Abstract 4482: A new quantification method for assessment of plasma concentrations of pemetrexed and its polyglutamate metabolites
Authors: Stoop Marcel , Visser Sabine , van Dijk Evert , Stricker Bruno , Luider Theo , Aerts Joachim 
Published: August 2015

Abstract
Pemetrexed (PMTX) is a multi-targeted antifolate drug applied in the treatment of diseases such as malignant pleural mesothelioma and non-small cell lung cancer. Assessment of therapeutic plasma drug and drug metabolite concentrations offers a useful tool for monitoring drug dosage, avoiding drug toxicity and obtaining therapeutic efficacy.
Stock solutions of synthesized PMTX and its (poly)-glutamyl metabolites (PMTXPG1-3), as well as their corresponding heavy labeled analogues (13C5, 15N (6 Da mass difference)) were prepared and spiked into plasma from healthy donors, and 2 M trichloroacetic acid (TCA) was added for protein precipitation. The samples were centrifuged at 5223 g and subsequently the supernatant was taken for measurement on a UHPLC-QqQ mass spectrometry set-up. After assessment of analytical factors such as linear response, stability, reproducibility and recovery, plasma of 4 non-small cell cancer patients undergoing PMTX treatment were measured to establish the methods ability for measuring clinically relevant concentrations.
The stability of patient samples stored at -80°C was monitored during a six month period, in which no statistically significant change in recovery between them (all recoveries were between 97-101%, no trend of decrease in time) was observed. The recovery of PMTX and its metabolites after the sample pretreatment procedure ranged between 45% and 79%. The relative standard deviations (RSD) in the biological replicates of the reproducibility analysis were 7.9% for PMTX, 5.9% for PMTXPG1, 7.2% for PMTXPG2 and 37.9% for PMTXPG3. For all analytes the linear response was determined over three orders of magnitude in concentration (0.2 to 500 nmol/L). Additionally, the limit of quantification (lowest quantifiable concentration (RSD &lt; 20%)) was determined for all analytes. For PMTX, PMTXPG1 and PMTXPG2 subnanomolar limits of quantification were determined. The linear response for the compounds was determined by calculation of the correlation coefficient, which was better than 0.99 for all four analytes. In all four PMTX-treated patients the plasma concentrations of PMTX, PMTXPG1 and PMTXPG2 could be detected and quantified. PMTXPG3 was not detected in any of the tested clinical samples of the four patients.
In conclusion, a LC-MS/MS-based method to measure PMTX and its polyglutamate metabolites with minimal sample pretreatment is described. To our knowledge this is the first time measurement method for pemetrexed metabolites in human plasma is reported. The method also has relatively short measurement times and good performance in being able to quantify the drug and two of its metabolites at clinically relevant concentrations in plasma of patients with lung carcinoma.
Citation Format: Marcel Stoop, Sabine Visser, Evert van Dijk, Bruno Stricker, Theo Luider, Joachim Aerts. A new quantification method for assessment of plasma concentrations of pemetrexed and its polyglutamate metabolites. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2015-4482