An Offline Automated Solid-Phase Extraction Method for Measurement of Serum 25-Hydroxyvitamin D for Clinical Research

Significance of the Topic

Accurate measurement of serum 25-hydroxyvitamin D (25(OH)D) is increasingly vital to support large-scale clinical research into vitamin D status and its links to diverse diseases. Automated, high-throughput methods reduce human error, improve consistency, and enable laboratories to process the volume of samples required for randomized trials investigating conditions such as cancer, cardiovascular disease, and diabetes.

Objectives and Study Overview

This study aimed to develop and validate an offline automated solid-phase extraction (SPE) workflow combined with UPLC-MS/MS for quantifying total 25(OH)D (sum of D2 and D3) in human serum. Key goals included minimizing manual intervention, demonstrating robust agreement with an established LC-MS/MS protocol, and ensuring analytical performance suitable for clinical research settings.

Methodology and Instrumentation

The workflow integrates protein precipitation and SPE on an Offline Automated Sample Preparation System (OASPS), streamlining sample cleanup before UPLC-MS/MS analysis.

• Sample pretreatment: Protein disruption using zinc sulfate and methanol, performed automatically with barcode tracking.
• Solid-phase extraction: Oasis HLB μElution plates conditioned and washed on the OASPS, elution in a two-step protocol eliminating evaporation.
• Chromatographic separation: Waters ACQUITY UPLC BEH Phenyl column (2.1 x 50 mm, 1.7 µm) at 35 °C, gradient elution from 65% to 85% methanol in 3 minutes.
• Mass spectrometry: Waters ACQUITY TQD TQ detector in positive ESI mode monitoring specific MRM transitions for 25(OH)D2, 25(OH)D3, and deuterated internal standards.

Used Instrumentation

• Waters ACQUITY UPLC System with BEH Phenyl Column
• Waters ACQUITY TQD Triple Quadrupole MS Detector
• Waters Offline Automated Sample Preparation System (OASPS)
• Oasis HLB μElution 96-well SPE Plates
• MassLynx 4.1 and TargetLynx Software for data acquisition and processing

Main Results and Discussion

The assay exhibited excellent linearity for both 25(OH)D2 (r2 > 0.997) and 25(OH)D3 (r2 > 0.998) across 2.5–220 ng/mL. Intra-assay precision (CV ≤ 7.7%) and inter-assay precision (CV < 12%) met stringent criteria. Recovery exceeded 80% throughout the analytical range. Comparison with an independent hexane extraction LC-MS/MS method on 30 anonymized samples showed strong agreement (Passing–Bablok slope 0.98, intercept 1.47 ng/mL; Bland–Altman bias +1.3 ng/mL). Matrix effects from phospholipids and plasticizers were effectively minimized by the SPE protocol.

Benefits and Practical Applications

• High throughput: 96 samples prepared in approximately two hours with minimal hands-on time.
• Reduced variability: Automated liquid handling and sample tracking ensure consistent extraction performance.
• Elimination of drying/reconstitution steps: μElution format accelerates turnaround and minimizes losses.
• Applicability: Method supports clinical trial workflows and large research studies requiring robust 25(OH)D quantification.

Future Trends and Potential Applications

Advances may include integration with online SPE for fully automated platforms, expansion to other vitamin D metabolites, and coupling to high-resolution mass spectrometry for improved specificity. Machine-learning algorithms applied to spectral data could further enhance throughput and data quality control. Broader adoption of standardized automated workflows will facilitate multi-center studies and inter-laboratory comparability.

Conclusion

The offline automated SPE-UPLC-MS/MS method delivers precise, accurate, and high-throughput quantification of serum 25(OH)D2 and 25(OH)D3. By reducing manual interventions and streamlining sample cleanup, it enables research laboratories to meet the demands of large-scale clinical studies investigating vitamin D status in health and disease.

References

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