title: Assessment of intracellular methotrexate and methotrexate-polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry.
abstract: A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients.
authors.affiliation: Department of Neurology, University Medical Center Rotterdam (ErasmusMC), Rotterdam, The Netherlands. r.meesters@erasmusmc.nl
authors.forename: Roland J W
authors.initials: RJ
authors.lastname: Meesters
authors.forename: Ethan
authors.initials: E
authors.lastname: den Boer
authors.forename: Robert
authors.initials: R
authors.lastname: de Jonge
authors.forename: Jan
authors.initials: J
authors.lastname: Lindemans
authors.forename: Theo M
authors.initials: TM
authors.lastname: Luider
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000188:drug therapy; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016014:Linear Models; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood* / Q000627:therapeutic use; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate