WS/T 677-2020Screening methods for vitamin D deficiency in the population (English PDF)

5.1 Collection and storage of blood samples

Follow the method specified in Appendix A.

5.2 The first method. liquid chromatography tandem mass spectrometry Follow the method specified in Appendix B.

5.3 Method 2.Chemiluminescence immunoassay

Follow the method specified in Appendix C.

5.4 The third method. enzyme-linked immunoassay Follow the method specified in Appendix D.

Appendix A

(Normative appendix) Collection and preservation of blood samples A.1 Specimen collection A.1.1 Collection of blood samples. fasting venous blood, the blood collection method is based on WS/T 225 and the requirements of the detection method to collect 1 mL of fasting venous blood.

A.1.2 Blood collection tube. According to the required serum or plasma, use different blood collection tubes for packaging.

A.2 Serum and plasma preservation the specimen can be stable for 7 days at 2 °C to 8 °C, and stable for 12 months at -20 °C. If the specimen cannot be tested in time, it is recommended Store specimens below -70 °C, freeze-thaw only once.

Appendix B

(Normative appendix) Liquid chromatography tandem mass spectrometry B.1 Principle the 25(OH)D2 and 25(OH)D3 in serum (plasma) were precipitated with methanol/acetonitrile, extracted with n-hexane, dried with nitrogen, and dried with initial the mobile phase is reconstituted, separated by liquid chromatography, detected by tandem mass spectrometry in multiple reaction monitoring mode (MRM), and quantified by isotope internal standard the method can measure 25(OH)D2 and 25(OH)D3 respectively.

B.2 Reagents and solutions Unless otherwise specified, all reagents in this method are analytically pure, the water complies with the first grade water regulations in GB/T 6682, and the solution complies with GB/T 603 Preparation.

B.2.1 Reagents

B.2.1.1 25(OH)D2 standard product (C28H44O2, CAS. 21343-40-8). purity > 98% or certified standard material.

B.2.1.2 25(OH)D3 standard substance (C27H44O2, CAS. 19356-17-3). Purity > 98% or certified standard substance.

B.2.1.3 Isotope internal standard [2H3]-25(OH)D2 standard product (C28H41 H3O2, CAS. 1217467-39-4). Purity > 98%.

B.2.1.4 Isotope internal standard [2H3]-25(OH)D3 standard (C27H41 H3O2, CAS. 140710-94-7). Purity > 98%.

B.2.1.5 Methanol (CH3OH). chromatographically pure.

B.2.1.6 n-hexane (C6H14). chromatographically pure.

B.2.1.7 Formic acid (HCOOH). chromatographically pure.

B.2.1.8 Bovine serum albumin (BSA). purity >=98%.

B.2.1.9 Nitrogen (N2). Purity >=99.9%.

B.2.1.10 Formic acid-water solution. 0.1%.

B.2.1.11 Formic acid-methanol solution. 0.1%.

B.2.1.12 Acetonitrile (CH3CN). chromatographically pure.

B.2.1.13 BSA bovine serum albumin aqueous solution. 1%.

B.2.1.14 Precipitant. methanol acetonitrile (11).

B.2.1.15 Standard stock solution (10 µg/mL). Weigh 1 mg of 25(OH)D2 and 25(OH)D3 standards (accurate to 0.01 mg) in in 2 100 mL volumetric flasks, dissolve with methanol and dilute to the mark. The concentration of 25(OH)D2 and 25(OH)D3 in this solution is 10 µg/mL. Melt After transferring the solution to the reagent bottle, store it in the dark at -20°C for later use.

B.2.1.16 Isotope internal standard stock solution (10 µg/mL). Weigh the isotope internal standards [2H3]-25(OH)D2 and [H3]-25(OH)D3 respectively 1 mg (accurate to 0.01 mg) in 2 100 mL volumetric flasks, dissolved in methanol and dilute to the mark. In this solution [2H3]-25(OH)D2 and the concentration of H3]-25(OH)D3 is 10 µg/mL. After the solution is transferred to the reagent bottle, store it in the dark at -20°C for later use.

B.2.1.17 Standard series solutions. accurately pipette 25(OH)D2 (10 µg/mL) and 25(OH)D3 (10 µg/mL) 5 µL, 10 µL, 25 µL, 50 µL, 125 µL, 250 µL and 500 µL were placed in 7 10 mL volumetric flasks, diluted with methanol and made up to the mark. This series of solutions the concentrations of 25(OH)D2 and 25(OH)D3 are 5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 125 ng/mL, 250 ng/mL and 500 ng/mL.

B.2.1.18 Mixed isotope internal standard solution (100 ng/mL). accurately draw the isotope internal standard [2H3]-25(OH)D2 stock solution (10 µg/mL) and [2H3]-25(OH)D3 stock solution (10 µg/mL) 100 µL each in a 10 mL volumetric flask, dilute with methanol and make up to the mark.

The isotope internal standard of this solution [2H3]-25(OH)D2 and [ the concentration of H3]-25(OH)D3 is 100 ng/mL. After the solution is transferred to the reagent bottle, Store in the dark at -20°C for later use.

B.2.2 Apparatus and equipment B.2.2.1 Analytical balance, sensitivity. 0.0001 g.

B.2.2.2 Analytical balance, sensitivity. 0.00001 g.

B.2.2.3 Liquid chromatography-tandem mass spectrometer.

B.2.2.4 Nitrogen blowing instrument or vacuum centrifugal dryer.

B.2.2.5 Centrifuge. centrifugal force >=12000×g.

B.2.2.6 Vortex mixer.

B.2.2.7 Pipette gun or pipette.

B.3 Analysis steps

B.3.1 Sample preparation Take.200 µL of serum (plasma) sample into a 2 mL centrifuge tube, add 20 µL of mixed isotope internal standard solution, and vortex for 30 s. plus Add 400 µL of precipitant (methanol acetonitrile, 1 1), vortex and shake at room temperature for 30 s; add 1.2 mL of n-hexane, and vortex at room temperature 5 min, then centrifuge for 5 min at 4 °C and a centrifugal force greater than 12000×g; pipette 1.0 mL of supernatant into a 1.5 mL centrifuge tube, at room temperature Blow to dryness with nitrogen; reconstitute with 100 µL of initial mobile phase, vortex for 30 s, centrifuge at 4°C and centrifugal force greater than 12000×g for 5 min, the supernatant was transferred to the sample bottle for LC-MS/MS analysis.

B.3.2 Preparation of standard working solution Take 7 2 mL centrifuge tubes and add 180 µL blank replacement serum sample (1% BSA bovine serum albumin aqueous solution) and 20 µL respectively Standard series solution (B.2.1) and 20 µL mixed isotope internal standard solution, this series is equivalent to the concentration range of 0.5 ng/mL~50 ng/mL.

Vortex for 30 s. The following operations are the same as in B.3.1.

B.3.3 Parameter settings of liquid chromatography-tandem mass spectrometry B.3.3.1 Reference conditions for liquid chromatography analysis.

a) Chromatographic column. C18 column (column length 100 mm, column inner diameter 2.1 mm, packing particle size 1.7 µm, or a column with equivalent column efficiency);

b) Mobile phase. Phase A. aqueous solution containing 0.1% formic acid; Phase B. methanol solution containing 0.1% formic acid;

c) Gradient elution. see Table B.1;

d) Flow rate. 0.30 mL/min;

e) Column temperature. 40 °C;

f) Injection volume. 10 µL.

B.3.3.2 Reference conditions for mass spectrometry determination.

a) Ionization method. electrospray ionization, positive ion mode (ESI);

b) Detection method. multiple reaction monitoring mode (MRM);

c) Curtain air. 0.25 MPa;

d) Spray voltage. 4000 V;

e) Desolvent gas temperature. 450 °C;

f) Atomizing gas. 0.35 MPa;

g) Desolvent gas. 0.35 MPa;

h) MRM ion pair parameters are shown in Table B.2.

B.3.4 Qualitative determination Perform the determination of standard solution and sample solution under the same color/mass spectrum conditions, if the retention time of the chromatographic peak detected in the sample solution it is consistent with the retention time of the chromatographic peak detected in the standard solution, the mass-to-charge ratio of the selected ion pair is also consistent, and the qualitative ionization in the sample solution the relative abundance of the sub-pairs is compared with the relative abundance of the qualitative ion pairs in the standard solution with the same concentration, and the relative deviation does not exceed those specified in Table B.3 Within the range, the substance can be determined in the sample solution.

B.3.5 Production of working curve Under the analysis conditions of liquid chromatography and tandem mass spectrometry in B.3.3, the standard working solution obtained in B.3.2 is processed from low concentration to high concentration.

For sample analysis, linear regression is performed based on the peak area ratio-concentration of 25(OH)D2 and 25(OH)D3 chromatographic peaks and their corresponding isotopic internal standard chromatographic peaks the linear fitting equation of the standard working solution is obtained, and the linear correlation coefficient should be greater than 0.99.

B.3.6 Sample determination Under the reference conditions of liquid chromatography and tandem mass spectrometry in B.3.3, the sample solution to be tested obtained in B.3.1 is injected and analyzed according to the internal standard method Calculate the mass concentration of the target substance in the solution to be tested. The mass concentration of the target substance in the sample solution to be tested should be within the linearity of the standard working curve Within the range, if it exceeds the linear range, the sample should be appropriately diluted before repeating the processing and determination.

B.3.7 Blank test

Replace the serum (plasma) sample with 1% BSA bovine serum albumin aqueous solution, and do a blank test according to the steps in B.3.1.

B.4 Quality control

B.4.1 Internal quality control the laboratory shall formulate test results quality control procedures, and clarify the content, methods and requirements of internal quality control. Need to be tested with the same product Determine quality control samples, draw quality control diagrams, observe the stability, system deviations and trends of the test work, find abnormal phenomena in time and collect Take corresponding improvement measures.

B.4.2 External quality control Laboratories should participate in proficiency testing activities organized by domestic and foreign laboratory accreditation agencies, and participate in laboratory comparisons between international and domestic counterparts test. Evaluate the quality of the inspection results based on the results of external review, proficiency verification, assessment, and comparison, and take corresponding improvement measures.

B.6 Precision

The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

B.7 Detection limit and quantification limit in this method, the detection limit of 25(OH)D2 and 25(OH)D3 are both 0.15 ng/mL, and the limit of quantification is 0.5 ng/mL.