Influence of a Composite Polylysine-Polydopamine-Quaternary Ammonium Salt Coating on Titanium on Its Ostogenic and Antibacterial Performance.

Thin oxide layers form easily on the surfaces of titanium (Ti) components, with thicknesses of <100 nm. These layers have excellent corrosion resistance and good biocompatibility. Ti is susceptible to bacterial development on its surface when used as an implant material, which reduces the biocompatibility between the implant and the bone tissue, resulting in reduced osseointegration. In the present study, Ti specimens were surface-negatively ionized using a hot alkali activation method, after which polylysine and polydopamine layers were deposited on them using a layer-by-layer self-assembly method, then a quaternary ammonium salt (QAS) (EPTAC, DEQAS, MPA-N+) was grafted onto the surface of the coating. In all, 17 such composite coatings were prepared. Against Escherichia coli and Staphylococcus aureus, the bacteriostatic rates of the coated specimens were 97.6 ± 2.0% and 98.4 ± 1.0%, respectively. Thus, this composite coating has the potential to increase the osseointegration and antibacterial performance of implantable Ti devices.

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.

OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Standardizing 25-hydroxyvitamin D data from the HunMen cohort.

Standardization of 25-hydroxyvitamin D (25OHD) values is still a challenge. We propose standardization by correction of the measured 25OHD values using the linear regression bias from the National Institute of Standards and Technology (NIST) 'total' target values reported by Vitamin D External Quality Assessment Scheme (DEQAS). Our approach could perhaps be a practical solution to the anomaly surrounding non-standardized 25OHD values. INTRODUCTION: Standardization of 25OHD values is still a challenge. We propose standardization by correction of the measured 25OHD values using the linear regression equation derived from the analysis of relationship between total 25OHD values measured by the methodology used by the laboratory and the NIST total target values (TV) reported by the DEQAS for all 5 of the DEQAS samples in a given survey. METHODS: We applied our approach to standardize total 25OHD values of the HunMen cohort. RESULTS: All 206 samples for the HunMen cohort were evaluated using the automated Liaison DiaSorin total 25OHD chemiluminescence immunoassay (CLIA). The timing of these measurements coincided with that of the October 2015 DEQAS survey using samples 481 to 485. Following standardization, the mean total 25OHD changed from 53 to 62 nmol/L and the prevalence of hypovitaminosis D (<75 nmol/L) decreased significantly from 84 to 72%. CONCLUSION: A simple approach readily applicable at the routine diagnostic laboratory could perhaps be a practical solution to the anomaly surrounding non-standardized 25OHD values.

Analytical variation concerning total 25-hydroxyvitamin D measurement, where are we now? A DEQAS review of current assay performance.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and other vitamin D metabolites. In this review an assessment of the state of the art in the performance of 25OHD methods is presented. This assessment is based on an analysis of data submitted by scheme participants for the 2021/22 distribution cycle, which comprised of four distributions each containing five DEQAS samples. These distributions enabled the assessment of performance across a wide concentration range and included samples containing endogenous 25OHD2. Overall analytical performance continues to improve, but there is still significant method variation and bias in some automated methods. These automated methods continue to be challenged in measuring 25OHD at the extremes of the measuring range and in the presence of 25OHD2. LC-MS/MS methods still show superior performance with regards to bias, but are out-performed by some automated methods in terms of assay variability. Through participating in an accuracy based EQA scheme, such as DEQAS, laboratories are able to assess the accuracy of their methods in comparison to a gold standard reference measurement procedure. It is crucial for all laboratories to be aware of the performance and limitations of their 25OHD assays and to educate their users accordingly in order to ensure reliable assessment of vitamin D status.

Lack of significant seasonal association between serum 25(OH)D concentration, muscle mass and strength in postmenopausal women from the D-FINES longitudinal study.

The aim of the present study was to assess the seasonal relationship between serum 25(OH)D concentration, lean mass and muscle strength. This was a secondary data analysis of a subgroup of 102 postmenopausal women participating in the 2006-2007 D-FINES (Vitamin D, Food Intake, Nutrition and Exposure to Sunlight in Southern England) study. The cohort was assessed as two age subgroups: <65 years (n=80) and ≥65 years (n=22). Outcome measures included lean mass (DXA), muscle strength (handgrip dynamometry) and serum 25(OH)D concentration (enzymeimmunoassay). Derived outcomes included appendicular skeletal muscle mass (ASM) and relative appendicular skeletal muscle index (RASM). Sarcopenia status was assessed using the European Working Group on Sarcopenia in Older People 2018 criteria. Non-parametric partial correlation using BMI as a covariate was used to evaluate the study aims. There were no statistically significant associations between total lean mass, ASM or RASM and 25(OH)D in any group at any season. There was a trend for handgrip strength to be positively associated with serum 25(OH)D concentration. There was a trend showing a higher prevalence of sarcopenia in women ≥65 years. Sarcopenia status appeared transient for five women. In conclusion, the present study found no significant association between vitamin D status and functional indicators of musculoskeletal health, which were additionally not affected by season.

Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD2, 24,25-(OH)2D3, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 µg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC-MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.

Vitamin D status in children and adults in Sweden: dietary intake and 25-hydroxyvitamin D concentrations in children aged 10-12 years and adults aged 18-80 years.

The study aimed to estimate vitamin D intake and plasma/serum 25-hydroxyvitamin D (25(OH)D) concentrations, investigate determinants of 25(OH)D concentrations and compare two 25(OH)D assays. We conducted two nationwide cross-sectional studies in Sweden with 206 school children aged 10-12 years and 1797 adults aged 18-80 years (n 268 provided blood samples). A web-based dietary record was used to assess dietary intake. Plasma/serum 25(OH)D was analysed by liquid chromatography-mass spectrometry (LC-MS) and immunoassay in adults and LC-MS/MS in children. Most participants reported a vitamin D intake below the average requirement (AR), 16 % of children and 33 % of adults met the AR (7⋅5 µg). In adults, plasma 25(OH)D below 30 and 50 nmol/l were found in 1 and 18 % of participants during the summer period and in 9 and 40 % of participants during the winter period, respectively. In children, serum 25(OH)D below 30 and 50 nmol/l were found in 5 and 42 % of participants (samples collected March-May), respectively. Higher 25(OH)D concentrations were associated with the summer season, vacations in sunny locations (adults), and dietary intake of vitamin D and use of vitamin D supplements, while lower concentrations were associated with a higher BMI and an origin outside of Europe. Concentrations of 25(OH)D were lower using the immunoassay than with the LC-MS assay, but associations with dietary factors and seasonal variability were similar. In conclusion, vitamin D intake was lower than the AR, especially in children. The 25(OH)D concentrations were low in many participants, but few participants had a concentration below 30 nmol/l.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Hydroxyvitamin D assays: An historical perspective from DEQAS.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

Vitamin D in the Middle East and North Africa.

PURPOSE: The Middle East and North Africa (MENA) region registers some of the lowest serum 25­hydroxyvitamin D [25(OH)D] concentrations, worldwide. We describe the prevalence and the risk factors for hypovitaminosis D, completed and ongoing clinical trials, and available guidelines for vitamin D supplementation in this region. METHODS: This review is an update of previous reviews published by our group in 2013 for observational studies, and in 2015 for randomized controlled trials (RCTs) from the region. We conducted a comprehensive search in Medline, PubMed, and Embase, and the Cochrane Library, using MeSH terms and keywords relevant to vitamin D, vitamin D deficiency, and the MENA region, for the period 2012-2017 for observational studies, and 2015-2017 for RCTs. We included large cross-sectional studies with at least 100 subjects/study, and RCTs with at least 50 participants per arm. RESULTS: We identified 41 observational studies. The prevalence of hypovitaminosis D, defined as a 25­hydroxyvitamin D [25(OH)D] level below the desirable level of 20 ng/ml, ranged between 12-96% in children and adolescents, and 54-90% in pregnant women. In adults, it ranged between 44 and 96%, and the mean 25(OH)D varied between 11 and 20 ng/ml. In general, significant predictors of low 25(OH)D levels were female gender, increasing age and body mass index, veiling, winter season, use of sun screens, lower socioeconomic status, and higher latitude.We retrieved 14 RCTs comparing supplementation to control or placebo, published during the period 2015-2017: 2 in children, 8 in adults, and 4 in pregnant women. In children and adolescents, a vitamin D dose of 1000-2000 IU/d was needed to maintain serum 25(OH)D level at target. In adults and pregnant women, the increment in 25(OH)D level was inversely proportional to the dose, ranging between 0.9 and 3 ng/ml per 100 IU/d for doses ≤2000 IU/d, and between 0.1 and 0.6 ng/ml per 100 IU/d for doses ≥3000 IU/d. While the effect of vitamin D supplementation on glycemic indices is still controversial in adults, vitamin D supplementation may be protective against gestational diabetes mellitus in pregnant women. In the only identified study in the elderly, there was no significant difference between 600 IU/day and 3750 IU/day doses on bone mineral density. We did not identify any fracture studies.The available vitamin D guidelines in the region are based on expert opinion, with recommended doses between 400 and 2000 IU/d, depending on the age category, and country. CONCLUSION: Hypovitaminosis D is prevalent in the MENA region, and doses of 1000-2000 IU/d may be necessary to reach a desirable 25(OH)D level of 20 ng/ml. Studies assessing the effect of such doses of vitamin D on major outcomes, and confirming their long term safety, are needed.

Concerning the vitamin D reference range: pre-analytical and analytical variability of vitamin D measurement.

Unlike other vitamins, the vitamin D concentration in blood varies cyclically over the course of the year in relation to genetic (gender, ethnicity, polymorphisms) and environmental factors (sunlight exposure, diet, food-related or direct vitamin D supplementation, skin pigmentation). Although the major diagnostics manufacturers have recently developed improved automated 25-hydroxy vitamin D immunoassays, the intra- and inter-laboratory variability is still high (especially at low vitamin D concentrations) which might lead to incorrect vitamin D deficiency/insufficiency diagnosis. Moreover, despite recent efforts to standardize the assay and minimize its variability, the current bias for measured vitamin D concentrations is often still above the desirable ± 10% criterion. Because the implications of low vitamin D concentrations in non-skeletal diseases are still partially unknown, international guideline recommendations for establishing meaningful ranges, at any time over the course of the year, irrespective not only of environmental and personal factors but also of instrumental variability, are needed. In this review, we discuss the main factors that influence the variability of vitamin D concentrations and whether a centile curve, individually calculated by a theoretical equation considering such factors, might be better suited than a fixed limit to assess abnormal vitamin D concentrations in otherwise healthy subjects. Vitamin D reference ranges during pregnancy, childhood, or diagnosed illnesses, which merit separate discussion, are beyond the scope of this review.

Vitamin D measurement standardization: The way out of the chaos.

Substantial variability is associated with laboratory measurement of serum total 25-hydroxyvitamin D [25(OH)D]. The resulting chaos impedes development of consensus 25(OH)D values to define stages of vitamin D status. As resolving this situation requires standardized measurement of 25(OH)D, the Vitamin D Standardization Program (VDSP) developed methodology to standardize 25(OH)D measurement to the gold standard reference measurement procedures of NIST, Ghent University and CDC. Importantly, VDSP developed protocols for standardizing 25(OH)D values from prior research based on availability of stored serum samples. The effect of such retrospective standardization on prevalence of "low" vitamin D status in national studies reported here for The Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) and the German Health Interview and Examination Survey for Children and Adolescents (KIGGS, 2003-2006) was such that in NHANES III 25(OH)D values were lower than original values while higher in KIGGS. In NHANES III the percentage with values below 30, 50 and 75 nmol/L increased from 4% to 6%, 22% to 31% and 55% to 71%, respectively. Whereas in KIGGS after standardization the percentage below 30, 50, and 70 nmol/L decreased from 28% to 13%, 64% to 47% and 87% to 85% respectively. Moreover, in a hypothetical example, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the 25(OH)D concentration which maximally suppressed PTH could vary from 20 to 35ng/mL. These examples underscore the challenges (perhaps impossibility) of developing vitamin D guidelines using unstandardized 25(OH)D data. Retrospective 25(OH)D standardization can be applied to old studies where stored serum samples exist. As a way forward, we suggest an international effort to identify key prior studies with stored samples for re-analysis and standardization initially to define the 25(OH)D level associated with vitamin D deficiency (rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, examples reported here highlight the importance of suspending publication of meta-analyses based on unstandardized 25(OH)D results.

Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

Developing vitamin D dietary guidelines and the lack of 25-hydroxyvitamin D assay standardization: The ever-present past.

Unstandardized laboratory measurement of 25-hydroxyvitamin D (25(OH)D) confounds efforts to develop clinical and public health vitamin D guidelines. The Vitamin D Standardization Program (VDSP), an international collaborative effort, was founded in 2010 to correct this problem. Nearly all published vitamin D research is based on unstandardized laboratory 25(OH)D measurements. While it is impossible to standardize all old data, it may be possible to identify a small subset of prior studies critical to guidelines development. Once identified it may be possible to calibrate their 25(OH)D values to the NIST and Ghent University reference measurement procedures using VDSP methods thereby permitting future guidelines to be based on standardized results. We simulated the calibration of a small set of ten clinical trials of vitamin D supplementation on achieved 25(OH)D under minimal sun exposure. These studies were selected because they played a prominent role in setting the 2010 vitamin D dietary reference intakes (DRI). Using random-effects meta-regression analysis, Vitamin D External Quality Assessment (DEQAS) data on assay bias was used to simulate the potential bias due to the lack of assay standardization by calibrating the achieved 25(OH)D levels from those 10 studies to: (1) the largest negative, and (2) the largest positive bias from the DEQAS all laboratory trimmed mean (ALTM) for the appropriate assay and year of analysis. For a usual vitamin D intake of 600IU/day the difference in mean achieved 25(OH)D values for those two options was 20nmol/L. However, without re-calibration of 25(OH)D values it is impossible to know the degree to which any of the current guidelines may have been biased. This approach may help stimulate the search for and standardization of that small subset of key studies and, in the cases where standardization is impossible, to identify areas of urgently needed vitamin D research.

25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4nmol/L), 24R,25(OH)2D3 (14.4nmol/L) and 24S,25(OH)2D3 (57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3.

A method based on automated on-line solid phase extraction coupled to two-dimensional liquid chromatography with tandem mass spectrometry detection (SPE-2DLC-MS/MS) is here reported for vitamin D metabolite profiling in human serum with absolute quantitation. Two-dimensional LC was configured with two complementary analytical columns, pentafluorophenyl (PFP) and C18 phases, for determination of 25 hydroxyvitamin D3 epimers and the resting bioactive metabolites of vitamin D (D3 and D2)-25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3. Quantitative determination was supported on the use of a stable isotopic labelled internal standard for each analyte and the resulting method was validated by analysis of a standard reference material certified by the National Institute of Standards & Technology (NIST-972a) and 5 samples provided by the vitamin D External Quality Assurance Scheme (DEQAS). The limits of detection were between 9 and 90pg/mL for the eight analytes, and precision, expressed as relative standard deviation, was lower than 11.6%. Two-dimensional LC has shown to be the key to discriminate between 25 hydroxyvitamin D3 epimers in a quantitative analysis also involving dihydroxyvitamin D metabolites.

Automated immunoassays for 25-hydroxyvitamin D (25-OHD): do plasticisers interfere?

The international quality assessment scheme for vitamin D metabolites (DEQAS) was established in 1989. The scheme involves the quarterly distribution of 5 serum samples prepared from blood collected in plain plastic bags. Following transfer of the donors to a clinic using different bags, sera were found to contain a contaminant that interfered in both the local LC-MS/MS assay and the NIST reference measurement procedure for 25-OHD. It seemed likely that the contaminant was a substance, possibly a plasticiser, leached from the plastic bag. It was subsequently suggested that the unidentified contaminant might also cause interference in certain automated non-extraction assays for 25-OHD. This was investigated in 3 automated immunoassays by comparing serum 25-OHD results from blood collected simultaneously into plain glass tubes and plastic bags. There was no significant difference in results, indicating that the leached substance had no effect on any of the 3 immunoassays examined.

Quantitative analytical method to evaluate the metabolism of vitamin D.

A method for quantitative analysis of vitamin D (both D2 and D3) and its main metabolites - monohydroxylated vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) and dihydroxylated metabolites (1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3) in human serum is here reported. The method is based on direct analysis of serum by an automated platform involving on-line coupling of a solid-phase extraction workstation to a liquid chromatograph-tandem mass spectrometer. Detection of the seven analytes was carried out by the selected reaction monitoring (SRM) mode, and quantitative analysis was supported on the use of stable isotopic labeled internal standards (SIL-ISs). The detection limits were between 0.3-75pg/mL for the target compounds, while precision (expressed as relative standard deviation) was below 13.0% for between-day variability. The method was externally validated according to the vitamin D External Quality Assurance Scheme (DEQAS) through the analysis of ten serum samples provided by this organism. The analytical features of the method support its applicability in nutritional and clinical studies targeted at elucidating the role of vitamin D metabolism.

Analytical measurement of serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC-MS/MS in infant and pediatric specimens.

OBJECTIVES: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D3 (25-OH-D3), 25-OH-vitamin D2 (25-OH-D2), and their C3-epimers. METHODS: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D3 and C3-epi-25-OH-D3, 407.2→371.3 for d6-25-OH-D3, 413.2→331.2 for 25-OH-D2 and C3-epi-25-OH-D2 and 419.2→337.1 for, d6-25-OH-D2. As a proof-of-principle, 25-OH-D3 and C3-epi-25-OH-D3 were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer. RESULTS: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D2/3 and <15% for C3-epi-25-OH-D3. C3-epi-25-OH-D3 was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D3 concentrations increased linearly, while C3-epi-25-OH-D3 concentrations remained constant. At 12 months of age, C3-epi-25-OH-D3 concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D3 supplements did not contain appreciable amounts of C3-epi-D3. CONCLUSIONS: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D3 metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.