GB 36898-2018Feed additive - D-biotin (English PDF)

4.1 Appearance and properties

The product is a white or almost white crystal or crystalline powder. It is soluble in dilute alkaline solutions, slightly soluble in dilute acid solutions, very slightly soluble in water and ethanol, and practically insoluble in acetonitrile, acetone and chloroform.

4.2 Technical indicators

The technical indicators shall meet the requirements of Table 1.

Table 1 - Technical indicators: D-biotin content (as C10H16N2O3S, dry basis), %: 97.5 to 101.0; Loss on drying, %: <= 0.5; Specific rotation, (°)·dm2/kg: +89.0 to +93.0; Melting point, °C: 229 to 232; Impurities, %: any single impurity <= 1.0, total impurities <= 2.0; Clarity of solution: passes the test; Residue on ignition, %: <= 0.1; Heavy metals (as Pb), mg/kg: <= 10; Total arsenic (as As), mg/kg: <= 2.

5.1 Appearance inspection

Place an appropriate amount of the sample in a clean, dry white porcelain dish and observe its colour and form under natural light.

5.2 Identification

5.2.1 Reagents or materials

5.2.1.1 Bromine water, 3 %

5.2.1.2 D-biotin reference substance

Purity >= 99.0 %.

5.2.2 Instruments and equipment

5.2.2.1 Analytical balance

Sensitivity 0.1 mg.

5.2.2.2 Infrared spectrometer

5.2.3 Identification tests

5.2.3.1

Test by the method specified in GB/T 6040-2002, preparing the sample by the potassium bromide pellet method; the infrared absorption spectrum of the sample shall be consistent with that of the D-biotin reference substance. The infrared absorption spectrum of the D-biotin reference substance is given in Figure A.1.

5.2.3.2

Take about 40 mg of the sample, add 20 mL of water and stir to make a saturated solution; add dropwise 0.1 mL of 3 % bromine water (5.2.1.1): the bromine water is immediately decolourised.

5.2.3.3

In the chromatogram recorded in the determination of content (5.6), the retention time of the main peak of the sample solution shall be consistent with the retention time of the D-biotin main peak of the standard solution.

5.3 Loss on drying

5.3.1 Instruments and equipment

5.3.1.1 Electrically heated constant-temperature drying oven.

5.3.1.2 Analytical balance: sensitivity 0.1 mg.

5.3.2 Test procedure

Take about 2 g of the sample (to the nearest 0.1 mg) in a weighing bottle previously dried to constant mass, dry at 105 °C for 4 h, take it out, cool to room temperature and weigh accurately.

5.3.3 Test data processing

The loss on drying w1, expressed as a mass fraction in %, is calculated according to formula (1):

w1 = (m1 - m0) / m2 × 100 % ... (1)

where:

m1 - mass of the sample and weighing bottle before drying, in grams (g);

m0 - mass of the sample and weighing bottle after drying, in grams (g);

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

The determination result is expressed as the arithmetic mean of parallel determinations, and the calculated result is retained to one decimal place.

5.4 Specific rotation

5.4.1 Instruments and equipment

5.4.1.1 Polarimeter.

5.4.1.2 Analytical balance: sensitivity 0.1 mg.

5.4.2 Reagents or materials

NaOH solution (0.1 mol/L): weigh 4 g of sodium hydroxide, dissolve in water and dilute to 1 L.

5.4.3 Test procedure

5.4.3.1 Preparation of the sample solution

Weigh about 2 g (to the nearest 0.1 mg) of the sample, dried at 105 °C for 4 h and cooled, into a 100 mL volumetric flask, dissolve in NaOH solution (5.4.2), make up to the mark at 20 °C ± 0.5 °C and shake well.

5.4.3.2 Measurement procedure

At 20 °C ± 0.5 °C, rinse the measuring tube several times with NaOH solution (5.4.2), then slowly fill it with NaOH solution (5.4.2) (taking care not to produce air bubbles), place it in the polarimeter and set the zero point. In the same way, place the sample solution (5.4.3.1) in the measuring tube, put it in the polarimeter and read the value, which is the optical rotation; rotation of the plane of polarised light to the right is dextrorotatory and is indicated by the sign "+", rotation to the left is laevorotatory and is indicated by the sign "-". Take 3 readings, to the nearest 0.01°, and calculate the mean.

5.4.4 Test data processing

The specific rotation alpham of D-biotin at 20 °C and the sodium D line (589.3 nm) is calculated according to formula (2):

alpham(20 °C, D) = 100alpha / (l × rho) ... (2)

where:

alpha - measured optical rotation, in degrees (°);

l - length of the polarimeter tube, in decimetres (dm);

rho - mass concentration of D-biotin per 100 mL of the test solution, in grams per millilitre (g/mL).

The determination result is expressed as the arithmetic mean of parallel determinations, and the calculated result is retained to one decimal place.

5.5 Melting point

5.5.1 Instruments and equipment

Melting point apparatus.

5.5.2 Test procedure

Take an appropriate amount of the sample, dried at 105 °C for 4 h and cooled, and determine in accordance with GB/T 21781.

5.6 Determination of content

5.6.1 Principle

The sample is dissolved in acetonitrile solution, separated by high performance liquid chromatography, detected with an ultraviolet detector or a diode array detector, and quantified by peak area using the external standard method.

5.6.2 Reagents or materials

5.6.2.1 Acetonitrile

Chromatographically pure.

5.6.2.2 D-biotin reference substance

Purity >= 99.0 %.

5.6.2.3 Diluent

Acetonitrile + water = 1 + 4.

5.6.2.4 Buffer solution

Weigh 1 g of sodium perchlorate monohydrate, dissolve in 500 mL of water, add 1 mL of phosphoric acid and dilute with water to 1 000 mL.

5.6.2.5 Standard solution

Weigh about 40 mg of the D-biotin reference substance (to the nearest 0.01 mg) into a 200 mL volumetric flask, add about 180 mL of diluent (5.6.2.3) and dissolve by ultrasonication at 50 °C for 5 min; cool, dilute to the mark with diluent (5.6.2.3), shake well and filter through a filter membrane (5.6.2.6) for HPLC analysis.

5.6.2.6 Filter membrane

Aqueous type, 0.45 µm.

5.6.3 Instruments and equipment

5.6.3.1 Analytical balance

Sensitivity 0.01 mg.

5.6.3.2 High performance liquid chromatograph

Equipped with an ultraviolet detector or a diode array detector.

5.6.4 Test procedure

5.6.4.1 Preparation of the sample solution

Weigh about 40 mg of the sample (to the nearest 0.01 mg), dried at 105 °C for 4 h and cooled, into a 200 mL volumetric flask, add an appropriate amount of diluent (5.6.2.3) and dissolve by ultrasonication at 50 °C for 5 min; cool, dilute to the mark with diluent (5.6.2.3), shake well and filter through a filter membrane (5.6.2.6) for HPLC analysis.

5.6.4.2 Reference chromatographic conditions

Column: C18, 150 mm long, 4.6 mm internal diameter, 5 µm particle size, or equivalent performance;

Mobile phase: acetonitrile + buffer solution (5.6.2.4) = 8.5 + 91.5;

Flow rate: 1.2 mL/min;

Detection wavelength: 210 nm;

Injection volume: 20 µL.

5.6.4.3 Determination

Inject the standard solution (5.6.2.5) and the sample solution (5.6.4.1) into the high performance liquid chromatograph, record the chromatograms and calculate by peak area using the external standard method. A chromatogram of the standard solution is given in Figure B.1.

5.6.5 Test data processing

The content w2, expressed as a mass fraction in %, is calculated according to formula (3):

w2 = (A2 × rho1) / (A1 × rho2) × 100 % ... (3)

where:

A2 - peak area of the D-biotin main peak obtained from the chromatographic analysis of the sample solution;

rho1 - mass concentration of D-biotin in the standard solution, in milligrams per millilitre (mg/mL);

A1 - peak area of the D-biotin main peak obtained from the chromatographic analysis of the standard solution;

rho2 - mass concentration of D-biotin in the sample solution, in milligrams per millilitre (mg/mL).

The determination result is expressed as the arithmetic mean of parallel determinations, and the calculated result is retained to one decimal place.

5.6.6 Precision

The absolute difference between two independent test results obtained in the same laboratory, by the same operator using the same equipment and the same test method, on the same test object within a short interval of time, shall not exceed 2 % of the arithmetic mean of the two determined values, on condition that cases exceeding 2 % of the arithmetic mean do not exceed 5 %.

5.7 Impurities

Take the sample solution (5.6.4.1), determine under the reference chromatographic conditions (5.6.4.2) and record the chromatogram up to twice the retention time of the main component peak. If impurity peaks (excluding solvent peaks) appear in the chromatogram of the sample solution, calculate by the peak area normalisation method: the largest single impurity shall not exceed 1.0 % and the total impurities shall not exceed 2.0 %.

5.8 Clarity of solution

Take 0.25 g of the sample (to the nearest 1 mg) and dissolve it in 25 mL of 0.1 mol/L sodium hydroxide solution (5.4.2); the solution shall be clear and colourless.

5.9 Residue on ignition

5.9.1 Reagents or materials

Warning - Sulfuric acid is a strongly corrosive liquid; operators shall wear protective goggles and gloves to prevent burns.

Sulfuric acid.

5.9.2 Instruments and equipment

5.9.2.1 Analytical balance: sensitivity 0.1 mg.