Systematic Optimization of an SLE‐DAPTAD LC–MS/MS Workflow for Simultaneous Quantification of Low‐Abundance Vitamin D 3 Metabolites in Plasma

Marzieh Amirmostofian; Michael D. Wiese; Paul H. Anderson; Peter Hoffmann

doi:10.1002/rcm.70158

Systematic Optimization of an SLE‐DAPTAD LC–MS/MS Workflow for Simultaneous Quantification of Low‐Abundance Vitamin D 3 Metabolites in Plasma - ResearchHub

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Systematic Optimization of an SLE‐DAPTAD LC–MS/MS Workflow for Simultaneous Quantification of Low‐Abundance Vitamin D 3 Metabolites in Plasma

Marzieh Amirmostofian, Michael D. Wiese, Paul H. Anderson, Peter Hoffmann

Rapid Communications in Mass Spectrometry2026doi:10.1002/rcm.70158

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Abstract

ABSTRACT Rationale Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of vitamin D 3 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) 2 D 3 . This study systematically compared extraction and derivatization strategies to develop a simplified workflow for simultaneous quantification of 25(OH)D 3 , 3‐epi‐25(OH)D 3 , 1,25(OH) 2 D 3 , and 24,25(OH) 2 D 3 in plasma. Methods 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. Results 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 ( r 2 > 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)D 3 , 1,25(OH) 2 D 3 , and 24,25(OH) 2 D 3 , and 0.88 ng/mL for 25(OH)D 3 . Conclusions The optimized SLE‐DAPTAD LC–MS/MS workflow provides a simple, sensitive, and automation‐compatible approach for simultaneous quantification of four vitamin D 3 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) 2 D 3 measurement.

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Systematic Optimization of an SLE‐DAPTAD LC–MS/MS Workflow for Simultaneous Quantification of Low‐Abundance Vitamin D 3 Metabolites in Plasma

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