Development and validation of LC-MS/MS assay for the simultaneous determination of methotrexate, 6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia: Correlation with genetic polymorphism.
Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt; Bioanalysis Research Group, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Electronic address: medhat.alghobashy@cu.edu.eg.
Medhat A
MA
Al-Ghobashy
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
Said A
SA
Hassan
Department of Clinical Pharmacy, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt.
Doaa H
DH
Abdelaziz
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
Noha M
NM
Elhosseiny
Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
Nirmeen A
NA
Sabry
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
Ahmed S
AS
Attia
Department of Pediatrics, Hematology-Oncology Division, Faculty of Medicine, Ain-Shams University, Cairo, Egypt.
Manal H
MH
El-Sayed
D000964:Antimetabolites, Antineoplastic / Q000097:blood*; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D015122:Mercaptopurine / Q000097:blood*; D008727:Methotrexate / Q000097:blood*; D011110:Polymorphism, Genetic; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000188:drug therapy* / Q000235:genetics; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D013866:Thioguanine / Q000097:blood*
D000964:Antimetabolites, Antineoplastic; D015122:Mercaptopurine; D013866:Thioguanine; D008727:Methotrexate
6-Mercaptopurine; Acute lymphoblastic leukemia; Individualized therapy; LC–MS/MS; Leukemia; Methotrexate