HPLC method for the quantification of methotrexate in plasma samples
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1
University of Thessaly, Laboratory of Pharmacology, Greece
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2
University of Thessaly, Laboratory of Biochemistry, Greece
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3
University of Thessaly, Oncology Clinic, Greece
Introduction. Methotrexate (MTX) is an antimetabolite and antifolate drug acting as a dihydrofolate reductase inhibitor. Low-dose MTX is well established for the treatment of rheumatic diseases, whereas high-dose regimens are widely used in the treatment of various kinds of cancer including osteosarcoma. The effectiveness of high dose MTX therapy has been greatly enhanced by the observation that patients at high risk of serious toxicity might be detected by monitoring serum MTX concentrations. Therefore, routine monitoring of drug plasma concentrations are imperative for both patient safety and MTX therapeutic concentration evaluation. Numerous analytical approaches, based, in their majority, on different binding assays for MTX monitoring have been suggested [1]. Potential antibody crossreactivity, however, with MTX metabolites still raises doubt about the accuracy and specificity of these assays.
Methods and results. In the present study an improved instrumental assay was developed for the quantification of MTX in plasma samples. The method is comprised of drug isolation from plasma by Solid Phase Extraction on Phenyl phase followed by HPLC analysis with UV detection of MTX at 307nm. The use of Phenyl phase assured highly specific and efficient (recovery 94-108%) isolation of MTX attributed to specific π-π interactions between MTX and solid phase. The method detection limit was established at 0.003 μΜ (S/Ν=3) and limit of quantitation was 0.01 μΜ (S/N=10). The detector response was linear (r2=0,9956) in the investigated MTX concentration range 0.05-5.0μΜ.
Conclusion. A sensitive HPLC method has been developed for monitoring MTX in patients under high dose drug therapy.
References
1. McCrudden and Tett, J. Chromatography B, 721: 87-92, 1999
Keywords:
Methotrexate,
plasma concentrations,
HPLC
Conference:
8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010.
Presentation Type:
Oral
Topic:
Novel techniques in xenobiotic studies
Citation:
Begas
E,
Tsakalof
A,
Papandreou
C,
Daliani
D and
Asprodini
E
(2010). HPLC method for the quantification of methotrexate in plasma samples.
Front. Pharmacol.
Conference Abstract:
8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010.
doi: 10.3389/conf.fphar.2010.60.00103
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Received:
28 Oct 2010;
Published Online:
04 Nov 2010.
*
Correspondence:
Dr. Eftihia Asprodini, University of Thessaly, Laboratory of Pharmacology, Thessaly, Greece, easpro@med.uth.gr