Development and Validation of a Highly Sensitive Liquid Chromatography/Mass Spectrometry Method for Simultaneous Quantification of Lenalidomide and Flavopiridol in Human Plasma

Abstract

Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are both active therapies for clinical use in genomic high risk chronic lymphocytic leukemia (CLL). A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile- (ACN) containing internal standard (IS), genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and IS were separated by reverse phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization (APCI) in positive-ion mode with single reaction monitoring on a triple quadrupole mass spectrometer were applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification (LLOQ) of lenalidomide and flavopiridol were 1nM and 0.3nM respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in CLL patients.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

“…The lenalidomide was extracted from these tablets using the procedures outlined in "Methods" and confirmed to have very high purity by NMR and liquid chromatography/mass spectrometry analysis performed by The Ohio State University Pharmacoanalytical Shared Resource using a highly sensitive assay. 22 The NMR spectra contained only lenalidomide resonance peaks and had no indication of contaminating materials. This same assay is used to measure lenalidomide plasma concentrations as part of ongoing clinical trials at The Ohio State University.…”

“…The purity of capsule-extracted lenalidomide was evaluated with nuclear magnetic resonance (NMR) and liquid chromatography/mass spectrometry in The Ohio State University Pharmacoanalytical Shared Resource as published. 22 The NMR spectra contained only lenalidomide resonance peaks and had no indication of contaminating materials. Powdered lenalidomide was then resuspended in phosphate-buffered saline (PBS), pH 1.4 (vehicle).…”

Development and validation of ultra-performance liquid chromatographic method with tandem mass spectrometry for determination of lenalidomide in rabbit and human plasma

“…The stock solutions and working standard of LND and IS were also stable for 15 days at refrigerator temperature (below 10°C) and for 12 h at room temperature. Therefore, LND was deemed to be stable upto 30 days at −80°C in spiked plasma and up to 15 days in aqueous solution in refrigerator, as reported previously [19]. …”

“…The first one employed liquid chromatography-mass spectrometry using single ion monitoring with a limit of quantitation of 5 ng/mL[18]. The second method included the simultaneous determination of LND and flavopiridol by liquid chromatography-mass spectrometry (LCMS/MS) using a single reaction monitoring (SRM) and a gradient elution [19]. Both methods have the disadvantages of long run time of 8 and 10 min respectively which does not meet the requirement of high throughput and speedy analysis of biosamples in clinical laboratories.…”

Therapeutic drug monitoring and LC–MS/MS

“…C18 analytical columns can be used for both these drugs. Although the structures of these drugs are similar, it has been shown that APCI negative ionisation provides the most sensitivity for thalidomide analysis [198], whereas APCI positive ionisation is preferred for lenalidomide [199].…”