1. Appearance: white powder

Weigh 1.00g of HMBCa sample, put it on a colorless and transparent glass sheet, and observe the color and properties of the sample.

2. High-performance liquid chromatography identification

Precisely weigh about 100mg CaHMB standard, put it in a 25mL volumetric flask, add phosphate buffer, shake until dissolved, and use the buffer to fix the volume to

the standard line. Place on an ultrasonicator and sonicate for 10 minutes. Aspirate about 1 mL into a centrifuge tube. Centrifuge at >1000 r/min for 10 min and aspirate the supernatant into the sample tube.

Chromatographic column: C18 250 × 4.6mm × 5μm Flow rate: 1mL/min

Detection wavelength: 210nm Injection volume: 20μL Standard curve plotting

Use the standard series of the product to inject the sample separately, and make the standard curve with the concentration of the standard sample against the peak area.

3.Water solubility: colorless and transparent solution

Weigh 50g of HMBCa sample and dissolve it in 200mL deionized water, the sample should be completely dissolved within 30min, and observe the color and transparency of the solution.

4.Alcohol solubility: colorless and transparent solution

Weigh 50g of HMBCa sample and dissolve it in 200mL anhydrous ethanol, the sample should be completely dissolved within 30min, and observe the color and transparency of the solution.

5. Heavy metals (as Pb): not more than 10 ppm (<0.001%)

Weigh 2.00g of HMBCa sample, put it in a dry 150mL beaker, add 45.0mL of deionized water, dissolve it and add 5.0mL of 2.8mol.L-1HCl solution dropwise with constant stirring, mix well, if HMB precipitates, filter out; divide 25mL of filtrate, put it in a 50mL colorimetric tube, add 0.50mL of 30% acetic acid solution, 10mL of After shaking well, put it in the dark place for 10min, compare with the standard lead colorimetric tube, and record the number of milliliters of standard solution contained in the standard tube with the same color as the sample solution, the result is not more than 1.00mL (10ppm). Preparation of standard lead solution colorimetric tube: accurately absorb 0.25mL, 0.50mL, 0.75mL, 1.00mL, 1.25mL, 1.50mL, 1.75mL, 2.00mL of standard lead solution with the concentration of 0.010mg.mL-1, put it in a

50mL colorimetric tube, dilute it into 25mL with water, add 0.50mL of 30% acetic acid solution, 10mL saturated hydrogen sulfide solution, shake well, put it in the dark for 10min, and then use it as the standard lead cuvette.

HeavyMetals(Pb%)=VmL×0.01mg/mLm×12×1000mg/g

Ammonia-ammonium chloride buffer solution with PH=10 (mixture of 17ml of 0.2mol/L of Ammonia and 3ml of of 0.2mol/L of Ammonium chloride)

5g/L chromium black T indicator

Apparatus: general laboratory operation apparatus

EDTA configuration: weigh 20g EDTA, heat and dissolve in 1000mL water, cool, shake well.

0.05 mol/L EDTA calibration: Weigh 1 g of ZnO cauterized at 800°C to constant weight, and weigh to 0.0002 g. Moisten with a little water, add 20% hydrochloric acid solution until the sample dissolves, transfer to a 250 ml volumetric flask, dilute to the scale, and shake well.

Shake well. Add 70mL of water and neutralize with ammonia solution (10%) to pH 7~8.

Add 10mL ammonia-ammonium chloride buffer solution (pH=10) and 5 drops of 5g/L chromium black T indicator solution, and titrate with the configured EDTA solution until the solution changes from purple to pure blue. The blank test was also done.

EDTA standard titration solution concentration calculation. C (EDTA) = m/[(V1-V2) * 0.08138]

m: the mass of zinc oxide (g)

V1: EDTA solution titration amount (mL) V2: blank test EDTA titration amount (mL)

0.08138: mass of ZnO expressed in grams equivalent to 1.00mL EDTA standard solution [C(EDTA) = 1.000mol/L].

Calcium content determination: weigh about 0.3g of HMBCa sample, add 100mL of water to dissolve, add 4mL of ammonia-ammonium chloride buffer solution, shake well, add 2~3 drops of chromium black T indicator, shake well, titrate with EDTA (0.05 mol/L) until the solution changes from purple to pure blue, each 1mL of EDTA (0.05 mol/L) is equivalent to 2.004mg of calcium.

Calculation of calcium content.

(Ca) = {V * 2.004 /[1000 * m]} * 100%

V: titration consumed EDTA volume (mL) m: weight of the weighed sample (g)

2.004mg: 1mL EDTA (0.05 mol/L) is equivalent to 2.004mg calcium

9.Product purity (HPLC)

9.1  Mobile phase

Dissolve 6.8g KH2PO4 in 900mL pure water, adjust the pH value to 3 withphosphoric acid, replenish the pure water to 950mL, add acetonitrile 50mL and mix, filter by 0.45μm microporous membrane, degas by ultrasonic for 30 minutes and set aside.

9.2  Sample preparation

Accurately weigh about 100mg of CaHMB standard, place it in a 25ml volumetric flask, add phosphate buffer, shake until dissolved,

Use buffer solution to fix the volume to the marking line. Put it on the ultrasound

instrument and conduct ultrasound for 10 minutes. Suck about 1ml into the centrifuge tube.> 1000r/min centrifugation

10 minutes, suck the supernatant into the sample tube for testing.

9.3  Sample preparation of standard solution

Precisely weigh about 100 mg of HMB-Ca standard in a 25 mL volumetric flask, add phosphate buffer, shake until dissolved, and fix the volume with buffer to the standard line. Place on an ultrasonicator and sonicate for 10 minutes. Aspirate about 1 mL into a centrifuge tube. Centrifuge at >1000r/min for 10 minutes, aspirate the supernatant into the sample tube, and wait for measurement.

9.3 Product sample preparation

Precisely weigh about 100mg of the product sample, put it in a 25mL volumetric flask, add phosphate buffer, shake until dissolved, and fix the volume with buffer to the standard line. Place on an ultrasonicator and sonicate for 10 minutes. Aspirate about 1mL into a centrifuge tube. Centrifuge at >1000r/min for 10 minutes, aspirate the supernatant into the sample tube, and set aside for measurement.

Chromatographic conditions

Chromatographic column: C18 250×4.6mm×5μm. Flow rate: 1 ml/min.

Detection wavelength: 210 nm. Injection volume: 20μL. Standard curve plotting

After using the standard liquid series of the product are injected, the standard curve is made by the concentration of the standard sample against the peak area.

Determination of samples

Inject the prepared sample solution into the sample. Qualify the peak of

β-hydroxy-β-methylbutyric acid in the sample according to the retention time of the standards. Based on the peak area of the sample, calculate the percentage content of β-hydroxy-β-methylbutyric acid by external standard method.

Calculation of results

Calculate the HMB content of the sample according to the following equation

HMBcontent%=Wstd×(100－Lstd)×0.01×KWsmp×(100－Lsmp)×0.01×AsmpAstd×100

In the formula:

K- the content of HMB in the standard sample of HMBCa (%)

Astd- peak area of HMB in the standard solution

Asmp- peak area of HMB in the sample solution

Wstd- weight of standard sample in standard solution HMBCa (g)

Wsmp- weight of HMBCa in the sample solution (g)Lstd- loss on drying of the standard sample (%)Lsmp- dry weight loss of the sample(%)

Calculate the HMB content of the sample according to the following equation (on dry base)