title: A rapid and simple determination of A77 1726 in human serum by high-performance liquid chromatography and its application for optimization of leflunomide therapy.
abstract: Leflunomide is a disease-modifying antirheumatic drug, which is bioactivated by formation of A77 1726. In this study a rapid and simple quantitative assay using a reversed phase HPLC-UV method is validated for detection of A77 1726 in human serum. The HPLC-UV method uses a mobile phase consisting of methanol and a KH2PO4-buffer (45 mM, pH = 3) (50:50,v/v), at a flow rate of 1 mL/min. A77 1726 is detected by UV-absorption at 295 nm with a retention time of 8.9 min. Demoxepam is used as internal standard. Validation showed lower and upper limits of quantitation of 0.5 and 100 mg/L, respectively. The assay was linear over the concentration range of 0.5-100 mg/L (r2 > 0.999). Intra- and inter-day precision showed coefficients of variation within 15% over the complete concentration range; accuracy was within 8%. Commonly prescribed drugs to treat rheumatoid arthritis like disease-modifying antirheumatic drugs, analgesics and corticosteroids, and their main metabolites, are separated from A77 1726 with a resolution >2. Serum levels of A77 1726 in 37 patients on leflunomide therapy were determined using this HPLC-UV method. Measured serum A77 1726 serum concentrations in patient samples showed large variability with a range of 3-176 mg/L.
authors.affiliation: Department of Clinical Pharmacy & Clinical Pharmacology, Medical Centre Leeuwarden, P.O. Box 888, 8901 BR Leeuwarden, The Netherlands. e.n.van.roon@znb.nl
authors.forename: E N
authors.initials: EN
authors.lastname: van Roon
authors.forename: J P
authors.initials: JP
authors.lastname: Yska
authors.forename: J
authors.initials: J
authors.lastname: Raemaekers
authors.forename: T L Th A
authors.initials: TL
authors.lastname: Jansen
authors.forename: M
authors.initials: M
authors.lastname: van Wanrooy
authors.forename: J R B J
authors.initials: JR
authors.lastname: Brouwers
mesh_terms: D000814:Aniline Compounds / Q000097:blood*; D018501:Antirheumatic Agents / Q000627:therapeutic use*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003437:Crotonates; D006801:Humans; D006885:Hydroxybutyrates / Q000097:blood*; D007555:Isoxazoles / Q000627:therapeutic use*; D000077339:Leflunomide; D009570:Nitriles; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014052:Toluidines
chemical_list: D000814:Aniline Compounds; D018501:Antirheumatic Agents; D002021:Buffers; D003437:Crotonates; D006885:Hydroxybutyrates; D007555:Isoxazoles; D009570:Nitriles; D014052:Toluidines; C527525:teriflunomide; D000077339:Leflunomide