Sensitive and Robust Measurement of Vitamin D Hydroxy Metabolites and Epimers in Serum Using the Agilent Ultivo Triple Quadrupole LC/MS

Importance of the Topic

Accurate measurement of 25-hydroxyvitamin D metabolites and their C3-epimers in serum is essential for reliable assessment of vitamin D status in clinical research and diagnostics. Conventional LC/MS methods may overestimate 25(OH)D2 and 25(OH)D3 when epimers coelute, leading to potential misinterpretation of a patient’s vitamin D sufficiency or deficiency.

Study Objectives and Overview

This study aimed to establish a 10-minute, high-throughput LC/MS/MS workflow on the Agilent Ultivo triple quadrupole system that provides baseline chromatographic separation of 25(OH)D2, 25(OH)D3 and their respective epimers. The method was evaluated for sensitivity, linearity, precision, accuracy, matrix effects, carryover, recovery, and long-term robustness, including performance with certified reference materials.

Methodology and Instrumentation

Serum samples and reference materials were protein-precipitated with cold acetonitrile containing formic acid and deuterated internal standards. After centrifugation, supernatants were directly injected onto an Agilent InfinityLab Poroshell 120 PFP column (3.0×100 mm, 2.7 µm) using a 10-minute gradient. Mass spectrometric detection employed MRM transitions optimized for each analyte and internal standard on an Agilent Ultivo LC/TQ with Jet Stream ESI. Calibration ranged from LOQ to 200 ng/mL using deuterated standards for quantitation.

Main Results and Discussion

The method achieved baseline separation of 25(OH)D2 and 25(OH)D3 from their epimers within 10 minutes. Limits of detection and quantitation were 0.4–1.0 ng/mL, with calibration curves showing R² ≥ 0.999. Intra- and inter-day precision met acceptance criteria (<5% area RSD, <0.5% retention time RSD) and accuracy ranged between 95–115%. Recoveries for QC levels were 92–109% (RSD ≤ 2%). Matrix suppression was minimal (>87% signal), carryover was <0.004%, and 500 continuous injections of MQC samples demonstrated robustness (concentration RSD <5%). Validation with ChromSystems certified reference standards confirmed performance within specified limits.

Benefits and Practical Applications

This robust and sensitive method enables reliable differentiation of vitamin D metabolites and epimers in clinical, research, and QA/QC laboratories. High throughput, minimal carryover, and extended stability support large sample batches, reducing downtime for maintenance and tune adjustments.

Future Trends and Applications

Advances may include expansion to additional vitamin D metabolites (e.g., 1,25-dihydroxyvitamin D), integration with high-resolution MS for structural confirmation, microflow or nanoflow LC for reduced solvent consumption, and automation of sample preparation. Multiplexed assays for broader lipidomic profiling could further enhance clinical and research applications.

Conclusions

The developed Agilent Ultivo LC/TQ method delivers specific, sensitive, and reproducible quantitation of 25(OH)D2, 25(OH)D3, and their epimers in serum. Baseline separation, excellent linearity, precision, accuracy, and proven robustness make it well suited for routine analysis in clinical and lipidomics laboratories.

Used Instrumentation

• Agilent 1260 Infinity II flexible pump and multisampler
• Agilent InfinityLab Poroshell 120 PFP column (3.0×100 mm, 2.7 µm)
• Agilent Ultivo triple quadrupole LC/TQ with Jet Stream ESI source