25-Hydroxyvitamin D Isomerizes to Pre-25-hydroxyvitamin D in Solution: Considerations for Calibration in Clinical Measurements

Reference standards for the vitamin D metabolites 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D3 were evaluated using liquid chromatography (LC) with ultraviolet (UV) absorbance and mass spectrometric (MS) detection to assess purity. The chromatograms for solutions of all three 25(OH)D compounds, obtained using a pentafluorophenyl (PFP) stationary phase, revealed peaks that increased in area over time and had MS spectra that were nearly identical to the parent compound, indicating isomers had formed in solution that were unrelated to the reference standard purity. However, when the purity evaluations were completed with a cyanopropyl stationary phase, the isomeric products coeluted with the parent compounds and were not observable. The rates of formation of the isomeric products were found to increase when heated and were confirmed to be pre-25-hydroxyvitamin D compounds using spectral information from both MS detection and nuclear magnetic resonance (NMR) spectroscopy. The rates of conversion of 25(OH)D3 to pre-25(OH)D3 was studied in solutions of ethanol and bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The solutions prepared with BSA/PBS were found to form twice as much pre-25(OH)D3 as the solutions in ethanol. The isomerization of 25(OH)D in solution has implications for calibration of 25(OH)D in clinical measurements, which are discussed.

维生素D代谢物25-羟基维生素D3、25-羟基维生素D2及3-表-25-羟基维生素D3的参考标准品，采用液相色谱（liquid chromatography，LC）-紫外（ultraviolet，UV）吸收检测与质谱（mass spectrometric，MS）检测联用技术进行纯度评估。采用五氟苯基（pentafluorophenyl，PFP）固定相获得的三种25(OH)D化合物溶液的色谱图显示，某些峰的面积随时间推移逐渐增大，且其质谱图与母化合物几乎一致，这表明溶液中形成了与参考标准品纯度无关的异构体。然而，使用氰丙基固定相进行纯度评估时，异构产物与母化合物共洗脱，因此无法被检测到。研究发现，加热会加快异构产物的生成速率；结合质谱检测与核磁共振（nuclear magnetic resonance，NMR）波谱的光谱信息，证实这些异构产物为前25-羟基维生素D（pre-25-hydroxyvitamin D）类化合物。研究人员在乙醇溶液及磷酸盐缓冲盐水（phosphate-buffered saline，PBS）中的牛血清白蛋白（bovine serum albumin，BSA）溶液中，考察了25(OH)D3向pre-25(OH)D3的转化速率。结果显示，BSA/PBS溶液中生成的pre-25(OH)D3量是乙醇溶液中的两倍。溶液中25(OH)D的异构化现象对临床检测中25(OH)D的校准具有重要意义，本文对此展开了讨论。