Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC-MS/MS and its application to a bioequivalence study

• Published in: Journal of pharmaceutical analysis Vol. 7; no. 6; pp. 417 - 422
• Main Authors: Contractor, Pritesh, Gandhi, Abhishek, Solanki, Gajendra, Shah, Priyanka A., Shrivastav, Pranav S.
• Format: Journal Article
• Language: English
• Published: China Elsevier B.V 01.12.2017; Xi'an Jiaotong University, Journal of Pharmaceutical Analysis; Bioanalytical Department, Veeda Clinical Research, Ahmedabad-387810, India%Bioanalytical Department, Veeda Clinical Research, Ahmedabad-387810, India%Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad-380009, India; Department of Chemistry, Kadi Sarva Vishwavidyalaya, Gandhinagar, Ahmedabad-382015, India; Xi'an Jiaotong University
• Subjects: 4-phenyl-1,2,4-triazoline-3,5-dione; Acetonitrile; Chromatography; Data processing; Diels-Alder reaction; Efficiency; Ergocalciferol; Formic acid; Human plasma; Ions; LC–MS/MS; Mass spectrometry; Mass spectroscopy; Metabolism; Metabolites; Methylamine; n-Hexane; Plasma; Quality control; Scientific imaging; Short Communication; Vitamin D; Vitamin D2; Vitamin deficiency; United States > US; Ergocalciferol; Human plasma; Diels-Alder reaction; 4-phenyl-1,2,4-triazoline-3,5-dione; LC–MS/MS
• ISSN: 2095-1779; 2214-0883
• DOI: 10.1016/j.jpha.2017.05.006

An accurate, sensitive and selective method is developed for determination of ergoealeiferol （vitamin D2） in human plasma using LC-MS/MS. After liquid-liquid extraction with n-hexane, ergoealeiferol was derivatized by reacting with 4-phenyl-l,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 Cls （100 mmx4.6 mm, 2.5 lam） 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 LIMSTM 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 ~tL） for processing. The method was linear in the concentration range of 0.10-100 ng/mL for ergoealciferol. 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 ergoealciferol capsules in 12 healthy subjects.