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Technical Briefs |
1 Institute of Clinical Chemistry, Hospital of the University of Munich, Munich, Germany;2 Roche Diagnostics GmbH, Penzberg, Germany
aaddress correspondence to this author at: Institute of Clinical Chemistry, Hospital of the University of Munich, D-81366 Munich, Germany; fax 49-89-7095-3240, e-mail Michael.Vogeser{at}med.uni-muenchen.de
Quantification of 25-hydroxyvitamin D3 (25-hydroxycholecalciferol) in serum is the best-established indicator of vitamin D status (1). Vitamin D3 (cholecalciferol) is absorbed from the diet, and, given sufficient ultraviolet irradiation, nutritionally adequate amounts of vitamin D3 are formed in the skin from its precursor, 7-dehydrocholesterol. In the liver, vitamin D3 undergoes hydroxylation to 25-hydroxyvitamin D3, which is further metabolized in the kidney to form the active metabolite 1,25-dihydroxyvitamin D3. Vitamin D2 (ergocalciferol) is derived solely from plant sources; relevant serum concentrations of 25-hydroxyvitamin D2 are observed only after ingestion of vitamin D2 drug preparations, and biological equivalence to vitamin D3 has never been demonstrated conclusively.
The prevalence of hypovitaminosis D has been recognized as substantial (2)(3), even in regions with high sun exposure (4), contributing not only to osteoporosis (5)(6) but possibly to a loss of muscle strength in aging as well (7).
Various assays are used for the quantification of circulating 25-hydroxyvitamin D3 that incorporate either vitamin D-binding globulin or anti-vitamin D antibodies for analyte recognition (8)(9)(10). Fully automated tests have become available during recent years (11). Several HPLC methods with ultraviolet detection have been described as well (12)(13), but their routine use is limited by complex sample preparation requirements.
A key problem for the quantification of circulating 25-hydroxyvitamin D3 is the strong binding of the molecule to vitamin D-binding globulin. Precipitation of serum constituents by use of organic solvents or acids can lead to variable coprecipitation of the analyte. Release of 25-hydroxyvitamin D3 from its bonds to the binding protein is technically challenging in automated assays in particular. In light of these analytical problems, a reference method for the quantification of 25-hydroxyvitamin D3 is desired to permit validation of routine immunoassays. Gas chromatography–mass spectrometry methods were developed years ago (14)(15)(16)(17), but they are extremely complex and did not gain use for quality-control programs or routine assay validation. Liquid chromatography–tandem mass spectrometry (LC-TMS) requires substantially less time-consuming sample clean-up and offers far shorter analytical run times than gas chromatography–mass spectrometry. The feasibility of vitamin D quantification by LC-TMS was demonstrated previously (18), but the procedure included derivatization, which is complex and labor-intensive. Reference systems based on LC-MS were accepted in clinical chemistry recently (19)(20), and we decided to develop a convenient and specific isotope-dilution LC-TMS method for the quantification of 25-hydroxyvitamin D3 in serum as a candidate reference method.
For use as an internal standard, stable-isotope-labeled 25-hydroxyvitamin D3 was synthesized as described previously (14)(17); the molecule contained three deuterium atoms and one 13C atom. 25-Hydroxyvitamin D3 was from Sigma Chemical Co.
A Waters Alliance 2690 HPLC module coupled with a split of 
1:10 to a Micromass Quattro LC TMS system was used.
For sample preparation, we added 30 µL of an internal standard working solution (570 nmol/L) to 200 µL of serum. After vigorous mixing, the samples were allowed to equilibrate at 37 °C for 2 h. We added 20 µL of a 1 mol/L sodium hydroxide solution to release the analyte and the internal standard from the protein bonds during a 20-min incubation, and then added 250 µL of acetonitrile for protein precipitation. During a 1-h incubation at 4 °C, the samples formed a nonliquid gel. After centrifugation for 20 min at 16 000g, a clear supernatant was obtained and transferred to HPLC vials.
For online solid-phase extraction, we used a Waters Oasis HLB® column [20 x 2.1 mm (i.d.); 25-µm bead size; Waters, Milford] in combination with a six-port high-pressure switching valve. Deproteinized sample (70 µL) was injected and then loaded on the extraction column in valve position A (Fig. 1
); the mobile phase was water–methanol (95:5 by volume) delivered at a flow rate of 3 mL/min. Potentially interfering compounds were washed into the waste. In parallel, the analytical column [LiCrospher® 100 RP-18 end-capped; 125 x 4 mm (i.d.); 5-µm bead size; Merck] was equilibrated with methanol–0.5 mmol/L ammonium acetate (90:10 by volume) delivered at a flow rate of 0.85 mL/min. After 3 min, the switching valve changed to position B. The analytes bound to the extraction column were then eluted in backflush mode onto the analytical column. After 2 min, the valve switched back to position A. During analytical chromatography into the mass spectrometer in position A, the extraction column was washed with acetonitrile–methanol (50:50 by volume) at a flow rate of 3 mL/min for 3.5 min and subsequently reequilibrated with water–methanol (95:5 by volume). The extraction and analytical columns were kept at 30 °C. The retention time of 25-hydroxyvitamin D3 and the stable isotope-labeled internal standard was 8.1 min after injection into the extraction column.
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Electrospray atmospheric pressure ionization in the positive mode was used; the source conditions were set to obtain the protonated quasimolecular ions of 25-hydroxyvitamin D3 and the labeled internal standard compound (m/z 401 and m/z 405, respectively). The instrument settings were as follows: capillary voltage, 3.0 kV; cone voltage, 20V; source temperature, 90 °C; desolvation temperature, 280 °C; nitrogen flow, 550 L/h; cone gas flow, 75 L/h; collision energy, 25 V. The following transitions were recorded: 25-hydroxyvitamin D3, m/z 401
159; labeled 25-hydroxyvitamin D3, m/z 405159.
For quantification, we performed a one-point calibration using a pure solution of 25-hydroxyvitamin D3 [25 nmol/L in methanol–water (1:1 by volume)].
To study the efficiency of the extraction procedure, we injected a pure solution of labeled 25-hydroxyvitamin D3 (325 nmol/L) by HPLC directly into the MS system without online extraction; we then subjected a serum sample containing the same quantity of labeled analyte as the internal standard to the complete extraction protocol and compared the respective peak areas of labeled 25-hydroxyvitamin D3. The mean (SD) amount of 25-hydroxyvitamin D3 measured after the extraction protocol, including deproteinization and online solid-phase extraction, was 91 (1.6)% (n = 3) of the initial concentration.
To verify the linearity of the method, to a low-concentration pool (original 25-hydroxyvitamin D3 concentration, 14.5 nmol/L) we added 25-hydroxyvitamin D3 to a concentration of 363 nmol/L. The enriched sample was serially diluted 1:1 with the low pool in four steps, and 25-hydroxyvitamin D3 was measured in duplicate in all samples. We obtained a correlation coefficient (r2) of 0.9996 between expected and observed 25-hydroxyvitamin D3 concentrations.
To study the imprecision of the method, we analyzed two serum pools five times in four independent analytical series to calculate the intraassay and total CV; for the "low pool", the total CV was 12% (mean analyte concentration, 14.5 nmol/L), and for the "normal pool", the CV was 7.8% (mean concentration, 66.0 nmol/L).
Pure solutions, each containing 25 000 nmol/L of the following compounds in methanol, were analyzed as samples to verify the specificity of the method: vitamin D3 (cholecalciferol); 1,25-(OH)2-vitamin D3; 24,25-(OH)2-vitamin D3; vitamin D2 (ergocalciferol); and 25-OH-vitamin D2. None of the compounds generated a signal in the multiple-reaction mode traces of 25-hydroxyvitamin D3 or the labeled internal standard.
In a method comparison with a RIA (Gamma B 25-Hydroxy Vitamin D RIA; IDS), MS gave a mean concentration of 56.8 nmol/L (range, 7.3–182 nmol/L) and RIA gave a concentration of 47.8 nmol/L by (n = 140 samples). Passing–Bablok regression analysis (21) produced the following equation:
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The principle of isotope dilution (ID) with stable-isotope-labeled internal standard compounds and mass spectrometric detection is generally accepted as yielding the highest attainable analytical accuracy. The physicochemical behavior of stable-isotope-labeled compounds with respect to sample preparation and signal generation is practically identical to that of the native unlabeled analyte, but labeled and unlabeled analyte can be clearly separated by mass spectrometric detection. Thus, any matrix effect of an assay is fully compensated by ID standardization. In the assay described here, samples to which labeled internal standard has been added are allowed to equilibrate over an extended period of time during which the labeled compound is bound to vitamin D-binding protein in the same manner as the target analyte. The characteristics of release from the binding protein can be assumed to be identical to that of native unlabeled 25-hydroxyvitamin D3 as well. In a method comparison study with a widely used RIA, we found a limited correlation, with lower concentrations reported by the immunoassay; this may be explained by incomplete release of the analyte from its protein bond during the precipitation step, which is identical in both methods. In contrast, in the MS method, such loss of analyte during sample preparation is fully compensated by the highly accurate principle of ID.
The ID-LC-TMS method described here is proposed for evaluation as a reference method for the quantification of 25-hydroxyvitamin D3. The semiautomated sample preparation protocol and excellent practicability of LC-TMS allow the method to be used with large validation series, but the method is also applicable in routine laboratory settings. A multicenter validation of the method is currently planned with the goal of implementing an international reference system for 25-hydroxyvitamin D3 measurement.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Knox, J. Harris, L. Calton, and A M. Wallace A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry Ann Clin Biochem, May 1, 2009; 46(3): 226 - 230. [Abstract] [Full Text] [PDF]
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G. D Carter and J. C Jones Use of a common standard improves the performance of liquid chromatography-tandem mass spectrometry methods for serum 25-hydroxyvitamin-D Ann Clin Biochem, January 1, 2009; 46(1): 79 - 81. [Abstract] [Full Text] [PDF]
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H. J. Roth, H. Schmidt-Gayk, H. Weber, and C. Niederau Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography-tandem mass spectrometry as a reference Ann Clin Biochem, March 1, 2008; 45(2): 153 - 159. [Abstract] [Full Text] [PDF]
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 G. L. Lensmeyer, D. A. Wiebe, N. Binkley, and M. K. Drezner HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays Clin. Chem., June 1, 2006; 52(6): 1120 - 1126. [Abstract] [Full Text] [PDF]
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Z. Maunsell, D. J. Wright, and S. J. Rainbow Routine Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D2 and D3 Clin. Chem., September 1, 2005; 51(9): 1683 - 1690. [Abstract] [Full Text] [PDF]
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