Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling

• Published in: Clinical biochemistry Vol. 45; no. 16-17; pp. 1491 - 1496
• Main Authors: Baecher, Silvia, Leinenbach, Andreas, Wright, Jo Anne, Pongratz, Stephan, Kobold, Uwe, Thiele, Roland
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.11.2012; Elsevier
• Subjects: 24R,25-Dihydroxyvitamin D3; 25-Hydroxyvitamin D 2 - blood; 25-Hydroxyvitamin D 2 - isolation & purification; 25-Hydroxyvitamin D2; 25-Hydroxyvitamin D3; 3-Epi-25-hydroxyvitamin D3; Biological and medical sciences; Blood Chemical Analysis - standards; Calibration; Chromatography, High Pressure Liquid; HPLC–MS/MS; Humans; Hydroxycholecalciferols - blood; Hydroxycholecalciferols - isolation & purification; Investigative techniques, diagnostic techniques (general aspects); Limit of Detection; Medical sciences; Reference Standards; Reproducibility of Results; Tandem Mass Spectrometry - standards; Vitamin D profiling; r2; 26,27-D6-25OH-D3; 24R,25(OH)2-D3; MRM; Vitamin D profiling; APCI; 3-epi-25OH-D3; SRM; QC; DBP; S/N; ESI; PFP; 25OH-D2; v/v; Rt; 25OH-D3; IS; LOQ; 25-Hydroxyvitamin D2; CV; HPLC–MS/MS; vitD; 25-Hydroxyvitamin D3; NIST; 24R,25-Dihydroxyvitamin D3; 3-Epi-25-hydroxyvitamin D3; RP; RAM; Human; Metabolite; 3-Epi-25-hydroxyvitamin D; HPLC chromatography; HPLC―MS/MS; 24R,25-Dihydroxyvitamin D; Vitamin D; Mass spectrometry MS/MS; Clinical biology; Serum; Colecalciferol; 25-Hydroxyvitamin D
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2012.06.030

For quantification of 25-hydroxyvitamin D3 (25OH-D3), 25-hydroxyvitamin D2 (25OH-D2), 3-epi-25-hydroxyvitamin D3 (3-epi-25OH-D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2-D3) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method was developed and validated. After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi‐reaction monitoring is used for analyte detection. Baseline separation of the analytes is achieved below 10min. The method is linear over the range 4.0–265.3nmol/L for 25OH-D3, 3.9–183.6nmol/L for 25OH-D2, 2.0–133.8nmol/L for 3-epi-25OH-D3 and 2.8–129.9nmol/L for 24R,25(OH)2-D3 (r2>0.998). The limit of quantification is 4.0nmol/L for 25OH-D3, 3.9nmol/L for 25OH-D2, 2.0nmol/L for 3-epi-25OH-D3 and 2.8nmol/L for 24R,25(OH)2-D3. The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D3 and 24R,25(OH)2-D3. This highly specific HPLC–MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D3 and 24R,25(OH)2-D3. Serum concentration of 24R,25(OH)2-D3 increases disproportionally with increasing concentration of 25OH-D3. [Display omitted] ► HPLC–MS/MS method for vitamin D profiling in human serum samples ► Simultaneous quantification of 25OH-D3, 3-epi-25OH-D3, 25OH-D2 and 24R,25(OH)2-D3 ► Chromatographic separation of 25OH-D3 and 3-epi-25OH-D3 below 10min ► Significant amounts of 3-epi-25OH-D3 and 24R,25(OH)2-D3 in serum samples detected. ► 24R,25(OH)2-D3 level increased disproportionally with increasing 25OH-D3 level.