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Endocrinology and Metabolism |
1 Department of Clinical Biochemistry, Northwick Park Hospital, North West London Hospitals NHS Trust, Harrow, United Kingdom.
aAddress correspondence to this author at: Department of Clinical Biochemistry, Northwick Park Hospital, North West London Hospitals NHS Trust, Watford Rd., Harrow HA1 3UL, United Kingdom. Fax 44-20-8869-2119; e-mail sandra.rainbow{at}nwlh.nhs.uk.
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Abstract |
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Methods: 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 µL) 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.
Results: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1
-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.
Conclusions: 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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vivo metabolism of vitD has been reviewed extensively, and its role and interactions with other hormones in calcium homeostasis are well understood (2). In addition to the calcium regulatory functions of vitD, several other tissues, including heart, stomach, pancreas, brain, skin, gonads, and activated T and B lymphocytes, have nuclear receptors for 1,25-dihydroxyvitamin D, and non–calcium-related functions for vitD have been described (3)(4).
VitD homeostasis is best assessed by the measurement of total 25-hydroxyvitamin D (25-OH D) (5) because the half-life of 25-OH D is 
3 weeks whereas the half-life of vitD is 24 h (6)(7). Dietary supplementation of food and vitamin tablets comes in the form of both vitD2 and vitD3; it is therefore essential that both analytes are measured equimolarly. Laboratory methods for 25-OH D have been critically reviewed recently (8). Mass spectrometric analysis has been used in research settings: in particular, gas chromatography–mass spectrometry (GC-MS) has been used for measuring vitD metabolites (9), and research assays using fast atom bombardment liquid chromatography–tandem MS (LC-MS/MS) after derivatization with Cookson-type reagents have been described (10). Two LC-MS/MS methods have been reported recently: a candidate reference method for the quantification of circulating 25-OH D3 in serum that uses column switching for on-line solid-phase extraction (11); and a method incorporating manual cartridge extraction before LC-MS/MS (12).
We report here the development of a routine isotope-dilution (ID) LC-MS/MS assay for the measurement of the 25-OH D2 and D3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Hydroxyvitamin D3 (2 mg/L) was a sterile pharmaceutical preparation (Leo Pharma). [2H6]25-OH D3 (26,26,26,27,27,27-hexadeutero-25-hydroxycholecalciferol) was purchased from as Vitas and used as internal standard (IS). Formic acid (AnalaR), hexane (Chromonorm), methanol (HyperSolv), propan-2-ol (HyperSolv), and water (HyperSolv) were obtained from VWR. Reversed-phase HPLC columns [BDS C8; 50 x 2.1 mm (i.d.); 3 µm particle size] were purchased from ThermoHypersil. The HPLC system was an Agilent 1100 system comprising a quaternary pump, a vacuum degasser, a temperature-controlled autosampler, and a temperature-controlled column oven. The API 3000TM tandem mass spectrometer and TurboIonSprayTM source were supplied by Applied Biosystems, and the system was controlled by AnalystTM software (Ver. 1.3; Applied Biosystems). The API 3000 was calibrated for mass accuracy by use of polybutylene glutarate calibrators (Applied Biosystems). Disposable 12 x 75 mm borosilicate tubes were purchased from VWR. Autosampler vials (2 mL) were purchased from Agilent UK. Nitrogen gas was supplied by a gas generator (Peak Scientific) and was used as the drying, nebulizing, curtain, and collision gas.
Calibration solutions of 25-OH D2 and 25-OH D3 (25 µmol/L) were individually prepared in ethanol. The absolute concentrations of the calibrators were checked by use of a Lambda 5 ultraviolet/visible spectrophotometer (Perkin-Elmer) and calculated using molar absorptivities of 19 400 and 18 300 AU · mol–1 · L–1 at 265 nm for 25-OH D2 and 25-OH D3, respectively (13). Working calibrators containing both analytes were prepared in methanol–water (50:50 by volume) over the range 4–250 nmol/L. The zero calibrator (no analyte) was methanol–water (50:50 by volume).
The API 3000 tandem mass spectrometer was operated in positive mode with a TurboIonSpray electrospray source operating at a voltage of +5kV and desolvation temperature of 350 °C. The conditions for the ion selection and collision-activated fragmentation of the molecular ions were optimized by continuous infusion (Harvard infusion pump) of pure compounds (25 µmol/L) at a flow rate of 10 µL/min. After optimization, the instrument was operated in multiple-reaction monitoring mode with the following transitions: m/z+ 413.5/395.4 for 25-OH D2; m/z+ 401.8/383.5 for 25-OH D3; and m/z+ 407.2/389.4 for [2H6]-25-OH D3.
Blood samples were collected into SST tubes (BD Vacutainer Systems) and were centrifuged at 1200g for 10 min; the serum thus obtained was stored at –20 °C before analysis.
sample preparation
Patient sera, controls, or calibrators (100 µL) were pipetted into disposable borosilicate tubes. To each of these, 75 µL of 60 nmol/L IS in methanol–propanol (80:20 by volume) was added, which acted as a protein-precipitating agent. The tubes were vortex-mixed for 10 s, and 500 µL of hexane was added to extract the 25-OH D2 and D3 metabolites and the IS. The tubes were revortexed for 10 s and centrifuged for 15 min at 1600g; finally, 400 µL of each hexane layer was transferred to a clean autosampler vial. The solvent was evaporated to dryness under a stream of nitrogen in a heating block (Grant Instruments) at 75 °C. The residue was reconstituted in 300 µL of methanol–water (70:30 by volume). Vials were sealed, vortex-mixed, and assayed by LC-MS/MS.
lc-ms/ms
Chromatographic separation was performed with a BDS C8 reversed-phase column [51 x 2.1 mm (i.d.); 3 µm particle size] equilibrated at 20 ± 0.1 °C. Two eluants were used in the mobile phase: methanol (eluant A) and 0.5 mL/L formic acid in water (eluant B). The flow rate was 300 µL/min, and 50 µL of extract was injected per assay. A methanol gradient was used to elute the analytes from the column. The composition of the mobile phase was 70% A:30% B for 3 min, which retained the 25-hydroxy metabolites on the column. The concentration of eluant A was increased to 95% (5% B) over 1 min and held constant for 1 min. The mobile phase was then returned to 70% A:30% B over 1 min, and the column was reequilibrated with 70% A:30% B for an additional 2 min. The total run time was 8 min per sample.
Comparative analysis of total 25-OH D was performed in batch mode by RIA (DiaSorin) after acetonitrile precipitation according to the manufacturer’s recommended procedures. The manufacturer reports that this method measures 25-OH D2 and 25-OH D3 equimolarly but also measures the dihydroxy metabolites.
interferences
A pharmaceutical preparation of 1-OH D3 was used to demonstrate that the 25-hydroxy metabolites of vitD could be resolved from the 1-hydroxy metabolite. Other monohydroxy metabolites of vitD are not commercially available and could not be tested. The dihydroxy metabolites do not have a molecular mass that would be detected by the transitions selected.
A computer search for compounds with the same molecular masses as the monohydroxides of vitD2 and D3 and the IS was performed with an internet search engine (14).
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Results |
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), that for 25-OH D3 was m/z+ 401.8 (Fig. 1A
), and that for hexadeuterated 25-OH D3 (IS) was m/z+ 407.2. The optimum conditions found were a declustering potential of 50V, focusing potential of 220V, and entrance potential of 10V. The nebulizer gas was set at 15 and the curtain gas at 11 on the instrument settings. The temperature of the drying gas was optimized at 350 °C, and the drying gas flow rate was 7 L/min. Molecular ions were individually fragmented in the collision cell, using a product ion scan with a ramp program to select a robust product ion. The molecular ions were easily fragmented, and the most intense and reproducible product ions were seen with the loss of H2O from each molecule with a low collision energy of 12V and collision gas flow of 8 (Table 1
). At the conditions selected, product ions for 25-OH D2 were seen at m/z+ 395.5, 377.0, 355.4, 337.4, 325.1, 271.1, and 255.4 (Fig. 1D
), and product ions for 25-OH D3 were seen at m/z+ 383.5, 365, 5, 257.4, 131.1, and 109.1 (Fig. 1B
). Finally, the conditions of the third quadrupole were optimized, and the cell exit potential was set at 12V. Once the instrument had been optimized, it was operated in multiple-reaction monitoring mode with the following transitions: 25-OH D2, m/z+ 413.5/395.5; 25-OH D3, m/z+ 401.8/383.5; and IS, m/z+ 407.2/389.4.
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chromatography
The eluant was optimized to retain the 25-hydroxy metabolites of vitD on the column while eluting ions suppressing moieties, with an isocratic solvent of 70:30 methanol–aqueous formic acid (by volume) for 3 min. Higher initial concentrations of organic phase led to broadening of the peaks. A fast gradient was used to elute the metabolites, and no attempt was made to resolve the metabolites chromatographically because the specificity of the mass selection and fragmentation gave the necessary compound specificity. The retention time of 25-OH D2 was fractionally, but not significantly, longer (5.91 min) than that for 25-OH D3 (5.88 min) and the IS (5.87 min); this allowed the use of a single IS (Fig. 2
).
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is
The IS was investigated in terms of chemical and isotopic purity. Chemical purity was assessed by running a 25 µmol/L IS solution in 500 mL/L methanol through the column. Monitoring of the transition m/z+ 407.7/389.5, which corresponded to the IS, revealed the presence of 2 impurity peaks after the main IS peak; however, these were easily separable from the IS by the liquid chromatography step. Monitoring of the transitions used to detect 25-OH D2 and 25-OH D3 revealed that there was none of either analyte in the solution. This was important because contamination of the IS with nondeuterated 25-OH D2 or D3 could lead to false calibration of the assay. To assess the isotopic purity of the IS, we monitored transitions m/z+ 407.7/389.5, 406.7/388.5, 405.7/387.5, and 404.7/386.5, which corresponded to the hexa-, penta-, tetra-, and trideuterated forms of the IS and subsequent loss of water to form the product ion. By comparing relative peak areas, we found that the material was >98% isotopically pure.
assay performance and validation
Assay calibration was achieved by means of 8-point calibration curves for 25-OH D2 and 25-OH D3 constructed from serial dilutions of the working calibrators in 500 mL/L methanol. Data are reported as ratios of the peak areas of 25-OH D2 or D3 to the peak area of the IS. These calibrators were prepared in the same way as patient samples, undergoing all stages of sample preparation. The concentrations in the calibrators ranged from 4.95 to 316 nmol/L for 25-OH D2 and 4.0 to 256 nmol/L for 25-OH D3. Linear responses were achieved over this range. A 0 calibrator (500 mL/L methanol) was included in assays for both analytes (Fig. 3
).
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We investigated the efficiency of the release of 25-OH D from binding proteins after protein precipitation by allowing 5 aliquots of the same sample to stand for 0, 2, 5, 10, and 30 min after addition of the protein precipitation reagent. Recoveries of the 25-hydroxy metabolites did not increase with increased equilibration time. Extraction of the 25-hydroxy metabolites into the hexane layer was used to reduce the ion suppression; a single extraction recovered >94%.
The limit of detection can be defined in MS as a peak with a signal-to-noise ratio >3, whereas the limit of quantification requires a signal-to-noise ratio >10 (15). The lowest calibrator, corresponding to 4.95 nmol/L 25-OH D2 and 4.0 nmol/L 25-OH D3, was analyzed and found to have signal-to-noise ratios of 12.4 and 10.7, respectively. It was therefore concluded that the limit of quantification was below these values.
To assess assay precision, both the intra- and interassay CVs were calculated for 25-OH D2 and 25-OH D3. We assessed the intraassay CV by extracting 3 patient samples independently 10 times and assaying them in a single run. Intraassay CVs were 6.2%, 3.5%, and 5.2% at 16, 35, and 76 nmol/L, respectively. We prepared an internal quality control by adding the metabolites to serum. For the internal quality control, 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.
To determine recoveries, we added high- and low-concentration solutions of 25-OH D2 and 25-OH D3 in methanol to 5 patient samples in various concentrations (endogenous total 25-OH D concentrations, 9, 16, 25, 66, and 83 nmol/L). We then added 5 µL of each to a 95-µL sample. The high-concentration solution contained 317 nmol/L 25-OH D2 and 256 nmol/L D3, and the low-concentration solution contained 158.5 nmol/L 25-OH D2 and 128 nmol/L 25-OH D3. The recoveries were 91%–110% for 25-OH D3 and 94%–108% for 25-OH D2.
We assessed method linearity by preparing 1:2 and 1:4 dilutions of patient samples (total 25-OH D concentrations, 9, 53, 62, and 83 nmol/L) in 50:50 methanol–water (by volume). These were all assayed by LC-MS/MS in the same run. Recoveries ranged from 89% to 113% (mean, 102%).
Interferences in this method were predicted to be minimal because of the nature of the assay. The analytical method uses 2 separation stages: chromatography based on polarity, followed by detection on the basis of mass-to-charge ratios. The MS/MS step is highly specific because fragments unique to the molecule of interest are detected. To identify potential interfering substances, we carried out molecular weight searches. Computer searches were performed on the molecular weights of 412.6 ± 0.5 (25-OH D2; 56 compounds listed) 400.6 ± 0.5 (25-OH D3; 59 compounds listed), and 406.8 ± 0.5 (IS; 82 compounds listed) (14). The majority of these were not compounds of pharmaceutical or metabolic interest. None of the compounds with listed molecular weights of 412.6 ± 0.5 and 406.8 ± 0.5 were considered to be of any biological significance. A few naturally occurring and pharmaceutical compounds of sterols and fatty acid derivatives were listed with a molecular weight of 400.6 (same as for 25-OH D3); the ones of metabolic or pharmaceutical interest were 1-OH D3; 7 -hydroxy-4-cholesten-3-one (a bile acid precursor), whose plasma concentration has been suggested as a marker of bile acid malabsorption (16); Campesterol (a phytosterol); colostolone [a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor (American Cyaphytnamid) that entered clinical trials in 1987 and has never been marketed]; and gefarnate (an unsaturated fatty acid, currently in phase 1 clinical trials in Japan). These were excluded from further examination. The only molecules that were considered to be potential interfering compounds with molecular weights of 400.6 ± 0.5 were 1-OH D3 (Alfacalcidol) and 7-hydroxy-4-cholesten-3-one, which was first identified as a large peak in chromatographic 25-OH D analysis and subsequently identified by GC-MS (17). A solution of 1-OH D3 was added to a calibration solution containing 250 nmol/L 25-OH D3, the mixture was then extracted into hexane and prepared as above. This was injected on the column and run on the mass spectrometer; the transition corresponding to 25-OH D3 was monitored. Two distinct peaks were observed; the first (retention time, 5.95 min) corresponded to 25-OH D3 and the second (retention time, 6.61min) to 1-OH D3 (Fig. 4
). The 2 metabolites, although not separable by MS alone, were clearly resolved by the HPLC step. A late-eluting peak (retention time, 6.36 min) was observed during routine analysis of some patient samples, particularly sera from patients with short bowel syndrome or intestinal failure; this was consistent with the retention time for 7-hydroxy-4-cholesten-3-one.
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method comparison
A method comparison was performed with samples (n = 185) submitted for routine analysis by RIA and ID-LC-MS/MS. Twenty-five patient results were excluded from the analysis because the 25-OH D result measured by RIA was below the detection limit (<13 nmol/L) of the assay. ID-LC-MS/MS results for 7 of the 25 samples were below the detection limit of the assay (<4 nmol/L for both 25-OH D2 and D3), whereas 14 samples had results between 4 and 12 nmol/L, of which the predominant metabolite was 25-OH D3. Four samples had measured concentrations of 15, 17, 19, and 19 nmol/L, and in all of these samples, 25-OH D2 accounted for >50% of the total 25-OH D. A total of 160 patient samples had total 25-OH D concentrations that were measurable by both assays. The Deming regression against the DiaSorin RIA was y = 0.97x – 2.7 nmol/L (r = 0.91). Bland–Altman analysis indicated that there were no concentration-dependent differences between the 2 methods (Fig. 5
). Of the samples analyzed, 18 had 25-OH D2 concentrations that were >60% of the total 25-OH D; the correlation against the DiaSorin RIA method for these samples was y = 0.93x + 2.9 nmol/L (r = 0.97).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Laboratory methods for the measurement of 25-OH D using immunologic techniques and chromatographic methods have been reviewed recently (8). The replacement of the traditional RIA with nonisotopically labeled assays has allowed automation of the analysis; however, recent studies have suggested that both the Nichols Advantage automated chemiluminescence protein-binding assay and, to a lesser extent, the IDS RIA underrecover 25-OH D2 compared with HPLC analysis (22)(23). Recent publications have highlighted the interlaboratory variability of 25-OH D analysis on patient samples measured by RIA and chemiluminescence assays (24) and quality assurance material(22). In view of this, some authors have suggested that there should be international standardization of assays and have suggested that until that is achieved, RIA techniques should be used for clinical analyses (24). However, the use of a routine LC-MS/MS method offers a real alternative and negates the use of radioactive tracers.
MS has until recently been the prerogative of research and reference laboratories and has rarely been applied to the routine quantification of analytes in the clinical laboratory. GC-MS has been used for research analysis of vitD metabolites in plasma, but the complexity of the analysis has precluded its use for routine analysis (9). Recent evaluations of LC-MS/MS have shown that this technique offers an alternative to the traditional immunoassay and potentially offers increased specificity and sensitivity for a variety of analytes (25)(26). LC-MS/MS methods have been described for metabolites of vitD, including a recent suggestion for a candidate reference method for circulating 25-OH D3 (11) that uses electrospray ionization and LC-MS/MS after extensive protein precipitation and column switching as a clean-up procedure. However, the measurement of 25-OH D3 alone is not sufficient for the clinical assessment of vitD status. Another recent report used atmospheric pressure chemical ionization and LC-MS/MS after protein precipitation and purification on Bond Elute C18 silica cartridges (12). Both of these methods use the transitions of molecular ions to small fragment ions: 25-OH D3, m/z+ 401/159 and tetradeuterated 25-OH D3 (IS), m/z+ 401/59 (11); 25-OH D3, m/z+ 401.1/257.0; 25-OH D2, m/z+ 413.4/355.4; and [2H6]25-OH D3, m/z+ 407.4/263.4 (12). We have chosen the less specific fragmentation ions (loss of water) for 25-OH D2 (m/z+ 413.5/395.4), 25-OH D3 (m/z+ 401.8/383.5), and [2H6]25-OH D3 (m/z+ 407.2/389.4), using low collision energy cell conditions, which in our hands always gave the most intense and reproducible signal (Table 1
).
We have developed an ID-LC-MS/MS assay for the routine measurement of the 25-hydroxy metabolites of vitD2 and vitD3. It has advantages over other previously reported LC-MS/MS methods in that it uses a small sample volume (100 µL) and the sample preparation is relatively simple and could be automated. The automated LC-MS/MS system allows up to 180 tests to be performed in a 24-h period. The method has been fully validated and is now in routine diagnostic use. We measured 25-OH D2 and D3 in 185 samples by ID-LC-MS/MS compared with a commercial RIA (25 samples had undetectable concentrations by RIA). The correlation (r) of total 25-OH D was 0.91 (y = 0.97x – 2.7 nmol/L; n = 160) for all samples and 0.97 (y = 0.93x + 2.9 nmol/L; n = 16) for the samples in which the 25-OH D2 concentration comprised >60% of the total 25-OH D, which supports the manufacturer’s claim that the RIA method does measure the 25-OH D2 metabolite equimolarly.
The system is able to independently quantify 25-OH D2 and D3 in serum and is free of interference from the dihydroxy metabolites of vitD because of differences in mass. Separation of the 25-hydroxy metabolites from the 1-hydroxy metabolites was achieved chromatographically, but other monohydroxy metabolites were not tested. The reported concentrations of these metabolites are extremely low (<6 nmol/L) in patients treated with small daily doses of vitD2, however, but can increase to 70 nmol/L in individuals treated with large daily doses (50 000 IU/day) of vitD2 (27).
The method has been in routine use in this laboratory for 9 months, and >6000 samples have been routinely analyzed by biomedical scientists. We therefore conclude that the assay is suitable for use in routine laboratory medicine departments.
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Footnotes |
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