Resistance to chloroquine by Plasmodium vivax in Irian Jaya, Indonesia.

Evidence of emerging resistance to chloroquine by Plasmodium vivax is described from Irian Jaya (Indonesian New Guinea). Sixteen of 24 residents in the village of Arso PIR II taking supervised weekly chloroquine prophylaxis (5 mg base/kg) had asexual parasitemia with P. vivax at least once during eight weeks of surveillance. An American working in the same village developed symptomatic P. vivax parasitemia despite chloroquine prophylaxis. Five days after therapy with 600 mg chloroquine base, the asexual parasitemia in the American increased 40-fold, but cleared after treatment with 1,500 mg chloroquine base. Serum samples were not available from many of the cases, but six local residents and the American had serum levels of chloroquine in excess of the ordinarily suppressive 15 ng/ml at the time of their asexual parasitemias (16-70 ng/ml). The weekly 300 mg base tablet of chloroquine, which has been the standard for prophylaxis against malaria for more than 40 years, was not effective against P. vivax in Arso PIR, Irian Jaya.

Effectiveness and tolerance of long-term malaria prophylaxis with mefloquine. Need for a better dosing regimen.

To measure the effectiveness and tolerance of long-term malaria prophylaxis with mefloquine, the incidence of Plasmodium falciparum malaria and of adverse reactions was compared in Peace Corps volunteers in West Africa who took mefloquine every 2 weeks and in volunteers who took chloroquine phosphate weekly. Mefloquine was only 63% more effective than chloroquine; the monthly incidence of P falciparum infections was one case per 100 volunteers who took mefloquine and 2.7 cases per 100 volunteers who took chloroquine. Using daily proguanil (chloroguanide) hydrochloride in addition to chloroquine did not provide additional protection. All mefloquine prophylaxis failures occurred during the second week of the every-2-weeks dosing regimen in volunteers who had used mefloquine for more than 2 months. Blood concentrations of mefloquine were lower during the second week of the alternate-week regimen than during the first week, suggesting that blood levels are too low during the second week to suppress parasitemia. No serious adverse reactions were observed. The results indicate that a dosing regimen of 250 mg of mefloquine weekly should be considered for travelers to areas with chloroquine-resistant P falciparum malaria.

Analysis of blood and urine samples from Macaca mulata for pyronaridine by high-performance liquid chromatography with electrochemical detection.

We describe a high-performance liquid chromatographic method with electrochemical detection for quantifying pyronaridine in rhesus monkey (Macaca mulata) blood and urine samples. The detection limit is 20 ng/ml at a signal-to-noise ratio of 4 in 0.5-ml samples of blood or urine. Blood analysis includes a liquid-liquid extraction and a subsequent solid-phase extraction that removes an interferent present in blood. For urine, a back-extraction is substituted for the solid-phase extraction step. The method uses an analogue of amodiaquine as internal standard, a 10-microns rigid macroporous styrene-divinylbenzene copolymer column and a mobile phase of 1% (v/v) triethylamine in methanol-water (34:66, v/v). The method was applied to samples of blood and urine from a monkey after a single intramuscular dose of pyronaridine tetraphosphate (160 mg as base).

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).

Adaptations of the Saker-Solomons test: simple, reliable colorimetric field assays for chloroquine and its metabolites in urine.

Two field-adapted colorimetric methods for measuring the antimalarial drug chloroquine in urine are described. Both are modifications of the method of Saker and Solomons for screening urine for phencyclidine and other drugs of abuse, using the colour reagent tetrabromophenolphthalein ethyl ester. One method is semiquantitative, detecting the presence of chloroquine (Cq) and its metabolites in urine with a 1 microgram/ml detection limit; it is more sensitive and reliable than the commonly used Dill-Glazko method and is as easy to apply in the field. The second method uses a hand-held, battery-operated filter photometer to quantify Cq and its metabolites with a 2 microgram/ml detection limit and a linear range up to 8 micrograms/ml. The first method was validated in the field using a published quantitative colorimetric method and samples from a malaria study in Nigeria. The second method was validated in the laboratory against high-performance liquid chromatographic results on paired samples from the Nigerian study. Both methods may be used in remote locations where malaria is endemic and no electricity is available.

Adaptations of the Saker-Solomons test: sample, reliable colorimetric field assays for chloroquine and its metabolites in urine

Two field-adapted colorimetric methods for measuring the antimalarial drug chloroquine in urine are described. Both are modifications of the method of Saker and Solomons for screening urine for phencyclidine and other drugs of abuse, using the colour reagent tetrabromophenolphthalein ethyl ester. One method is semiquantitative, detecting the presence of chloroquine (Cq) and its metabolites in urine with a 1 ug/ml detection limit; it is more sensitive and reliable than the commonly used Dill-Glazko method and is as easy to apply in the field. The second method uses a hand-held, battery-operated filter photometer to quantity Cq and its metabolites with a 2 ug/ml detection limit and a linear range up to 8 ug/ml. The first method was validated in the field using a published quantitative colorimetric method and samples from a malaria study in Nigeria. The second method was validated in the laboratory against high-performance liquid chromatographic results on paired samples from the Nigerian study. Both methods may be used in remote locations where malaria is endemic and no electricity is available(AU)

Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method using fluorescence detection is described for the quantification of hydroxychloroquine (HCQ) and three of its metabolites in blood and urine samples. The method is selective, permitting quantification of analytes without interferences from chloroquine or quinine in the sample. Detection limits for HCQ, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylchloroquine are 10, 30, 5, and 5 ppb, respectively, for a 100-microliters blood or urine sample. The internally standardized method requires only one extraction step and utilizes normal-phase HPLC conditions including an amine modifier in the mobile phase. These conditions facilitate fluorescence detection, selective separation, and acceptable peak shapes. A mobile phase of 0.5% n-butylamine in methanol-hexane-methyl tert. butyl ether (1:1:1) is used in the analysis. Analysis of blood and urine samples from two healthy volunteers given 400 mg of Plaquenil (310 mg of HCQ base) weekly for four weeks provided data on HCQ metabolism for the two persons during the recommended chemoprophylactic regimen for malaria.

Field application of a colorimetric method of assaying chloroquine and desethylchloroquine in urine.

In a study in western Kenya of malaria-infected adult women who had been treated with chloroquine, we compared the level of chloroquine and its principal metabolite, desethylchloroquine, in urine, measured using a newly developed modified Haskins test, with the level of chloroquine in whole blood, determined by high-performance liquid chromatography. Over a 28-day follow-up period, 277 matched urine and blood samples from 81 women were evaluated. A high correlation was observed between the level of chloroquine in whole blood (in mug/l) and that of chloroquine + desethylchloroquine in urine (in mg/l). The test was easily performed and may be useful for monitoring use of chloroquine in a community and determining pre-study or post-treatment ingestion or absorption of the drug in in vivo studies of parasite sensitivity.

Field application of a colorimetric method of assaying chloroquine and desethylchloroquine in urine

In a study in western Kenya of malaria-infected adult women who had been treated with chloroquine, we compared the level of chloroquine and its principal metabolite, desethylchloroquine, in urine, measured using a newly developed modified Haskins test, with the level of chloroquine in whole blood, determined by high-performance liquid chromatography. Over a 28-day follow-up period, 277 matched urine and blood samples from 81 women were evaluated. A high correlation was observed between the level of chloroquine in whole blood (in ug/l) and that of chloroquine+desethylchloroquine in urine (in ug/l). The test was easily performed and may be useful for monitoring use of chloroquine in a community and determining a pre-study or post-treatment ingestion or absorption of the drug in in vivo studies of parasite sensitivity

Field-adapted method for high-performance thin-layer chromatographic detection and estimation of chloroquine and desethylchloroquine in urine.

A high-performance thin-layer chromatographic (HPTLC) method which can be used in remote field locations lacking electricity has been developed for selective and sensitive detection and estimation of chloroquine (Cq), desethylchloroquine (DECq) and two other antimalarial compounds in urine. The method requires a single extraction step and has a detection limit of 0.25 micrograms/ml for both Cq and DECq. The HPTLC method was coupled with a colorimetric field assay for Cq and metabolites in urine and a field-interfaced, laboratory-based, high-performance liquid chromatographic assay of Cq and DECq in finger-stick blood to survey antimalarial drug use practices in Esmeraldas Province in northwestern Ecuador. Fourteen of 66 patients from whom urine samples were obtained were found to have detectable Cq and DECq. Results of the three assays are compared for these individuals, and the role of the HPTLC method in field studies is assessed.

Colorimetric and thin-layer chromatographic methods for field assay of chloroquine and its metabolites in urine.

Three field-adapted methods for the quantification of the antimalarial drug chloroquine are described. Two of the methods are modifications of the Haskins test and are based on ion-pair formation between chloroquine and methyl orange in either dichloromethane or chloroform. Absorbance values measured at 420 nm with a hand-held, battery-operated filter photometer were linearly related to chloroquine concentrations in urine up to 100 mumol/l (32 mug/ml) for both methods. The contribution of the desethylchloroquine metabolite to the measured absorbance for both methods is less than that of chloroquine; the relative sensitivity for this metabolite is about 50% of that of chloroquine for both methods. The detection limit for modification I is 1 mumol/l (0.3 mug/ml), while that for modification II is 3 mumol/l (1 mug/ml). A single dose of chloroquine diphosphate (300 mg as base) administered to each of three volunteers yielded detectable levels by modification I of chloroquine in the urine for 28 days after dosing. Results for the colorimetric methods correlated well with the liquid chromatographic reference method used. The related thin-layer chromatographic method confirmed the presence of chloroquine and desethylchloroquine in the urine and permitted independent estimation of the concentration of these two compounds if desired. The two colorimetric methods may be used in remote locations where no electricity is available.

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.