Analytical Unreliability of 25 Hydroxy Vitamin D Measurements in Pre-Term Neonates.

BACKGROUND: Vitamin D supplementation is common practice for neonates and infants due to limited stores of vitamin D at birth. Although not commonly encountered, vitamin D toxicity can occur due to over-supplementation. However, toxic concentrations are often not included in method validation experiments, and assays often are not validated in the neonatal population. METHODS: We compared serial 25 hydroxy vitamin D [25(OH)D] measurements in pre-term neonates receiving 25(OH)D supplementation and identified 12 patients wherein concentrations of 25(OH)D were above 50 ng/mL (125 nM) that required additional investigations as the 25(OH)D results did not match the clinical picture. Available samples were compared across 4 immunoassay platforms (LIAISON XL, Roche Cobas e602, Abbott Alinity i, and Siemens Centaur XP) and LC-MS/MS. RESULTS: Concentrations of 25(OH)D observed on one individual immunoassay platform (LIAISON XL) fluctuated substantially between subsequent blood draws in select neonates with elevated concentrations. Serum samples from these patients showed variable agreement between LC-MS/MS and other immunoassay platforms. These fluctuations were not explained by the presence of 3-epimer-25(OH)D or 24,25(OH)2D. CONCLUSIONS: Although we were unable to identify a cause for the variable elevated results, our findings suggest that neonatal 25(OH)D measurements alone should not be used for assessment of nutritional monitoring, and that clinical correlation and other laboratory parameters including ionized calcium should be considered.

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.

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.