Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry

Abstract Objectives Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples. Results The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)2D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,20S(OH)2D3) was consistently achieved in human serum samples. Conclusions This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.

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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

A Critical Evaluation of Vitamin D - Basic Overview ◽

10.5772/64518 ◽

2017 ◽

Author(s):

Lars Kattner ◽

Dietrich A. Volmer

Keyword(s):

Mass Spectrometry ◽

Vitamin D ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Human Serum ◽

Risk Prediction ◽

Tandem Mass ◽

Vitamin D Metabolites ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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Circulating conjugated and unconjugated vitamin D metabolite measurements by liquid chromatography mass spectrometry

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab708 ◽

2021 ◽

Author(s):

Carl Jenkinson ◽

Reena Desai ◽

Malcolm D McLeod ◽

Jonathan Wolf Mueller ◽

Martin Hewison ◽

...

Keyword(s):

Mass Spectrometry ◽

Vitamin D ◽

Liquid Chromatography ◽

Human Serum ◽

Phase Ii ◽

Enzyme Hydrolysis ◽

Vitamin D Metabolites ◽

Vitamin D Status ◽

Liquid Chromatography Mass Spectrometry ◽

Chromatography Mass Spectrometry

Abstract Context Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D’s diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms, however the clinical significance of circulating phase II vitamin D metabolites remains uncertain. In this study we analysed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum. Methods An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3 and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorised by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction. Results As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18-53%) were a higher proportion than glucuronide conjugates (ranging between 2.7-11%). The proportion of conjugated 25OHD3 (48±9%) was higher compared to 25OHD2 conjugates (29.1±10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all four vitamin D metabolites (r=0.85 to 0.97). Conclusions Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggests a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

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Improved analysis of vitamin D metabolites in plasma using liquid chromatography tandem mass spectrometry, and its application to cardiovascular research

Biomedical Chromatography ◽

10.1002/bmc.3208 ◽

2014 ◽

Vol 28 (6) ◽

pp. 913-917 ◽

Cited By ~ 5

Author(s):

Jatinderpal K. Sandhu ◽

Janica Auluck ◽

Leong L. Ng ◽

Donald J. L. Jones

Keyword(s):

Mass Spectrometry ◽

Vitamin D ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Tandem Mass ◽

Vitamin D Metabolites ◽

Cardiovascular Research ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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Detection of Vitamin D Metabolites in Breast Milk: Perspectives and challenges for measurement by Liquid Chromatography Tandem-Mass Spectrometry

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2021.08.003 ◽

2021 ◽

Author(s):

Carlie Tuddenham ◽

Ronda F Greaves ◽

Anushi E. Rajapaksa ◽

John D. Wark ◽

Rosita Zakaria

Keyword(s):

Mass Spectrometry ◽

Vitamin D ◽

Liquid Chromatography ◽

Breast Milk ◽

Tandem Mass Spectrometry ◽

Tandem Mass ◽

Vitamin D Metabolites ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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A Novel, Rapid, and Low-Volume Assay for Therapeutic Drug Monitoring of Posaconazole and Other Long-Chain Azole-Class Antifungal Drugs

mSphere ◽

10.1128/msphere.00623-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 2

Author(s):

Gregory R. Wiedman ◽

Yanan Zhao ◽

David S. Perlin

Keyword(s):

Liquid Chromatography ◽

Therapeutic Drug Monitoring ◽

Human Serum ◽

Drug Monitoring ◽

Fungal Infections ◽

Antifungal Drugs ◽

Therapeutic Drug ◽

Serum Samples ◽

Human Serum Samples ◽

Long Chain

ABSTRACT Clinicians need a better way to accurately monitor the concentration of antimicrobials in patient samples. In this report, we describe a novel, low-sample-volume method to monitor the azole-class antifungal drug posaconazole, as well as certain other long-chain azole-class antifungal drugs in human serum samples. Posaconazole represents an important target for therapeutic drug monitoring (TDM) due to its widespread use in treating invasive fungal infections and well-recognized variability of pharmacokinetics. The current “gold standard” requires trough and peak monitoring through high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Other methods include bioassays that use highly susceptible strains of fungi in culture plates or 96-well formats to monitor concentrations. Currently, no method exists that is both highly accurate in detecting free drug concentrations and is also rapid. Herein, we describe a new method using reduced graphene oxide (rGO) and a fluorescently labeled aptamer, which can accurately assess clinically relevant concentrations of posaconazole and other long-chain azole-class drugs in little more than 1 h in a total volume of 100 µl. IMPORTANCE This work describes an effective assay for TDM of long-chain azole-class antifungal drugs that can be used in diluted human serum samples. This assay will provide a quick, cost-effective method for monitoring concentrations of drugs such as posaconazole that exhibit well-documented pharmacokinetic variability. Our rGO-aptamer assay has the potential to improve health care for those struggling to treat fungal infections in rural or resource-limited setting.

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