| Objective:The aim of this research is to develop an isotope dilution high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) for measuring serum lα,25-dihydroxy vitamin D2 (lα,25(OH)2D2) and lα,25-dihydroxy vitamin D3(lα,25(OH)2D3).Method:We perform method establishment and evaluation in this study. Serum samples were mix with 1,25(OH)2D3-D3 and extracted with 90% hexane/10% ethyl acetate. After centrifugation, the supernatants were transferred to a new tube and centrifuged in vacuum to dryness and reconstituted with methanol. Then the residuals were analyzed by LC/MS/MS system in positive electro-spray ionization mode and multiple reaction monitor (MRM) mode. A series of standard calibration curve and isotope internal stand method were introduced for quantification of 1,25(OH)2D2 and 1,25(OH)2D3. Pooled serum samples spiked with 1,25(OH)2D at three levels were aliquoted and stored at-70℃ until analysis. The precision of the method was determined by running those samples multiple times within a batch over multiple days. In addition, the recovery rates were analyzed with serum samples added with different levels of stands.173 serum samples were collected and measured with LC-MS/MS and CLIA method respectively for 1α,25(OH)2D3 method comparison. Excel 2007 was used for data recording and processing. Serum samples from 155 apparently healthy individuals aged 18-65 were collected and measured with LC-MS/MS method for reference interval research.2.5%-97.5%percentile value range estimated by nonparametric method was reported as reference interval.Results:Serum 1α,25(OH)2D2 and 1α,25(OH)2D3 measured by LC-MS/MS were separated well from several interference. The retention time of 1,25(OH)2D and 1,25(OH)2D3-D3 was about 4.7 min and the analysis time was 10 min per injection. The correlation coefficients between peak area ratio and 1,25(OH)2D2 as well as 1,25(OH)2D3 concentrations were both higher than 0.990. The within-run and total coefficients of variation (CV) of 1,25(OH)2D2 were 3.88%-6.35% and 5.43%-9.41%; and the within-run and total CV of 1,25(OH)2D3 were 2.34%-6.79% and 5.37%-8.40%. The analytical recoveries of la,25(OH)2D2 and la,25(OH)2D3 were 95.80% -104.21% and 95.91%-99.83%, respectively. The limit of detection of la,25(OH)2D2 and la,25(OH)2D3 were both 5 pg/mL, and the lower limit of quantification were both 10 pg/mL.The within-run CV of CLIA for 1α,25(OH)2D3 quantification were 2.7%,5.0% and 5.0%, and the total CV were 13.8%,15.0% and 13.5%. The method comparison resulted in coefficient of correlation of R2= 0.8041, a slope of 0.9614 and a intercept of 4.3977. 5.78%(10/173) of the samples falls out of the limit of agreement of two methods. The maximum absolute bias of two methods was 26.1 pg/mL. Reference interval of serum la,25(OH)2D3 concentration for LC-MS/MS method obtained from 155 apparently healthy individuals was 32.9 pg/mL-l 12.5 pg/mL.Conclusions:A simple specific and sensitive LC-MS/MS method has been established for serum la,25(OH)2D2 and la,25(OH)2D3 quantification. This LC-MS/MS method was better than commercial CLIA method and a reference interval for apparent healthy individuals was preliminaly estabilished with LC-MS/MS method. |
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