Determination of Free Indomethacin in Human Plasma Using HPLC with UV Detection

Determination of free indomethacin concentration in human plasma is an issue of great importance for the drug safety monitoring and the efficiency of the drug therapy, especially in the case of patent ductus arteriosus treatment (PDA) in neonates. The presented study describes a simple and sensitive method of free indomethacin analysis in plasma samples using high performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection. In the developed method the unbound drug separation was performed by means of the ultrafiltration method on Amicon MPS units, utilizing the YM-10 membranes of 10 kDa molecular mass cutoff. The sample preparation step involved liquid–liquid extraction with ethyl acetate, providing very good absolute recovery of the analyte (ca. 97%, n = 7). Chromatographic separation was achieved on a C18 column using 63% acetonitrile and 37% water (pH = 2.0, adjusted with 0.2% orthophosphoric acid) as the mobile phase. The external standard calibration curves showed good linearity (R2 > 0.9995) in the concentration range from 10 to 5000 ng/mL (total drug determination) and from 10 to 200 ng/mL (free drug determination). The intra-day and inter-day precision and accuracy were satisfactory, with relative standard deviations not exceeding 10%. The limit of detection (LOD) and the limit of quantitation (LOQ) were 3 ng/mL and 10 ng/mL, respectively. The presented analytical approach constitutes substantial improvement over previously reported methods for indomethacin analysis and thus seems to be the method of choice for determination of free indomethacin in plasma.