Simultaneous Quantification of 25-Hydroxyvitamin D3, 25-Hydroxyvitamin D2, and 24,25-Dihydroxyvitamin D3 in Clinical Research Studies By UPLC-MS/MS

Importance of the Topic

In clinical and preclinical research, accurate quantification of vitamin D metabolites is critical for assessing nutritional status and metabolic flux. 25-hydroxyvitamin D2 and D3 (25(OH)D2 and 25(OH)D3) serve as the primary biomarkers of vitamin D status, while the catabolic product 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) reflects the activity of the CYP24A1 enzyme and clearance rate of 25(OH)D3. Reliable multi-analyte measurement in low-volume serum samples accelerates studies in humans, rodents, and in vitro models and supports investigations into vitamin D metabolism and its clinical implications.

Objectives and Study Overview

This work aimed to develop a sensitive and robust UPLC-MS/MS method enabling simultaneous quantification of 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 from just 100 µL of serum. By integrating liquid–liquid extraction with chemical derivatization using DMEQ-TAD and a high-resolution ACQUITY UPLC system coupled to a Xevo TQ-S mass spectrometer, the protocol intends to streamline vitamin D metabolite profiling for clinical research applications.

Methodology and Instrumentation

• Sample preparation:
  • Serum aliquots (100 µL) were acidified and precipitated with ZnSO4 and methanol.
  • Organic extraction was performed with hexane and MTBE, followed by nitrogen drying.
  • Derivatization used DMEQ-TAD in ethyl acetate, incubated sequentially for enhanced adduct formation.
• Chromatographic conditions:
  • Instrument: Waters ACQUITY UPLC system with BEH-Phenyl column (2.1 × 50 mm, 1.7 µm) at 40 °C.
  • Mobile phases: 2 mM ammonium acetate/0.1% formic acid in water (A) and in methanol (B).
  • Gradient: 35:65 A:B to 10:90 over 5 minutes at 400 µL/min; total run time 6 minutes.
  • Injection: 10 µL in full-loop mode; column temperature 40 °C; sample at 4 °C.
• Mass spectrometry:
  • Detector: Xevo TQ-S operated in positive electrospray ionization (ESI+) with MRM transitions targeting DMEQ-TAD adducts.
  • Key transitions: m/z 746.6>468.3 for 25(OH)D3, 758.6>468.3 for 25(OH)D2, and 762.6>468.3 for 24,25(OH)2D3.
  • Desolvation temp 650 °C, gas flows at 1000 L/h (desolvation) and 150 L/h (cone).
• Data analysis:
  • MassLynx Software v4.1 with TargetLynx for quantification and calibration curve processing.

Main Results and Discussion

The DMEQ-TAD derivatization improved ionization efficiency and reduced background signals by adding 336 Da to analyte mass. Chromatographic separation resolved 3-epi-25(OH)D3 from 25(OH)D3, preventing isobaric interference. Calibration curves showed linearity (r2 ≥ 0.997) over clinically relevant ranges: 3.8–148 ng/mL for 25(OH)D3, 4.9–120 ng/mL for 25(OH)D2, and 0.4–11.6 ng/mL for 24,25(OH)2D3. Limits of quantification were around 0.1–0.2 ng/mL, with detection limits down to 0.04 ng/mL based on S/N ≥ 3. Precision across 14 days yielded within-run CVs of 3–4% and between-run CVs of 4–7%. Accuracy against DEQAS reference samples returned mean biases of −2% for 25(OH)D3 and −5% for 25(OH)D2.

Benefits and Practical Application

• High sensitivity and specificity enable reliable detection of low-abundance metabolites with minimal sample volume.
• Rapid analysis (6-minute run) increases throughput for clinical and preclinical studies.
• Resolved chromatographic peaks prevent cross-interference from epimers and isobaric compounds.
• Method adapts to rodent and cell culture matrices, broadening its research utility.

Future Trends and Opportunities

As the role of vitamin D metabolites in health and disease continues to expand, further developments may include:

• Enhanced multiplexing to include additional hydroxylated or conjugated vitamin D forms.
• Automation of sample preparation to support large-scale epidemiological studies.
• Integration with high-resolution accurate-mass spectrometry for discovery of novel metabolites.
• Miniaturized platforms for point-of-care or field applications.

Conclusion

The presented UPLC-MS/MS method combining DMEQ-TAD derivatization and minimal-volume sample handling provides a robust tool for simultaneous quantification of key vitamin D metabolites. Its accuracy, precision, and versatility support diverse clinical research needs, from human cohorts to animal and in vitro models.

Reference

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