| With better understanding of hazards of vitamin D deficiency, there is increased vitamin D testing worldwide and increasing evidence of deficiency in various populations.The 25-hydroxyvitamin D (25(OH)D) which consist of liver metabolites 25-hydroxyvitamin D3 and D2 is the primary circulating form of vitamin D and is generally used to estimate vitamin D status. Traditionally,25(OH)D in serum has been measured by immunoassay, however, these methods often have problems with specificity, accuracy and sensitivity because of the limit of the antibodies cross-reactivity and nonequimolar recognition of the D2 and D3 forms of the 25(OH)D metabolite. In contrast to immunoassays, the Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is also high specificity and sensitivity in measuring serum 25(OH)D, eliminating most technical problems of immunoassays.We developed a novel LC-MS/MS assay for the accurate quantification of 25(OH)D2 and 25(OH)D3 in serum. The process included solid phase extraction, liquid chromatography and mass spectrometry. According to FDA’s guideline (Guidance for Industry Bioanalytical Method Validation), we comducted the general method performance verification. The results showed:①Linear of 25(OH)D2 and 25(OH)D3 were between 6.25 and 500 nmol/L, respectively.②The LC-MS/MS assay had a limit of quantitation of 2.5 nmol/L for 25(OH)D2 and a limit of quantitation of 1.25 nmol/L for 25(OH)D3.③Intra- and inter-assay precision of 25(OH)D2 and 25(OH)D3 were<4% and<6%, respectively. ④Recovery of 25(OH)D from serum samples ranged between 93.26 and 123%.⑤The result of DEQAS interlaboratory quality assessment had a bias<10%. The results showed that the established LC-MS/MS’s basic performance meet FDA evaluation standards and the assay is accurate and precise at physiologically relevant 25(OH)D concentrations.One hundred ninety randomly selected serum samples of healthy people were prepared and 25(OH)D was measured by LC-MS/MS, electro-chemiluminescence immunoassay assay(ECLIA), micropaticle immunoassay(MEIA) and enzyme linked immunosorbent(ELISA).The LC-MS/MS assay was compared with an ECLIA, a MEIA and an ELISA. Results:①Correlations between the ECLIA, MEIA and ELISA to LC-MS/MS were 0.79,0.69 and 0.56, respectively.② The 25(OH)D2 cross-reactivity of ECLIA, MEIA and ELISA was 42.42%,24.05%,9.22%, respectively. ③All the immunoassays showed an increasingly negative bias with increasing D2%.The result showed that the accuracy of 25(OH)D detected by immunoassay still had question.It would obviously underestimate 25(OH)D particularly supplement of the vitamin D2.Two hundreds and seventy-one serum samples of hepatitis C and two hundreds and eighteen serum samples of healthy people were collected. The concentration of 25(OH)D and HCVRNA of hepatitis C patients and score of Fibretest were detected. We compared the result of the 25(OH)D in group of hepatitis C patients and the healthy one,and analyzed the relevance between the concentration of 25(OH)D and virus load of HCV and Fibretest. Following were the results:①The average concentration of the 25(OH)D in hepatitis C patients and healthy people were 49.31±1.39 nmol/L VS 60.42±1.34 nmol/L, with a significant difference. (P<0.01)②Comparing with the healthy group, the percent of vitamin D deficiency and severe deficiency in hepatitis C group were 27.98%(61/218) Vs41.33% (112/271),3.67%(8/218)Vs 14.4%(39/271) respectively. (P<0.01)③No relevance was found between the HCNRNA or Fibrotes and the concentration of the 25(OH)D.(P>0.05) We concluded the trend of vitamin D deficiency in hepatitis C patients was high, and advised to supple vitamin D for them.Comparing with the traditional immunology methods, determination of serum 25(OH)D by LC-MS/MS was a novel method of sensitivity and specificity. We developed a simple and fast method allowing the accurate quantification of 25(OH)D3 and 25(OH)D2 in human serum. It could be available for evaluation the status of vitamin D and support of the relevance study. |
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