Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies.

A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs.

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma.

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.

An alternative high-performance liquid chromatographic method for determination of clindamycin in plasma.

A simple, sensitive, selective and reproducible method based on a reversed-phase chromatography was developed for the determination of clindamycin in human plasma. Clindamycin was separated from the internal standard (phenobarbital) on a Luna C18 column (250 x 4.6 mm, 5 mm particle size: Phenomenex, USA), with retention times of 5.6 and 14.2 minutes, respectively. Ultraviolet detection was set at 210 nm. The mobile phase consisted of a solution of 0.02 M disodiumhydrogenphosphate (pH 2.8) and acetonitrile (76:24 v/v), running through the column at a flow rate of 1.0 ml/min. The chromatographic analysis was operated at 25 degrees C. Sample preparation (1 ml plasma) was done by a single step liquid-liquid extraction with water saturated ethylacetate. Calibration curves in plasma at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 microg/ml 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) was below 15% (% coefficient of variations: %CV). Good accuracy was observed for both the intra-day and inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +/- 15%). Limit of quantification was accepted as 0.07 microg using 1 ml plasma sample. The mean recovery for clindamycin and the internal standard were greater than 95%. The method was free from interference from fosmidomycin, including commonly used drugs, antimalarials and antihelminthics. The method appears to be robust and has been applied to a pharmacokinetic study of clindamycin in a patient with malaria following oral doses of clindamycin at 10 mg/kg body weight given twice daily for 7 days.

The pharmacokinetics of eflornithine (alpha-difluoromethylornithine) in patients with late-stage T.b. gambiense sleeping sickness.

OBJECTIVE: To investigate the plasma, cerebrospinal fluid (CSF) levels and pharmacokinetics of eflornithine (DFMO) in patients with late-stage T.b. gambiense sleeping sickness who were treated with an oral DFMO at 100 mg/kg or 125 mg/kg body weight every 6 h for 14 days. METHODS: Plasma and CSF concentrations of DFMO were measured during day 10 and day 15 in patients following oral DFMO at 100 mg/kg (group I: n=12) and 125 mg/kg (group II: n=13) body weight every 6 h for 14 days. Clinical and parasitological assessments were performed at 24 h after the last dose of DFMO and at 12 months. RESULTS: Patients in each group had a good initial response, but relapse was observed in six patients (three patients for each group) during 12 months follow-up. Plasma DFMO concentrations did not increase proportionally to doses when the dose increased from 100 mg/kg to 125 mg/kg body weight given every 6 h (60-70% of the expected increase). In most cases, concentration-time profiles of DFMO in each group were best fit using a two-compartment open model with first-order input, with absorption lag-time and first-order elimination. Average trough (C(ss-min)) and average (C(ss-ave)) plasma DFMO concentrations during steady state varied between 189-448 nmol/ml and 234-528 nmol/ml, following 100 mg/kg and 125 mg/kg dose group, respectively. C(max), t(max) and AUC(0- infinity ) values following the last dose were 296-691 nmol/l, 2-3 h, and 2911-6286 nmol h/ml, respectively. V(z)/F, CL/F and t(1/2z) values were 0.47-2.66 l/kg, 0.064-0.156 l/h/kg, and 3.0-16.3 h, respectively. CSF concentrations at steady state varied between 22.3 nmol/ml and 64.7 nmol/ml. Patients who had treatment failure tended to have lower plasma and CSF DFMO concentrations than those who had successful treatment. CONCLUSION: Oral DFMO at the dose of 125 mg/kg body weight given every 6 h for 14 days may not produce adequate therapeutic plasma and CSF levels for patients with late-stage T.b. gambiense sleeping sickness.

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.

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography.

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C18) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1-5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1-4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.