreign particles floating in the reagent；
e）a wavy reagent baseline.
The PAHBAH solution can also be prepared as follows：place the 250cm3 0.5mol/L HCl solution in a beaker，warm it to 45℃and under constant stirring add the PAHBAH and the citric acid monohydrate to the HCl solution. Let the solution cool down，transfer it to a volumetric flask and dilute to volume. It has been observed that preparation of the PAHBAH solution following this procedure prevents the formation of a precipitate in the analytical stream.
3.7 D-Glucose （C6 H12O6，p. a.）for the Preparation of Standards.
Store in a desiccator.
3.8 Standard Glucose Solutions
3.8.1 Stock Solution：Weigh，to the nearest 0.0001g，approximately 10.0g of glucose （3.7），dissolve in 800cm3 of 5%acetic acid （3.4）and dilute to 1 dm3 in a volumetric flask with 5% acetic acid （3.4） . This solution contains approximately 10mg of glucose per cm3. Store in a refrigerator. Prepare a fresh solution every month.
3.8.2 Working Standards：From the stock solution produce a series of at least five calibration solutions （in 5% acetic acid）whose concentrations cover the range expected to be found in the samples e. g.0.2～1.8mg glucose per cm3. Calculate the exact concentration for each standard. Store in a refrigerator. Prepare fresh solutions every two weeks.
4. APPARATUS
4.1 The necessary general laboratory equipment，for the preparation of samples，standards and reagents.
4.2 Continuous flow analyzer （see diagram 1）consisting of：
Sampler
Proportioning pump
Dialyser
Heating bath
Delay coils
Colorimeter （or equivalent）with 410nm filter （s）
Recorder
5. ANALYSIS OF TOBACCO SAMPLES
5.1 Prepare the tobacco samples for analysis by grinding （the sample should totally pass through a 1 mm sieve）and determine the moisture content. If the tobacco is too wet for grinding it can be dried at a temperature not exceeding 40℃.
5.2 Weigh，to the nearest 0.0001g，approximately 250mg of the tobacco in-to a 50cm3 dry conical flask. Add 25cm3 of 5%acetic acid （3.4），stopper the flask and shake for 30 minutes.
5.3 Filter the extract through a Whatman N°40 （or equivalent）filter paper， reject the first few cm3 of the filtrate，then collect the filtrate in an analyzer cup.
5.4 Run the samples and standards through the system in the normal manner （e.g. priming with 6 tobacco extracts，calibration standards and samples with 1 intermediate calibration solution after every 6 samples）. If sample concentrations lie outside the range of the standards，the samples shall be diluted and run again.
6. CALCULATION
6.1 Plot a graph of peak height against equivalent glucose concentrations for all the calibration solutions.
6.2 Calculate the percentage reducing carbohydrates （expressed as glucose） （dry weight basis）in the tobacco using the formula：
c is the reducing carbohydrates concentration，expressed in milligrams per millilitre，obtained from the calibration curve （6.1）；
V is the volume，in millilitres，of extract prepared （5.2）（normally 25 millilitres）；
m is the masss，in milligrams，of the sample （5.2）；
M is the moisture content，expressed as percentage by mass，of the tobacco （5.1）.
The test result shall be expressed to one decimal place.
Note 4：If this method is performed simultaneously with CORESTA Recommended Method N°35 or CORESTA Recommended Method N°36 combined standards may be prepared.
7. REPEATABILITY AND REPRODUCIBILITY
7.1 An international collaborative study involving 13 laboratories and 3 samples conducted in 1993 showed that when single grades of tobacco were analyzed by this method，the following values for repeatability （r）and reproducibility （R）were obtained.
The difference between two single results found on different extractions by one operator using the same apparatus within a short time interval （the time it takes to analyze 40 sample cups）and without recalibration of the equipment during the time of analysis will exceed the repeatability value （r）on average not more than once in 20 cases in the normal and correct operation of the method.
Single results reported by two laboratories will differ by more than the reproducibility value （R）on average not more than once in 20 cases in the normal and correct operation of the method.
Data analysis gave the estimates as summarized in Table 1.
Table 11993 Study
For the purpose of calculatingr andR，one test result was defined as the yield obtained from analyzing a single extract once.
7.2 During 2005 the CORESTA Scientific Commission sanctioned the CORESTA Routine Analytical Chemistry Sub-group to carry out a collaborative study to confirm theser&R values. This international study involved 11 laboratories and 4 samples and was conducted during 2006. The resulting data are to be found in Table 2.
Table 2 Results from the 2006 RAC Collaborative Study
A plot comparing this data to that of the original study can be found below：
Comparison of r and R Results from the 1993 and 2006 Studies
APPENDIX 1
An injection fitting with a large internal diameter （2mm）shall be used when introducing the PAHBAH solution into the analytical stream in order to prevent precipitation of PAHBAH. In addition the concentration of the PAHBAH solution may be reduced as long as it is ascertained that the PAHBAH is in excess in the analytical stream. This prevents precipitation as well.
It is preferable to use on-line mixing of PAHBAH/NaOH （see diagram 1）. If however a precipitate forms on-line it is possible to pre-mix daily the PAHBAH/NaOH solutions and introduce the combined reagent to the analytical stream. Experiments have shown that similar results are obtained provided the combined reagent is not kept longer than 8 hours. If a combined reagent is used baseline correction may be required due to increased background signal.
二、烟草中总植物碱的测定
2.1 CORESTA Recommended Method N°35
Determination of Total Alkaloids （as Nicotine)in Tobacco
by Continuous Flow Analysis
（CRM developed into ISO 15152：2003)
0. INTRODUCTION
Studies carried out by a CORESTA Task Force between 1989 and 1993 have shown that the two procedures for determination of total alkaloids in tobacco as described in CORESTA Recommended Method N°20 and the present Method may not produce identical results for some dark tobaccos or those containing significant levels of alkaloids other than nicotine.
The studies have indicated that these differences may be due to the fact that the recoveries and detection sensitivities of the two methods towards the alkaloids other than nicotine are different.(www.xing528.com)
Therefore，when reporting results it is important to specify the method used.
1. FIELD OF APPLICATION
This method is applicable to leaf samples and tobacco blends.
2. REFERENCES
CORESTA Recommended Method N°20：1968
Determination of alkaloids in manufactured tobacco.
CORESTA Recommended Method N°39：1994
Determination of the purity of nicotine and nicotine salts by gravimetric analysis-Tungstosilicic acid method.
3. PRINCIPLE
An aqueous （see note 1）extract of the tobacco is prepared and the total alkaloids （as nicotine）content of the extract is determined by reaction with sulphanilic acid and cyanogen chloride.
Cyanogen chloride is generated in situ by the reaction of potassium cyanide and chloramine T （see appendix 1）. The developed colour is measured at 460nm.
Note 1：Collaborative studies have shown that this method gives equivalent results for water and 5% acetic acid extracts. It is recommended that 5% acetic acid extracts should be used if total alkaloids （as nicotine）and reducing substances （see CORESTA Recommended Method N°37）or reducing carbohydrates （see CORESTA Recommended Method N°38）analysis are to be carried out simultaneously.
4. SAFETY PRECAUTIONS
Potassium cyanide is poisonous and irritant，thus all safety precautions must be observed when handling this material. Solutions shall be prepared by a designated re-sponsible person. Gloves and safety glasses shall always be used when making up solutions and bottles of the made-up reagent shall always be carried in a suitable safety carrier. To prevent the escape of vapour into the laboratory， reagent pick-up tubes shall pass through a soda-lime trap into the reagent bottle （see diagram 2）.
The cyanide neutralising agents A and B are pumped as shown in the flow diagram （see diagram 1）and mixed in a 2dm3 Buchner flask with magnetic stirring （see diagram 3）. All waste solutions containing cyanogen chloride are run into this flask where conversion to the “Prussian Blue”complex occurs. The contents of the Buchner flask are allowed to over-flow into a storage flask，the contents of which are stored overnight in a fume cupboard and then disposed of as waste.
Suitable cyanide poisoning treatment kits are available from laboratory suppliers and shall be located in the vicinity of the analyzer to be used by a competent person.
5. REAGENTS
All reagents shall be used according to good laboratory practice and existing national regulations.
5.1 Brij-35 Solution （Polyoxyethylene Lauryl Ether）
Add 1 dm3 distilled water to 250g Brij-35，warm and stir until dissolved.
5.2 Buffer Solution A
Dissolve 2.35g sodium chloride （NaCl） and 7.60g sodium tetraborate （Na2B4O3·10H2O）in distilled water. Transfer to a 1dm3 volumetric flask，add 1 cm3 Brij-35 solution （5.1）and dilute to volume with distilled water. Filter the solution through a Whatman N°1 （or equivalent）filter paper before use.
5.3 Buffer Solution B
Dissolve 26g anhydrous disodium hydrogen orthophosphate （Na2 HPO4），10.4g citric acid [COH（COOH）（CH2COOH）2·H2O]and 7g sulphanilic acid （NH2C6H4SO3H）2 in distilled water，transfer to a 1 dm3 volumetric flask， add 1 cm3 Brij-35 solution （5.1）and dilute to volume with distilled water. Filter the solution through a Whatman N°1 （or equivalent）filter paper before use
5.4 Chloramine T Solution （N-chloro-4-methyl benzenesulphonamide so-dium salt），[CH3C6H4SO2N（Na）Cl·3H2O]
Dissolve 8.65g chloramine T in distilled water，transfer to a 500cm3volumetric flask and dilute to volume with distilled water. Filter the solution through a Whatman N°1 （or equivalent）filter paper before use.
5.5 Cyanide Neutralising Solution A
Dissolve 1g citric acid （5.3）and 10g ferrous sulphate （FeSO4·7H2O）in distilled water and dilute to 1 dm3.
5.6 Cyanide Neutralising Solution B
Dissolve 10g anhydrous sodium carbonate （Na2 CO3）in distilled water and di-lute to 1 dm3.
5.7 Potassium Cyanide Solution （KCN）
CARE：POTASSIUM CYANIDE IS EXTREMELY TOXIC！SEE SAFETY PRECAUTIONS.
In a fume cupboard，weigh 2g potassium cyanide into a 1dm3 beaker. Add 500cm3 distilled water and stir （magnetic stirrer）until all of the solid has dis-solved. Store in a brown glass bottle.
5.8 Nicotine Hydrogen Tartrate [C10 H14 N2 （C4H6O6）2·2H2O] for the Preparation of Standards
5.9 Standard Nicotine Solutions
Check the purity of the nicotine hydrogen tartrate according to CORESTA Recommended Method N°39.
5.9.1 Stock Solution：Weigh，to the nearest 0.0001g，approximately 1.3g of nicotine hydrogen tartrate in distilled water and dilute to 250cm3 in a volumetric flask. This solution contains approximately 1.6mg nicotine per cm3. Store in a refrigerator. Prepare a fresh solution every month.
5.9.2 Working Standards：From the stock solution produce a series of at least five calibration solutions whose concentrations cover the range expected to be found in the samplese.g.0.04～0.80mg nicotine per cm3. Calculate the exact concentration for each standard taking into account the purity of the nicotine hydrogen tartrate. Store in a refrigerator. Prepare fresh solutions every two weeks.
Note 2：The method can also be standardized by using nicotine or other nicotine salts of known purity. In this case an amount equivalent to the above used nicotine hydrogen tartrate shall be used.
6. APPARATUS
6.1 The necessary general laboratory equipment，for the preparation of sam-ples，standards and reagents.
6.2 Continuous flow analyzer （see diagram 1）consisting of：
Sampler
Proportioning pump
Dialyser
Delay coils
Colorimeter （or equivalent）with 460nm filter （s）
Recorder
Coil for cyanogen chloride generation
A commercially available microbore mixing coil can be used for thein situ generation of cyanogen chloride. Alternatively a five turn mixing coil can be prepared （see appendix 2）
7. ANALYSIS OF TOBACCO SAMPLES
7.1 Prepare the tobacco for analysis by grinding （the sample should totally pass through a 1 mm sieve）and determine the moisture content. If the tobacco is too wet for grinding it can be dried at a temperature not exceeding 40℃.
7.2 Weigh，to the nearest 0.0001g，approximately 250mg of the tobacco in a 50cm3 dry conical flask. Add 25cm3 distilled water，stopper the flask and shake for 30 minutes.
7.3 Filter the extract through a Whatman N°40 （or equivalent）filter paper， reject the first few cm3 of the filtrate，then collect the filtrate in an analyzer cup.
7.4 Run the samples and standards through the system in the normal manner （e.g. priming with 6 tobacco extracts，calibration standards and samples with 1 intermediate calibration solution after every 6 samples）. If sample concentrations lie outside the range of the standards，the samples shall be diluted and run again.
8. CALCULATION
8.1 Plot a graph of peak height against equivalent nicotine concentrations for all the calibration solutions.
8.2 Calculate the percentage nicotine （dry weight basis）in the tobacco using the formula：
c is the nicotine concentration，expressed in milligrams per millilitre，obtained from the calibration curve （8.1）；
V is the volume，in millilitres，of extract prepared （7.2）（normally 25 millilitres）；
m is the mass，in milligrams，of the sample （7.2）；
M is the moisture content，expressed as percentage by mass，of the tobacco （7.1）.
The test result shall be expressed to two decimal places.
Notes 3：When using 5% acetic acid extracts the standard nicotine solutions （5.9）must be made up with 5% acetic acid and the wash cycle must be with 5%acetic acid.
Notes 4：If this method is performed simultaneously with CORESTA Recommended Method N°36，CORESTA Recommended Method N°37 or CORESTA Recommended Method N°38 combined standards may be prepared.
9. REPEATABILITY AND REPRODUCIBILITY
9.1 An international collaborative study involving 12 laboratories and 3 samples conducted in 1993 showed that when single grades of tobacco were analyzed by this method，the following values for repeatability （r）and reproducibility （R）were obtained.
The difference between two single results found on different extractions by one operator using the same apparatus within a short time interval （the time it takes to analyze 40 sample cups）and without recalibration of the equipment during the time of analysis will exceed the repeatability value （r）on average not more than once in 20 cases in the normal and correct operation of the method.
Single results reported by two laboratories will differ by more than the reproducibility value （R）on average not more than once in 20 cases in the normal and correct operation of the method.
Data analysis gave the estimates as summarized in table 1 and 2.
Table 1 Extraction with Water （1993 Data)
Table 2 Extraction with 5% Acetic Acid （1993 Data)
For the purpose of calculatingr andR，one test result was defined as the yield obtained from analyzing a single extract once.
9.2 During 2005 the CORESTA Scientific Commission sanctioned the CORESTA Routine Analytical Chemistry Sub-group to carry out a collaborative study to confirm theser&R values. This international study involved 17 laboratories and 6 samples and was conducted during 2006. The resulting data are to be found in Table 3.
Table 3 Results from the 2006 RAC Collaborative Study
NOTE：This CRM recommends that equivalent results are obtained when either water or 5% acetic acid are used as the extraction solvents and therefore the results from this study were not segregated in the subsequent data analysis. A plot comparing this data to that of the original study can be found below：
Comparison of r and R Results from the 1993 and 2006 Studies
APPENDIX 1
Several collaborative studies，carried out by a CORESTA Task Force during 1989 and 1990，have shown that two other procedures give equivalent results to the Recommended Method. It may be necessary to use one of these alternative procedures，if so，the following comments should be considered before use：
Cyanogen chloride can be alternatively generated in situ by the reaction of potassium thiocyanate and sodium hypochlorite. In order for this reaction to be successful the sodium hypochlorite must have an available chlorine content of 10%～14%（m/m）. It has been found that sodium hypochlorite with this amount of available chlorine is sometimes difficult to obtain.
Cyanogen bromide in reaction with aniline can also be used in the determination of total alkaloids. Because of the hazardous nature of the cyanogen bromide，some countries have found problems with the importation and use of this substance.
APPENDIX 2
Preparation of a Microbore Five Turn Mixing Coil
1. Loop a standard orange-white （0.64mm id）pump tube 5 times around a glass tube （e.g. test tube，glass rod）with an external diameter of approximately 12mm.
2. While holding the loops in place brush them thoroughly with cyclohexanone.
3. Use adhesive tape to hold the loops in place while the cyclohexanone sets the tubing，（about 10 hours）.
4. Remove the glass tube from the coil.
The apparatus consists of a 2dm3 Buchner flask on a magnetic stirrer，with a 2cm diameter PVC tube inserted into it，through a rubber bung，such that the tube is just above the magnetic follower in the flask.Four holes are dilled in the tube and nipples attached by gluing into position.The pullback line and the debubble line containing the cyanogen chloride are attached to the nipples，together with the two neutralising agents.This arrangement ensures that the cyanogen chloride has to pass down the tube and through the bulk of the flask before overflowing to waste，thus ensuring complete neutralisation.
2.2 CORESTA Recommended Method N°85
Tobacco-Determination of the content of total alkaloids
as nicotine-continuous flow analysis method using KSCN/DCIC
（April 2017)
0. INTRODUCTION
In 2014 the CORESTA Routine Analytical Chemistry Sub-Group （RAC）undertook a collaborative study of two methods for the determination of total alkaloids in tobacco （as nicotine）by segmented continuous-flow analysis. The two methods are CRMNo.35 （CRM35）（basis for ISO 15152）and a new method proposed by China National Tobacco Quality Supervision&Test Center. In CRM35 cyanogen chloride is gener-atedin situ by the reaction of potassium cyanide and chloramine T. The proposed method eliminates the use of the potassium cyanide （KCN）by employing potassium thiocyanate （KSCN） with sodium dichloroisocyanurate dihydrate （DCIC） for colour development. Each method was tested using water extracted tobacco and 5%acetic acid extracted tobacco. Calibration standards were prepared with the same extraction solutions.
1. FIELD OF APPLICATION
This CRM specifies a method for the determination of the content of total alka-loids as nicotine in tobacco by continuous-flow analysis.
This method is applicable to leaf samples，stems，reconstituted tobacco sheet materials and tobacco blends.
2. NORMATIVE REFERENCES
ISO 13276，Tobacco and tobacco products—Determination of nicotine purity—Gravimetric method using tungstosilicic acid
ISO 3696，Water for analytical laboratory use—Specification and test methods
3. PRINCIPLE
An aqueous extract （see below）of the tobacco is prepared and the total alkaloids content （as nicotine）of the extract is measured by reaction of sodium citrate and cyanogen chloride.
Cyanogen chloride is producedin situ by reaction of KSCN and DCIC. The developed brown colour is measured at 460nm.
Collaborative studies1 have shown that the method gives equivalent results for water and 5%acetic acid extracts. It is recommended that 5%acetic acid extracts should be used if total alkaloids （as nicotine）and reducing substances （see ISO 15153）or reducing carbohydrates （see ISO 15154）are to be carried out simultaneously.
Note 1：Routine Analytical Chemistry Sub-Group Technical Report，2014 Collaborative Study Comparing CRM35 for the Determination of Total Alkaloids （as Nicotine） in Tobacco by Continuous Flow Analysis to a New Method with Safer Chemistry-Project 52.
4. REAGENTS
Use only reagents of recognized analytical grade. All reagents shall be used according to good laboratory practice and existing national regulations. Water must be high quality distilled or deionized （DI） water， free from organic contamination，e.g. Grade 1 as defined in ISO 3696.
4.1 Polyoxyethylene lauryl ether （Brij-35TM，30% w/w solution），CAS # 9002-92-0
4.2 Sodium phosphate dibasic dodecahydrate，Na2 HPO4·12H2O，CAS # 10039-32-4
4.3 Sodium phosphate monobasic dihydrate， NaH2 PO4· 2H2O， CAS #13472-35-0
4.4 Sodium citrate dihydrate，C6H5Na3O7·2H2O，CAS #6132-04-3
4.5 Sulphanilic acid，NH2C6H4SO3H，CAS #121-57-3
4.6 Potassium thiocyanate （KSCN），CAS #333-20-0
4.7 Sodium dichloroisocyanurate （DCIC），C3Cl2N3NaO3，CAS #51580-86-0
4.8 Sodium carbonate，Na2 CO3，CAS #497-19-8
4.9 Iron （II）sulphate heptahydrate，FeSO4·7H2O，CAS #7782-63-0
4.10 Citric acid monohydrate，C6 H8O7·H2O，CAS #5949-29-1
4.11 Nicotine hydrogen tartrate，C10H14N2（C4H6O6）·2H2O，CAS #6019-06-3
5. PREPARATION OF SOLUTIONS
Use distilled or deionized water，free from organic contamination，e.g. Grade 1 as defined in ISO 3696. To reach the performance levels stated，reagents and sam-pler wash must be free of solids and dissolved air.
For best results vacuum filter all reagents through a 0.45μm filter2 （see Figure 1）. If necessary，vacuum filter all DI water used in the preparation of standards and for the sampler wash，otherwise degas the water in another way.
Figure 1 Example vacuum filter set-up
Note 2：Millipore XX1604700 | MilliSolve Kit，complete with 2L flask is an example of a suitable product available commercially. This information is given for the convenience of the users of this recommended method and does not constitute an endorsement by CORESTA of this product.
5.1 System wash solution
Add 1mL of Brij-35，30% solution to about 800mL DI water and mix. Then dilute to 1000mL with DI water. Do not store the solution longer than a week and use a clean bottle for the fresh solution.
5.2 Sampler wash solution
Use the extraction solution，DI water or 5% acetic acid as sampler wash solution.
5.3 Potassium thiocyanate solution
Dissolve 2.88g of potassium thiocyanate in DI water. Dilute to 250mL with DI water and mix well.
5.4 Sodium dichloroisocyanurate （DCIC）solution
Dissolve 2.20g of sodium dichloroisocyanurate and dilute to 250mL with DI water. Prepare a fresh solution each day of measurement. Neutralisation solution A Dissolve 1 g of citric acid monohydrate and 10g of ferrous sulfate in about 500mL of DI water.
Dilute to 1000mL with DI water and mix well.
5.5 Neutralisation solution B
Dissolve 10g of sodium carbonate in about 500mL of DI water. Dilute to 1000mL with DI water and mix well.
5.6 Buffer solution A
Dissolve 71.6g of sodium phosphate dibasic dodecahydrate and 11.76g of sodium citrate dihydrate in about 500mL of DI water. Dilute to 1000mL with DI water， add 1mL of Brij-35，30% solution mix thoroughly.
5.7 Buffer solution B
Dissolve 71.6g of sodium phosphate dibasic dodecahydrate，6.2g of sodium phosphate monobasic dihydrate，11.76g of sodium citrate dihydrate and 7.0g of sulphanilic acid in about 800mL of DI water. Dilute to 1000mL with DI water，add 1mL of Brij-35，30% solution mix thoroughly.
6. PREPARATION OF STANDARDS
Check the purity of the nicotine hydrogen tartrate according to ISO 13276. The method can also be standardized by using nicotine or other nicotine salts of known purity. In this case，an amount equivalent to the above used nicotine hydrogen tartrate should be used.
6.1 Nicotine stock solution
Weigh 3.75g （to the nearest 0.0001g）of nicotine hydrogen tartrate in DI water and dilute to 500mL in a volumetric flask. The solution contains approximately 2.5mg nicotine permL，Store in a refrigerator （0～4）℃. Prepare a fresh solution every month.
6.2 Working standards
From the nicotine stock solution and extraction solution （water or 5% acetic acid solution），prepare a series of at least 5 calibration solutions according to the nicotine concentration which is expected to be found in the test samples （e.g.0.5%～15% （w/w））. Calculate the exact concentration for each standard taking into account the purity of the nicotine hydrogen tartrate.
Store in a refrigerator at （0～4）℃. Prepare fresh solutions every two weeks.
7. APPARATUS
The laboratory needs the usual laboratory apparatus and，in particular，the following items.
Continuous-flow analyser，consisting of
-Autosampler
-Peristaltic pump
-Chemistry manifold with dialyser and delay coils
-Photometric detector equipped with a 460nm filter
-Data acquisition system or recorder
See Annex A for examples of suitable flow diagrams.
8. PROCEDURE
8.1 Preparation of samples for analysis
Prepare the tobacco for analysis by grinding （the sample should totally pass a 1 mm sieve）and determine the moisture content. If the tobacco is too wet for grinding it can be dried at a temperature not exceeding 40℃.
8.2 Test portion
Weigh to the nearest 0.1 mg，approximately 250mg，of the ground tobacco into a 50mL conical flask. Add 25mL of the extraction solution （water or 5%acetic acid solution）. Stopper and shake for 30 minutes at＞150rpm.
8.3 Preparation of test extract
Filter the extract through a quantitative filter paper such as Whatman No 403 （or equivalent ashless，quantitative filter paper）filter paper，rejecting the first fewmL of the filtrate，then collect the filtrate.
Run the sample and standards through the system in the normal manner （e.g. priming with 6 tobacco extracts，calibration standards and samples with 1 intermediate calibration solution after every 6 samples.）. If sample concentration lies outside the range of the standards，the sample shall be diluted and run again.
When using 5% acetic acid extracts， the wash solution shall be 5% acetic acid.
NOTE：If this method is performed simultaneously with the methods described in ISO 15154 or ISO 15517，combined standards may be prepared. Combined stock solutions may precipitate after about two weeks.
Note 3：Whatman No.40 is an example of a suitable product available commercially. This information is given for the convenience of the users of this recommended method and does not constitute an endorsement by CORESTA of this product.
9. CALCULATION
9.1 Plot a graph of peak height against equivalent nicotine concentration for all of the calibration solutions.
9.2 Calculate the percentage of nicotine，w，on a dry weight basis，in the tobacco using the formula
where
c is the nicotine concentration，expressed in milligrams per millilitre，obtained from the calibration curve （8.3）；
V is the volume，in millilitres，of the sample （see 8.2），normally 25mL；
m is the mass，in milligrams，of the sample （see 8.2）；
M the moisture content，expressed as percentage by mass，of the tobacco （see8.1）
The test result shall be expressed to two decimal places.
10. REPEATABILITY AND REPRODUCIBILITY
In 2014 an international collaborative study4 involving 19 laboratories and eight samples （four straight grade tobaccos，a fire-cured cigarette，a blended cigarette， CM7，and 3R4F）was conducted. The repeatability limit （r）and reproducibility limit （R）were calculated for this new KSCN/DCIC method and CRM35 using both water and 5% acetic acid extractions （see Tables 1 &2）.
The difference between two single results，found on different extractions by one operator using the same apparatus within a short time interval （the time it takes to analyse～40 sample cups）and without recalibration of the equipment during the time of analysis，will exceed the repeatability limit （r）on average not more than once in 20 cases in the normal and correct operation of the method.
Single results reported by two laboratories will differ no more than the reproducibility limit （R）on average not more than once in 20 cases in the normal and correct operation of the method.
Note 4：Routine Analytical Chemistry Sub-Group Technical Report，2014 Collaborative Study Comparing CRM35 for the Determination of Total Alkaloids （as Nicotine） in Tobacco by Continuous Flow Analysis to a New Method with Safer Chemistry-Project 52
Table 1 Extraction with Water
续表
Table 2 Extraction with 5% Acetic Acid
续表
11. BIBLIOGRAPHY
ISO 15152，Tobacco—Determination of the content of total alkaloids as nico-tine—Continuousflow analysis method
CRM No.35，Determination of Total Alkaloids （as Nicotine）in Tobacco by Continuous Flow Analysis
APPENDIX A-SUITABLE FLOW DIAGRAMS
Figure 2 Suggested Flowchart for Macro Flow （mL/min）Systems
Figure 3 Suggested Flowchart for Micro Flow （μL/min）Systems
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