Simultaneous quantification of fat- and water-soluble vitamins in serum by valve-mediated dual LC-MS/MS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the benchmark platform for multiplex vitamin quantification, yet simultaneous determination of chemically disparate fat-soluble (FSVs) and water-soluble vitamins (WSVs) remains analytically challenging. We introduce a pragmatic dual-stream workflow that converts the intrinsic waste segments of any LC gradient into productive analytical time, delivering complete FSV and WSV profiles. Two 5-min chromatographic separations, optimized for fat-soluble and water-soluble vitamins respectively, are staggered through a six-port divert valve: while one column eluted its analytes to the mass spectrometer, the other was shunted to waste. This seamless overlap effectively doubles the throughput within 5 min. And every WSV elution window remained fully enclosed within the waste segment of the FSV gradient, with CVs < 10 % for the internal-standard peak areas across both vitamin classes. Moreover, method comparison between dual LC-MS/MS and single LC-MS/MS across twenty patient sera yielded Spearman's rho values ranging from 0.950 to 0.997, Passing-Bablok slopes spanning 0.884-1.128, and Bland-Altman biases below 8.3 %, confirming clinical concordance. Owing to its simple equipment requirements and setup process, this method should be highly accessible for other laboratories.