Metabolic profiling of major vitamin D metabolites using Diels–Alder derivatization and ultra-performance liquid chromatography–tandem mass spectrometry

Abstract

Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1alpha,25-dihydroxyvitamin D(3) in human serum (15-60 pg/mL). Here, we demonstrate that liquid-liquid or solid-phase extraction of vitamin D metabolites in combination with Diels-Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)-electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1alpha,25-dihydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(2), 24R,25-dihydroxyvitamin D(3), 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6-4.8 % and 5-16 % for 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3), respectively, using solid-phase extraction.

Assessment of vitamin D status – a changing landscape

“…Such a derivatization can overcome the main problems related to the measurement of vitamin D metabolites: a very low analyte concentration, thermal instability and low polarity of the target compounds. The most common Cooksontype reagent is PTAD [171,[175][176][177][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193][194], but other similar reactants such as MBOTAD [187], DMEQ-TAD [188] or DAPTAD [168,188,190] have also been tested. Today, the commercial Amplifex Diene from ABSciex is increasingly used [191,192] since it provides better sensitivity compared to PTAD.…”

Cuantificación De Vitamina D: De La Investigación a La Práctica Clínica

“…Varios autores continúan recomendando los inmunoensayos que se ejecutan en plataformas automatizadas como la técnica de preferencia, dado la comodidad y alto rendimiento de la prueba, en especial cuando se requiere analizar con regularidad un gran número de muestras. Mientras estos autores sugieren que la cuantificación de 25(OH)D por inmunoensayo sigue siendo el método de elección por razones de conveniencia, velocidad y costo (12), otros autores argumentan que estos ensayos, si bien sencillos y de alto rendimiento, han sacrificado el rigor analítico de la cuantificación y, por lo tanto, sólo proporcionan datos para indicar el estado relativo de vitamina D en términos semicuantitativos (25,32 (33)(34)(35)(36). Por consiguiente, este método es considerado hoy el recomendado para la medición del estado de VD, tanto en investigaciones como en la asistencia clínica; y los expertos recomiendan que debe llevarse a cabo en un laboratorio con experiencia adecuada, competente y con historia registrada de buen rendimiento (31).…”

Genetic, Environmental, and Disease-Associated Correlates of Vitamin D Status in Children with CKD

“…Intact parathyroid hormone (iPTH) was measured with the Elecsys ECLIA (Roche Diagnostics, Indianapolis, IN), and C-terminal c-terminal fibroblast growth factor was measured with the Immutopics ELISA (San Clemente). Vitamin D metabolites were measured via liquid chromatography-mass spectrometry/mass spectrometry using an Acquity UPLC coupled to a Quattro Premier Mass Spectrometer (Waters, Milford, MA) as described by Aronov et al (26). The lower limit of quantification was 25 pg/ml for all vitamin D metabolites.…”