24-25-dihydroxyvitamin-d-3 and 4-(2-(6-7-dimethoxy-4-methyl-3-oxo-3-4-dihydroquinoxalinyl)ethyl)-1-2-4-triazoline-3-5-dione

The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D].. We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 μL of serum with a 5-minute run time.. The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter.. We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models.. The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia.. The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Blood Chemical Analysis; Chromatography, Liquid; Dietary Supplements; Female; Humans; Hypercalcemia; Mice; Mice, Knockout; Predictive Value of Tests; Quinoxalines; Tandem Mass Spectrometry; Triazoles; Vitamin D; Vitamin D Deficiency

The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.

Topics: 24,25-Dihydroxyvitamin D 3; Calcifediol; Chromatography, High Pressure Liquid; Fluorescent Dyes; Humans; Quinoxalines; Spectrometry, Fluorescence; Triazoles

The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.

Topics: 24,25-Dihydroxyvitamin D 3; Calcifediol; Chromatography, High Pressure Liquid; Fluorescent Dyes; Fluorometry; Quinoxalines; Triazoles; Vitamin D