This study was undertaken to design and set up a rather simple, reliable, and less expensive high-performance liquid chromatography (HPLC)-based method to assay 25(OH)D as a diagnostic tool for vitamin D assessment. Serum proteins were precipitated using ethanol and, after 10 minutes incubation at room temperature, methanol:isopropanol. The extraction was performed using hexane followed by evaporation under nitrogen flow. The sediment was then reconstituted in methanol and passed through a polypropylene filter. To run the chromatographic analysis, 20 microL of the filtrate was injected to the column. Peaks of 25(OH)D2 and 25(OH)D3 were both detected using a UV detector set at 265 nm. With a flow rate of 1.2 mL/minute, peaks of D3 and D2 vitamers were detected around 9.5 and 10.7 minutes, respectively. The intra- and inter-assay variations were 8.1% and 12.6%, respectively, and the recovery percent was found to be 100 +/- 5%. To compare the procedure with conventional methods, 90 serum samples from subjects (48 females and 42 males) aged 40.5 +/- 13.9 yrs, were analyzed for 25(OH)D using HPLC, competitive protein-binding assay (CPBA), and radioimmunoassay (RIA). Generally, CPBA and RIA assays both showed over-estimation of serum 25(OH)D, compared to HPLC. Though all three methods correlated significantly with each other, with the strongest between HPLC and RIA (r = 0.87, p < 0.001), both RIA and CPBA were found unreliable in detection of some deficient samples.

Determination of serum 25-hydroxy cholecalciferol using high-performance liquid chromatography: a reliable tool for assessment of vitamin D status.

Concentrations of vitamin D3 and 25-hydroxyvitamin D3 in raw and cooked New Zealand beef and lamb

There are some reports of decreased serum levels of 25(OH)D in the subjects with impaired glucose tolerance and type 2 diabetes mellitus (T2DM). To assess vitamin D status of the Iranian diabetics, a pilot study was conducted on 90 subjects with either type 1 diabetes mellitus (T1DM) (n= 30), T2DM (n= 30), or apparently healthy subjects (n= 30) during fall and winter of 2005. Serum samples were analyzed for 25-hydroxycholecalciferol using three different methods: high-performance liquid chromatography (HPLC), competitive protein-binding assay (CPBA) and radioimmunoassay (RIA). In this study serum levels of 25(OH)D were categorized as follows: sufficient ≥ 37 nmol/L; 25 nmol/L ≤ mild deficiency P= 0.024). Moreover, both CPBA and RIA showed some over-estimation of serum 25(OH)D, compared to HPLC. Our findings suggest that, at least in the cold seasons, vitamin D status of the healthy subjects may not be higher than that of T1DM patients.

/pdf/iranian-diabetics-may-not-be-vitamin-d-deficient-more-than-44acwx24jb.pdf

Iranian diabetics may not be vitamin d deficient more than healthy subjects

Determination of 25-hydroxyvitamin d by competitive protein-binding assay and 125i-based radioimmunoassay method: a validation study

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Determination of 25-hydroxyvitamin d by competitive protein-binding assay and 125i-based radioimmunoassay method: a validation study