Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: A sample-multiplexed high-throughput assay for 25-hydroxyvitamin D₂and D₃

BACKGROUND: The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D₂ and D₃ [25(OH)D₂ and 25(OH)D₃], which increased sample throughput 5-fold. METHODS: After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D₂ and 25(OH)D₃ for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS: TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/ h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D₂], 2.2 nmol/L [0.9μg/L, 25(OH)D₃], and 10 nmol/L [4μg/L, 25(OH)D₂], 5 nmol/L [2 μg/L, 25(OH)D₃], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D₂ (n = 1733) and 25(OH)D₃ (n = 7614) (R²=0.96 and 0.97), respectively. CONCLUSIONS: Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D₂ and 25(OH)D₃ allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.