High-performance liquid chromatographic method for determination of 2-difluoromethyl-DL-ornithine in plasma and cerebrospinal fluid

A simple, sensitive, selective and reproducible method based on anion-exchange liquid chromatography with post-column derivatisation was developed for the determination of eflornithine (2-difluoromethyl-DL-ornithine; DFMO) in human plasma and cerebrospinal fluid. The 1-alkylthio-2-alkyl-isoindoles fluorescent derivative of the drug was separated from the internal standard (MDL 77246A) on an anion-exchange column (PRP-X300, 250x2.1 mm, 7-microm particle size: Hamilton, USA), with retention times of 6.9 and 10.7 min, respectively. Fluorescence detection was set at 430/340 nm (emission/excitation wavelength). The elution solvent consisted of a solution of 30 mM potassium dihydrogen phosphate buffer (pH 2.2) and acetonitrile (50:50, v/v), running through the column at a flow-rate of 0.3 ml/min. The chromatographic analysis was operated at 37 degrees C. Sample preparation for either plasma or CSF (100 microl) was done by single-step protein precipitation with 20% trichloroacetic acid after incubation at 4 degrees C for 1 h. Calibration curves for plasma (100, 200, 400, 600, 800 and 1200 nmol/100 microl, and 10, 20, 40, 80, 120 and 160 nmol/100 microl for the high and low concentration range curves, respectively) and CSF (1, 2, 4, 8, 16, 32 nmol/100 microl) were all linear with correlation coefficients better than 0.999. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) at high concentration range was below 15%, whereas at low concentration range was below 20% (% coefficient of variations: %C.V.) Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +/-15 and +/-20% at high and low concentration range, respectively. The limit of quantification was accepted as 0.1 nmol using 100-microl samples. The mean recovery for DFMO and the internal standard were greater than 95%. The method was free from interference from commonly used drugs including antimalarials and antihelminthics. The method appears to be robust and has been applied to a pharmacokinetic study of DFMO in patients with African trypanosomiasis following oral doses of Ornidyl (Aventis Pharma, Frankfurt, Germany) at 500 mg/kg body weight (125 mg q.i.d.) for 14 days.