HPLC-UV method development for fentanyl determination in rat plasma and its application to elucidate pharmacokinetic behavior after i.p. administration to rats

A simple, rapid and validated high performance liquid chromatography method with UV detection for the quantification of an opioid agonist, fentanyl (FEN), in rat plasma was developed. The assay procedure involved chromatographic separation using a ZIC-HILIC SeQUANT column (250 mm × 4.6 mm, i.d., 5 μm) and a mobile phase of acetonitrile and acetate buffer (pH 3.4, 20 mM) of ratio (=65:35, v/v) at a flow rate of 1.2 mL/min and detection wavelength of 201 nm. Plasma sample (100 μL) pretreatment was based on simple deprotienization by acetonitrile spiked with clonidine as an internal standard (I.S.) of 20 ng/mL followed by extraction with tert-butyl methyl ether and centrifugation. The organic layer was evaporated under N2 gas and reconstituted with 100 μL of acetate buffer (pH 3.4, 20 mM), and 50-μL portions of reconstituted sample were injected onto the column. Sample analysis including sample pretreatment was achieved within 35 min. Calibration curve was linear (r ≥ 0.998) from 5 to 100 ng/mL. Both intra- and inter-day assay precisions that are presented through RSD were lower than 12.6% for intra-day and lower than 12.0% for inter-day assessment. Limit of detection was 0.8 ng/mL at S/N of 3. This method was omitting the use of expensive solid phase extraction and time consuming liquid extraction procedures. Moreover, the present method was successfully applied to study pharmacokinetic parameters of FEN after intraperitoneal administration to male Wistar rat. Pharmacokinetic parameters estimated by using moment analysis were T1/2 198.3 ± 44.7 min, Tmax 28.3 ± 2.9 min and AUC0–180 15.6 ± 2.9 (×102) ng min/mL.