Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry.

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC-MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography-tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D4 and 5- methyl tetrahydrofolic acid 13 C5 , were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651-883 and 518-635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3-10.5 and 9.2-17.6 h, respectively.

Overcoming the Charged Ion Competition in Triple Quad Chemical Ionization Ion Source for Estimation of Celecoxib in Biological Matrix and its Application to Bioequivalence Study.

The purpose of the study was to develop and validate a sensitive, selective and reproducible method for the estimation of Celecoxib in human plasma. During the development, ion competition phenomenon observed in electrospray ionization ion source of liquid chromatography mass spectrometer system and in order to overcome it, different approaches applied. The Celecoxib and Celecoxib D7 in plasma were extracted by liquid-liquid extraction and separated in less than 4.0 min on a reverse phase C18 column, using isocratic elution with a binary mobile phase in the ratio of 70:30 v/v (Acetonitrile: 0.1% Ammonia Solution). Mass spectrometer was used as detector to quantitate the analytes in positive ion mode, using Atmospheric Pressure Chemical Ionization mode. Since the method was developed to perform the pharmacokinetic end point study for United States and Europe market, the method was fully validated, complying Food and Drug Administration, European Medicines Agency guideline and recommendations of American Association of Pharmaceutical Scientists white papers.