GB 7295-2018Feed additive - Thiamine hydrochloride (Vitamin B1) (English PDF)

4.1 Appearance and properties

The product is a white crystal or crystalline powder with a slight characteristic odour and a bitter taste. It is readily soluble in water, slightly soluble in ethanol and insoluble in ether.

4.2 Technical indicators

The technical indicators of thiamine hydrochloride (vitamin B1) shall meet the requirements of Table 1.

Table 1 - Technical indicators: Thiamine hydrochloride (as C12H17ClN4OS·HCl, dry basis), %: 98.5 to 101.0; Loss on drying, % <= 5.0; Residue on ignition, % <= 0.1; Acidity: 2.7 to 3.4; Sulfate (as SO4 2-), % <= 0.03; Heavy metals (as Pb), mg/kg <= 10.0; Total arsenic (as As), mg/kg <= 2.0; Colour of solution: not deeper than 0.012 g/L potassium dichromate solution.

5.1 Appearance inspection

Place 10 g to 20 g of the sample in a clean white porcelain dish of 25 cm × 30 cm and assess it by the senses under normal lighting, in a well-ventilated environment free from foreign odours.

5.2 Identification tests

5.2.1 Reagents or materials

5.2.1.1 Sodium hydroxide solution

43 g/L.

5.2.1.2 Potassium ferricyanide solution

100 g/L. Prepare when needed.

5.2.1.3 n-Butanol

5.2.1.4 Manganese dioxide

5.2.1.5 Sulfuric acid

5.2.1.6 Potassium iodide

5.2.1.7 Starch indicator

Weigh 0.5 g of soluble starch, add 5 mL of water and stir well, then pour slowly into 100 mL of boiling water while stirring. Continue boiling for 2 min, allow to cool and take the supernatant. Prepare when needed.

5.2.1.8 Potassium iodide starch test paper

Immerse strips of filter paper in 100 mL of freshly prepared starch indicator containing 0.5 g of potassium iodide; after wetting, take them out and dry them in the shade.

5.2.2 Instruments and equipment

5.2.2.1 Analytical balance

Sensitivity 0.1 mg.

5.2.2.2 Infrared spectrometer

Scanning range 4 000 cm-1 to 400 cm-1, maximum resolution >= 4.0 cm-1.

5.2.3 Identification procedure

5.2.3.1

Weigh 0.005 g of the sample and dissolve it in sodium hydroxide solution (5.2.1.1); add 0.5 mL of potassium ferricyanide solution (5.2.1.2) and 5 mL of n-butanol, shake vigorously for 2 min and allow the layers to separate. The upper layer shows an intense blue fluorescence; when the solution is made acidic the fluorescence disappears, and when it is made alkaline again the fluorescence reappears.

5.2.3.2

Identification reaction for chloride: weigh 0.5 g of the sample into a dry test tube, add 0.5 g of manganese dioxide and mix, moisten with sulfuric acid and heat gently; chlorine is evolved, which turns moistened potassium iodide starch test paper blue.

5.2.3.3

Determine by infrared spectrophotometry: the infrared absorption spectrum of the sample shall be consistent with the reference spectrum (see Annex A for the reference spectrum).

5.3 Determination of thiamine hydrochloride content

5.3.1 Silicotungstic acid precipitation method (referee method)

5.3.1.1 Reagents or materials

5.3.1.1.1 Hydrochloric acid.

5.3.1.1.2 Silicotungstic acid solution: 100 g/L.

5.3.1.1.3 Hydrochloric acid solution: dilute 5 mL of hydrochloric acid (5.3.1.1.1) with water to 100 mL.

5.3.1.1.4 Acetone.

5.3.1.2 Instruments and equipment

Analytical balance: sensitivity 0.1 mg.

5.3.1.3 Test procedure

Weigh 0.1 g of the sample, previously dried to constant mass at 105 °C, to the nearest 0.000 2 g. Dissolve in 50 mL of water, add 2 mL of hydrochloric acid (5.3.1.1.1) and bring to the boil; immediately add dropwise 4 mL of silicotungstic acid solution (5.3.1.1.2) and continue boiling for 2 min. Filter through a No. 4 sintered-glass crucible previously dried to constant mass at 80 °C. Wash the precipitate first with 20 mL of boiling hydrochloric acid solution (5.3.1.1.3) in several portions, then once with 10 mL of water, and finally twice with 5 mL portions of acetone (5.3.1.1.4). Dry the precipitate at 80 °C to constant mass.

5.3.1.4 Test data processing

The thiamine hydrochloride content w1, expressed as a mass fraction in %, is calculated according to formula (1):

w1 = (m1 × 0.193 9) / m2 × 100 ... (1)

where:

m1 - mass of the precipitate after drying to constant mass, in grams (g);

0.193 9 - factor for converting thiamine silicotungstate into thiamine hydrochloride;

m2 - mass of the sample, in grams (g).

The test result is the arithmetic mean of the results of parallel determinations, retained to three significant figures.

5.3.1.5 Precision

The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 0.5 %.

5.3.2 Non-aqueous titration method

5.3.2.1 Principle

The thiamine hydrochloride (vitamin B1) molecule contains a basic primary amine group and a quaternary ammonium group, which react with perchloric acid in non-aqueous solution (in the presence of mercuric acetate); the thiamine hydrochloride content is calculated from the volume of perchloric acid standard solution consumed.

5.3.2.2 Reagents or materials

5.3.2.2.1 Glacial acetic acid.

5.3.2.2.2 Mercuric acetate solution: 50 g/L in glacial acetic acid.

5.3.2.2.3 Quinaldine red - methylene blue mixed indicator solution: 0.3 g of quinaldine red and 0.1 g of methylene blue, made up to 100 mL with methanol.

5.3.2.2.4 Perchloric acid standard solution: c(HClO4) = 0.1 mol/L.

5.3.2.3 Instruments and equipment

Analytical balance: sensitivity 0.1 mg.

5.3.2.4 Test procedure

Weigh 0.15 g of the sample, previously dried to constant mass at 105 °C, to the nearest 0.000 2 g, into a conical flask. Add 20 mL of glacial acetic acid and dissolve with gentle heating; allow to cool to room temperature, then add 5 mL of mercuric acetate solution and 2 drops of quinaldine red - methylene blue mixed indicator solution. Titrate with perchloric acid standard solution to a sky-blue colour that does not fade within 30 s of vigorous shaking, which is the end point. Correct the titration result with a blank test.

5.3.2.5 Test data processing

The thiamine hydrochloride content w2, expressed as a mass fraction in %, is calculated according to formula (2):

w2 = ((V1 - V0) × c × M) / (m3 × 1 000) × 100 ... (2)

where:

V1 - volume of perchloric acid standard solution consumed by the sample, in millilitres (mL);

V0 - volume of perchloric acid standard solution consumed by the blank test, in millilitres (mL);

c - concentration of the perchloric acid standard solution, in moles per litre (mol/L);

M - molar mass of thiamine hydrochloride, in grams per mole (g/mol) [M(1/2 C12H17ClN4OS·HCl) = 168.6 g/mol];

m3 - mass of the sample, in grams (g).

The test result is the arithmetic mean of the results of parallel determinations, retained to three significant figures.

5.3.2.6 Precision

The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 0.5 %.

5.4 Examination of solution colour

5.4.1 Reagents or materials

5.4.1.1 Potassium dichromate: primary standard grade.

5.4.1.2 Potassium dichromate solution (0.012 g/L): on an analytical balance with a sensitivity of 0.000 1 g, accurately weigh 0.400 0 g of potassium dichromate, dissolve and make up to 500 mL. Store in a brown bottle.

5.4.2 Test procedure

Weigh 1.0 g of the sample into a 50 mL Nessler colorimetric tube, dissolve in 10 mL of water and compare with the same volume of reference solution (0.6 mL of the potassium dichromate solution for colorimetry, made up to 40 mL with water); the colour shall not be deeper.

5.5 Acidity

5.5.1 Instruments and equipment

5.5.1.1 Analytical balance: sensitivity 0.1 mg.

5.5.1.2 pH meter: measuring range pH 0 to 14, accuracy 0.02 pH unit.

5.5.2 Test procedure

Weigh 0.50 g of the sample to the nearest 0.000 2 g into a 100 mL beaker, dissolve in 50 mL of water and measure the pH with the pH meter.

5.6 Determination of sulfate

5.6.1 Reagents or materials

5.6.1.1 Hydrochloric acid solution: 1 + 10.

5.6.1.2 Barium chloride solution: 250 g/L.

5.6.1.3 Potassium sulfate standard solution: rho(SO4 2-) = 0.1 g/L.

5.6.2 Instruments and equipment

Analytical balance: sensitivity 0.1 mg.

5.6.3 Test procedure

Weigh 1.0 g of the sample to the nearest 0.000 2 g and dissolve in water to 20 mL; if the solution is turbid, filter it. Transfer to a 50 mL Nessler colorimetric tube, dilute with water to 25 mL and add 1 mL of hydrochloric acid solution (5.6.1.1). Add 3 mL of barium chloride solution (5.6.1.2), shake well and allow to stand for 10 min; any turbidity that appears shall not be greater than that of a reference solution prepared in the same way with 3 mL of potassium sulfate standard solution (5.6.1.3).

5.7 Determination of loss on drying

5.7.1 Instruments and equipment

Analytical balance: sensitivity 0.1 mg.

5.7.2 Test procedure

Weigh 1 g of the sample to the nearest 0.000 2 g into a weighing dish previously dried to constant mass, place it in an electric drying oven at 105 °C ± 2 °C with the lid of the weighing dish open and dry for 3 h. Take it out, close the lid, place in a desiccator, cool to room temperature and weigh. Repeat drying for 1 h at a time and weighing until constant mass is reached.

5.7.3 Test data processing

The loss on drying of thiamine hydrochloride w3, expressed as a mass fraction in %, is calculated according to formula (3):

w3 = (m4 - m5) / m6 × 100 ... (3)

where:

m4 - mass of the sample and weighing dish before drying, in grams (g);

m5 - mass of the sample and weighing dish after drying, in grams (g);

m6 - mass of the sample, in grams (g).