Development of a HPLC method for the determination of methotrexate (MTX) and MTX-polyglutamates of red blood cells: A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours at Lincoln University

Abstract

Methotrexate (MTX) low-dose therapy has been increasingly used for the treatment of patients with rheumatoid arthritis (RA). Patient response to, the drug and development of side effects are poorly understood. There is large a variation in clinical response between patients and this influences the dose requirement. Therefore, there is a need for therapeutic drug monitoring to ascertain clinical efficacy and drug toxicity. Plasma monitoring of MTX is of little value. Drug activity depends on the accumulation of MTX and active metabolites, MTX polyglutamates (glu)s in the cells. MTX(glu)s are retained in red blood cells (RBC) and have a half-life of 4 to 6 weeks. Measuring these metabolites is relevant as a steady RBC MTX and MTX(glu) level can be achieved following 6 to 8 weeks continuous MTX treatment. A simple, rapid high-performance liquid chromatographic (HPLC) assay to determine MTX and MTX(glu)s in RBC is described. After extraction of MTX(glu)s from RBCs, chromatographic separation was achieved using a C₁₈ reversed-phase column, followed by ultraviolet (UV) detection at 254 nm. RBC standards for MTX and MTX(glu)s were linear over the concentration range of 0 - 500 ng/200 μI. The minimum detection limit of MTX and MTX(glu)s was 15.6 ng/200 μI. MTX, MTX(glu)2 & MTX(glu)3 were detected in the RBCs of two out of five patients treated for RA. This method requires further validation, including intra- and inter-day coefficients of variation. This method for the determination of MTX and MTX(glu)s in RBCs would provide clinicians more information on pharmacokinetics of MTX and thus allow them to make better judgement in the clinical evaluation of patients undergoing MTX lowdose therapy.