Quantitative NMR (qNMR) spectroscopy based investigation of the absolute content, stability and isomerization of 25-hydroxyvitamin D2/D3 and 24(R),25-dihydroxyvitamin D2 in solution phase.

Vitamin D is an important parameter, in serum/plasma based diagnostic analysis, for the determination of optimal regulation of calcium and phosphate homeostases in the human body, vital for the monitoring/progression of osteomalacia and rickets. Particularly, the quantification of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D in blood is an excellent indicator for the vitamin D status of a patient. For this purpose, LC-MS/MS methods, based on appropriate vitamin D reference standards, are considered to be 'gold standard' for such measurements. We have utilized quantitative NMR spectroscopy to determine the absolute content of these molecules, available as non-certified chemicals, and have determined the stability of these callibrators in borderline polar solvents at room temperature. We have observed significant isomerization of the analytes, which can play a big role in quantification of these analytes by hyphenated LC and GC analytical techniques. Appropriate explanations are given for the observation of new impurities with time in solution phase. The spin system selected for quantitation was determined using relevant 1D and 2D NMR pulse sequences. The advantage of the qNMR approach is that it is based on the quantification of atoms rather than molecular properties (e.g., quantitation by LC/UV, GC, etc.). Since the signals in an NMR spectrum are different nuclear spin-systems dispersed precisely in a magnetic environment, with the intensity being directly proportional to the amount of a particular type of nuclear spin, this technique delivers unparalleled information about the chemical structure and the absolute content.

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