A rapid and simple determination of A77 1726 in human serum by high-performance liquid chromatography and its application for optimization of leflunomide therapy

J Pharm Biomed Anal. 2004 Sep 21;36(1):17-22. doi: 10.1016/j.jpba.2004.05.019.


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.

Publication types

  • Validation Study

MeSH terms

  • Aniline Compounds / blood*
  • Antirheumatic Agents / therapeutic use*
  • Buffers
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Hydroxybutyrates / blood*
  • Isoxazoles / therapeutic use*
  • Leflunomide
  • Sensitivity and Specificity
  • Spectrophotometry, Ultraviolet
  • Time Factors


  • Aniline Compounds
  • Antirheumatic Agents
  • Buffers
  • Hydroxybutyrates
  • Isoxazoles
  • teriflunomide
  • Leflunomide