A Direct LC/MS/MS Method for Quantitative Determination of 25-Hydroxyvitamin D2 and D3 in Human Plasma

Importance of the topic

Vitamin D is critical for bone health and disease prevention. Serum levels of its main circulating metabolites 25-hydroxyvitamin D2 and D3 reflect nutritional status and deficiency risk. Standard LC/MS/MS methods require extensive sample preparation, hindering routine screening. A direct, high-sensitivity approach can improve clinical throughput.

Objectives and overview of the study

This work aimed to develop and validate a streamlined LC/MS/MS assay for simultaneous quantification of 25-OH-D2 and 25-OH-D3 in human plasma, reducing sample volume and preparation time while achieving low detection limits and robust performance across the 1–100 ng/mL range.

Methodology and instrumentation

• Sample preparation: Spike plasma (125 µL) with standards and deuterated internal standards, precipitate proteins by adding 370 µL acetonitrile/methanol (1:1), vortex, centrifuge at 13 000 rpm for 10 min, filter through 0.2 µm nylon, and inject 10 µL.
• Liquid chromatography: Kinetex 1.7 µm C18 column (100×2.1 mm), 45 °C, mobile phases A=0.1% formic acid in water, B=0.1% formic acid in methanol, 0.5 mL/min, 15 min gradient.
• Mass spectrometry: Shimadzu LCMS-8050 with APCI positive mode, interface 400 °C, desolvation 200 °C, collision gas Ar at 270 kPa, nitrogen nebulizing and drying gases.
• Quantitation: Three MRM transitions per analyte; primary transition based on dehydration ion used for quantitation, additional transitions for confirmation.

Main results and discussion

• Calibration curves (pre- and post-spiked plasma) exhibited linearity (R2 >0.996) over 1–100 ng/mL; 25-OH-D3 showed a non-zero intercept due to residual endogenous levels (~5.1–5.4 ng/mL).
• Lower limits of detection and quantification estimated at ~1 ng/mL and 3 ng/mL, respectively, with a 10 µL injection.
• Matrix effects, recovery, and process efficiency were within ±15% for most concentration levels; a single instance of >20% process efficiency was observed at 1 ng/mL for D3.
• Repeatability (n=7) yielded RSD <7.5% for concentrations ≥5 ng/mL; higher RSDs (10–22%) occurred at the 1 ng/mL level.
• Specificity achieved through retention time matching and MRM ratio criteria ensured confident analyte identification in plasma matrices.

Benefits and practical applications of the method

• Minimal sample volume (125 µL) and simple protein precipitation accelerate throughput in clinical and research laboratories.
• Direct injection without further cleanup reduces labor and consumables cost.
• Sensitivity and specificity meet the requirements for vitamin D status screening and pharmacokinetic studies.

Future trends and possibilities of use

Advances in high-throughput LC/MS/MS and microflow chromatography may further reduce sample requirements and analysis time. Integration with automated robotics could enable large-scale screening. Expanding the panel to additional vitamin D metabolites and conjugates can provide comprehensive assessments of vitamin D metabolism in health and disease.

Conclusion

The direct LC/MS/MS approach on the Shimadzu LCMS-8050 offers a robust, sensitive, and streamlined assay for 25-OHD2 and 25-OHD3 in human plasma. Validation data support its applicability for clinical screening and research applications, with reliable performance across a clinically relevant concentration range.

Reference

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