Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry
2015; Elsevier BV; Volume: 56; Issue: 6 Linguagem: Inglês
10.1194/jlr.d058511
ISSN1539-7262
AutoresGerhard Liebisch, Silke Matysik,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoIn 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 > 365.3208D6-25(OH)D22.1–3202.13-Epi-25(OH)D2413.34 > 395.3314413.34 > 377.3208D6-25(OH)D27.0–4607.0D6-25(OH)D3407.38 > 389.3691407.38 > 371.3585−−D6-25(OH)D2419.38 > 401.3691419.38 > 383.3585−− Open table in a new tab Method validation was performed on the basis of the US Food and Drug Administration (18.US Food and Drug Administration (FDA).Guidance for Industry: Bioanalytical Method Validation. FDA, US Department of Health and Human Services, Washington, DC. 2001; Google Scholar) and the European Medicines Agency (19.Committee for Medicinal Products for Human Use (CHMP).Guideline on Bioanalytical Method Validation. CHMP, European Medicines Agency, London. 2014; Google Scholar) guidelines on bioanalytical method validation (see supplementary Experimental Details). The aim of the current study was to develop 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 core-shell pentafluorophenyl column with polar and aromatic selectivity was selected to separate the isobaric 3β-25(OH)D and 3α-25(OH)D (= epi) isomers as described in previous studies (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). We could achieve almost baseline separation of 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2 within a run time of 5 min (Fig. 1A). Due to the fact that HR-MS offers increased specificity compared with unit resolution usually provided by quadrupoles, [M+H-H2O]+ ions were selected as sensitive fragment ions for all analytes (Table 1). During the first tests including the ISs D6-25(OH)D3 and D6-25(OH)D2, an immediate drop of the signal was recorded for 3-epi-25(OH)D3 (supplementary Fig. 1). Product ion spectra displayed a mass shift for the m/z 383 product ion beyond the ±5 ppm mass window of the [25(OH)D3+H-H2O]+ from 3.68 min to 3.70 min (supplementary Figs. 1 and 2). An increase of the mass resolution settings from 17,500 to 35,000 separated two ions, m/z 383.3314 [M+H-H2O]+ of 25(OH)D3 and m/z 383.3585 [M+H-2H2O]+ of D6-25(OH)D2, resulting from an in-source fragmentation (supplementary Table 1 and supplementary Fig. 3). With the mass resolution setting of 35,000, no signal drop could be observed for 3-epi-25(OH)D3 (supplementary Fig. 3). The specificity of the method was investigated using qualifier ions (Table 1) in six different patient samples including potential interferences from hemolysis, icterus, and lipemia. 25(OH)D2 and 3-epi-25(OH)D2 were not present in the tested patient samples above LoQ, and 3-epi-25(OH)D3 was present below or close to LoQ. Therefore, these analytes were evaluated only in spiked samples while 25(OH)D3 specificity was also checked in unspiked samples. The ion ratios quantifier/qualifier correspond to those of authentic standards with a maximum deviation of ±15% for all analytes (except for one sample with a low concentration of 25(OH)D3 and the low spike of 25(OH)D2; data not shown). Moreover, ion ratios are evaluated for all patient samples during routine diagnostic with an acceptance criterion of ±20% as the maximum relative deviation. We only observed a failure of the ion ratio criterion for concentrations close to LoQ due to qualifier intensities at or below limit of detection (signal dropout of qualifier ions was partly due to a mass shift outside of the ±5 ppm mass window). Similarly, matrix effects were evaluated in patient samples with potential interferences at low and high spike concentrations. For all patient samples, the IS-normalized matrix factor was within a window of 100 ± 15% (data not shown). D6-25(OH)D3 was used as IS 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. Linear calibration lines were found for all analytes within the analyzed range (Table 1). Calibration lines were weighted 1/x, and back calculated concentrations were found within ±15% of the nominal values (supplementary Figs. 4–7). Determination of LoQ for LC-MS/MS usually involves calculation of signal-to-noise ratios. Due to its high specificity, we did not observe baseline noise in LC-MS/HR-MS for 25(OH)D species. Therefore, LoQ was determined by functional testing with serial dilutions of control and calibrator samples (supplementary Fig. 8). LoQs for all analytes were found below 10 nM (Table 1). Typically, signals below the LoQ showed signal dropouts due to reduced mass precision and target masses outside the ±5 ppm mass window as shown for 25(OH)D2 (Fig. 1B). Commercial serum controls traceable to NIST 972a reference material were used to evaluate imprecision and trueness including a low-level prepared by dilution with physiological human albumin solution. For medium and high levels, CVs were below 10% and trueness was between 90% and 110% for all analytes (Table 2). Imprecision and trueness of the low-level control were significantly higher with up to 16% and between 85% and 115%, respectively. The low level of 3-epi-25(OH)D2 was below LoQ and displayed a trueness of ∼130%. A serum pool, used as a quality control during routine analysis for 25(OH)D3 (mean 55.5 nM) and 3-epi-25(OH)D3 (mean 3.33 nM), showed an impressive between-run precision (n = 10) of 3.9% and 3.8% CV, respectively. Results obtained for 10 samples of the Vitamin D External Quality Assessment Scheme analyzed by NIST reference methods were in good agreement with LC-MS/HR-MS concentrations (supplementary Table 2).TABLE 2Trueness and imprecisionAnalyteLevelTarget (nmol/ml)Within-Run Trueness (%)Within-Run CV (%)Between-Run Trueness (%)Between-Run CV (%)25(OH)D3Low8.9115.27.499.613.6Medium44.5107.43.9101.02.3High112105.82.8102.65.225(OH)D2Low8.02107.29.785.716.0Medium40.1101.82.897.34.4High89.7101.54.3103.42.53-epi-25(OH)D3Low6.86100.57.8106.111.1Medium34.3100.21.199.63.2High57.3104.03.298.73.03-epi-25(OH)D2Low6.16132.68.5133.711.7Medium30.898.64.295.66.1High51.599.92.195.93.2Within-run and between-run trueness and imprecision were calculated each from five replicates. Open table in a new tab Within-run and between-run trueness and imprecision were calculated each from five replicates. The fraction of 3-epi-25(OH)D3 related to total 25(OH)D was calculated in patient samples submitted to routine diagnostics at the University Hospital Regensburg (Fig. 2). As expected, children younger than 1 year displayed the highest fraction with a median of 14% and a maximum >30% (Fig. 2A). An age-dependent decrease was observed with a median of 4.3% in adult patients. 3-Epi-25(OH)D3 concentrations above LoQ were detected in ∼60% of the samples of adult patients, and >10% of these samples contained a 3-epi-25(OH)D3 fraction above 10% of total 25(OH)D (Fig. 2B). Here, we present a fast and accurate method for the quantification of 25(OH)D species using LC-MS/HR-MS. Two studies showed an increased analytical response of 3-epi-25(OH)D3 compared 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). Consequently, only chromatographic separation of these isobaric analytes avoids overestimation of 25(OH)D3 concentrations in samples with significant 3-epi-25(OH)D3 fractions. Our method has a run time of 5 min, which is acceptable for routine analysis and comparable to the fastest published methods by van den Ouweland et al. with 6.5 min (20.van den Ouweland J.M. Beijers A.M. van Daal H. 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.Clin. Chem. 2011; 57: 1618-1619Crossref PubMed Scopus (53) 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 multiplexing of LC channels may increase the throughput up to 5-fold. The standard instruments for tandem mass spectrometric quantitation are triple-quadrupole instruments that offer unit mass resolution. In this study, we used a hybrid instrument quadrupole-Orbitrap that offers high mass resolution with up to 140,000 at m/z 200. Based on their high specificity, these instruments have also been applied in full scan or targeted selected ion monitoring (SIM) (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). We tested these analysis modes together with MS/HR-MS (data not shown). However, despite high mass resolution, SIM displayed base line noise and unspecific signals comparable to the data by Bruce 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 decided to used MS/HR-MS with a mass resolution setting of 35,000 that permits separation of quasi-isobaric product ions at m/z 383 generated from 25(OH)D3 and D6-25(OH)D2 (supplementary Fig. 3). Without high mass resolution, D6-25(OH)D2 may interfere with 25(OH)D3 isomers in case of a coelution (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). Consequently, analysis by low mass resolution instruments requires either an alternative (potentially more expensive) IS for 25(OH)D2 like D3-25(OH)D2 or other fragment ions or has to avoid coelution of D6-25(OH)D2 and 25(OH)D3 isomers. HR-MS permits the use of [25(OH)D+H-H2O]+ fragment ions, which are prominent and therefore sensitive but generally considered as unspecific. However, in our experiments the extracted ion chromatograms of MS/HR-MS spectra contain no baseline noise, which impressively demonstrates the specificity of the method. Thus, LoQ has to be evaluated by functional testing but not by signal-to-noise measurement (supplementary Fig. 8). LoQ for all analytes is below 10 nM and sufficient for testing of vitamin D status. Moreover, the presented LC-MS/HR-MS method exhibits LoQs for 25(OH)D comparable to that reported from triple-quadrupole instruments in the lower nanomolar range (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, 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). The fraction of epimer detected in patients of the University Hospital Regensburg fits very well with the literature data (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). Previous studies observed a median of 3% to 6% of the 3-epi-25(OH)D3 fraction in adult patients, which is in good agreement with 4.3% found in our patients (Fig. 2B). In conclusion, the presented method shows for the first time the feasibility of LC-MS/HR-MS for small-molecule quantification in laboratory routine testing. The high specificity of this technique may be also useful for other analytes, for example, steroid hormone quantification. The authors thank Simone Düchtel and Doreen Müller for expert technical assistance. 25-hydroxy-vitamin D 25-hydroxy-ergocalciferol 25-hydroxy-cholecalciferol butylated 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
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