Analysis of vitamin D metabolic markers by mass spectrometry: Current techniques, limitations of the “gold standard” method, and anticipated future directions

Abstract

Vitamin D compounds belong to a group of secosteroids, which occur naturally as vitamin D3 in mammals and D2 in plants. Vitamin D is vital for bone health but recent studies have shown a much wider role in the pathologies of diseases such as diabetes, cancer, autoimmune, neurodegenerative, mental and cardiovascular diseases. Photosynthesis of vitamin D in the human skin and subsequent hepatic and renal metabolism generate a wide range of transformation products occurring over a large dynamic range spanning from picomolar to nanomolar levels. This necessitates selective and sensitive analytical methods to quantitatively capture these low concentration levels in relevant tissues such as blood. Ideally, vitamin D assessment would be performed using a universal and standardized analytical method available to clinical laboratories that provides reliable and accurate quantitative results for all relevant vitamin D metabolites with sufficiently high throughput. At present, LC-MS/MS assays are the most promising techniques for vitamin D analysis. The present review focuses on developments in mass spectrometry methodologies of the past 12 years. It will highlight detrimental influences of the biological matrix, epimer contributions, pitfalls of specific mass spectrometry data acquisition routines (in particular multiple reaction monitoring, MRM), influence of ionization source, derivatization reactions, inter-laboratory comparisons on precision, accuracy, and application range of vitamin D metabolites.

Quantum‐Assisted Metrology of Neutral Vitamins in the Gas Phase

“…In particular, vitamins in the gas phase have recently received renewed theoretical interest,2 and these ubiquitous but thermally sensitive particles have been experimentally studied in the gas phase by mass spectrometry3 and microwave spectroscopy 4. Herein, we utilized the benefits of near‐field quantum interference to measure the optical polarizabilities and electric susceptibilities of molecules in the same setup.…”

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

“…R eliable quantification of vitamin D in biological samples remains difficult as even selective mass spectrometry assays exhibit many possibilities for errors and interferences [1][2][3]. While high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on triple quadrupole (QqQ) instruments is currently considered the optimum method for quantifying the vitamin status marker 25-hydroxyvitamin D 3 (25(OH)D 3 ) [4,5], the technique is not as straightforward to implement for vitamin D compounds as is the case for other endogenous compounds [1,2].…”

“…While high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on triple quadrupole (QqQ) instruments is currently considered the optimum method for quantifying the vitamin status marker 25-hydroxyvitamin D 3 (25(OH)D 3 ) [4,5], the technique is not as straightforward to implement for vitamin D compounds as is the case for other endogenous compounds [1,2]. LC-MS/MS requires skilled personnel and carefully optimized instrumental parameters to avoid interferences and to provide sufficient sensitivity.…”

“…As recently demonstrated [6], there is a large number of isobaric endogenous and exogenous compounds present during the LC-MS/MS analysis of 25(OH)D 3 . Furthermore, additional complications arise from isomeric contributions, where the C-3 epimer of 25(OH)D 3 presents a particular challenge [2]. Whether or not the epimeric species are biologically relevant is currently unknown [7]; regardless, chromatographic separation is essential to avoid bias in the analysis [1,2,8,9].…”

“…Several experimental approaches have been developed to overcome some of these problems, including derivatization techniques to improve ionization efficiency [10][11][12], ion mobility spectrometry to remove isobars [6], isotope standards, and certified reference materials to improve intra-and interlaboratory precision and accuracy [2], and high resolution mass spectrometry (HRMS) [2]. Mass analyzers of medium/high mass resolving power such as time-of-flight (TOF), Orbitrap, and Fourier transform ion cyclotron resonance (FTICR) have not yet routinely entered clinical practice, even though their potential has been clearly recognized [13,14].…”