Separation and identification of monoglucuronides of vitamin D3 metabolites in urine by derivatization-assisted LC/ESI-MS/MS using a new Cookson-type reagent.

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed using a new Cookson-type reagent, 4-[4-(1-pipelidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD), to analyze the monoglucuronides (Gs) of vitamin D3 metabolites in human urine. The G of 23S,25-dihydroxyvitamin D3 [23,25(OH)2D3] was previously found as a major metabolite of vitamin D3 in the urine, but its conjugation position remained undetermined. Determination of the position was an important research issue to clarify the whole picture of the excretion of surplus 25-hydroxyvitamin D3 [25(OH)D3, the circulating form of vitamin D3] in humans. After the pretreated urine sample was derivatized with PIPTAD, the peak corresponding to the G of 23,25(OH)2D3 was satisfactorily separated from the urine-derived interfering substances on reversed-phase LC, which could not be achieved by using the previous analogous reagent, DAPTAD. The PIPTAD-derivatized Gs of the vitamin D3 metabolites provided characteristic product ions useful for identifying the conjugation positions during the MS/MS. Accordingly, we successfully determined the glucuronidated position of 23,25(OH)2D3 to be the C23-hydroxy group. The developed method also enabled the simultaneous detection of Gs of 25(OH)D3 and 24R,25-dihydroxyvitamin D3 as well as 23,25(OH)2D3-23-G without interference from the urine components.

Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry.

CONTEXT: Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D's diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms; however, the clinical significance of circulating phase II vitamin D metabolites remains uncertain. OBJECTIVE: In this study we analyzed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum. METHODS: An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3, and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorized by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction. RESULTS: As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18% and 53%) were a higher proportion than glucuronide conjugates (ranging between 2.7% and 11%). The proportion of conjugated 25OHD3 (48 ± 9%) was higher than 25OHD2 conjugates (29.1 ± 10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all 4 vitamin D metabolites (r = 0.85 to 0.97). CONCLUSION: Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggest a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Multiplex LC-MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D3, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio.

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC-MS/MS to measure 25-(OH)D3, 25-(OH)D2, 24,25-(OH)2D3, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC-MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC-MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.

Development and application of a LC-MS/MS assay for simultaneous analysis of 25-hydroxyvitamin-D and 3-epi-25-hydroxyvitamin-D metabolites in canine serum.

Hypovitaminosis D and hypervitaminosis D are well recognised disorders in dogs. Hypovitaminosis D can occur following consumption of a diet inadequately supplemented with vitamin D or as a sequelae of severe intestinal disease. Hypervitaminosis D may occur as a result of consuming proprietary dog foods over-supplemented with vitamin D or through ingestion of vitamin D containing medicinal products or rodenticides. Consequently, there is a clear need to establish a methodology that can accurately quantify vitamin D metabolites across a broad dynamic range in dogs. The existence of C3-epimers of vitamin D metabolites has yet to be elucidated in dogs, yet are known to interfere with the analysis of vitamin D and have unknown biological activity in other species. Here, we describe the development and validation of a sensitive, specific and robust analytical liquid chromatography tandem mass spectrometry (LC-MS/MS) assay capable of separating and accurately measuring 25-hydroxyvitamin-D2/3 (25(OH)D2/3) and 3-epi-25-hydroxyvitamin-D2/3 (3-epi-25(OH)D2/3). We describe a simplified workflow utilising supported liquid extraction (SLE) without derivatization that provides good linearity (mean r > 0.996) and accuracy across a broad dynamic range of 4-500 nmol/L for D3 metabolites and 7.8-500 nmol/L for D2 metabolites. Upon application of this assay to 117 canine serum samples, 25(OH)D3 was detectable in all samples with a median concentration of 82.1 nmol/L (inter-quartile range (IQR) 59.7-101.8 nmol/L). 3-epi-25(OH)D3 could be detected in 87.2 % of the study population, with a median concentration of 5.2 nmol/L (2.4-8.1 nmol/L). However, 3-epi-25(OH)D3 was quantified below the LLOQ in 40.2 % of these samples. 3-epi-25(OH)D3 contributed on average 6.3 % to 25(OH)D3 status (contribution ranges from 0 to 23.8%) and a positive correlation was detected between 25(OH)D3 and 3-epi-25(OH)D3 concentrations. Free 25(OH)D was also measured using an immunoassay with a median concentration of 15.2 pmol/L (12.5-23.2 pmol/L), and this metabolite was also positively correlated to both 3-epi-25(OH)D3 and 25(OH)D3 concentrations. D2 metabolites were not detected in canine serum as expected. Vitamin D metabolite concentrations were variable between individuals, and research into the causes of this variation should include factors such as breed, age, sex and neuter status to determine the impact of genetic and hormonal factors. Given the clinical importance of vitamin D in dogs, and the immense potential for utilising this species as a model for human disease, further elucidation of the vitamin D pathway in this species would provide immense clinical and research benefit.

Utilizing cooled liquid chromatography and chemical derivatization to separate and quantify C3-epimers of 25-hydroxy vitamin D and low abundant 1α,25(OH)2D3: Application in a pediatric population.

There is need for a single assay able to quantify the most biologically active metabolite, 1α,25-dihydroxy-vitamin-D3, and the recently discovered biologically distinct C3-epimers of 25OHD, in addition to traditional vitamin D metabolites. We developed a method of chromatographic separation and absolute quantification of the following ten forms of vitamin D: 3-epi-25OHD3, 25OHD3, 3-epi-25OHD2, 25OHD2, 1α,25(OH)2D3, 24R,25(OH)2D3, 23R,25(OH)2D3, 1a,25(OH)2D2, D3, and D2 by single extraction and injection. Chemical derivatization followed by liquid chromatography using a charged surface hybrid C18 column and subsequent tandem mass spectrometry was utilized to detect and quantify each metabolite. This method is remarkable as a cooled column was required to achieve chromatographic resolution of epimers. Validation of each metabolite was performed at four concentrations and revealed inter- and intra-day precision and accuracy below 15% across three consecutive days of analysis. After validation, this method was applied to analyze the blood plasma from 739 samples from 352 subjects (8mo to 20 yr), 79 pooled plasma samples, and 10 NIST SRM972a samples. Healthy control samples (n = 357) were used to investigate developmentally associated changes in vitamin D metabolite concentrations during early life. This method yields excellent linearity (R2 ≥ 0.99) across concentrations encompassing the biological range of many metabolites including 1α,25(OH)2D3. Concentrations of 25OHD2 and 24R,25(OH)2D3 were significantly (q ≤0.05) lower in infants compared to both children and adolescents. The percentage of 3-epi-25OHD3 in total 25OHD3 was significantly lower (q ≤ 0.009) in post-puberty subjects. Here we present a single assay capable of separating and quantifying ten vitamin D metabolites including C3-epimers of 25OHD, and quantifying 1α,25-dihydroxy-vitamin-D3 at and below concentrations observed in human plasma (LLOQ < 10 pM).

Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography.

A column-switching system, composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was developed for the analysis of vitamin D in oily and fatty matrices. The SFC with the similar retention behavior as normal-phase liquid chromatography (NPLC), was applied for an on-line clean-up of oily and fatty samples, then followed by separation and detection using a reverse-phase LC-MS/MS. Three SFC columns packed with materials of different functional groups (Silica, NH2, Diol) were compared and the column with diol groups, on which the retention time of vitamin D was the longest, was finally selected for purification of the samples. 100% methanol was chosen to carry vitamin D from the clean-up column to the pre-treatment column. It was also used as the mobile phase for the separation of vitamin D on a reverse phase C18 column. Vitamin D2 and D3 were baseline separated by using this system. The linearity was calculated with a value of coefficient of determination (r2) ≥ 0.998. The linear range is from 20 ng/mL to 200 ng/mL. Two kinds of liquid vitamin D3 supplements (Baby Ddrops and Vitamin AD drops) were directly analyzed using this system without any fussy preparation procedure. The limit of detection (LOD) for vitamin D3 in the two oily samples was estimated to be 10 ng/mL. The relative standard deviations (RSD) of intra- and inter-day precision, repeatability were 1.47%, 2.43% and 1.59% for Baby Ddrops and 5.76%, 8.24% and 5.86% for Vitamin AD drops. The recoveries vary between 84.3% and 102.8% with 7.1% RSD for Baby Ddrops and 90.8-109.6% with 5.83% RSD for Vitamin AD drops, respectively. These results suggest that the method based on the SFC-RPLC/MS column-switching system is simple and suitable for analysis of vitamin D in oily and fatty samples.

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

Ultrasonic-microwave method in preparation of polypyrrole-coated magnetic particles for vitamin D extraction in milk.

In this study, a nanocomposite of polypyrrole-coated magnetite nanoparticles (Fe3O4@PPy) was prepared by ultrasonic-microwave technique, and employed as magnetic solid-phase extraction (MSPE) sorbent for extraction of vitamin D from milk samples. The term of the synthesis by ultrasonic-microwave technique was dramatically shortened within 4h compared to 20h by conventional stirring-heating method. The resultant composites incorporating the π-π bonding (between PPy coating and the analytes) and magnetic separation can be applied for vitamin D analysis in complicated samples. Without saponification or protein precipitation, vitamin D2 and vitamin D3 could be captured directly from milk samples by Fe3O4@PPy, and separated by magnetic field with only 0.5mL desorption solvent. The total preparation time was completed within 15min. A method for the determination of vitamin D in milk samples by the Fe3O4@PPy extraction coupled with high performance liquid chromatography (HPLC) was developed. The LODs of vitamin D2 and vitamin D3, based on signal-to-noise ratio (S/N) of 3, were 0.02ng/mL and 0.05ng/mL respectively. The recoveries of vitamin D2 and vitamin D3 from milk samples were in the range of 71.9-90.3%, with relative standard deviations ranging between 3.6%-9.9%. The results indicated that the Fe3O4@PPy can be favorably used for the extraction of the vitamin D in milk samples.

High-Throughput Serum 25-Hydroxy Vitamin D Testing with Automated Sample Preparation.

Serum from bar-coded tubes, and then internal standard, are pipetted to 96-well plates with an 8-channel automated liquid handler (ALH). The first precipitation reagent (methanol:ZnSO4) is added and mixed with the 8-channel ALH. A second protein precipitating agent, 1 % formic acid in acetonitrile, is added and mixed with a 96-channel ALH. After a 4-min delay for larger precipitates to settle to the bottom of the plate, the upper 36 % of the precipitate/supernatant mix is transferred with the 96-channel ALH to a Sigma Hybrid SPE(®) plate and vacuumed through for removal of phospholipids and precipitated proteins. The filtrate is collected in a second 96-well plate (collection plate) which is foil-sealed, placed in the autosampler (ALS), and injected into a multiplexed LC-MS/MS system running AB Sciex Cliquid(®) and MPX(®) software. Two Shimadzu LC stacks, with multiplex timing controlled by MPX(®) software, inject alternately to one AB Sciex API-5000 MS/MS using positive atmospheric pressure chemical ionization (APCI) and a 1.87 min water/acetonitrile LC gradient with a 2.1 × 20 mm, 2.7 µm, C18 fused core particle column (Sigma Ascentis Express). LC-MS/MS through put is ~44 samples/h/LC-MS/MS system with dual-LC channel multiplexing. Plate maps are transferred electronically from the ALH and reformatted into LC-MS/MS sample table format using the Data Innovations LLC (DI) Instrument Manager middleware application. Before collection plates are loaded into the ALS, the plate bar code is manually scanned to download the sample table from the DI middleware to the LC-MS/MS. After acquisition-LC-MS/MS data is analyzed with AB Sciex Multiquant(®) software using customized queries, and then results are transferred electronically via a DI interface to the LIS. 2500 samples/day can be extracted by two analysts using four ALHs in 4-6 h. LC-MS/MS analysis of those samples on three dual-channel LC multiplexed LC-MS/MS systems requires 19-21 h and data analysis can be done by two analysts in 4-6 h.

Metrological approaches to organic chemical purity: primary reference materials for vitamin D metabolites.

Given the critical role of pure, organic compound primary reference standards used to characterize and certify chemical Certified Reference Materials (CRMs), it is essential that associated mass purity assessments be fit-for-purpose, represented by an appropriate uncertainty interval, and metrologically sound. The mass fraction purities (% g/g) of 25-hydroxyvitamin D (25(OH)D) reference standards used to produce and certify values for clinical vitamin D metabolite CRMs were investigated by multiple orthogonal quantitative measurement techniques. Quantitative (1)H-nuclear magnetic resonance spectroscopy (qNMR) was performed to establish traceability of these materials to the International System of Units (SI) and to directly assess the principal analyte species. The 25(OH)D standards contained volatile and water impurities, as well as structurally-related impurities that are difficult to observe by chromatographic methods or to distinguish from the principal 25(OH)D species by one-dimensional NMR. These impurities have the potential to introduce significant biases to purity investigations in which a limited number of measurands are quantified. Combining complementary information from multiple analytical methods, using both direct and indirect measurement techniques, enabled mitigation of these biases. Purities of 25(OH)D reference standards and associated uncertainties were determined using frequentist and Bayesian statistical models to combine data acquired via qNMR, liquid chromatography with UV absorbance and atmospheric pressure-chemical ionization mass spectrometric detection (LC-UV, LC-ACPI-MS), thermogravimetric analysis (TGA), and Karl Fischer (KF) titration.

Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry.

In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D3], 25-hydroxy-ergocalciferol [25(OH)D2], and their C3-epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D6-25(OH)D2 with 3-epi-25(OH)D3. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.

Quantitation of 1α,25-dihydroxyvitamin D by LC-MS/MS using solid-phase extraction and fixed-charge derivitization in comparison to immunoextraction.

BACKGROUND: The objective of this study was to present analysis of 1α,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques. METHODS: Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra- and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing. RESULTS: This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, R2=0.996, -0.2% bias). CONCLUSIONS: The study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.

Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D.

BACKGROUND: Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice. METHODS: 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD2 recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD3 cross-reactivity and precision of the automated assays were evaluated. RESULTS: The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD2 recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD2 under-recovery, a trend consistent with 3-epi-25-OHD3 cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD3 cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations. CONCLUSIONS: Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.

Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids - an application for 25-hydroxyvitamin D.

BACKGROUND: Already available sample preparation technologies for liquid chromatography-tandem mass spectrometry have substantial shortcomings with respect to automation. A novel approach is based on gel-like polymeric material with defined absorption chemistry, which is immobilized in micro-plate wells. It is referred to as Tecan Immobilized Coating Extraction™ (TICE™) technology and it enables easy automation on liquid handling systems. We aimed to study the performance of Tecan AC Extraction Plate™ based on this principle by addressing 25-hydroxyvitamin D (25OHD) as an exemplary analyte. METHODS: A protocol for extraction of 25OHD from serum samples based on TICE™ technology was implemented on a robotic liquid handling system Freedom EVO® (Tecan). An isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was used for quantification. Performance was tested according to a comprehensive protocol. RESULTS: Linearity was found over a range from 4.3 to 65.8 ng/mL for 25OHD3. The coefficients of variation for the intra-day and inter-day precision were <6% and accuracy ranged between 96.9% and 99.8% for 25OHD3. Recovery was 84% and efficient control of matrix effects was verified. High sample throughput could be observed with 96 samples prepared in <60 min. Close agreement of results was found for clinical samples analyzed with a second tandem mass spectrometry method based on protein precipitation and two-dimensional ultra-performance liquid chromatography for sample preparation (r=0.988, n=73). CONCLUSIONS: The new TICE™ technology was found to be a useful process for sample preparation in clinical mass spectrometry. Full automation suited for routine analysis was achieved.

Total 25-hydroxyvitamin D determination by an entry level triple quadrupole instrument: comparison between two commercial kits.

OBJECTIVE: 25-hydroxyvitamin D2/D3 (25-OHD2/D3) determination is a reliable biomarker for vitamin D status. Liquid chromatography-tandem mass spectrometry was recently proposed as a reference method for vitamin D status evaluation. The aim of this work is to compare two commercial kits (Chromsystems and PerkinElmer) for 25-OHD2/D3 determination by our entry level LC-MS/MS. DESIGN AND METHODS: Chromsystems kit adds an online trap column to an HPLC column and provides atmospheric pressure chemical ionization, isotopically labeled internal standard, and 4 calibrator points. PerkinElmer kit uses a solvent extraction and protein precipitation method. This kit can be used with or without derivatization with, respectively, electrospray and atmospheric pressure chemical ionization. For each analyte, there are isotopically labeled internal standards and 7 deuterated calibrator points. RESULTS: Performance characteristics are acceptable for both methods. Mean bias between methods calculated on 70 samples was 1.9 ng/mL. Linear regression analysis gave an R (2) of 0.94. 25-OHD2 is detectable only with PerkinElmer kit in derivatized assay option. CONCLUSION: Both methods are suitable for routine. Chromsystems kit minimizes manual sample preparation, requiring only protein precipitation, but, with our system, 25-OHD2 is not detectable. PerkinElmer kit without derivatization does not guarantee acceptable performance with our LC-MS/MS system, as sample is not purified online. Derivatization provides sufficient sensitivity for 25-OHD2 detection.

An approach for quantitative analysis of vitamins D and B9 and their metabolites in human biofluids by on-line orthogonal sample preparation and sequential mass spectrometry detection.

An approach for quantitative analysis of two vitamins with different polarities (vitamins D and B9) and their metabolites is presented here. The approach is based on an experimental setup based on hyphenation of an automated workstation for preparation of liquid samples and an LC-MS/MS system with a triple quadrupole mass spectrometer. This configuration enabled development of an orthogonal protocol for sequential SPE retention of analytes with different polarities for subsequent elution and chromatographic separation prior to detection. The resulting method was validated by application to three human biofluids. Estimation of recovery factors in the SPE step led to values from 85.2 to 100% for vitamin D and metabolites and from 93.1 to 100% for vitamin B9 and metabolites (folic acid and folates). The influence of sample matrix variability by analysis of human serum, urine and breast milk was minimized with a complete optimization of the SPE step. The utility of the proposed configuration is shown by the sensitivity and precision of the method, expressed as limits of detection (between 0.2 and 0.30 ng mL(-1) or 4 and 60 pg on-column) and within-laboratory reproducibility (lower than 6.7%, as relative standard deviation). The present application represents an example of determination methods involving targeted analysis of compounds with different polarities using a single aliquot of the sample.

Vitamin D and 25-hydroxyvitamin D determination in meats by LC-IT-MS.

This paper reports a method for the rapid, sensitive and simultaneous analysis of vitamin D (Vit D) and 25-hydroxyvitamin D (25OH-Vit D) in meats. Samples were saponified and underwent solid phase extraction with analysis by normal phase liquid chromatography (LC) with ion trap mass spectroscopy (IT-MS), using positive polarity atmospheric pressure chemical ionisation (APCI). Limits of detection (LOD) and quantification (LOQ) for Vit D and 25OH-Vit D were 0.03 and 0.05 µg/100g respectively. Deuterium labelled Vit D and 25OH-Vit D internal standards were added as surrogates prior to saponification, correcting for extraction inefficiencies and potential MS matrix enhancement or suppression effects. Recoveries using internal/surrogate standard correction ranged from 80% to 100% for all vitamers. Measurement uncertainty ranged from 6% to 15% for all vitamers in this method. This process required only 7.5 g of sample per extraction and a batch of 28 extractions could be completed in six hours.

Highly selective isolation and separation of 25-hydroxyvitamin D and 3-epi-25-hydroxyvitamin D metabolites from serum.

BACKGROUND: Increasing health concerns related to vitamin D deficiency including Alzheimer's and immune diseases, along with various cancers, have heightened awareness of the nutrient. As the associated health concerns grow, so does the need for fast and accurate analytical testing for diagnostics and treatment. Established immunoassay methods have been challenged for accuracy caused largely by endogenous interferences. This has driven the interest in more specific LC-MS/MS methodology where specificity is gained through chromatographic and MS resolution. RESULTS: Herein, a pentafluorophenyl stationary phase is shown to provide superior selectivity for the separation of the closely related 25-hydroxyvitamin D(3) and 3-epi-25-hydroxyvitamin D(3) as compared with many methods reported in the literature. To increase robustness and reliability, a novel protein precipitation/phospholipid removal device is also utilized. The novel approach was applied to human samples with a comparison with established clinical LC-MS/MS services for measuring 25-hydroxyvitamin D in adults and for infants (<1 year old). CONCLUSION: The data showed a good correlation between the routine service for adults and infant patient samples and illustrated the need to resolve the epimers. The unique selectivity of the pentafluorophenyl phase combined with the selective protein depletion and phospholipid enable a fast, accurate and robust analysis of 25-hydroxyvitamin D and related forms, which are otherwise unattainable with commonly used sample preparation and reversed-phase HPLC approaches.

Development and optimization of simplified LC-MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE).

25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC-MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid-liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC-MS/MS technique permits the measurement of both 25-hydroxyvitamin D(2) (25-OH D(2)) and the 25-hydroxyvitamin D(3) (25-OH D(3)) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 µM column with a 2.1 mm × 20 mm C18 3.5 µM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D(3) and 25-OH D(2).The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with either pure triple stripped blank serum or diluted in 6% phosphate buffer saline at pH 7.2. Linearity was tested up to 160 nmol/L for 25-OH D(3) and 75 nmol/L for 25-OH D(2). Low limit of quantification (LLOQ) were established at 3 nmol/L for 25-OH D(2) and 4 nmol/L for 25-OH D(3). Inter-assay and intra-assay precision (CV%) was determined using 3 levels of commercial controls (Utak, CA, USA) for 25-OH D(2) and 25-OH D(3). Results obtained for intra-assay and inter-assay precision (CV%) were 1.1-3.4% and 5-8.9% respectively for the PPE/centrifugation technique and 2.0-3.1% and 4.6-6.6% for the PPE/filtration technique. Accuracy was estimated with the same commercial controls: % bias was -11.2 to 4.9% with PPE/centrifugation and -3.2 to 6.1% with PPE/filtration. 25-OH D(2) and 25-OH D(3) concentrations in human serum with LLE were compared to the new extraction methods using either PPE/centrifugation or PPE/filtration. Correlations comparing the two methods revealed a slope approximately 1.0±0.3 with R≥0.98 with a bias<1 nmol/L. In summary, the new LC-MS/MS method described in this report using an on-line SPE technique with a simple off-line pre-treatment is faster, cost-effective, more reliable and more robust than current and widely used LLE/centrifugation methods coupled with LC-MS/MS.