Frederick C. Churchill

Amodiaquine as a prodrug: importance of metabolite(S) in the antimalarial effect of amodiaquine in humans

Existing analytical methods for assaying the 4-aminoquinoline antimalarial amodiaquine in body fluids are nonspecific and obscure the fact that little or no amodiaquine is present in the blood of dosed persons. We have isolated four metabolites of amodiaquine. The two major metabolites have been identified; one is desethylamodiaquine, and the other has been tentatively identified on the basis of proton nuclear magnetic resonance spectroscopy as 2-hydroxydesethylamodiaquine. We developed a reverse-phase high-performance liquid chromatographic (HPLC) method that separates the two major metabolites from each other and from amodiaquine, allowing separate quantification. The impact of these findings on in vitro sensitivity testing and blood analysis of persons dosed with amodiaquine is discussed.

Analysis of filter-paper-absorbed, finger-stick blood samples for chloroquine and its major metabolite using high-performance liquid chromatography with fluorescence detection

Methodology has been developed to facilitate the collection, transport, and analysis of blood samples in studies of chloroquine absorption and metabolism. The method utilizes high-performance liquid chromatography (HPLC) with fluorescence detection to quantify chloroquine and its major metabolite, desethylchloroquine, in 100-microliters quantities of blood collected on filter paper. Detection limits are 5 ng/ml for both analytes. No loss of either analyte occurred from filter-paper-collected blood spots stored over a twelve-weeks period at room temperature. Filter-paper-collected, finger-stick blood spots give values for each analyte comparable to corresponding determinations on venous, whole-blood samples. The HPLC mobile phase selected has general applicability to the separation of antimalarial drugs. The methodology permits effective assessment of chloroquine prophylaxis compliance and parasite drug resistance in remote, malaria-endemic regions.

High-performance liquid chromatographic assay for the simultaneous monitoring of mefloquine and its acid metabolite in biological samples using protein precipitation and ion-pair extraction

A high-performance liquid chromatographic (HPLC) method is presented for the simultaneous determination of mefloquine and its acid metabolite in plasma and whole blood. Plasma and whole blood are deproteinized with a combination of zinc and acetonitrile before extraction. Mefloquine and its acid metabolite are extracted simultaneously at pH 4 by methyl tert.-butyl ether, where mefloquine is extracted as an ion pair with heptanesulphonate. After evaporation of the organic phase, the residue is dissolved in mobile phase and injected on to the chromatographic column. A reversed-phase column (Spherisorb ODS-1) is used with acetonitrile-phosphate buffer (0.1 mol/l, pH 2.5) (42:58) containing 40 mmol/l perchlorate as the mobile phase. N,N-Dioctylamine was added to the mobile phase to give a concentration of 0.1% and the pH was adjusted to 2.3-2.7 with concentrated phosphoric acid. The method permits the determination of 0.10 mumol/l (30 ng/ml) mefloquine and its acid metabolite in plasma. The coefficient of variation was 5-6% at the therapeutic level (mefloquine 1-4 mumol/l, its carboxylic metabolite 2-6 mumol/l) in 0.5-ml samples. An alternative method is also described with a similar clean-up procedure that uses protein precipitation with zinc-acetonitrile as a sample pretreatment for therapeutic monitoring of mefloquine and metabolite in plasma and whole blood. Using this method, 0.25 mumol/l mefloquine and its metabolite can be determined. The results from the two methods correlate well.

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.

Isolation, characterization and standardization of a major metabolite of amodiaquine by chromatographic and spectroscopic methods

The amodiaquine metabolite 2-hydroxydesethylamodiaquine (designated metabolite II), one of the two major human metabolites of this antimalarial prodrug, is characterized by chromatographic and spectroscopic methods. This metabolite has been isolated in milligram quantities from the urine of an amodiaquine-dosed individual by extraction and preparative high-performance liquid chromatography (HPLC) and standardized using nuclear magnetic resonance spectroscopy with internal standardization. Aliquots of this standard provided accurately known amounts of the compound for spectroscopic characterization, for use as an HPLC standard and for assessment of in vitro activity against malaria parasites. Knowledge of the structure of the two major metabolites of amodiaquine (the other is desethylamodiaquine) permits speculation as to the presence of three additional human metabolites, chromatographic confirmation for one of which is demonstrated. The in vitro activity of metabolite II is shown to be 1% that of amodiaquine for two chloroquine-sensitive Plasmodium falciparum strains. Should this relationship hold generally, desethylamodiaquine is the only chemical species resulting from oral dosing with amodiaquine which contributes significantly to antimalarial activity in the blood.

Sensitive high-performance liquid chromatographic analysis for chloroquine in body fluids application to studies of drug resistance in plasmodium falciparum

A high-performance liquid chromatographic method has been developed for the sensitive determination of chloroquine in body fluids. THe method has been applied to quality-control assay of World Health Organization (WHO) In-Vitro, Macro-Test Kits for the assessment of susceptibility of Plasmodium falciparum to chloroquine. Experiments utilizing [14C] chloroquine demonstrated that water was not capable of efficiently desorbing chloroquine from the inside surfaces of kit vials. The addition of blood to the vials effectively desorbs chloroquine. Subsequent addition of the blood to aqueous base followed by hexane extraction permits quantitation by reversed-phase, ion-pair high-performance liquid chromatography utilizing ultraviolet detection at 344 nm. The method is capable of determining as little as 20 ng of chloroquine per vial. This method, utilizing the methyl ether of 9-anthra cenemethanol as internal standard, can quantify chloroquine in 1 ml of blood or urine with a minimum detection limit of 20 ppb (ng/ml). Measurement of blood levels of chloroquine in persons contracting falciparum malaria while following a prophylactic regimen complements in-vitro drug susceptibility measurements in characterizing resistant strains of the parasite.

Detection and determination of antimalarial drugs and their metabolites in body fluids

This review of methods for determining antimalarial drugs in biological fluids has focused on the various analytical techniques for the assay of chloroquine, quinine, amodiaquine, mefloquine, proguanil, pyrimethamine, sulphadoxine, primaquine and some of their metabolites. The methods for determining antimalarials and their metabolites in biological samples have changed rapidly during the last eight to ten years with the increased use of chromatographic techniques. Chloroquine is still the most used antimalarial drug, and various methods of different complexity exist for the determination of chloroquine and its metabolites in biological fluids. The pharmacokinetics of chloroquine and other antimalarials have been updated using these new methods. The various analytical techniques have been discussed, from simple colorimetric methods of intermediate selectivity and sensitivity to highly sophisticated, selective and sensitive chromatographic methods applied in a modern analytical laboratory. Knowledge concerning the method for a particular study is determined by the type of application and the facilities, equipment and personnel available. Often is it useful to apply various methods when conducting a clinical study in malaria-endemic areas. Field-adapted methods for the analysis of urine samples can be applied at the study site for screening, and corresponding blood samples can be preserved for subsequent analysis in the laboratory. Selecting samples for laboratory analysis is based on clinical, parasitological and field-assay data. The wide array of methods available for chloroquine permit carefully tailored approaches to acquire the necessary analytical information in clinical field studies concerning the use of this drug. The development of additional field-adapted and field-interfaced methods for other commonly used antimalarials will provide similar flexibility in field studies of these drugs.

High-performance liquid chromatographic method for quantitation of pentamidine in blood serum

A high-performance liquid chromatographic procedure has been developed for the determination of pentamidine concentrations in serum samples. A microbore, reversed-phase column was used with a mobile phase consisting of methanol and water with sodium heptanesulfonate and triethylamine as modifiers. Pentamidine could be extracted from serum only by the addition of an ion-pairing agent, di(2-ethylhexyl) phosphoric acid, to the chloroform used for extraction. The method can be used to reliably detect levels as low as 5 ng/ml. The pentamidine concentration in the serum of eleven patients 24 h after their tenth daily dose of pentamidine averaged 60 +/- 34 ng/ml.

Determination of mefloquine in blood by supercritical fluid chromatography with electron-capture detection

Supercritical fluid chromatography (SFC) with electron-capture detection is described for the sensitive quantification of mefloquine in 0.1-ml blood samples. The method is internally standardized and incorporates partitioning into methyl tert.-butyl ether (MTBE) from aqueous base, back-extraction into dilute aqueous acid and final partitioning into MTBE from aqueous base. SFC conditions include a silica-gel-packed, glass-lined steel column and a mobile phase of 0.15% n-butylamine and 1% methanol in supercritical n-pentane. The method has a detection limit of 7.5 ng/ml in 0.1-ml blood samples and exhibits good linearity and precision. The method compares favorably with a published high-performance liquid chromatographic procedure in the analysis of blood from volunteers who received mefloquine hydrochloride (15 mg as base per kg body weight).

High-performance liquid chromatographic separation and extraction investigation for the simultaneous determination of mefloquine and its carboxylic acid metabolite

Distribution ratios of mefloquine ion pairs with perchlorate and heptanesulphonate as counter-ions, the base distribution for mefloquine, the acid distribution for the carboxylic acid metabolite and the ion-pair distribution with quaternary ammonium ions as counter ions have been determined. Differences in retention characteristics with several commercially available supports bearing the same octadecyl label were found. The effect on the retention time of the pH, the percentage acetonitrile and the addition of an amine to the mobile phase were studied. Other antimalarial drugs present concurrently in the sample showed no chromatographic interference with mefloquine and its metabolite. A suitable internal standard for use in the analytical method was also tested.