Vitamin D biomarkers in plasma

Importance of the Topic

Vitamin D status is routinely monitored as a critical indicator of bone health and calcium homeostasis in clinical settings. Measurement of circulating 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 provides a reliable assessment of total vitamin D status due to their higher blood concentrations and longer half life compared to the active hormone form.

Objectives and Study Overview

The aim of this work was to develop a rapid, sensitive and reproducible method for quantifying 25-hydroxyvitamin D2 and D3 in human plasma. This assay leverages a novel hydrophobic retention SPE product, SOLA HRP 30 mg, in conjunction with a UHPLC MS MS platform to improve sensitivity and throughput for clinical research applications.

Methodology and Sample Preparation

A small volume of plasma was protein precipitated with acetonitrile and fortified with an isotopically labeled internal standard. Samples were processed on SOLA HRP 30 mg SPE cartridges using a water methanol wash and methanol elution. Extracts were dried and reconstituted in a water acetonitrile mixture prior to analysis.

Used Instrumentation

• Vanquish Horizon UHPLC system with binary pump split sampler and column compartment
• Hypersil GOLD VANQUISH C18 UHPLC column, 1.9 micrometer, 2.1 by 50 millimeter
• TSQ Quantiva triple stage quadrupole mass spectrometer with HESI II source
• Chromeleon Chromatography Data System software, version 7.2.10

Key Results and Discussion

• Calibration was linear from 5 to 1000 nanograms per milliliter for D2 and from 2.5 to 1000 for D3 with correlation coefficients above 0.99
• Lower limits of quantification were 5.0 nanograms per milliliter for D2 and 2.5 for D3
• Intra day precision (CV) did not exceed 9.6 percent and accuracy bias was within ±8.2 percent at quality control levels
• Recovery at mid QC was approximately 100 percent for both analytes
• Matrix effects were minimal with internal standard normalization factors near unity
• No carryover was observed in blank injections following the highest calibration standard

Benefits and Practical Applications

• Enhanced sensitivity due to higher SPE bed mass and reduced breakthrough
• High throughput processing compatible with 96 well plate format
• Low sample and solvent consumption
• Robust method suitable for large scale clinical research and routine monitoring

Future Trends and Opportunities

• Extension to measurement of additional vitamin D metabolites
• Automation of sample preparation to increase throughput and reproducibility
• Integration into routine clinical laboratory workflows for population studies
• Investigation of lower quantification limits using larger sample volumes or enhanced SPE formats

Conclusion

The combination of SOLA HRP 30 mg SPE and UHPLC MS MS on a Vanquish TSQ Quantiva platform yields a fast, robust and sensitive assay for 25-hydroxyvitamin D2 and D3 in human plasma, offering excellent reproducibility and minimal matrix interference for clinical research.

Reference

• Holick MF, Binkley NC, Bischoff Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH, Weaver CM. Evaluation, treatment, and prevention of vitamin D deficiency an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(7):1911–1930.
• Institute of Medicine Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington DC National Academy Press; 2010.