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.

High-performance liquid chromatographic method for the determination of ivermectin in plasma.

A simple, sensitive, selective and reproducible method based on reversed-phase chromatography was developed for the determination of ivermectin in human plasma. The internal standard (moxidectin) was separated from ivermectin on a Hypersil Gold C18 column (150 x 4.6 mm, 5 microm particle size), with retention time of 3.7 and 7.0 minutes, respectively. Fluorescence detection was set at an excitation and emission wavelength of 365 and 475 nm, respectively. The mobile phase consisted of acetonitrile, methanol and distilled water (50:45:5, v/v/v), running through the column at a flow rate of 1.5 ml/minute. The chromatographic analysis was operated at 25 degrees C. Sample preparation (100 microl plasma) was done by a single step protein precipitation with acetonitrile, followed by derivatization with 100 microl of N-methylimidazole solution in acetonitrile (1:1, v/v) and 150 microl of trifluoroacetic anhydrous solution in acetonitrile (1:2, v/v). Calibration curve over the concentration range of 20-8000 ng/ml plasma was linear with correlation coefficient better than 0.995. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (coefficient of variation) Good accuracy was observed for both 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 0.02 ng using 100 microl sample. The mean recovery for ivermectin and the internal standard was greater than 90%. The method was free from interference from endogenous substances and commonly used drugs. The method appears to be robust and has been applied to the investigation of plasma concentration vs time profile of ivermectin in five healthy Thai volunteers following a single oral dose of 200 microg ivermectin/kg body weight.

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.

Simple and sensitive high-performance liquid chromatographic.

A rapid, selective, sensitive, and reproducible reversed-phase HPLC procedure for the quantitative determination of mycophenolic acid (MPA)--an active plasma metabolite of the immunosuppressant mycophenolate mofetil (MMF) in plasma is described. The procedure involves one-step extraction of MPA and the internal standard, standard [RS-60461-000: (E)-6-[1,3-dihydro-4-(4-carboxy-butoxy)-6-methoxy-7-methyl-3-oxo-5-is obenzo-furanyl-4-methyl-4-hexenoic acid] with dichloromethane-dichloroethane (1:1, v/v) at acidic pH. Chromatographic separation consisted of the mobile phase [acetonitrile-0.05% phosphate buffer, pH 3.4 (45:55, v/v)] running through the column (Techopak-10 C18) at flow-rate of 0.8 ml/min. Detection was at UV wavelength of 254 nm. The mean recoveries of MPA and the internal standard at concentrations of 0.1 and 20 microg/ml were 89-98%, and 90-96%, respectively. The within-day coefficients of variation for MPA were 0.3-7.8% and the day-to-day coefficients of variation were 1.1-2.0%. The minimum detectable concentrations for both MPA and the internal standard in plasma were 0.005 microg/ml. The method was found to be suitable for use in clinical pharmacokinetic study.

Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection.

A rapid, selective, sensitive and reproducible HPLC with reductive electrochemical detection for quantitative determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: alpha and beta isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a microBondapak CN column. The method was capable of separating the two isomeric forms of DHA (alpha, beta). The retention times of alpha-DHA, beta-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80-640 ng/ml were 86-93%. The coefficients of variation were below 10% for all three drugs (ART, alpha-DHA, ARN). The minimum detectable concentrations for ART and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.

Plasma quinine concentrations in falciparum malaria with acute renal failure.

Plasma quinine (Qn) monitoring was performed in 32 patients with severe falciparum malaria (10 with acute renal failure (ARF) and 22 with other severe manifestations) who were treated with the standard regimen of 10 mg/kg body weight Qn dihydrochloride, with a loading dose of 20 mg/kg body weight. Median plasma Qn concentrations prior to the first dose on each day were approximately 10-30% higher in ARF patients than in non-ARF patients during acute infection. Seven patients underwent haemodialysis; 2 died after 2 cycles. There were no significant changes in plasma Qn concentrations in patients with ARF during haemodialysis. No Qn was detectable in haemodialysate fluids. This suggests that dosage adjustment of Qn during haemodialysis is unnecessary. Cardiotoxity of Qn must be of concern in malaria patients with ARF after 3 days of Qn therapy, and ECG monitoring during Qn infusion is recommended in all severe malaria patients with persistent ARF. If there is any arrhythmia, the infusion should be discontinued. However, in some hospitals where ECG facilities are not available, reduction in Qn dosage in persistent ARF patients should be considered after the third day of therapy. The appropriate dosage reduction should be further studied. Monitoring of total plasma Qn concentrations (which has been used routinely) is of no value for predicting the cardiotoxicity in ARF patients; monitoring of free Qn would be more appropriate. However, ECG seems to be the practical procedure to monitor cardiotoxicity of Qn. It may be possible to use the QTc interval to estimate the Qn concentration in severe malaria patients without ARF, but not in patients with persistent ARF.

Pharmacokinetics of quinine in patients with chronic renal failure.

METHODS. We investigated the pharmacokinetics of quinine (Qn) following administration of a single oral dose of 600 mg Qn sulphate in six male Thai patients with a moderate degree of chronic renal failure (CRF), and six male Thai subjects with normal renal function. RESULTS. The drug was well tolerated in both groups of subjects; no major adverse reactions were observed. A marked alteration in the pharmacokinetics of Qn was found in patients with CRF compared to healthy subjects; there were six significant changes in the pharmacokinetic parameters. Absorption was delayed, but increased in CRF (tmax 4.5 vs 1.6 h, Cmax 6.17 vs 3.45 micrograms.ml-1). Total clearance was significantly reduced (0.94 vs 2.84 ml.min-1.kg-1, whereas Vz/f remained unchanged (1.82 vs 2.78 l. kg-1). This resulted in the increased values of AUC and prolongation of the t1/2z and MRT in the patients (AUC 181.5 vs 61.8 micrograms.min-1.ml-1, t1/2z 26 vs 9.7 h, MRT 36.4 vs 11.3 h). Median concentrations of plasma unbound fraction of Qn collected at 4 h after drug administration in patients and healthy subjects were 7.3 vs 9.8%, respectively.

Pharmacokinetics of quinine in chronic liver disease.

The pharmacokinetics of quinine were investigated in a) six healthy male Thai subjects, and b) nine male Thai patients with a moderate degree of chronic liver disease, after a single oral dose of 600 mg quinine sulphate. tmax and t1/2.2 were significantly prolonged in patients (median [range] tmax 2 [1-5] vs 1.6 [0.8-2] h; t1/2,z 23.4 [17.4-41.7] vs 9.7 [7.8-17.2] h), and Vz/F was significantly larger (median [range] 4.21 [2.33-15.87] vs 2.78 [1.49-3.38] 1 kg-1). Median (range) concentration of the plasma unbound Qn fraction collected from the patients at 4 h after drug administration was 17 (8.4-17.8)% of total drug concentration.

Pharmacokinetics of mefloquine when given as a single and two divided-dose regimens.

The pharmacokinetics of mefloquine at 1250 mg, when given as a single oral dose or as 2 divided doses of 750 and 500 mg at 6-h intervals, was investigated in 18 Thai male patients with acute uncomplicated P. falciparum malaria. The pharmacokinetics of each of these two treatment regimens, expressed as the mean followed by the S.D. in brackets, were found to be similar. Maximum concentrations of 2302 (750) and 2399 (418) ng/ml were achieved at 15.9 (4.5) and 17.1 (3.1) h after a single and a divided-dose regimen respectively. Other parameters were also comparable between the 2 regimens of mefloquine [AUC: 21.78 (5.99) vs 20.7 (604) micrograms.day/ml; Vdz/f: 23.7 (3.4) vs 24.9 (3.7) L/kg; CL/f: 0.899 (0.23) vs 1.02 (0.51) ml/min/kg; t1/2z: 12.5 (3.2) vs 11.4 (2.1) days; MRT: 16.2 (2.2) vs 16.8 (3.1) days]. In areas where P. falciparum is highly resistant to mefloquine, the elevated dose of 1250 mg may prove beneficial when given as the 2 divided doses at a 6-h interval.

Pharmacokinetics of mefloquine, when given alone and in combination with artemether, in patients with uncomplicated falciparum malaria.

The pharmacokinetics of mefloquine at a single oral dose of 750 mg, when given alone or 24 hours after a single oral dose of artemether (300 mg) was investigated in 27 Thai patients with acute uncomplicated falciparum malaria (17 with mefloquine alone, 10 with the combination). The oral bioavailabiiity of mefloquine was significantly decreased when administered 24 hours after an oral dose of artemether. This was evident by the significantly lower values of Cmax, AUC[0-24 h], AUC[0-48 h], AUC[0-72 h], as well as total AUC[Cmax: 1,290 (827-2,619) vs 1,820 (1,283-2,531) ng.ml-1; AUC[0-24 h]: 0.99 (0.64-1.41) vs 1.33 (1.07-1.95) micrograms.day.ml-1; AUC[0-48 h]: 1.78(1.23-2.58) vs 2.67 (2.09-3.84) micrograms.day.ml-1; AUC[0-72 h]: 2.74 (1.63-3.6) vs 4.54 (2.88-5.38) micrograms.day.ml-1; AUC: 11.11 (6-20.96) vs 15.29 (9.3-36.71) micrograms.day.ml-1]. Tmax was also delayed with the combination regimen [14 (5-24) vs 6 (4-16) h]. Terminal elimination half-lives were comparable [t1/2z: 11.1 (6.8-14.3) vs 13.4 (10.5-19.1) h].

Mefloquine monitoring in acute uncomplicated malaria treated with Fansimef and Lariam.

Mefloquine levels were compared between Plasmodium falciparum malaria patients with sensitive response and those with treatment failure who received 3 drug regimens of mefloquine (46 patients with MSP 3 tablets (Fansimef), 38 and 34 with mefloquine (Lariam) 750 mg and 1,250 mg). Mefloquine concentrations on Day-1 in any regimens in patients with treatment failure were significantly lower than those from the sensitive response, whereas there was no difference in the concentrations on Day-7. However, MIC values of mefloquine prior to drug treatment were comparable in both groups. The study suggests that pre-treatment in vitro sensitivity testing was a non-reliable indicator of clinical outcome. Mefloquine concentration on the first day after treatment is a better predictor of the treatment outcome.

Pharmacokinetics of prophylactic mefloquine.

The pharmacokinetics of the prophylactic dose of mefloquine (Lariam: 500 mg every 4 weeks, with a loading dose of 750 mg on the first week) was studied in six healthy Thai male volunteers. Mefloquine was well tolerated during the study period of 16 weeks. The only side-effects found were nausea and diarrhea in 2 volunteers after the first dose of mefloquine. The mean minimum concentration of mefloquine at steady state ranged from 290 to 460 ng/ml. The maximum concentration on week 16 after the last dose was 1558 +/- 48 ng/ml at the mean time of 38 +/- 19 hours. The other pharmacokinetic parameters obtained were: absorption half life = 6.6 +/- 3.0 hours; distribution = 5.1 +/- 3.1 days; terminal half life = 12.9 +/- 2.2 days; apparent volume of distribution = 10.5 +/- 2.3 l/kg; area under the concentration-time curve = 26.9 +/- 2.2 mg/dl. Although this prophylaxis regimen is ideal when considering the compliance, the minimum concentration obtained was much too low for optimum therapeutic concentration. We therefore suggest that weekly prophylaxis schedule should be a better regimen as the difference between minimum and maximum mefloquine concentration would be smaller.