Journal of Chromatography B, 877 (2009) 3850–3856 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum Panagiotis Koufopantelis a , Sophia Georgakakou a , Michael Kazanis a , Costas Giaginis a , Alexandra Margeli b , Sophia Papargiri b , Irene Panderi a,∗ a University of Athens, School of Pharmacy, Division of Pharmaceutical Chemistry, Panepistimiopolis, Zografou 157 71, Athens, Greece b Children’s Regional General Hospital “Agia Sofia”, Thivon & Papadiamantopoulou, 11527 Goudi, Athens, Greece article info Article history: Received 22 May 2009 Accepted 24 September 2009 Available online 2 October 2009 Keywords: Liquid chromatography/mass spectrometry Methotrexate Folinic acid Folic acid Ondansetron Human serum abstract A rapid liquid chromatography/positive ion electrospray mass spectrometric assay (LC/ESI-MS) was developed for the quantitation of methotrexate, folinic acid, folic acid and ondansetron in human serum. The assay was based on 100 L serum samples, following acetonitrile precipitation of proteins and filtra- tion that enabled direct injection into the LC/MS system. All analytes and the internal standard, alfuzosin, were separated by using a Zorbax Eclipse XDB–C 8 analytical column (2.1 mm × 150.0 mm i.d., particle size 3.5 m) with isocratic elution. The mobile phase was composed of a mixture of water/acetonitrile contain- ing 0.1%, v/v formic acid (75:25, v/v), pumped at a flow rate of 0.15 mL min -1 . Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ion- ization interface. The assay was found to be linear in the concentration range of 0.01–25.00 g mL -1 for methotrexate and 0.01–5.00 g mL -1 for folic acid, folinic acid and ondansetron. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 7.0 min for each sample made it possible to analyze a large number of human serum samples per day. The method can be used to quantify methotrexate, folinic acid, folic acid and ondansetron in human serum covering a variety of clinical studies and it was applied to the analysis of human serum samples obtained from children with acute lymphoblastic leukemia. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Methotrexate, (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl- methylamino]benzoyl] amino] pentanedioic acid, is a folate analog that inhibits folate-dependent synthetic reactions, leading to inhibition of DNA synthesis [1]. It is used to treat childhood acute lymphoblastic leukemia (ALL) and a number of other malignant and non-malignant diseases [2]. Major drawbacks of methotrexate therapy are the large inter-patient variability in clinical response and the unpredictable appearance of a large spectrum of side effects [3]. Folate supplementation, in the form of folic acid or folinic acid, alleviates gastrointestinal and liver toxicity, allowing patients with rheumatoid arthritis to continue a low-dose oral methotrexate therapy [4] though a recent study advised caution and suggested that folate supplementation may decrease the efficacy of methotrexate [5]. For the treatment of several types of leukemia a high dose of methotrexate is administered intra- ∗ Corresponding author. Tel.: +30 210 7274820; fax: +30 210 7274747. E-mail address: ipanderi@pharm.uoa.gr (I. Panderi). venously, followed by rescue therapy with folinic acid (leucovorin). Serum levels of methotrexate must be monitored frequently both during and after the administration of folinic acid and the doses of both compounds must be adjusted individually as methotrexate dosing may be suboptimal in many patients, while an overdose of folinic acid put at risk the antileukemic effect of methotrexate and increases the danger for relapse [6,7]. A variety of methods exist that focuses on the determina- tion of methotrexate in biofluids [8]. Fluorescence polarization immunoassay [9], radioimmunoassay [10], capillary zone elec- trophoresis [11] have been used to quantify methotrexate. There have been numerous HPLC methods that utilize laborious and time-consuming sample preparation procedures with either UV or derivative fluorescence detection [12–19]. In addition, hyphenated techniques, such as LC–MS/MS have also been applied to the deter- mination of methotrexate in human plasma [20,21] mouse brain tissues [22] and environmental samples for occupational expose monitoring [23–25]. Spectral studies of methotrexate with nucleic acids have been recently reported [26]. Recently, liquid chromatog- raphy tandem mass spectrometric procedures have been proposed for the quantitation of folates in biological matrices [27–29]. The 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.09.034