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

Abstract

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 13C5, 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.

Towards voltammetric point of care detection of leucovorin

“…Current methodologies used for the detection of LV in blood and human serum include high-performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS), liquid chromatography mass spectrometry (LC-MS). 24–27,37 However, these present challenges for portable, point-of-care detection and monitoring. These methods offer excellent detection limits and can be considered the “gold standard” for quantification and identification but are hindered by size, cost, complexity, sample preparation and time to results for point-of-care applications.…”

“…Various analytical methodologies have been developed to detect and determine LV in pharmaceutical formulations and biological samples. 24–27 These primarily include fluorescence and chromatographic approaches which due to a variety of reasons are not always suitable for portable or point-of-care applications. Efficient clinical management hinges upon accurate and timely LV level measurements, demanding the development and optimization of sensitive and portable diagnostic tools.…”

Sensitive and rapid quantitation of dexamethasone in human plasma using liquid chromatography coupled with triple quadrupole mass spectrometer

“…Recognition of the importance of matrix effects in LC-MS/MS methods has been highlighted. 22,23 The matrix effect was found to be 3.01% for dexamethasone, and 2.5% for dexamethasone D4. The IS normalized factor for dexamethasone was 4.25%, and 4.95% for dexamethasone D4.…”

“…Our task was to develop a robust and rugged method for a 20 mg dose for which there was no LCMSMS method available in the public domain. The main, and utmost requirement was the selection of a stable labeled internal standard, as it helps to track 18 the analyte performance in multi-step extraction, and compensate any loss, and the other factor for internal standard selection was to see whether any isotopic interference 19,20 of the parent was causing the contribution to the selected internal standard. Dexamethasone with a deuterium exchange greater than three hydrogens would have been a good choice, as upto two deuterium exchanges there is an isotopic interference of 0.4% from the parent molecule itself, 21 hence dexamethasone-d4 was selected as an internal standard for development work.…”

Development and Validation of Total Levothyroxine and Total Liothyronine in Human Serum using Chemiluminescence Micro Particle Immunoassay and Its Application to Bioequivalence Study

“…ISR is the way to assess any bioanalytical method reproducibility for the unknown samples and numerous of regulatory recommendations and various publications suggest repeat of random study samples as the incurred samples [18][19][20][21][22][23][24][25][26] . A validated method, which is reproducible demonstration and proper evaluation of incurred sample reproducibility and stability is essential to produce a reliable study data.…”