Systematic Optimization of an SLE-DAPTAD LC-MS/MS Workflow for Simultaneous Quantification of Low-Abundance Vitamin...

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vitamin D₃ metabolites is analytically challenging because these compounds are structurally similar, strongly matrix-associated, and have poor ionization efficiency. These limitations are particularly important for low-abundance metabolites such as 1,25(OH)₂D₃. This study systematically compared extraction and derivatization strategies to develop a simplified workflow for simultaneous quantification of 25(OH)D₃, 3-epi-25(OH)D₃, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ in plasma.

Five sample-preparation approaches-simple protein precipitation, liquid-liquid extraction, salting-out assisted liquid-liquid extraction, solid-phase extraction, and supported liquid extraction-were evaluated under comparable conditions. Two derivatization reagents, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS), were compared. The selected workflow was validated according to bioanalytical method validation criteria.

Supported liquid extraction using ethyl acetate/methyl tert-butyl ether (1:1, v/v) provided the best overall balance of signal intensity, reproducibility, simplicity, and suitability for high-throughput processing. DAPTAD derivatization produced a substantially higher MS response than FMP-TS and was selected for the final method. The optimized SLE-DAPTAD LC-MS/MS workflow showed good linearity (r² > 0.99), acceptable accuracy and precision, recovery of 72.5%-104%, and limits of quantification of 0.08 ng/mL for 3-epi-25(OH)D₃, 1,25(OH)₂D₃, and 24,25(OH)₂D₃, and 0.88 ng/mL for 25(OH)D₃.

The optimized SLE-DAPTAD LC-MS/MS workflow provides a simple, sensitive, and automation-compatible approach for simultaneous quantification of four vitamin D₃ metabolites in plasma. The method is positioned as an analytical workflow for method development and low-volume plasma analysis, rather than as a fully standardized routine diagnostic assay for endogenous 1,25(OH)₂D₃ measurement.