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. 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. It is increasingly recognized that an adequate vitamin D status, besides being important for the regulation of bone and calcium-phosphate metabolism, seems to be protective against a number of diseases including diabetes; cancer; musculoskeletal disorders; cardiovascular, infectious, and autoimmune diseases; and dementia (1.Pludowski P. Holick M.F. Pilz S. Wagner C.L. Hollis B.W. Grant W.B. Shoenfeld Y. Lerchbaum E. Llewellyn D.J. Kienreich K. et al.Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality'a review of recent evidence.Autoimmun. Rev. 2013; 12: 976-989Crossref PubMed Scopus (590) Google Scholar). 25-Hydroxy-vitamin D [25(OH)D] is widely accepted as a reliable indicator of the vitamin D status. There is an ongoing debate about the standardization and specificity of 25(OH)D immunoassays (2.Enko D. Fridrich L. Rezanka E. Stolba R. Ernst J. Wendler I. Daniel F. Hauptlorenz S. Halwachs-Baumann G. 25-Hydroxy-vitamin D status: limitations in comparison and clinical interpretation of serum-levels across different assay methods.Clin. Lab. 2014; 60: 1541-1550Crossref PubMed Scopus (51) Google Scholar, 3.Su Z. Narla S.N. Zhu Y. 25-Hydroxyvitamin D: analysis and clinical application.Clin. Chim. Acta. 2014; 433: 200-205Crossref PubMed Scopus (32) Google Scholar, 4.Cavalier E. Lukas P. Crine Y. Peeters S. Carlisi A. Le G.C. Gadisseur R. Delanaye P. Souberbielle J.C. Evaluation of automated immunoassays for 25(OH)-vitamin D determination in different critical populations before and after standardization of the assays.Clin. Chim. Acta. 2014; 431: 60-65Crossref PubMed Scopus (60) Google Scholar). Thus, sources of inaccuracy may be related to variations in the levels of vitamin D binding protein (5.Heijboer A.C. Blankenstein M.A. Kema I.P. Buijs M.M. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration.Clin. Chem. 2012; 58: 543-548Crossref PubMed Scopus (292) Google Scholar) and cross-reactivity to 24,25-dihydroxy-vitamin D (6.Wallace A.M. Gibson S. de la Hunty A. Lamberg-Allardt C. Ashwell M. Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedures, performance characteristics and limitations.Steroids. 2010; 75: 477-488Crossref PubMed Scopus (248) Google Scholar). Therefore, a number of laboratories introduced LC-MS/MS methods for the quantification of 25(OH)D (reviewed in Ref. 7.van den Ouweland J.M. Vogeser M. Bacher S. Vitamin D and metabolites measurement by tandem mass spectrometry.Rev. Endocr. Metab. Disord. 2013; 14: 159-184Crossref PubMed Scopus (83) Google Scholar). Moreover, global standardization is advanced with LC-MS/MS reference methods (8.Tai S.S. Bedner M. Phinney K.W. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry.Anal. Chem. 2010; 82: 1942-1948Crossref PubMed Scopus (227) Google Scholar, 9.Stepman H.C. Vanderroost A. Van U.K. Thienpont L.M. Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry.Clin. Chem. 2011; 57: 441-448Crossref PubMed Scopus (193) Google Scholar) and the availability of National Institute of Standards and Technology (NIST) reference material (10.Phinney K.W. Bedner M. Tai S.S. Vamathevan V.V. Sander L.C. Sharpless K.E. Wise S.A. Yen J.H. Schleicher R.L. Chaudhary-Webb M. et al.Development and certification of a standard reference material for vitamin D metabolites in human serum.Anal. Chem. 2012; 84: 956-962Crossref PubMed Scopus (116) Google Scholar). Application of LC-MS/MS allows the separation of different 25(OH)D species, that is, 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. Meanwhile, it is recognized that accurate quantification of 25(OH)D by LC-MS/MS requires LC separation of 25(OH)D epimers due to an increased analytical response of the epimers (11.van den Ouweland J.M. Beijers A.M. van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014; 967: 195-202PubMed Google Scholar, 12.Flynn N. Lam F. Dawnay A. Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays.Ann. Clin. Biochem. 2014; 51: 352-359Crossref PubMed Scopus (20) Google Scholar) and significant concentrations of epimers in not only infants but also adults (13.Bailey D. Veljkovic K. Yazdanpanah M. Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.Clin. Biochem. 2013; 46: 190-196Crossref PubMed Scopus (160) Google Scholar, 14.Cashman K.D. Kinsella M. Walton J. Flynn A. Hayes A. Lucey A.J. Seamans K.M. Kiely M. The 3 epimer of 25-hydroxycholecalciferol is present in the circulation of the majority of adults in a nationally representative sample and has endogenous origins.J. Nutr. 2014; 144: 1050-1057Crossref PubMed Scopus (39) Google Scholar). In general, quantification of small molecules involves triple-quadrupole instruments (QQQs) operated at unit mass resolution. Until now, there have been only two methods published for 25(OH)D quantification by high-resolution mass spectrometry (HR-MS). One applied LC-HR-MS (15.Bruce S.J. Rochat B. Beguin A. Pesse B. Guessous I. Boulat O. Henry H. Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry – a method comparison and validation.Rapid Commun. Mass Spectrom. 2013; 27: 200-206Crossref PubMed Scopus (62) Google Scholar) and the second performed LC-HR-MS3 of derivatized 25(OH)D3 species with an ion trap-Orbitrap mass spectrometer (16.Abdel-Khalik J. Crick P.J. Carter G.D. Makin H.L. Wang Y. Griffiths W.J. Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation.Biochem. Biophys. Res. Commun. 2014; 446: 745-750Crossref PubMed Scopus (12) Google Scholar). Here, we present a novel method for the fast and accurate quantification of 25(OH)D3, 25(OH)D2, and their C3-epimers by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) using a quadrupole-Orbitrap instrument. Ammonium acetate analytical grade, formic acid analytical grade, ethanol absolute EMSURE, and isopropanol LiChrosolv were purchased from Merck (Darmstadt, Germany). Chloroform ROTISOLV® was purchased from Carl Roth GmbH (Karlsruhe, Germany), and methanol LC-MS Chromasolv from Fluka (Buchs, Switzerland). 25(OH)D3, 25(OH)D2, D6-25(OH)D3, D6-25(OH)D2, and 3-epi-25(OH)D3 were purchased from Toronto Research Chemicals (Toronto, Canada). Butylated hydroxytoluene (BHT), 3-epi-25(OH)D2, iso-octane ACS reagent, and Zone-Free Films were from Sigma Aldrich (München, Germany). Assay validation was performed with serum controls MassCheck® 3-epi-25-OH-D3/D2 and 25-OH-D3/D2 Level I (medium) and II (high), purchased from Chromsystems (München, Germany). A low-level control was prepared by 5-fold dilution of level I with physiological human albumin solution ALBUNORM 5% (Octapharma, Langenfeld, Germany). Additionally, pooled serum was used as an in-house quality control. Calibrators were prepared by standard addition from methanolic solutions of authentic standards to pooled human serum. ALBUNORM was used as analyte free level and did not contain any detectable 25(OH)D species (see supplementary Figs. 4–7, Cal 0). The level I calibrator was prepared by 4-fold dilution of a serum pool with ALBUNORM. The in-house calibrators (blank + 5 levels; see supplementary Figs. 4–7) were calibrated by repeated quantification (n = 6) using NIST traceable serum calibrators 3PLUS1® Multilevel Serum Calibrator Set 3-epi-25-OH-D3/D2 and 25-OH-D3/D2 (four calibration levels including a low level) obtained from Chromsystems. Liquid-liquid extraction was used as described by Midttun et al. (17.Midttun Ø. Ueland P.M. Determination of vitamins A, D and E in a small volume of human plasma by a high-throughput method based on liquid chromatography/tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2011; 25: 1942-1948Crossref PubMed Scopus (58) Google Scholar). In brief, 100 µl serum/control/calibrator was placed into 96-well deep-wells (2 ml Costar Assay Block; Corning, Amsterdam, The Netherlands) and deproteinized by 200 µl of an ethanolic solution containing 50 ng/ml each D6-25(OH)D3, D6-25(OH)D2, and 1 g/l BHT. Extraction was performed with 600 µl iso-octane-chloroform (3:1, v/v). Four hundred microliters of the upper phase was recovered using a Tecan Genesis (Männedorf, Switzerland) and transferred to another 96-well deep-well plate. Solvent was removed by vacuum-centrifugation. The samples were redissolved in 50 µl methanol containing 1 g/l BHT and sealed with Zone-Free Film. 25(OH)D analysis was performed by LC-MS/HR-MS. The LC consisted of an UltiMate 3000 XRS quaternary UHPLC pump, an UltiMate 3000 RS column oven, and an UltiMate 3000 isocratic pump (Thermo Fisher Scientific Waltham, MA) connected to a PAL HTS-xt autosampler (CTC Analytics, Zwingen, Switzerland) and a hybrid quadrupole-Orbitrap mass spectrometer QExactive (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization source. Five microliters of the redissolved samples was injected and separated on a Kinetex™ 2.6 µm PFP (50 × 2.1 mm; Phenomenex, Aschaffenburg, Germany) equipped with a 0.5 µm inline filter (Vici Valco, Schenkon, Switzerland) at a column temperature of 40°C. Mobile phase A consisted of methanol-water (5:95, v/v), mobile phase B was 100% methanol, both containing 0.1% formic acid and 2 mM ammonium acetate. Gradient elution started at 100% A with a flow rate of 500 µl/min, a linear increase to 68% B in 0.1 min, followed by an increase to 73% B until 4 min. For column cleaning, the methanol percentage and flow were increased to 100% and 800 µl/min within 0.1 min, respectively. After flushing for 0.5 min, the solvent composition was changed to 100% A within 0.1 min and held until 5 min at a flow rate of 800 µl/min. To minimize contamination of the mass spectrometer, the column flow was directed only from 3.0 to 4.0 min into the mass spectrometer using a divert valve. Otherwise methanol with a flow rate of 200 µl/min was delivered into the mass spectrometer. The ion source was operated in the positive ion mode using the following settings: ion spray 3,500 V, sheath gas 53, aux gas 14, sweep gas 3, and aux gas heater temperature of 250°C. Capillary temperature was set to 269°C, and the S-lens RF level to 55. Data were collected from 3.0 to 4.0 min in the targeted MS2 mode with the following settings: resolution 35,000, AGC target: 5e5, maximum IT 100 ms with a multiplex of 2 and quadrupole isolation window of m/z 0.8. Data analysis was performed with TraceFinder 3.1 Clinical (Thermo Fisher Scientific), a software module that extracts target ions (Table 1) within ±5 ppm mass window, generates calibration lines (supplementary Figs. 4–7), and checks quality controls and ion ratios of quantifier to qualifier ions (Table 1).TABLE 1Mass transitions, internal standards (IS) used for quantitation, calibration range, and limit of quantification (LoQ) of LC-MS/HR-MSAnalyteMass Transitions Quantifier (m/z)Mass Transitions Qualifier (m/z)ISCalibration Range (nM)LoQ (nM)25(OH)D3401.34 > 383.3314401.34 > 365.3208D6-25(OH)D35.6– 4205.625(OH)D2413.34 > 395.3314413.34 > 377.3208D6-25(OH)D27.3–3207.33-Epi-25(OH)D3401.34 > 383.3314401.34 > > 395.3314413.34 > > > > > in a validation was performed on the of the US Food and Drug Administration Food and Drug Administration for US of and Google Scholar) and the European Medicines Agency for for on European Medicines 2014; Google Scholar) on method validation (see supplementary The of the current was to an accurate and fast method for the quantification of 25(OH)D species by LC-MS/HR-MS using a quadrupole-Orbitrap hybrid mass spectrometer. A column with and was to separate the and as described in den Ouweland J.M. Vogeser M. Bacher S. Vitamin D and metabolites measurement by tandem mass spectrometry.Rev. Endocr. Metab. Disord. 2013; 14: 159-184Crossref PubMed Scopus (83) Google Scholar). separation of 25(OH)D3 and 3-epi-25(OH)D3 as as and within a run time of 5 min to the that increased specificity with unit resolution by ions were as ions for analytes (Table the including the and D6-25(OH)D2, an of the signal was for 3-epi-25(OH)D3 (supplementary ion a mass for the m/z ion the ±5 ppm mass window of the from min to min (supplementary Figs. 1 and increase of the mass resolution from to separated two m/z of 25(OH)D3 and m/z of D6-25(OH)D2, from an (supplementary 1 and supplementary the mass resolution of 35,000, signal be for 3-epi-25(OH)D3 (supplementary The specificity of the method was using qualifier ions (Table 1) in different samples including from and and were not present in the samples and 3-epi-25(OH)D3 was present below to Therefore, analytes were only in samples 25(OH)D3 specificity was also in The ion ratios to those of authentic standards with a maximum of for analytes for sample with a low concentration of 25(OH)D3 and the low of not Moreover, ion ratios are for samples routine with an of as the maximum only a of the ion for concentrations to due to qualifier at below limit of of qualifier ions was due to a mass of the ±5 ppm mass effects were in samples with at low and For the was within a window of 100 not was used as for 25(OH)D3, and D6-25(OH)D2 was used for 25(OH)D2, 3-epi-25(OH)D3 and 3-epi-25(OH)D2. calibration lines were for analytes within the (Table lines were and concentrations were within of the (supplementary Figs. Determination of for LC-MS/MS involves of to its we did not in LC-MS/HR-MS for 25(OH)D Therefore, was determined by testing with of control and calibrator samples (supplementary for analytes were below 10 (Table below the showed signal due to mass and target the ±5 ppm mass window as for serum controls traceable to NIST reference material were used to imprecision and trueness including a low-level prepared by dilution with physiological human albumin For and were below 10% and trueness was between 90% and for analytes (Table and trueness of the low-level control were with to and between and respectively. The low level of was below and a trueness of A serum used as a quality control routine analysis for 25(OH)D3 and 3-epi-25(OH)D3 showed an (n = of and respectively. obtained for 10 samples of the Vitamin D by NIST reference methods were in good agreement with LC-MS/HR-MS concentrations (supplementary and and trueness and imprecision were each from in a and trueness and imprecision were each from The of 3-epi-25(OH)D3 related to 25(OH)D was in samples to routine at the 1 the with a of and a maximum was with a of in concentrations were in of the samples of and of samples a 3-epi-25(OH)D3 10% of 25(OH)D Here, we present a fast and accurate method for the quantification of 25(OH)D species using LC-MS/HR-MS. showed an increased analytical response of 3-epi-25(OH)D3 with 25(OH)D3 (11.van den Ouweland J.M. Beijers A.M. van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014; 967: 195-202PubMed Google Scholar, 12.Flynn N. Lam F. Dawnay A. Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays.Ann. Clin. Biochem. 2014; 51: 352-359Crossref PubMed Scopus (20) Google Scholar). only separation of analytes overestimation of 25(OH)D3 concentrations in samples with significant 3-epi-25(OH)D3 method has a run time of 5 min, is for routine analysis and to the published methods by van den Ouweland et al. with min den Ouweland J.M. Beijers A.M. van Daal H. separation of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 in human serum by liquid chromatography-tandem mass of 3-epi-25-hydroxyvitamin D3 in and Chem. 2011; 57: PubMed Scopus Google Scholar) and 5 min (11.van den Ouweland J.M. Beijers A.M. van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014; 967: 195-202PubMed Google Scholar). Moreover, because only a 1 min window of the LC run was introduced into the mass spectrometer, a of LC may increase the to The standard instruments for tandem mass spectrometric are triple-quadrupole instruments that unit mass resolution. In we used a hybrid quadrupole-Orbitrap that mass resolution with to at m/z on their instruments have also been applied in targeted ion (15.Bruce S.J. Rochat B. Beguin A. Pesse B. Guessous I. Boulat O. Henry H. Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry – a method comparison and validation.Rapid Commun. Mass Spectrom. 2013; 27: 200-206Crossref PubMed Scopus (62) Google Scholar). analysis with not mass and to the by et al. (15.Bruce S.J. Rochat B. Beguin A. Pesse B. Guessous I. Boulat O. Henry H. Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry – a method comparison and validation.Rapid Commun. Mass Spectrom. 2013; 27: 200-206Crossref PubMed Scopus (62) Google Scholar). Therefore, we to used with a mass resolution of that separation of quasi-isobaric ions at m/z from 25(OH)D3 and D6-25(OH)D2 (supplementary mass D6-25(OH)D2 may with 25(OH)D3 in of a den Ouweland J.M. Vogeser M. Bacher S. Vitamin D and metabolites measurement by tandem mass spectrometry.Rev. Endocr. Metab. Disord. 2013; 14: 159-184Crossref PubMed Scopus (83) Google Scholar). analysis by low mass resolution instruments requires an for other ions has to of D6-25(OH)D2 and 25(OH)D3 the of are and but as in the ion of contain the specificity of the Thus, has to be by testing but not by measurement (supplementary for analytes is below 10 and for testing of vitamin D status. Moreover, the LC-MS/HR-MS method for 25(OH)D to that from triple-quadrupole instruments in the den Ouweland J.M. Vogeser M. Bacher S. Vitamin D and metabolites measurement by tandem mass spectrometry.Rev. Endocr. Metab. Disord. 2013; 14: 159-184Crossref PubMed Scopus (83) Google Scholar, D. Veljkovic K. Yazdanpanah M. Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.Clin. Biochem. 2013; 46: 190-196Crossref PubMed Scopus (160) Google Scholar). The of epimer in of the with the (13.Bailey D. Veljkovic K. Yazdanpanah M. Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.Clin. Biochem. 2013; 46: 190-196Crossref PubMed Scopus (160) Google Scholar, 14.Cashman K.D. Kinsella M. Walton J. Flynn A. Hayes A. Lucey A.J. Seamans K.M. Kiely M. The 3 epimer of 25-hydroxycholecalciferol is present in the circulation of the majority of adults in a nationally representative sample and has endogenous origins.J. Nutr. 2014; 144: 1050-1057Crossref PubMed Scopus (39) Google Scholar). a of to of the 3-epi-25(OH)D3 in is in good agreement with in In conclusion, the method for the time the of LC-MS/HR-MS for quantification in laboratory routine The specificity of may be also for other for The and for 25-hydroxy-vitamin D 25-hydroxy-ergocalciferol 25-hydroxy-cholecalciferol hydroxytoluene high-resolution tandem mass spectrometry internal standard liquid chromatography coupled to high-resolution tandem mass spectrometry limit of quantification National Institute of Standards and Technology

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.