Rapid liquid chromatography-tandem mass spectrometry method for determination of 24,25(OH)2D and 25OHD with efficient separation of 3-epi analogs.

This study establishes and validates a rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization steps to simultaneously measure of 24,25(OH)2D2, 24,25(OH)2D3, 25OHD2, and 25OHD3, while efficiently separating the 3-epi analogs. Samples were prepared by precipitation and liquid-liquid extraction. The linearity, precision, accuracy, recovery and matrix effect of the method were thoroughly evaluated according to the Clinical & Laboratory Standards Institute guidelines. Additionally, the four vitamin D metabolites in the serum of 38 apparently healthy Chinese volunteers were evaluated. The total analysis time was 8.0 min, with efficient separation of 3-epi 24,25(OH)2D3 and 3-epi 25OHD3, without interference from isomers such as 23,25(OH)2D3 or 1,25(OH)2D2, 1,25(OH)2D3. Good reproducibility was obtained for all four metabolites with within-run coefficient variations (CVs) of 4.07%-6.55%, 4.26%-7.84%, 2.46%-7.21%, and 4.90%-6.87% for 25OHD3, 25OHD2, 24,25(OH)2D3, and 24,25(OH)2D2, respectively, and the total CVs were 4.29%-6.64%, 6.14%-7.84%, 4.33%-7.21%, 5.82%-9.90%, respectively. The limit of quantification was 0.625 ng/mL for 25OHD3 and 25OHD2, and 0.5 ng/mL for 24,25(OH)2D3 and 24,25(OH)2D2. The relative bias of the LC-MS/MS method compared to the certified results of SRM 972a for 25OHD3, 25OHD2 and 24,25(OH)2D3 was -2.21% to 1.01%, 3.38% to 6.73%, and -7.72% to -3.9%, respectively. The mean±SD values for 25OHD, 24,25(OH)2D and 25OHD/24,25(OH)2D in the volunteers were 13.5±4.4 ng/mL(range:7.6-27.5 ng/mL), 0.84±0.42 ng/mL (range:0.26-2.1 ng/mL), and 18±7(range:8-37), respectively. Thus, a simple, precise LC-MS/MS method for appropriate retention and separation of vitamin D metabolites and their epi analogs was developed.

Improved accuracy of an tandem liquid chromatography-mass spectrometry method measuring 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay.

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150µL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.

On-line SPE sample treatment as a tool for method automatization and detection limits reduction: Quantification of 25-hydroxyvitamin D3/D2.

The development and approbation of new, automated UHPLC-DAD method for the quantification of 25-hydroxyvitamin D3/D2 (25OH-D3/D2) metabolites in plasma/serum for the evaluation of patient's vitamin D status are presented. The method was developed on the Ultimate 3000 UHPLC dual gradient system supplied with the on-line SPE-concentration column coupled through six port switching valve to analytical column. This configuration and materials selected enable large volume sample injection (500µL) and on-line sample preconcentration, clean up and subsequent selective metabolites transfer onto the analytical column. The new method abrogates main conventional time consuming and error source off-line steps of analysis and thus simplifies analysis. The large volume injection increases the sensitivity of instrumental analysis by about ten-fold on-line pre-concentration of metabolites. The instrument response is linear (R>0.99) in the investigated concentration range 10-100ngmL-1 which covers all the possible vitamin D status from serious deficiency (<12ngmL-1) to excess. The method detection limits (S/N=3) are LOD (25OH-D3)=0.94ngmL-1 and LOD (25OH-D2)=2.4ngmL-1. The method performance was assessed with the use of certified reference samples and perfect agreement between certified and measured values is demonstrated. The method was applied to human samples previously analyzed for total vitamin D by Competitive Protein-binding assay and findings of the two methods are compared.

A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.

Non-classical roles of fat-soluble vitamins (FSVs) in many pathologies including cancer have been identified. There is also evidence of hormonal interactions between two of these vitamins, A and D. As a result of this enhanced clinical association with disease, translational clinical research and laboratory requests for FSV measurement has significantly increased. However there are still gaps in the analytical methods available for the measurement of these vitamins. This study aimed to develop a method for simultaneous quantification of 25-hydroxyvitamin-D2 (25-OHD2), 25-hydroxyvitamin-D3 (25-OHD3) and its 3-epimer (epi-25-OHD3), retinol and α-tocopherol in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure was developed and validated across two LC-MS/MS platforms, using commercial calibrators referenced to certified reference materials, controls, and deuterated internal standards. The samples were prepared by liquid-liquid extraction prior to injection and LC separation (using a Pursuit-PFP column) on two Agilent MS/MS systems (6410 and 6490) in electrospray ionisation positive mode with multiple reaction monitoring. Identification and quantification of 25-OHD3 from its 3-epimer as well as 25-OHD2, retinol and α-tocopherol were achieved. The dynamic ranges were 4-160 nmol/L for 25-OHD2 and epi-25-OHD3, 4-200 nmol/L for 25-OHD3, 0.1-4.0µmol/L for retinol and 4-70µmol/L for α-tocopherol with correlation (r(2)) of 0.997-0.998. Based on participation in an external quality assurance program, the overall performance of the simultaneous methods were: imprecision (CV%) and inaccuracy (average bias) 3.0% and 3.2 nmol/L, respectively, for 25-OHD3; 5.0% and 0.04µmol/L, respectively, for retinol; and 4.7% and 0.2µmol/L, respectively, for α-tocopherol. In summary, two simple LC-MS/MS methods were successfully developed and validated for the simultaneous quantification of the three vitamin D metabolites (25-OHD2, 25-OHD3 and 3-epimer of 25-OHD3), vitamin A (retinol) and vitamin E (α-tocopherol) in serum.

Chromatographic separation of PTAD-derivatized 25-hydroxyvitamin D3 and its C-3 epimer from human serum and murine skin.

The detection of 25-hydroxyvitamin D at low levels in biological samples is facilitated by the use of chemical derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in concert with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This mode of analysis is notably hampered by chromatographic co-elution of 25-hydroxyvitamin D3 (25OHD3) and its C-3 epimer (C3epi). The objective of this work was to improve upon current LC-MS/MS methods used for the analysis of PTAD-derivatized 25-hydroxyvitamin D3 by resolving it from C3epi. Additionally, the applicability of this method in human serum and murine skin was investigated. C18 columns of increasing length and varying particle sizes were assessed for performance using a mixed standard of PTAD-derivatized 25OHD3 and C3epi. Serum samples were processed using solid phase extraction, and skin was powdered and extracted for lipophilic compounds. The samples were derivatized with PTAD and subsequently analyzed using isotope dilution LC-MS/MS with atmospheric pressure chemical ionization operated in positive mode. Near baseline resolution of PTAD-25OHD3 from PTAD-C3epi was achieved on a 250mm C18 column with 3µm sized particles. This separation allowed for detection and quantification of both metabolites in serum and skin samples. PTAD-C3epi represented a significant confounding analyte in all samples, and comprised up to 20% of the status measurement in skin. This method is a significant improvement on the chromatography of PTAD-derivatized vitamin D metabolites that could greatly influence the assessment of vitamin D status and C3epi biology in low abundance samples.

A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots.

BACKGROUND: Low levels of 25 hydroxyvitamin D (25OHD) during early development is associated with a range of adverse health outcomes. While a number of methods exist to measure 25OHD in sera, none have been specifically developed to examine dried blood spots (DBS). METHODS: We describe an assay where 25 hydroxyvitamin D(3) (25OHD3) and 25 hydroxyvitamin D(2) (25OHD2) are extracted from 3.2 mm DBS punches, derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis with LC/MS/MS. We assessed assay precision, relative accuracy and examined the impact of storage conditions in samples stored for up to 22 years. RESULTS: The new assay had good accuracy and precision, and was highly sensitive, being capable of detecting <1 nmol/l 25OHD3 and 2 nmol/l 25OHD2. CDER sensitivity criteria were slightly higher at 7.7 nmol/l for 25OHD3 and 10.7 nmol/l for 25OHD2. The mean 25OHD3 concentration in 118 archived DBS was 20.8+/-11.4, (4.8 to 67.8 nmol/l). 25OHD2 was detected in only two of these samples. 25OHD3 concentrations were significantly higher in DBS collected in summer compared to winter (p<0.0001). CONCLUSION: Both 25OHD3 and 25OHD2 can be reliably quantified in archived 3.2 mm dried blood spots. We can not be certain that the levels we measure in archived samples are exactly the same as when they were collected. However, the fact that the DBS levels reflect the well-known seasonal variation in this vitamin and when corrected for sera, values fall within the normal range for 25OHD3, means that DBS are a useful tissue repository for testing a range of hypotheses linking developmental hypovitaminosis D and adverse health outcomes.

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.

Retention behavior of conjugated metabolites of vitamin D and related compounds in high-performance liquid chromatography.

The retention behavior of sulfates or glucuronides of provitamin D, vitamin D, and 25-hydroxyvitamin D3, together with its fluorescent derivatives, are examined with reversed-phase high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as a mobile-phase additive is also used for this purpose. Conventional reversed-phase high-performance liquid chromatography clearly separates the positionally isomeric conjugates of 25-hydroxyvitamin D3. The addition of the host compound to the mobile phase is effective in separating the pairs of fluorescent derivatives of vitamin-D3 and -D2 conjugates or provitamin-D3 and -D2 conjugates.

The use of octadecyl-bonded microparticulate silica in the separation of free and bound fractions during saturation analysis of vitamin D metabolites.

The use of octadecyl-bonded microparticulate silica to separate free and bound fractions during the saturation analysis of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D has been investigated. A slurry of octadecyl-bonded silica in an appropriate incubation buffer was prepared and used in parallel with a conventional dextran-coated charcoal suspension in several assay procedures. Standard curves, non-specific binding and plasma values were compared. A competitive protein binding assay for 25-hydroxyvitamin D and two radioreceptor assays and one radioimmunoassay for 1,25-dihydroxyvitamin D were investigated. In most cases the octadecyl-bonded silica preparation gave the more favourable results; its action was rapid, time- and temperature-independent, and it produced low non-specific binding and higher B0 values in all the assays examined. It was in our hands easier to use than dextran-coated charcoal. The use of octadecyl-bonded silica is recommended as an efficient agent for the separation of free and bound fractions in the saturation analysis of vitamin D metabolites.

Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat.

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.

Amobarbital interactions with 25-hydroxycholecalciferol: effects on the extraction, quantification, and competitive protein binding in vitro.

Amobarbital has been found to coelute with 25-hydroxycholecalciferol (25OHD3) on a normal phase high performance liquid chromatographic system and cause subsequent interference in the UV detection and plasma transport competitive protein binding assay for this vitamin D metabolite. Concentrations of 25OHD3 were overestimated by 95% in the presence of 0.4 mg amobarbital in the competitive protein binding assay; as little as 0.1 mg amobarbital caused a 22% overestimation in the concentration of 25OHD3 in the assay. Separation of 25OHD3 from amobarbital on a reverse phase high performance liquid chromatographic system allowed for proper quantification without interference. Because of the similarity of chemical structures, other barbital-based compounds may cause similar interactions with 25OHD3 or other vitamin D metabolites as well.

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3.

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2-3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3. They metabolize 25-hydroxyvitamin D3 to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D3 and the other as its cis isomer. These two "metabolites" have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D3 on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.

Simultaneous determination of 25-hydroxy- and 1,25-dihydroxyvitamin D from a single sample by dual-cartridge extraction.

With this dual-cartridge system we extract 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] from a single serum sample by using a nonpolar octadecylsilanol silica cartridge to adsorb the vitamin D metabolites and other nonpolar substances; the polar substances wash through the cartridge. The eluted material is then applied to a second alkylamine cartridge, which adsorbs the relatively polar hydroxylated metabolites; the less-polar substances are washed from the second cartridge. Elution from the second cartridge purifies and also separates 25(OH)D and 1,25(OH)2D with analytical recoveries near 90%. The monohydroxyl metabolites are determined by "high-performance" liquid chromatography (HPLC); the dihydroxyl metabolites are further purified by HPLC and determined by radioreceptor assay according to established procedures. Mean (+/- SD) winter normal values (34 subjects of both sexes; blood drawn in mid-April) were 18 +/- 5 micrograms/L for 25(OH)D and 25 +/- 7 ng/L for 1,25(OH)2D. In nine laboratory volunteers, the mean increase in the serum 25(OH)D3 value 5 h after ingestion of 50 micrograms of 25-hydroxycholecalciferol (Calderol) was 9 (SD 4) micrograms/L.

Isolation, identification, and metabolism of (23S,25R)-25-hydroxyvitamin D3 26,23-lactol. A biosynthetic precursor of (23S,25R)-25-hydroxyvitamin D3 26,23-lactone.

23S,25R,26-Trihydroxyvitamin D3 and (23S,25R)-25-hydroxyvitamin D3 26,23-lactol were chemically synthesized, and the metabolism of the two compounds to (23S,25R)-25-hydroxyvitamin D3 26,23-lactone in chick kidney homogenates was studied. 23S,25R,26-trihydroxyvitamin D3 was efficiently metabolized to the lactone in kidney homogenates from vitamin D-supplemented chicks, but not from vitamin D-deficient chicks. In contrast, the (23S,25R)-25-hydroxyvitamin D3 26,23-lactol was converted to the lactone in kidney homogenates regardless of the vitamin D status of the animals used. A new metabolite was isolated in pure form from the incubation mixture of 23S,25R,26-trihydroxyvitamin D3 with kidney homogenates prepared from vitamin D-supplemented chicks. The metabolite was identified as (23S,25R)-25-hydroxyvitamin D3 26,23-lactol by its ultraviolet and mass spectra and by derivatization. The structure was confirmed by direct comparison with an authentic sample on high pressure liquid chromatography. The evidence suggests that the stereochemistries of the isolated lactol at the 23- and 25-positions are S and R, respectively.

Chromatographic separation of 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone using a cyano-bonded phase packing.

A high-performance liquid chromatographic system is described for the baseline resolution of 25-hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, the three principal circulating metabolites of vitamin D3 in the vitamin D-replete animal. The system is based upon a cyano-bonded phase packing and the solvent hexane--isopropanol--methanol (94:5:1). Of particular interest is the strong retention of carbonyl-containing vitamin D metabolites. The new system can be used for unequivocal analysis of vitamin D metabolites in plasma samples from clinical and animal studies and in the separation and identification of renal metabolites generated in vitro.

19-nor-10-ketovitamin D derivatives: unique metabolites of vitamin D3, vitamin D2, and 25-hydroxyvitamin D3.

Three metabolites were isolated after incubation of vitamin D3, vitamin D2, or 25-hydroxyvitamin D3 with bovine rumen microbes. They are identified as 5(E)-19-nor-10-ketovitamin D3, 5(E)-19-nor-10-ketovitamin D2, and 5(E)-19-nor-10-keto-25-hydroxyvitamin D3, respectively. The identifications were based on ultraviolet absorbance, mass spectroscopy, and chemical reactivity. All 5(E)-19-nor-10-ketovitamin D derivatives examined had an absorbance maximum at 312 nm and a characteristic fragment in their mass spectra corresponding to loss of 43 amu from their molecular ions. The vitamin D3 metabolite was identical in all essential spectral and chromatographic aspects with authentic synthetic 5(E)-19-nor-10-ketovitamin D3. These metabolites represent a unique pathway of vitamin D metabolism and the first characterized products of microbial vitamin D metabolism. The conversion of vitamin D and its metabolites to their 19-nor-10-keto forms likely represents a detoxification mechanism.

Isolation and identification of 1 alpha- and 23-hydroxylated metabolites of 25-hydroxy-24-oxovitamin D3 from in vitro incubates of chick kidney homogenates.

Five major metabolites (peaks I-V) of 25-hydroxy-24-oxovitamin D3 (25(OH)24-oxo-D3) have been isolated in pure form from in vitro incubates containing kidney homogenates of vitamin D-deficient chicks and chicks given 65 nmol of vitamin D3; peaks II, III, and V are from vitamin D-deficient chicks and peaks I, II, and IV are from vitamin D-supplemented birds. The structures of the metabolites were unequivocally identified as 23,25-dihydroxy-24-oxo-vitamin D3 (peak I), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) (peak II), 1 alpha, 25-dihydroxy-24-oxovitamin D3 (peak III), 23,24,25-trihydroxyvitamin D3 (peak IV), and 1 alpha,24,25-trihydroxyvitamin D3 (peak V) by means of ultraviolet absorption spectrometry, mass spectrometry, and specific chemical reactions. It is concluded that 25(OH)24-oxo-D3 is further hydroxylated at the 1 alpha-position in the kidney of vitamin D-deficient chicks and at the 23-position in that of vitamin D-supplemented animals. Formation of 24,25(OH)2D3 from 25(OH)24-oxo-D3 in both vitamin D-deficient and -supplemented animals provides evidence for the presence of an enzyme to reduce the 24-oxo group irrespective of the vitamin D status.