Time-course analysis of 3-epi-25-hydroxyvitamin D3 shows markedly elevated levels in early life, particularly from vitamin D supplementation in preterm infants.

BACKGROUND: An epimeric form of 25-hydroxyvitamin D3 (25(OH)D3) has recently been detected in clinical samples, with relatively high levels in infants. Little is known on 3-epi-25(OH)D3 formation and physiological function. Our objective was to study dynamics of 3-epi-25(OH)D3 formation during infancy. METHODS: 25(OH)D3 and 3-epi-25(OH)D3 levels were measured by liquid chromatography-tandem mass spectrometry in 22 preterm (aged 34-37 wk), 15 early preterm (aged <34 wk), and 118 term infants up to 2 y of age. All infants were prescribed vitamin D 400 IU/day after the first week of life. RESULTS: At birth, 3-epi-25(OH)D3 levels were 3 (1-7) nmol/l, <10% of total 25(OH)D3. From the second week to 3 mo of age, both 25(OH)D3 and 3-epi-25(OH)D3 increased, with highest 3-epi-25(OH)D3 contribution in early preterm infants (up to 55% of total 25(OH)D3 vs. 36% in term infants, P < 0.0001). After 3 mo of age, 3-epi-25(OH)D3 normalized to <10% in all infants. CONCLUSIONS: At birth, all infants showed low contribution of 3-epi-25(OH)D3, increasing the week after starting vitamin D supplementation, until 3 mo of age. Highest levels of 3-epi-25(OH)D3 were found in early preterm infants, supporting the hypothesis that hepatic immaturity plays a role in 3-epi-25(OH)D3 formation.

Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.

BACKGROUND: An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. METHODS: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. RESULTS: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. CONCLUSION: This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.

Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys Vitamin D Total protein binding assay.

BACKGROUND: Presence of the 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] metabolite affects accurate determination of 25(OH)D3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys). METHODS: Neonatal samples, containing up to 58% of 3-epi-25(OH)D3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D3 and 3-epi-25(OH)D3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D3. RESULTS: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D3 recognition by the CPB assay when present as the natural endogenous metabolite. CONCLUSIONS: The automated CPB assay displays minimal 3-epi-25(OH)D3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D3. Exogenous 3-epi-25(OH)D3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno- or protein binding assays.

Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay.

The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum. Hexadeuterium labelled 25-OH vitamin D3 internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's < or = 6% for both 25-OH vitamin D3 and 25-OH vitamin D2. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D3 and 82.7 and 100.3% for 25-OH vitamin D2. Our results for the DEQAS serum pools averaged -7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r(2)=0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r(2)=0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum.

Plasma citrulline measurement using UPLC tandem mass-spectrometry to determine small intestinal enterocyte pathology.

Citrulline is a nonessential free amino acid, detectable in various biological fluids such as plasma, urine and cerebrospinal fluid. The plasma citrulline concentration is increasingly considered to be a reliable biomarker of enterocyte function. Current analysis usually involves lengthy HPLC separations as a part of classical amino acid profiling, or mass spectrometry usually in combination with derivatization. We employed UPLC-HILIC-tandem mass-spectrometry (MS/MS) of acetonitrile-derived supernatants from plasma samples of control subjects and of patients who had received myeloablative chemotherapy. Detection was achieved by the selected reaction monitoring of transitions: m/z 176-->70 and 180-->74 (for the deuterated standard), respectively. The method was precise and accurate with inter-day CV<3.9% (n=30), recoveries ranging from 98.0 to 100.3% and high linearity from 0.3 to at least 2,000 micromol/L. The results for 202 plasma samples agreed well with those obtained by the classical HPLC-fluorescence method. By a simple protein precipitation/extraction step and the UPLC separation the result can be available within 30 min of receipt with a capacity of at least 12 assays per hour. Citrulline in blood and plasma or serum was stable for at least 2 days at room temperature which would permit postal transport to the laboratory. The UPLC-MS/MS method for measuring plasma citrulline concentrations is fast and robust and is therefore an ideal tool for monitoring the intestinal enterocyte capacity of patients with various pathological conditions.