Johannes M.W. van den Ouweland

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 assays 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 CVs < 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.

The role of liquid chromatography-tandem mass spectrometry in the clinical laboratory.

Liquid chromatography coupled to mass spectrometry (LC-MS/MS) is increasingly used as a routine methodology in clinical laboratories for the analysis of low molecular weight molecules. The high specificity in combination with high sensitivity and multi-analyte potential makes it an attractive complementary method to traditional methodology used for routine applications. Its strength and weaknesses in this context will be discussed and examples of successful clinical applications will be given. For LC-MS/MS to truly fulfil its promise in clinical diagnosis, the prerequisite steps being sample pre-treatment, chromatographic separation and detection by selected reaction monitoring must become more integrated as they are in conventional clinical analysers. The availability of ready-to-use reagents kits, eliminating efforts needed for method development and extensive validation, are likely to contribute to a wider acceptance of LC-MS/MS in clinical laboratories. Growing applicability of LC-MS/MS in the clinical laboratory field is expected from quantitative protein analysis.

Fast Separation of 25-Hydroxyvitamin D3 from 3-Epi-25-Hydroxyvitamin D3 in Human Serum by Liquid Chromatography–Tandem Mass Spectrometry: Variable Prevalence of 3-Epi-25-Hydroxyvitamin D3 in Infants, Children, and Adults

To the Editor: Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is becoming increasingly popular for the measurement of 25-hydroxyvitamin D [25(OH)D] in human serum. A limitation of most LC-MS/MS methods is the potential interference from coeluting isomeric compounds having identical elemental composition but different structure, leading to overestimation of true 25(OH)D concentrations. Of particular interest is the C3 epimer of 25(OH)D, 3-epi-25(OH)D, which can be present at relatively high concentrations in sera from infants, but can also be found in sera from adults, albeit at lower concentrations (1, 2). To separate 3-epi-25(OH)D from 25(OH)D, current procedures require lengthy chromatographic run times varying from 12 to 40 min (3), which makes these methods unsuitable for clinical laboratories that must deal with increasing numbers of vitamin D requests. We describe a modification of an established LC-MS/MS method for measurement of 25(OH)D3 and 25(OH)D2 (4) that allows fast separation of 25(OH)D3 from 3-epi-25(OH)D3 in human serum. Sample preparation, assay calibration, and instrument operation were carried out as described (4), with minor modifications. 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 were from Sigma Aldrich, and the internal standard …

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.

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.

Abstract 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.

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.