Analysis of 25-Hydroxyvitamin D in Serum for Clinical Research

Importance of the topic

The accurate measurement of serum 25-hydroxyvitamin D (25OHD) is essential for clinical research into bone health, immune function and chronic diseases. Immunoassays often lack equal sensitivity to both 25OHD2 and 25OHD3, leading to adoption of LC-MS/MS for reliable quantification and differentiation of these analytes.

Objectives and overview of the study

This work describes the development of a semi-automated, high-throughput LC-MS/MS method for simultaneous quantification of 25OHD2 and 25OHD3 in human serum. The goal was to simplify sample preparation, ensure full traceability from primary tube to data export, and demonstrate performance suitable for large clinical trials.

Methodology and sample preparation

Serum samples, calibrators and QCs were spiked with stable-labeled internal standards and protein-precipitated with zinc sulfate and methanol. Following centrifugation, supernatants were processed on an Oasis HLB µElution 96-well plate using a Tecan Freedom Evo 100/4 liquid handler. After conditioning and washing, analytes were eluted in a methanol/IPA solution, diluted and transferred to the UPLC autosampler. Chromatographic separation was achieved on a BEH Phenyl column with a 4.2-minute gradient. Detection was conducted by XEVO TQD in positive ESI mode using MRM transitions optimized for each analyte and internal standard.

Used Instrumentation

• ACQUITY UPLC I-Class system with BEH Phenyl column
• Xevo TQD triple quadrupole mass spectrometer
• Oasis HLB µElution Plate for SPE
• Tecan Freedom Evo 100/4 liquid handling platform
• MassLynx software with TargetLynx and LIMS interfaces

Main results and discussion

Precision over 20 non-consecutive days (n=80 at each level) showed total RSD ≤7.3% and repeatability ≤6.5% across 20–300 nmol/L. Limits of detection and quantification were 2.56/3.60 nmol/L for 25OHD2 and 4.07/5.56 nmol/L for 25OHD3. The method was linear from 7 to 450 nmol/L (r2>0.996), with recoveries of 95.7–104.6%, no significant carryover or matrix interferences. Comparison to 29 DEQAS samples gave a Deming regression y=1.02x+0.07, indicating negligible bias.

Benefits and practical applications

This semi-automated workflow eliminates evaporation and reconstitution, reduces hands-on time and operator variability, and supports up to 192 samples per shift. Full barcode-based tracking and seamless data transfer to a LIMS enhance sample integrity and audit trails, making the method suitable for high-volume clinical research laboratories.

Future trends and opportunities

Emerging directions include integration of automated workflows with electronic laboratory notebooks, expansion to additional vitamin D metabolites and epimers, miniaturized SPE formats, and coupling with high-resolution MS for broader metabolomic profiling. Enhanced automation and software connectivity will further streamline clinical studies and large-scale epidemiology.

Conclusion

The described semi-automated UPLC-MS/MS method delivers robust, high-throughput quantification of 25OHD2 and 25OHD3 in serum with excellent precision, sensitivity and agreement with external quality assessments. Adoption of µElution SPE and liquid-handling automation significantly reduces manual steps and supports reliable data generation for clinical research.