4 high-performance liquid chromatography (HPLC)

4.1 principle:

chromatograph test sample, in turn, add water and methanol, 25 hydroxy vitamin D3, ultrasonic extraction, is measured by high performance liquid chromatograph external standard method. 

4.2 Reagents or Materials

4.2.1 Water: GB/T 6682 Grade 1

4.2.2 Methanol: HPLC grade

4.2.3 85% Methanol Solution: Take 85 mL of methanol (4.2.2), dilute with water to 100 mL, and mix well.

4.2.4 Standard Stock Solution (1 mg/mL): Accurately weigh 25 mg of 25-hydroxyvitamin D3 standard (C₂₇H₄₄O₂·H₂O, CAS No.: 63283-36-3, purity not less than 98%), place it in a 25 mL brown volumetric flask, dissolve it with methanol (4.2.2), and dilute to the mark. Store at -20°C and it is valid for 2 months.

4.2.5 Standard Intermediate Solution I (100 ug/mL): Accurately transfer 2.00 mL of the 25-hydroxyvitamin D3 standard stock solution (4.2.4) into a 20 mL brown volumetric flask and dilute to volume with methanol (4.2.2). Store at 4°C and keep for up to 2 weeks.

4.2.6 Standard Intermediate Solution (25 ug/mL): Accurately transfer 5.00 mL of Standard Intermediate Solution I (4.2.5) into a 20 mL brown volumetric flask and dilute to volume with methanol (4.2.2). Prepare fresh before use.

4.2.7 Standard Series Solutions: Accurately transfer appropriate amounts of Standard Intermediate Solution II (4.2.6) into brown volumetric flasks and dilute with 85% methanol solution (4.2.3) to prepare standard series solutions with concentrations of 0.05 ug/mL, 0.5ug/mL, 1 ug/mL, 5 ug/mL, 10 ug/mL, and 25 ug/mL. Prepare fresh before use.

4.2.8 Micro-porous Filter Membrane: 0.22 um, organic system.

4.3 Instruments and Equipment

4.3.1 High Performance Liquid Chromatograph: Equipped with a UV detector/diode array detector.

4.3.2 Analytical Balance: Sensitivity 0.1 mg and 0.01 mg.

4.3.3 Centrifuge: Rotational speed not less than 9000 r/min.

4.3.4 Ultrasonic Cleaner: Power not less than 280 W.

4.4 Sample Preparation

Samples shall be prepared in accordance with the provisions of GB/T 20195, with a minimum weight of 200g. The samples shall be ground to pass through a 0.42mm analytical sieve, thoroughly mixed, and placed in a ground-mouth bottle for storage in the dark, ready for use.

4.5 Test Procedure

4.5.1 Preparation of Sample Solution

Two parallel tests shall be conducted. Weigh 3g of the sample accurately to 0.0001g and place it in a 50mL centrifuge tube. Add 10mL of water, sonicate for 10 minutes, and shake once during the process to prevent the sample from adhering to the bottom of the tube. Then add 30mL of methanol (4.2.2), shake well, and sonicate for 20 minutes. Centrifuge at 6000r/min for 5 minutes. Transfer the supernatant to a 50mL brown volumetric flask, make up to the mark with methanol (4.2.2), shake well, and pass through a 0.22um filter before use.

4.5.2 Reference Conditions for Liquid Chromatography

Chromatographic column: Thermo AQUASIL C18 column, 150mm in length, 3mm in inner diameter, 3um particle size, or equivalent.

Column temperature: 30℃

Detection wavelength: 264nm

Mobile phase: Phase A is water, phase B is methanol (4.2.2), and the gradient elution program is shown in Table 1.

Table 1  Gradient Elution Procedure

Flow rate: 0.6 mL/min

Injection volume: 50 ul

4.5.3 Measurement

4.5.3.1 Determination of standard series solutions and sample solutions

Under the optimal conditions of the instrument, the standard series solutions (4.2.7) and the sample solutions (4.5.1) were measured respectively. The liquid chromatogram of the 25-hydroxyvitamin D3 standard solution is shown in Appendix A.

4.5.3.2 Qualitative determination

Qualification was determined by retention time. The retention time of 25-hydroxyvitamin D3 in the sample solution should be consistent with that of 25-hydroxyvitamin D3 in the standard series solutions of the same concentration, with a relative deviation within ±2.5%.

4.5.3.3 Quantitative Analysis

Plot the concentration of 25-hydroxyvitamin D3 on the x-axis and the chromatographic peak area (response value) on the y-axis. Draw a standard curve, and the correlation coefficient should be no less than 0.999. The concentration of 25-hydroxyvitamin D3 in the sample solution should be within the linear range of the standard curve. If it exceeds the range, dilute the sample solution with 85% methanol solution (4.2.3) (n times) and re-determine. For single-point calibration, the difference between the concentration of 25-hydroxyvitamin D3 in the sample solution and the standard solution concentration should not exceed 30%.

4.6 Data Processing for the Test

The content of 25-hydroxyvitamin D3 (C₂₇H₄₄O₂·H₂O) in the sample is expressed as a mass fraction. For multi-point calibration, calculate according to formula (1), and for single-point calibration, calculate according to formula (2).

In the formula:

W1 - The numerical value of 25-hydroxyvitamin D3 (C₂₇H₄₄O₂·H₂O) content in the sample, with the unit of milligrams per kilogram (mg/kg)

P1 - The numerical value of the mass concentration of 25-hydroxyvitamin D3 in the sample solution obtained from the standard curve, with the unit of micrograms per milliliter (ug/ml)

V1 - The numerical value of the volume of the sample solution, with the unit of milliliters (ml)

n - The dilution factor of the sample solution

M - The numerical value of the sample mass, with the unit of grams (g)

In the formula:

--- Chromatographic peak area of 25-hydroxy vitamin D3 in the sample solution

A_s --- Peak area of 25-hydroxy vitamin D3 in the standard solution

P_s --- Numerical value of the mass concentration of 25-hydroxy vitamin D3 in the standard solution, unit is micrograms per milliliter (ug/mL)

V₁ --- Numerical value of the volume of the sample solution, unit is milliliters (mL)

The measurement result is expressed as the arithmetic mean of parallel determinations, and retains 3 significant digits

4.7 Precision

Under repeatability conditions, when the content of 25-hydroxy vitamin D3 in the sample is ≥ 20mg/kg, the absolute difference between the two independent determination results and their arithmetic mean is not greater than 10% of the arithmetic mean; when the content of 25-hydroxy vitamin D3 in the sample is < 20mg/kg, the absolute difference between the two independent determination results and their arithmetic mean is not greater than 15% of the arithmetic mean

Appendix A

(Informative)

HPLC chromatogram of 25-hydroxyvitamin D3 standard solution (5 μg/mL)

The liquid chromatogram of the standard solution of 25-hydroxyvitamin D3 (5 μg/mL) is shown in Figure A.1