High Throughput UHPLC-MS/MS Method for the Simultaneous Quantification of Six Vitamin D Metabolites: Application for Vitamin D Determination in Patient after Liver or Kidney Transplantation
Vitamin D metabolites are fat-soluble vitamins that regulates a broad spectrum of physiological and pathological processes. Accurate and high-throughput methods for the detection of vitamin D metabolites are essential to elucidate their body functions. In this study, a sensitive and high throughput ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was proposed for the accurate quantification of six vitamin D metabolites, including vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamine D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3. Through optimization of the chromatographic and mass spectrometry conditions, only 20 μL serum or plasma can satisfy the quantification of six vitamin D metabolites. The limit of detections (LODs) were determined to be 0.02-0.05 pg/mL. Method validation was carried out following the International Council for Harmonization (ICH) guidelines. All the quantification performances, including linearity, accuracy, precision, extraction recovery and matrix effect were all investigated and were good enough for accurate detection of vitamin D metabolites. As the main organs for vitamin D metabolism, liver or kidney disease could lead to abnormal levels of vitamin D. Here, the established UHPLC-MS/MS method was further used for determination of vitamin D levels in plasma samples of patients after liver or kidney transplantation. Thirty-three liver transplant recipients (LTRs) and 63 kidney transplant recipients (KTRs) were included in this study. Vitamin D deficiency or insufficiency is common in KTRs and LTRs, with prevalence more than 99%.