Development and validation of an automated solid phase extraction and liquid chromatographic method for the determination of piperaquine in urine

A sensitive and specific bioanalytical method for determination of piperaquine in urine by automated solid-phase extraction (SPE) and liquid chromatography (LC) has been developed and validated. Buffered urine samples (containing internal standard) were loaded onto mixed phase (cation-exchange and octylsilica) SPE columns using an ASPEC XL SPE robot. Chromatographic separation was achieved on a Chromolith Performance RP-18e (100mm×4.6mm I.D.) LC column with phosphate buffer (pH 2.5; 0.1mol/L)–acetonitrile (92:8, v/v). Piperaquine was analysed at a flow rate of 3mL/min with UV detection at 347nm. A linear regression model on log–log transformed data was used for quantification. Within-day precision for piperaquine was 1.3% at 5000ng/mL and 6.6% at 50ng/mL. Between-day precision for piperaquine was 3.7% at 5000ng/mL and 7.2% at 50ng/mL. Total-assay precision for piperaquine over 4 days using five replicates each day (n =20) was 4.0%, 5.2% and 9.8% at 5000, 500 and 50ng/mL, respectively. The lower limit of quantification (LLOQ) was set to 3ng/mL using 1mL of urine, which could be lowered to 0.33ng/mL when using 9mL of urine and an increased injection volume.