Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and 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 [0.9 µg/L, 25(OH)D3], and 10 nmol/L [4 µg/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.

Serum 25-hydroxyvitamin D3 levels and flares of systemic lupus erythematosus: a longitudinal cohort analysis.

The aim of this study is to evaluate the relationship between baseline serum 25-hydroxyvitamin D3 levels and SLE activity/flares over time. This is a longitudinal study of 276 patients who fulfilled ≥ 4 ACR criteria for SLE and recruited in the year 2011. Serum samples were collected at baseline and assayed for 25-hydroxyvitamin D3 at the end of a mean follow-up of 32.5 months. Participants were stratified into three groups according to baseline 25-hydroxyvitamin D3 levels: group I (< 15 ng/ml, deficiency), group II (15-30 ng/ml, insufficiency), and group III (> 30 ng/ml, adequate). Baseline and summated SLE disease activity index (SLEDAI) score over time and the annual incidence of lupus flares were compared among these groups. 25-hydroxyvitamin D3 levels of < 15, 15-30, and > 30 ng/ml were present in 26, 54, and 20% of the recruited patients, respectively. Group I had significantly higher baseline SLEDAI scores. After a follow-up of 32.5 ± 5.5 months, 153 mild/moderate and 91 severe flares developed. The mean summated SLEDAI was 3.2 ± 2.0 in group I, 2.4 ± 1.9 in group II and 2.7 ± 2.1 in group III patients (P = 0.02). The annual incidence of mild/moderate and severe flares was 0.26 ± 0.39 and 0.20 ± 0.45 (group I); 0.20 ± 0.33 and 0.09 ± 0.22 (group II); and 0.20 ± 0.32 and 0.14 ± 0.46 (group III), respectively (P > 0.05). In a subgroup of 73 patients who were clinically and serologically quiescent at baseline, a similar trend of more flares was observed in group I patients. Vitamin D deficiency was frequent in Chinese SLE patients and was associated with more active disease at baseline and over time, as well as a trend of more severe lupus flares.