Determination of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum using liquid chromatography with tandem mass spectrometry

• We established a method for simultaneous quantification of serum 1,25-dihydroxy vitamin D3 and 1,25-dihydroxy vitamin D2.
• Sample preparation in this assay was much simpler. No immunoextraction or solid phase extraction procedure was needed.
• The low limits of quantitation obtained were 10 pg/mL for both 1,25-dihydroxy vitamin D3 and 1,25-dihydroxy vitamin D2 without any derivation procedure.

Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH)2D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) is considered to be the “gold standard” for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC–MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and 1α,25-dihydroxy vitamin D2 (1α,25(OH)2D2). The analytes were extracted from the matrix by liquid–liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH)2D. The assay was sensitive with a low limit of quantitation of 10.0 pg/mL for both 1α,25(OH)2D3 and 1α,25(OH)2D2 using a 0.5 mL sample aliquot. Linearity was obtained over the range of 10.0 pg/mL to 500 pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100 ± 5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH)2D3 and 1α,25(OH)2D2 concentrations.