Influence of Experimental Conditions on the Ratio of 25-Hydroxyvitamin D₃ Conformers for Validating a Liquid Chromatography/Ion Mobility-Mass Spectrometry Method for Routine Quantitation

Abstract

The utility of adding ion mobility (IM) to liquid chromatography/mass spectrometry (LC/MS) for quantitation of 25hydroxyvitamin D 3 (25OHD3) is evaluated. Sodiated 25OHD3 ions adopt both closed and open conformations, whereas the stereoisomer 3-epi-25-hydroxyvitamin D (epi25OHD), when sodiated, adopts only a closed gas-phase conformation. The unique open conformation for sodiated 25OHD3 permits unambiguous quantitation. Nonetheless, the ratio of open versus closed gas-phase conformations for sodiated 25OHD3 can vary with instrumental conditions; conversion from the open to the closed conformer is attributed to radio frequency (rf) heating within the ion accumulation trap. Ion heating becomes significant when space charge in the trap forces ions to larger radii where the rf field is higher. To avoid biasing quantitation, an isotopically labeled internal standard must be used to account for changes in conformer ratio. Thirty-three serum extract samples were evaluated by LC/IM-MS and were found to not be biased by changes in ion conformer ratios, permitting reliable quantitation of 25OHD3 without interference from the epimer.

Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/drift Tube Ion Mobility-Mass Spectrometry

“…In this work, one of two possible conformers of the same ion is quantified under the assumption that the unique conformer is representative of the solution concentration. Work in our lab has shown that the ratio of the conformers depends on the instrument settings and ion flux within the mass spectrometer where the unique open conformer converts to the closed conformer, but potential bias from interconversion can be avoided with the use of an isotopically labeled internal standard, as shown in our previous work 25 .…”

“…As seen in Figure 3, under ideal conditions, approximately 10% of 25OHD3 remains in the closed conformer. Additionally, it has been shown that the open conformer readily converts to the closed conformer under high-energy conditions 25 . Strategies that reduce the abundance of the closed conformer of 25OHD such as alternative cation adducts 40 , could be implemented to improve simultaneous quantitation of the epimers, but introduction of alternative cations into the LC/IM-HRMS method is not straightforward and the utility of rapid quantitation of the epimers is low.…”

“…Previous work in our lab proposed that sample-to-sample variations in total ion intensity could drive conversion of the open conformer into the closed conformer leading to suppression of the analyte, but could be compensated by similar interconversion of the isotopically-labeled internal standard 25 ; therefore, it is important to consider the degree to which the ion mobility separation affects variation in the internal standard response. In this study, variation in the internal standard response is observed across a 46-sample run including 30 patient samples, 6 commercial QC samples, 2 blanks, and 8 calibrators.…”

Magnesium Klorida (MgCl2): Karakteristik dan Dinamika Molekuler Pada MgCl2

“…Mobilitas ion (221)(222)(223)(224)(225) dapat dihitung dengan pengukuran sebenarnya jarak yang ditempuh setiap ion dalam waktu tertentu dengan metode batas bergerak.…”

“…Mobilitas ion (221)(222)(223)(224)(225) merupakan kecepatan ion pada beda potensial antara kedua elektroda atau bias dibilang perbandingan antara laju dan kuat medan. Mobilitas Ionik dalam air pada suhu 298 K. (243)(244)(245)(246)(247)(248)(249)(250)(251)(252)…”