A Simple, Rapid Atmospheric Pressure Chemical Ionization Liquid Chromatography Tandem Mass Spectrometry Method for the Determination of 25‐Hydroxyvitamin D2 and D3

Abstract

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods.

Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC–MS/MS and its application to a bioequivalence study

“…Similarly, the incubation time (10–60 min) for completion of the derivatization reaction was also examined. The optimum conditions for derivatization established were 20 µL, 100 µg/mL of PTAD solution and an incubation time of 20 min at room temperature, which is much less compared to previous reports [9] , [11] . The derivatized product was easily ionized in the positive ESI-MS due to higher proton affinitive tendency of triazoline moiety and thus increased the ionization efficiency.…”

“…It has been reported earlier that two epimers, 6 S and 6 R, are formed when vitamin D analogues are derivatized with PTAD as it reacts with s-cis -diene moiety from the α- and β-sides. 6S-isomer is observed in greater proportion compared to 6R-isomer and the ratio of 6S/6R is ~5.0:1.0 [9] . Thus, two peaks are generally observed in the MRM ion chromatograms if the two epimers are well resolved under the optimized chromatographic conditions, and the major peak is used for quantitation purpose.…”

“…Several methods are available in literature for the determination of 25OH D 2/3 [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] and 1,25OH D 2/3 metabolites [9] , [12] , [13] , [17] , [20] , [21] , which are potential markers for vitamin D deficiency and function. However, few methods are reported for the determination of ERG (vitamin D 2 ) in biological samples [18] , [19] .…”

A review of chromatographic methods for the determination of water‐ and fat‐soluble vitamins in biological fluids

“…Reversed‐phase chromatography is a well‐suited technique for analysis of these compounds. Mobile phases are composed mainly of organic solvents, such as methanol or acetonitrile , because of the different solubilities of fat‐soluble vitamins and their reversed‐phase retention properties. Low water content is used to enhance separation of the analytes.…”

“…The method was more sensitive than UV diode array detection and allowed for the determination of a wide range of 25‐OH‐D 3 and 25‐OH‐D 2 concentrations (5–2400 nM, which refers to 2–990 ng/mL) in human samples. Recently, the combination of HPLC–MS/MS has become a commonly used technique for the determination of fat‐soluble vitamins . The increased analytical specificity provided by the MS/MS detection offers the potential for high accuracy and overcomes many of the difficulties associated with other techniques, such as inadequate sensitivity in samples of patients with vitamin deficiency and interferences of the analytes with matrix components or 3‐epi‐25‐OH‐D 3 , which leads to overestimation of 25‐OH‐D 3 .…”