Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This part is drafted in accordance with the rules given in the GB/T 1.1-2009
This part replaces GB/T 14700-2002 Determination of Vitamin B1 in Feeds.
The following technical deviations have been made with respect to the GB/T 14700-2002 (the previous edition):
— modification of the Scope (see Clause 1; Clause 1 of Edition 2002);
— deletion of the molecular formula of thiamine (see Clause 3; 3.1 of Edition 2002);
— addition of the specific preparation process of acidic ethanol solution (20%) (see Clause 3; 3.2 of Edition 2002);
— addition of the requirements of “additive premix shall not be ground” in the specimen preparation (see Clause 4; 4.4 of Edition 2002);
— change of the extract of compound premix to acidic ammonium chloride methanol solution to to distinguish it from vitamin premix (see Clause 4; 4.2.9 of Edition 2002);
— addition of the chromatograms and spectrograms of thiamine (see Annex A).
This standard was proposed by and is under the jurisdiction of SAC/TC 76 (National Technical Committee 76 on Feed Industry of Standardization Administration of China).
The previous editions of this standard are as follows:
— GB/T 14700-1999, GB/T 14700-2002.
Determination of Thiamine in Feed
1 Scope
This standard specifies two methods for the determination of thiamine content in feed, namely by fluorescence spectrophotometry and high-performance liquid chromatography.
Method 1 of this standard is applicable to the determination of thiamine in feedstuffs, formula feed and concentrate. The quantitative limit of Method 1 is 1 mg/kg (this method is not applicable in the presence of adsorbent thiamine or interfering substances that affect thiochrome fluorescence). Thiamine determined by this method includes the sum of endogenous and additive amounts.
Method 2 of this standard is applicable to the determination of thiamine in compound premix and vitamin premix. The detection limit of Method 2 is 3 mg/kg, and the quantitative limit is 15 mg/kg.
2 Normative References
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 6682 Water for Laboratory Use — Specifications and Test Methods
GB/T 14699.1 Animal Feeding Stuffs — Sampling
GB/T 20195 Animal Feeding Stuffs —Preparation of Test Samples
3 Method 1: Fluorescence Spectrophotometry
3.1 Principle
Thiamine in the specimen is decomposed by dilute acid and digestive enzyme, and separated and purified by adsorbent, then oxidized by potassium ferricyanide to produce fluorochrome-thiochrome under alkaline conditions, and extracted by n-butanol. The fluorescence intensity of thiochrome in n-butanol is directly proportional to the content of thiamine in the specimen, and the content can be determined quantitatively.
3.2 Reagent or solution
Unless otherwise specified, the reagents used in this standard shall be of analytical grade, and the water for chromatographic use shall meet the requirements of Grade 1 water specified in GB/T 6682. The water for laboratory use shall meet the requirements of Grade 3 water specified in GB/T 6682.
3.2.4 Amylase suspension emulsion (100 g/L): suspend 10 g amylase preparation with sodium acetate solution (3.2.3), dilute to 100 mL, and prepare on the day of use.
3.2.5 Potassium chloride solution: 250 g/L.
3.2.6 Acidic potassium chloride solution: add 8.5 mL concentrated hydrochloric acid to the potassium chloride solution (3.2.5) and dilute to 1000 mL.
3.2.7 Sodium hydroxide solution: 150 g/L
3.2.8 Potassium ferricyanide solution: 10 g/L.
3.2.9 Alkaline potassium ferricyanide solution: take 4.00 mL of potassium ferricyanide solution (3.2.8) and mix with sodium hydroxide solution (3.2.7) to make 100 mL. This solution is used within 4h.
3.2.10 Glacial acetic acid solution: 30 mL/L.
3.2.11 Acidic ethanol solution (20%): take 80 mL of water, adjust the pH to 3.5 to 4.3 with hydrochloric acid solution (3.2.1), and mix with 20 mL of anhydrous ethanol.
3.2.12 Synthetic zeolite [0.25 mm to 0.18 mm (60 to 80 mesh)]: before use should be activated, the method is as follows: put a suitable amount of synthetic zeolite in a beaker, add 10 times volume of glacial acetic acid solution (3.2.10) which had been heated up to 60 ℃ to 70 ℃; evenly stirring for 10 min with a glass rod to make the zeolite suspended in the glacial acetic acid solution; after the settlement of the zeolite, the upper layer of glacial acetic acid solution is discarded; repeat the above procedures twice. Add 5 times volume of potassium chloride solution (3.2.5) which had been heated up to 60 ℃ to 70 ℃, stirring and cleaning twice (15 min for each time). Then wash it with hot glacial acetic acid solution for 10min. Finally, the zeolite is washed in hot water to chlorine-free (tested wih 10g/L silver nitrate solution). Filter with brinell funnel and dry at 105 ℃, store in a ground bottle for 6 months.
Prior to use, check the recovery rate of zeolite to the thiamine standard solution. If the recovery rate is less than 92%, the zeolite shall be reactivated.
Note: Check the recovery rate of thiamine by zeolite: Take 2 mL of thiamine standard intermediate solution (3.2.13.2) and dilute the volume to 100 mL with acidic potassium chloride solution (3.2.6). It shall be oxidated according to the procedures 3.5.4.1 to 3.5.4.3 and taken as the external standard. Take 25 mL of another thiamine standard intermediate solution (3.2.13.3) and repeat the column chromatography operation specified in 3.5.3.1 to 3.5.3.3. It shall be oxidated according to the procedures 3.5.4.1 to 3.5.4.3. The fluorescence intensity of the two solutions shall be determined synchronously. The calculated result of Formula (1) is converted into a percentage, which is the recovery rate of thiamine by zeolite.
3.2.13 Thiamine standard solution
3.2.13.1 Thiamine standard stock solution: take standard thiamine nitrate (purity > 99%) and dry it in a phosphorus pentoxide dryer for 24h. Weigh 0.01 g (to the nearest 0.0001g), dissolve it in acidic ethanol solution (20%) (3.2.11) and dilute the volume to 100mL. Put it in a brown bottle and store it in refrigerator at 2℃ to 8℃ for 3 months. The solution with a thiamine content of 0.1 mg/mL.
3.2.13.2 Thiamine standard intermediate solution: take 10 mL of thiamine standard stock solution (3.2.13.1) and dilute the volume to 100 mL with acidic ethanol solution (20%) (3.2.11). Put it in a brown bottle and store it in refrigerator at 2℃ to 8℃ for 48 h. The solution with a thiamine content of 10 μg/mL.
3.2.13.3 Thiamine standard working solution: take 2 mL of thiamine standard intermediate solution (3.2.13.2) mixed with 65 mL of hydrochloric acid solution (3.2.1) and 5mL of sodium acetate solution (3.2.3), and dilute the volume to 100 mL. Prepare before analysis. The solution with a thiamine content of 0.2 μg/mL.
3.2.14 Quinine sulphate solution
3.2.14.1 Quinine sulphate stock solution: Weigh 0.1 g of quinine sulphate (to the nearest 0.001 g), dissolve it with sulfuric acid solution (3.2.2) and dilute the volume to 1 000 mL. Store it in a brown bottle and refrigerated. If the solution is cloudy, it shall be reformulated.
3.2.14.2 Quinine sulphate working solution: take 3 mL of quinine sulphate stock solution (3.2.13.1) and dilute the volume to 1 000 mL with sulfuric acid solution (3.2.2).Store it in a brown bottle and refrigerated. The solution with a quinine sulphate content of 0.3 μg/mL.
3.2.15 N-butanol: the fluorescence intensity shall not exceed 4% of quinine sulphate working solution (3.2.14.2), otherwise it shall be re-distilled by all-glass distiller, and take the fraction at 114℃ to 118℃.
3.3 Apparatus
3.3.1 Normal laboratory glassware.
3.3.2 Analytical balance: with a sensibility of 0.0001 g and 0.001 g.
3.3.3 Autoclave, the operating temperature is 121℃ to 123℃ or the pressure reaches 15kg/cm2.
3.3.4 Electric thermostat, 45℃ to 50℃.
3.3.5 Adsorption separation column: the total length is 235 mm, the outer diameter × length is as follows: the size of upper tank is 35 mm × 70 mm, and the capacity is about 50 mL; the middle adsorption tube is 8 mm × 130 mm; the lower end is drawn into a capillary tube of 35 mm.
3.3.6 Centrifugal tube with stopper: 25 mL.
3.3.7 Fluorescence spectrophotometer with 1cm quartz cuvette.
3.3.8 Syringe: 10 mL.
3.4 Specimen
Representative feed specimens shall be sampled according to GB/T 14699.1 and the quartering method is used to reduce the specimen. The specimens shall be prepared according to GB/T 20195, ground and filtered with a 0.425 mm aperture sieve, and thoroughly mixed.
3.5 Test procedures
3.5.1 Weighing
Weigh the feedstuffs, formula feed and concentrate 1 g to 2 g, to the nearest 0.001g, and place them in 100mL brown conical bottle.
3.5.2 Preparation of sample solution
3.5.2.1 Hydrolysis: add 65 mL of hydrochloric acid solution (3.2.1) to a conical flask, place the solution in a boiling water bath and heat for 30 min [or heat in a autoclave (3.3.2) for 30 min] after the filling. Start heating for 5 min to 10min, and shake the conical bottle from time to time to prevent caking.
3.5.2.2 Enzymatic hydrolysis: cool the conical flask to below 50℃, add 5 mL of amylase suspension (3.2.4), and mix well. The solution had a pH of about 4.0 to 4.5. The conical flask is placed in an electric thermostat (3.3.4) at 45℃ to 50℃ for 3h, and then taken out for cooling. The pH is adjusted to 3.5 with hydrochloric acid solution (3.2.1), and then transferred to a 100mL brown volumetric flask. Dilute the volume to 100 mL with water and mix well.
3.5.2.3 Filtration: filter all test solutions through ash-free filter paper, discard 5mL of the initial filtrate, and collect the filtrate as the sample solution.
3.5.3 Purification of sample solution
3.5.3.1 Preparation of adsorption column: weigh 1.5 g activated artificial zeolite (3.2.12) and place it in a small 50 mL beaker; add glacial acetic acid solution (3%) (3.2.10) and soak it with the liquid surface of solution can completely submerge the zeolite.Place the absorbent cotton at the bottom of the adsorption separation column (3.3.5) and press lightly with a glass rod. Then wash all zeolites soaked in acetic acid and transfer them into the column (do not dehydrate the adsorption column) and the flow rate of column should be controlled at 1 mL/min. Use 10mL of near-boiling water to wash the column once more.
3.5.3.2 Take 25 mL of sample solution (3.5.2.3), slowly add it into the prepared adsorption column, discard the filtrate, and use each 5 mL near-boiling water to wash the column for 3 times and discard the detergent. Conduct parallel test at the same time.
3.5.3.3 Add 25 mL of 60℃ to 70℃ acidic potassium chloride solution (3.2.6) into the adsorption column for 3 consecutive times, and collect the eluent in a 25 mL volumetric bottle; after cool down, dilute to the volume with acidic potassium chloride solution and mix well.
3.5.3.4 Use 25mL thiamine standard working solution (3.2.13.3) at the same time. Repeat the procedures 3.5.3.1 to 3.5.3.3, and take it as the external standard.
3.5.4 Oxidation and extraction
Warning: The following procedures shall be conducted in a dark place.
3.5.4.1 Respectively add 5 mL of eluent (3.5.3.3) into two centrifugal tubes (with stoppers) (3.3.6), and labeled as A and B, respectively.
3.5.4.2 Add 3 mL of sodium hydroxide solution (3.2.7) to tube B, and then add 3 mL of alkaline potassium ferricyanide solution (3.2.9) to tube A, and gently rotate. Immediately add 15 mL of n-butanol (3.2.15) to tube A and cover it with stopper, and then shake it violently for 15s; then add 15 mL of n-butanol to tube B, shake it together for 90s, and lay them in static stratification.
3.5.4.3 Suck the lower aqueous phase with a syringe (3.3.8), add about 2 g of anhydrous sodium sulfate to each tube, and shake it for testing.
3.5.4.4 At the same time, take 5 mL of eluent (3.5.3.4) as the external standard, and add into two other centrifugal tubes with stopper, and labeled as C and D, respectively. Conduct the procedures according to 3.5.4.1 to 3.5.4.3.
3.5.5 Determination
3.5.5.1 Adjust the fluorometer with quinine sulphate working solution (3.2.14.2) to fix it at a certain value as the fixed condition of the instrument.
3.5.5.2 Fluorescence intensity of the extract in tubes A, B, C and D are measured at excitation wavelength 365 nm and emission wavelength 435 nm.
3.6 Processing of test data
The thiamine determined by this method is calculated as thiamine nitrate; or calculated as thiamine hydrochloride, if necessary, it is converted to 1 mg thiamine hydrochloride containing 1.03 mg thiamine nitrate.
The content of thiamine in the specimen can be calculated by using Formula (1):
(1)
Where,
wi — the content of thiamine in the specimen, in milligrams per kilogram (mg/kg);
T1 — the fluorescence intensity of test solution in tube A;
T2 — the fluorescence intensity of test solution blank in tube B;
T3 — the fluorescence intensity of standard solution in tube C;
T4 — the fluorescence intensity of standard solution blank in tube D;
ρ — the concentration of thiamine standard solution solution, in micrograms per milliliter (μg/mL);
V0 — the total volume of extract, in milliliters (mL);
V1 — the volume of sample solution passing through the column, in milliliters (mL);
V2 — the volume of eluent of acidic potassium chloride, in milliliters (mL);
m — the mass of test specimen, in grams (g).
Express the determination result with the arithmetic mean through parallel determination and to three decimal places.
3.7 Repeatability
For feeds with thiamine content less than 5 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 15% of the arithmetic mean value of the two test values under the repeatability conditions.
For feeds with thiamine content greater than 5 mg/kg but less than 50 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 10% of the arithmetic mean value of the two test values under the repeatability conditions.
For feeds with thiamine content greater than 50 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 5% of the arithmetic mean value of the two test values under the repeatability conditions.
After ultrasonic extraction of the acid extract of specimen, the filtered and centrifugated test solution is injected into a high-performance liquid chromatography (HPLC) system for separation. Ultraviolet (or diode matrix detector) is used for detection. The content of thiamine can be calculated by external standard method.
4.2 Reagent or solution
Unless otherwise specified, the reagents used shall be of analytical grade, and the water for chromatographic use shall meet the requirements of Grade 1 water specified in GB/T 6682.
Foreword II 1 Scope 2 Normative References 3 Method 1: Fluorescence Spectrophotometry 4 Method 2: High-performance Liquid Chromatography Annex A (Informative) Standard Chromatogram and Spectrogram of Thiamine
Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This part is drafted in accordance with the rules given in the GB/T 1.1-2009
This part replaces GB/T 14700-2002 Determination of Vitamin B1 in Feeds.
The following technical deviations have been made with respect to the GB/T 14700-2002 (the previous edition):
— modification of the Scope (see Clause 1; Clause 1 of Edition 2002);
— deletion of the molecular formula of thiamine (see Clause 3; 3.1 of Edition 2002);
— addition of the specific preparation process of acidic ethanol solution (20%) (see Clause 3; 3.2 of Edition 2002);
— addition of the requirements of “additive premix shall not be ground” in the specimen preparation (see Clause 4; 4.4 of Edition 2002);
— change of the extract of compound premix to acidic ammonium chloride methanol solution to to distinguish it from vitamin premix (see Clause 4; 4.2.9 of Edition 2002);
— addition of the chromatograms and spectrograms of thiamine (see Annex A).
This standard was proposed by and is under the jurisdiction of SAC/TC 76 (National Technical Committee 76 on Feed Industry of Standardization Administration of China).
The previous editions of this standard are as follows:
— GB/T 14700-1999, GB/T 14700-2002.
Determination of Thiamine in Feed
1 Scope
This standard specifies two methods for the determination of thiamine content in feed, namely by fluorescence spectrophotometry and high-performance liquid chromatography.
Method 1 of this standard is applicable to the determination of thiamine in feedstuffs, formula feed and concentrate. The quantitative limit of Method 1 is 1 mg/kg (this method is not applicable in the presence of adsorbent thiamine or interfering substances that affect thiochrome fluorescence). Thiamine determined by this method includes the sum of endogenous and additive amounts.
Method 2 of this standard is applicable to the determination of thiamine in compound premix and vitamin premix. The detection limit of Method 2 is 3 mg/kg, and the quantitative limit is 15 mg/kg.
2 Normative References
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 6682 Water for Laboratory Use — Specifications and Test Methods
GB/T 14699.1 Animal Feeding Stuffs — Sampling
GB/T 20195 Animal Feeding Stuffs —Preparation of Test Samples
3 Method 1: Fluorescence Spectrophotometry
3.1 Principle
Thiamine in the specimen is decomposed by dilute acid and digestive enzyme, and separated and purified by adsorbent, then oxidized by potassium ferricyanide to produce fluorochrome-thiochrome under alkaline conditions, and extracted by n-butanol. The fluorescence intensity of thiochrome in n-butanol is directly proportional to the content of thiamine in the specimen, and the content can be determined quantitatively.
3.2 Reagent or solution
Unless otherwise specified, the reagents used in this standard shall be of analytical grade, and the water for chromatographic use shall meet the requirements of Grade 1 water specified in GB/T 6682. The water for laboratory use shall meet the requirements of Grade 3 water specified in GB/T 6682.
3.2.1 Hydrochloric acid solution: c(HCl) = 0.1 mol/L.
3.2.2 Sulfuric acid solution: c(1/2H2SO4) = 0.05 mol/L.
3.2.3 Sodium acetate solution c(CH3COONa) = 2.0mol/L.
3.2.4 Amylase suspension emulsion (100 g/L): suspend 10 g amylase preparation with sodium acetate solution (3.2.3), dilute to 100 mL, and prepare on the day of use.
3.2.5 Potassium chloride solution: 250 g/L.
3.2.6 Acidic potassium chloride solution: add 8.5 mL concentrated hydrochloric acid to the potassium chloride solution (3.2.5) and dilute to 1000 mL.
3.2.7 Sodium hydroxide solution: 150 g/L
3.2.8 Potassium ferricyanide solution: 10 g/L.
3.2.9 Alkaline potassium ferricyanide solution: take 4.00 mL of potassium ferricyanide solution (3.2.8) and mix with sodium hydroxide solution (3.2.7) to make 100 mL. This solution is used within 4h.
3.2.10 Glacial acetic acid solution: 30 mL/L.
3.2.11 Acidic ethanol solution (20%): take 80 mL of water, adjust the pH to 3.5 to 4.3 with hydrochloric acid solution (3.2.1), and mix with 20 mL of anhydrous ethanol.
3.2.12 Synthetic zeolite [0.25 mm to 0.18 mm (60 to 80 mesh)]: before use should be activated, the method is as follows: put a suitable amount of synthetic zeolite in a beaker, add 10 times volume of glacial acetic acid solution (3.2.10) which had been heated up to 60 ℃ to 70 ℃; evenly stirring for 10 min with a glass rod to make the zeolite suspended in the glacial acetic acid solution; after the settlement of the zeolite, the upper layer of glacial acetic acid solution is discarded; repeat the above procedures twice. Add 5 times volume of potassium chloride solution (3.2.5) which had been heated up to 60 ℃ to 70 ℃, stirring and cleaning twice (15 min for each time). Then wash it with hot glacial acetic acid solution for 10min. Finally, the zeolite is washed in hot water to chlorine-free (tested wih 10g/L silver nitrate solution). Filter with brinell funnel and dry at 105 ℃, store in a ground bottle for 6 months.
Prior to use, check the recovery rate of zeolite to the thiamine standard solution. If the recovery rate is less than 92%, the zeolite shall be reactivated.
Note: Check the recovery rate of thiamine by zeolite: Take 2 mL of thiamine standard intermediate solution (3.2.13.2) and dilute the volume to 100 mL with acidic potassium chloride solution (3.2.6). It shall be oxidated according to the procedures 3.5.4.1 to 3.5.4.3 and taken as the external standard. Take 25 mL of another thiamine standard intermediate solution (3.2.13.3) and repeat the column chromatography operation specified in 3.5.3.1 to 3.5.3.3. It shall be oxidated according to the procedures 3.5.4.1 to 3.5.4.3. The fluorescence intensity of the two solutions shall be determined synchronously. The calculated result of Formula (1) is converted into a percentage, which is the recovery rate of thiamine by zeolite.
3.2.13 Thiamine standard solution
3.2.13.1 Thiamine standard stock solution: take standard thiamine nitrate (purity > 99%) and dry it in a phosphorus pentoxide dryer for 24h. Weigh 0.01 g (to the nearest 0.0001g), dissolve it in acidic ethanol solution (20%) (3.2.11) and dilute the volume to 100mL. Put it in a brown bottle and store it in refrigerator at 2℃ to 8℃ for 3 months. The solution with a thiamine content of 0.1 mg/mL.
3.2.13.2 Thiamine standard intermediate solution: take 10 mL of thiamine standard stock solution (3.2.13.1) and dilute the volume to 100 mL with acidic ethanol solution (20%) (3.2.11). Put it in a brown bottle and store it in refrigerator at 2℃ to 8℃ for 48 h. The solution with a thiamine content of 10 μg/mL.
3.2.13.3 Thiamine standard working solution: take 2 mL of thiamine standard intermediate solution (3.2.13.2) mixed with 65 mL of hydrochloric acid solution (3.2.1) and 5mL of sodium acetate solution (3.2.3), and dilute the volume to 100 mL. Prepare before analysis. The solution with a thiamine content of 0.2 μg/mL.
3.2.14 Quinine sulphate solution
3.2.14.1 Quinine sulphate stock solution: Weigh 0.1 g of quinine sulphate (to the nearest 0.001 g), dissolve it with sulfuric acid solution (3.2.2) and dilute the volume to 1 000 mL. Store it in a brown bottle and refrigerated. If the solution is cloudy, it shall be reformulated.
3.2.14.2 Quinine sulphate working solution: take 3 mL of quinine sulphate stock solution (3.2.13.1) and dilute the volume to 1 000 mL with sulfuric acid solution (3.2.2).Store it in a brown bottle and refrigerated. The solution with a quinine sulphate content of 0.3 μg/mL.
3.2.15 N-butanol: the fluorescence intensity shall not exceed 4% of quinine sulphate working solution (3.2.14.2), otherwise it shall be re-distilled by all-glass distiller, and take the fraction at 114℃ to 118℃.
3.3 Apparatus
3.3.1 Normal laboratory glassware.
3.3.2 Analytical balance: with a sensibility of 0.0001 g and 0.001 g.
3.3.3 Autoclave, the operating temperature is 121℃ to 123℃ or the pressure reaches 15kg/cm2.
3.3.4 Electric thermostat, 45℃ to 50℃.
3.3.5 Adsorption separation column: the total length is 235 mm, the outer diameter × length is as follows: the size of upper tank is 35 mm × 70 mm, and the capacity is about 50 mL; the middle adsorption tube is 8 mm × 130 mm; the lower end is drawn into a capillary tube of 35 mm.
3.3.6 Centrifugal tube with stopper: 25 mL.
3.3.7 Fluorescence spectrophotometer with 1cm quartz cuvette.
3.3.8 Syringe: 10 mL.
3.4 Specimen
Representative feed specimens shall be sampled according to GB/T 14699.1 and the quartering method is used to reduce the specimen. The specimens shall be prepared according to GB/T 20195, ground and filtered with a 0.425 mm aperture sieve, and thoroughly mixed.
3.5 Test procedures
3.5.1 Weighing
Weigh the feedstuffs, formula feed and concentrate 1 g to 2 g, to the nearest 0.001g, and place them in 100mL brown conical bottle.
3.5.2 Preparation of sample solution
3.5.2.1 Hydrolysis: add 65 mL of hydrochloric acid solution (3.2.1) to a conical flask, place the solution in a boiling water bath and heat for 30 min [or heat in a autoclave (3.3.2) for 30 min] after the filling. Start heating for 5 min to 10min, and shake the conical bottle from time to time to prevent caking.
3.5.2.2 Enzymatic hydrolysis: cool the conical flask to below 50℃, add 5 mL of amylase suspension (3.2.4), and mix well. The solution had a pH of about 4.0 to 4.5. The conical flask is placed in an electric thermostat (3.3.4) at 45℃ to 50℃ for 3h, and then taken out for cooling. The pH is adjusted to 3.5 with hydrochloric acid solution (3.2.1), and then transferred to a 100mL brown volumetric flask. Dilute the volume to 100 mL with water and mix well.
3.5.2.3 Filtration: filter all test solutions through ash-free filter paper, discard 5mL of the initial filtrate, and collect the filtrate as the sample solution.
3.5.3 Purification of sample solution
3.5.3.1 Preparation of adsorption column: weigh 1.5 g activated artificial zeolite (3.2.12) and place it in a small 50 mL beaker; add glacial acetic acid solution (3%) (3.2.10) and soak it with the liquid surface of solution can completely submerge the zeolite.Place the absorbent cotton at the bottom of the adsorption separation column (3.3.5) and press lightly with a glass rod. Then wash all zeolites soaked in acetic acid and transfer them into the column (do not dehydrate the adsorption column) and the flow rate of column should be controlled at 1 mL/min. Use 10mL of near-boiling water to wash the column once more.
3.5.3.2 Take 25 mL of sample solution (3.5.2.3), slowly add it into the prepared adsorption column, discard the filtrate, and use each 5 mL near-boiling water to wash the column for 3 times and discard the detergent. Conduct parallel test at the same time.
3.5.3.3 Add 25 mL of 60℃ to 70℃ acidic potassium chloride solution (3.2.6) into the adsorption column for 3 consecutive times, and collect the eluent in a 25 mL volumetric bottle; after cool down, dilute to the volume with acidic potassium chloride solution and mix well.
3.5.3.4 Use 25mL thiamine standard working solution (3.2.13.3) at the same time. Repeat the procedures 3.5.3.1 to 3.5.3.3, and take it as the external standard.
3.5.4 Oxidation and extraction
Warning: The following procedures shall be conducted in a dark place.
3.5.4.1 Respectively add 5 mL of eluent (3.5.3.3) into two centrifugal tubes (with stoppers) (3.3.6), and labeled as A and B, respectively.
3.5.4.2 Add 3 mL of sodium hydroxide solution (3.2.7) to tube B, and then add 3 mL of alkaline potassium ferricyanide solution (3.2.9) to tube A, and gently rotate. Immediately add 15 mL of n-butanol (3.2.15) to tube A and cover it with stopper, and then shake it violently for 15s; then add 15 mL of n-butanol to tube B, shake it together for 90s, and lay them in static stratification.
3.5.4.3 Suck the lower aqueous phase with a syringe (3.3.8), add about 2 g of anhydrous sodium sulfate to each tube, and shake it for testing.
3.5.4.4 At the same time, take 5 mL of eluent (3.5.3.4) as the external standard, and add into two other centrifugal tubes with stopper, and labeled as C and D, respectively. Conduct the procedures according to 3.5.4.1 to 3.5.4.3.
3.5.5 Determination
3.5.5.1 Adjust the fluorometer with quinine sulphate working solution (3.2.14.2) to fix it at a certain value as the fixed condition of the instrument.
3.5.5.2 Fluorescence intensity of the extract in tubes A, B, C and D are measured at excitation wavelength 365 nm and emission wavelength 435 nm.
3.6 Processing of test data
The thiamine determined by this method is calculated as thiamine nitrate; or calculated as thiamine hydrochloride, if necessary, it is converted to 1 mg thiamine hydrochloride containing 1.03 mg thiamine nitrate.
The content of thiamine in the specimen can be calculated by using Formula (1):
(1)
Where,
wi — the content of thiamine in the specimen, in milligrams per kilogram (mg/kg);
T1 — the fluorescence intensity of test solution in tube A;
T2 — the fluorescence intensity of test solution blank in tube B;
T3 — the fluorescence intensity of standard solution in tube C;
T4 — the fluorescence intensity of standard solution blank in tube D;
ρ — the concentration of thiamine standard solution solution, in micrograms per milliliter (μg/mL);
V0 — the total volume of extract, in milliliters (mL);
V1 — the volume of sample solution passing through the column, in milliliters (mL);
V2 — the volume of eluent of acidic potassium chloride, in milliliters (mL);
m — the mass of test specimen, in grams (g).
Express the determination result with the arithmetic mean through parallel determination and to three decimal places.
3.7 Repeatability
For feeds with thiamine content less than 5 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 15% of the arithmetic mean value of the two test values under the repeatability conditions.
For feeds with thiamine content greater than 5 mg/kg but less than 50 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 10% of the arithmetic mean value of the two test values under the repeatability conditions.
For feeds with thiamine content greater than 50 mg/kg, the difference between the two independent test results and the arithmetic mean value is no more than 5% of the arithmetic mean value of the two test values under the repeatability conditions.
4 Method 2: High-performance Liquid Chromatography
4.1 Principle
After ultrasonic extraction of the acid extract of specimen, the filtered and centrifugated test solution is injected into a high-performance liquid chromatography (HPLC) system for separation. Ultraviolet (or diode matrix detector) is used for detection. The content of thiamine can be calculated by external standard method.
4.2 Reagent or solution
Unless otherwise specified, the reagents used shall be of analytical grade, and the water for chromatographic use shall meet the requirements of Grade 1 water specified in GB/T 6682.
Contents of GB/T 14700-2018
Foreword II
1 Scope
2 Normative References
3 Method 1: Fluorescence Spectrophotometry
4 Method 2: High-performance Liquid Chromatography
Annex A (Informative) Standard Chromatogram and Spectrogram of Thiamine
