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

Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC–MS/MS methods not separating both metabolites as determined by an LC–MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum

“…Of all published LC-MS/MS methods for simultaneous measurement of 25(OH)D 3 , 25(OH)D 2 and their respective C 3 -epimers [6][7][8][13][14][15][16][17][18][19][20] it has not always been clear how the C 3 -epimer concentration is calculated. Some reported relative percentages ([C 3 -epi peak area/ (C 3 -epi + 25(OH)D) peak areas] × 100%) as 3-epi-25(OH)D 3 calibration curves were lacking [7,19], others reported absolute concentrations 3-epi-25(OH)D 3 , however, without giving details on whether separate 3-epi-25(OH)D 3 calibrators were used [6,8,[13][14][15][16][17][18]20].…”

Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin D

“…It was hypothesized that hepatic immaturity may play a role in epimer production (22 ), but substantial concentrations have also been found in adults (33 D (27 ); it was discovered that the concentrations of both compounds were significantly lower in the patients compared to healthy individuals. (18,27,40,49 ). For all methods, it is important to consider that the natural (R)-form and not the (S)-epimer is metabolically active (50 …”

Synthesis of Low Abundant Vitamin D Metabolites and Assaying Their Distribution in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) as a New Tool for Diagnosis and Risk Prediction of Vitamin DRelated Diseases

“…The vitamin D metabolites generally elute in the order trihydroxylated < dihydroxylated < monohydroxylated metabolites, that is, for vitamin D 3 -related molecules, the order of chromatographic retention is 1,25(OH) 2 Possibly more interesting-from a chromatography point of view-are the biochemically formed isomers after stereochemical reversal (β→α) at C-3; for example, the epimers 25(OH)D 3 and 3-epi-25(OH)D 3 . The subtle diastereomeric differences between these species can be distinguished using specialized columns, such as combined C18/chiral [50], pentafluorophenyl (PFP) [51][52][53], and cyano (CN) [54][55][56][57].…”