D3 In Human Serum Research Articles

LC-MS/MS analysis of vitamin D3 metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization

LC-MS/MS has recently emerged as the best-practice for simultaneous analysis of vitamin D metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3 in human serum. These three metabolites were extracted from 50 μL of serum by acetonitrile protein precipitation followed by salting-out of acetonitrile. DAPTAD (4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione) was used to derivatize the extracted metabolites and their deuterated isotope internal standards. Chromatographic separation was achieved on a UPLC C18 column (Waters® ACQUITY 100 × 2.1 mm, 1.7 µm) utilizing 0.1% formic acid and acetonitrile as mobile phases. Limits of quantification were 1 ng/mL for 25(OH)D3 and 0.1 ng/mL for 24,25(OH)D3 and 3-epi-25(OH)D3. In-house and external Vitamin D External Quality Assessment Scheme (DEQAS) quality control sample analysis revealed satisfactory method accuracy. Within-analytical batch and between analytical batches precision were <15%. Extraction recovery for the three analytes were all ˃ 85% and all showed adequate autosampler, bench-top and freeze–thaw stability. Inter-methodological comparison of 25(OH)D3 results in patient serum samples revealed systematic and proportional differences between our method and DiaSorin® Liaison immunoassay, however a good agreement with an independent LC-MS/MS method was found.

Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography: An economical and validated method with bovine serum albumin

A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

Rapid Quantification of 25-Hydroxyvitamin D3 in Human Serum by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

LC-MS/MS is widely utilized today for quantification of vitamin D in biological fluids. Mass spectrometric assays for vitamin D require very careful method optimization for precise and interference-free, accurate analyses however. Here, we explore chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) as a rapid alternative for quantitative measurement of 25-hydroxyvitamin D3 in human serum, and compare it to results from LC-MS/MS. The method implemented an automated imaging step of each MALDI spot, to locate areas of high intensity, avoid sweet spot phenomena, and thus improve precision. There was no statistically significant difference in vitamin D quantification between the MALDI-MS/MS and LC-MS/MS: mean ± standard deviation for MALDI-MS-29.4 ± 10.3ng/mL-versus LC-MS/MS-30.3 ± 11.2ng/mL (P = 0.128)-for the sum of the 25-hydroxyvitamin D epimers. The MALDI-based assay avoided time-consuming chromatographic separation steps and was thus much faster than the LC-MS/MS assay. It also consumed less sample, required no organic solvents, and was readily automated. In this proof-of-concept study, MALDI-MS readily demonstrated its potential for mass spectrometric quantification of vitamin D compounds in biological fluids. Graphical Abstract ᅟ.

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum.

We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease. Graphical Abstract ᅟ.

Candidate Reference Measurement Procedure for the Determination of (24R),25-Dihydroxyvitamin D3 in Human Serum Using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry.

The two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D species, and then further hydroxylated in the kidney to various dihydroxyvitamin D species. (24R),25-Dihydroxyvitamin D3 ((24R),25(OH)2D3) is a major catabolite of 25-hydroxyvitamin D metabolism and is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. The National Institute of Standards and Technology has recently developed a reference measurement procedure for the determination of (24R),25(OH)2D3 in human serum using isotope-dilution LC-MS/MS. The (24R),25(OH)2D3 and added deuterated labeled internal standard (24R),25(OH)2D3-d6 were extracted from serum matrix using liquid-liquid extraction prior to LC-MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmospheric pressure chemical ionization in the positive ion mode and multiple reaction monitoring were used for LC-MS/MS. The accuracy of the measurement of (24R),25(OH)2D3 was evaluated by recovery studies of measuring (24R),25(OH)2D3 in gravimetrically prepared spiked samples of human serum with known (24R),25(OH)2D3 levels. The recoveries of the added (24R),25(OH)2D3 averaged 99.0% (0.8% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of ∼1%. The limit of quantitation at a signal-to-noise ratio of ∼10 was 0.2 ng/g. Potential isomeric interferences from other endogenous species and from impurity components of the reference standard were investigated. LC baseline resolution of (24R),25(OH)2D3 from these isomers was achieved within 35 min. This method was used for value assignment of (24R),25(OH)2D3 in Standard Reference Materials of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.

Simultaneous quantification of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in human serum by LC-MS/MS

Introduction. Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D. Method. An LC-MS/MS method was developed and validated to quantify vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in the eluate were derivatized by 4-phenyl-1,2,4-triazoline-3,5-dione to improve sensitivity in the following LC-MS/MS analysis. Results. Using only 100 μL serum the limit of quantification was < 0.2 ng/mL for vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D3, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D3 and up to 200 ng/mL (499 nmol/L) for 25-hydroxyvitamin D3. Precision was < 6.5% for vitamin D3 and 25-hydroxyvitamin D3 and < 10.2% for 24,25-dihydroxyvitamin D3. Conclusion. We demonstrate that a method including not only serum 25-hydroxyvitamin D3 but also vitamin D3 and 24,25-dihydroxyvitamin D3 could easily be implemented in most modern biochemical laboratories. The method could be used to study the metabolism of endogenous synthesized vitamin D3 as well as vitamin D3 in intervention studies.

The Determination of 25-OH Vitamin ( D2 / D3 ) in Human Serum by Liquid Chromatography Tandem Mass Spectrometry with Comparison to IDS Enzyme Immunoassay

The proper assessment of the status of vitamin D requires the accurate measurement of both 25-OH vitamin D2 and 25-OH vitamin D3, which collectively constitute 25-OH vitamin D, the best indicator of vitamin D status. Currently, numerous assay methods are available for 25-OH vitamin D measurement but their comparability is uncertain. We employed isotope dilution liquid chromatography coupled with tandem mass spectrometry (ID-LC-MS/MS) to quantify 25-OH vitamin D2 and 25-OH vitamin D3 in human serum. Hexadeuterium labeled 25-OH vitamin D3 internal standard was added to calibrators, controls prepared in 6% bovine serum albumin in phosphate buffered saline, and patients’ sera. Zinc sulphate was added to release 25-OH vitamin D metabolites for vitamin D binding protein, followed by a precipitation step withthe addition of acetonitrile. Subsequent online phase extraction by trap column followed by chromatographic separation on a C-8 column using a water/acetonitrile gradient was employed. Detection was performed using Atmospheric Pressure Chemical Ionization (AP-CI) in a Multiple Reaction Monitoring (MRM) mode. The method was linear from 4 to 70 ng/mL. The intra and inter-day CV% were ≤ 10 for both 25-OH vitamin D2 and 25-OH vitamin D3. Recoveries ranged between 39.09 % to 64.31 % for 25-OH vitamin D2 and 30.44 % to 58.66 % for 25-OH vitamin D3, while recoveries from hexadeuterium 25-OH vitamin D3 ranged from 44.11 % to 67.5%.We compared the newly validated LC-MS/MS with a commercial Enzyme Immunoassay from Immunodiagnostic Systems (IDS EIA) in terms of inter-method agreement, correlation, and impact of assay variation on the diagnostic categorization of vitamin D status through the measurement of 182 subjects’ sera. The mean bias % of the IDS EIA relative to the LC-MS/MS was -34.28 ± 10.15 (mean ± std) with 95% CI [-24.13 to 44.43]. The two methods were ingood agreement with reasonable correlation (r²=0.82, P value = 0.000). Inter-method diagnostic categorization was variableand depended on the type of assay method and the applied cut offs used. Cross calibration and standardization of vitamin D assay methods is crucial for proper clinical assessment of vitamin D status. This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D2 and 25-OH vitamin D3 in human serum.

Stability of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum

Stability of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum

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 …

Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum

BackgroundAn ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) and the C-3 epimer of 25OHD3 (epi-25OHD3) in human serum. MethodsTri- and hexa-deuterated internal standards were added to serum (100μl) to monitor recovery. Liquid–liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8–100nmol/L 25OHD2, 12–150nmol/L 25OHD3, and 4–50nmol/L epi-25OHD3] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4ml/min, run time was 14min per sample; 25OHD3 and epi-25OHD3 were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD2, m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD3, m/z 383.3>365.3 (105.1 qualifier); d3-25OHD2, m/z 398.3>380.3; and d6-25OHD3, m/z 389.3>371.3. ResultsRecovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20nmol/l. Bias averaged <5%. Detection limits were <5nmol/l. Median (nmol/l) 25OHD2, 25OHD3 and epi-25OHD3 were quantitated in 98 blood donors (<LOD, 56.0, <LOD) and 35 pregnant women (<LOD, 87.6, 3.70). ConclusionsThis method is highly accurate, precise and specific.

Metabolic profiling of major vitamin D metabolites using Diels–Alder derivatization and ultra-performance liquid chromatography–tandem mass spectrometry

Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1alpha,25-dihydroxyvitamin D(3) in human serum (15-60 pg/mL). Here, we demonstrate that liquid-liquid or solid-phase extraction of vitamin D metabolites in combination with Diels-Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)-electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1alpha,25-dihydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(2), 24R,25-dihydroxyvitamin D(3), 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6-4.8 % and 5-16 % for 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3), respectively, using solid-phase extraction.

Routine Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D2 and D3

Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. We used isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope-labeled internal standard (IS). Serum samples (100 microL) were denatured with methanol-propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode. 25-OH D2 and D3 and the IS nearly coeluted, whereas 1alpha-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y=0.97x - 2.7 nmol/L (r=0.91). The analytical system can assay 100 samples in 12.5 h. This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.

Determination of 25-hydroxyvitamin D3 in human serum by fluorescence labelling and high-performance liquid chromatography

A method is described for the determination of 25-hydroxyvitamin D3 in human blood serum. The problems of sensitivity and selectivity encountered with previous techniques were avoided by the formation of a highly fluorescent Diels-Alder adduct following solid-phase extraction of the vitamin. After excess of reagent had been eliminated, quantification was achieved by high-performance liquid chromatography. The recovery of the vitamin from serum was 76.4 +/- 1.76%. The precision of the method was determined, and the relative standard deviations were 8.38% at a concentration of 47.0 x 10(-9) mol dm-3, 6.74% at a concentration of 99.8 x 10(-9) mol dm-3 and 3.79% at a concentration of 146.8 x 10(-9) mol dm-3. The detection limit for the adduct was 2.93 x 10(-14) mol injected, for a signal-to-noise ratio of 3:1, and serum concentrations of 0.25 x 10(-9) mol dm-3 could easily be quantified. No interference from endogenous or exogenous substances was observed.

24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.

We describe a precise, specific method for measuring 24,25-dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by "high-performance" liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.

Seasonal variation of vitamin D3 and 25-hydroxy vitamin D3 in human serum.

Seasonal variation of vitamin D3 and 25-hydroxy vitamin D3 in human serum.

Assay of 1,25-dihydroxy vitamin D3 by isotope dilution--mass fragmentography.

We describe a specific assay of 1,25-dihydroxy vitamin D3 in human serum, based on isotope dilution-mass fragmentography. We added [26-2H3]-1,25-dihydroxy vitamin D3 to a fixed amount of serum. The steroids were extracted with chloroform/methanol and purified by liquid chromatography. The purified materal was converted into the trimethylsilyl ether and analyzed. Unlabeled 1,25-dihydroxy vitamin D3 was quantitated from the ratio between the tracings at m/e 452 and 455. The two ions used correspond to loss of two trimethylsilyloxo functions from the molecular ion. Essentially the same results, with some problems of interference, were obtained when we used the more intense ions at m/e 131 and 134 (corresponding to cleavage between C-24 and C-25). The detection limit was about 5 ng/L of serum; the coefficient of variation was about 6%. The accuracy of the method was assessed by recovery experiments. 1,25-Dihydroxy vitamin D3 in sera from 15 healthy subjects was found to average 55 +/- 10 ng/L(+/- SD). We believe this represents the first determination of 1,25-dihydroxy vitamin D3 in serum by use of a method not based on radioimmunoassay or receptor assay. Slightly lower values have been reported by those latter techniques.