Contractor Pritesh, Gandhi Abhishek, Solanki Gajendra, Shah Priyanka A, Shrivastav Pranav S
Department of Chemistry, Kadi Sarva Vishwavidyalaya, Gandhinagar, Ahmedabad-382015, India.
Bioanalytical Department, Veeda Clinical Research, Ahmedabad-387810, India.
J Pharm Anal. 2017 Dec;7(6):417-422. doi: 10.1016/j.jpha.2017.05.006. Epub 2017 May 19.
An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D) in human plasma using LC-MS/MS. After liquid-liquid extraction with hexane, ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C (100 mm×4.6 mm, 2.5 µm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring (MRM). Entire data processing was done using Watson LIMS™ software which provided excellent data integrity and high throughput with improved operational efficiency. The major advantage of this method includes higher sensitivity (0.10 ng/mL), superior extraction efficiency (≥83%) and small sample volume (100 µL) for processing. The method was linear in the concentration range of 0.10-100 ng/mL for ergocalciferol. The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects.
建立了一种准确、灵敏且具选择性的方法，用于采用液相色谱-串联质谱法（LC-MS/MS）测定人血浆中的麦角钙化醇（维生素D）。用己烷进行液-液萃取后，麦角钙化醇通过与4-苯基-1,2,4-三唑啉-3,5-二酮（PTAD，一种基于狄尔斯-阿尔德反应的强亲双烯体）反应进行衍生化。麦角钙化醇及其氘代内标物麦角钙化醇-d6在X Select CSH C18（100 mm×4.6 mm，2.5 µm）色谱柱上，使用乙腈和含0.14%甲胺的0.1%（v/v）甲酸水溶液，在梯度洗脱模式下于6.0分钟内进行分析。采用正离子模式的串联质谱通过多反应监测（MRM）对麦角钙化醇进行定量。整个数据处理使用Watson LIMS™软件完成，该软件提供了出色的数据完整性和高通量，并提高了操作效率。该方法的主要优点包括更高的灵敏度（0.10 ng/mL）、卓越的萃取效率（≥83%）以及用于处理的小样本体积（100 µL）。该方法在麦角钙化醇浓度范围为0.10 - 100 ng/mL时呈线性。批内和批间的准确度和精密度（%CV）值分别在97.3%至109.0%以及1.01%至5.16%之间变化。该方法成功应用于支持12名健康受试者中1.25 mg麦角钙化醇胶囊的生物等效性研究。
