Characterization of specific monoclonal antibodies for detection of mefloquine in body fluids.

Specific monoclonal antibodies (MAbs) to mefloquine conjugated to bovine serum albumin (mefloquine-BSA) were produced by hybridoma technology. The mefloquine-BSA was synthesized by converting mefloquine into hemisuccinate followed by convalently linked to bovine serum albumin (BSA) and coupling with N,N' disuccinimidyl carbonate (DSC). The conjugate was purified by Sephadex G-75 gel filtration using 0.01 M PBS pH 7.2. An average of 19.34 molecules of mefloquine were conjugated to each molecule of protein determined by differential UV absorption spectra of hapten and protein carrier. Sixteen monoclones producing antibody specific to mefloquine were screened by indirect ELISA using homologous antigens. The specificity of MAbs was determined by reacting with BSA and the structurally related antimalarial drug, quinine. Three, three, five and two MAbs belonged to IgG1, IgG2a, IgG2b and IgG3, respectively. Most of the MAbs slightly reacted with quinine-BSA due to the closely related structure of mefloquine to quinine. The selected MAb designated 11F9(G5)G9 which showed no cross reaction with quinine-BSA gave high reactivity with blood samples from malaria patients previously treated with mefloquine when compared to normal blood by indirect ELISA. The preliminary results indicated that such specific MAb could be used as antibody probe for detection of mefloquine in biological fluids.

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography.

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 microl of plasma or whole blood, the limit of determination was 0.2 microg/ml with UV detection for both enantiomers. The limit of determination in 500 microl of urine was 0.08 microg/ml with UV detection.

High-performance liquid chromatographic method for the enantioselective analysis of mefloquine in plasma and urine.

An HPLC method for analysis of the enantiomers of the antimalarial drug mefloquine is presented. A complete resolution of (-)-(11S,2'R) and (+)-(11R,2'S) erythro-mefloquine from plasma and urine was obtained on a commercial AGP column. Mefloquine enantiomers were detected by UV at 222 nm. The separation factor (alpha) at +20 degrees C was 1.50. The limit of determination (coefficient of variation 4.0%) for the enantiomeric ratio (11S,2'R)/(11R,2'S) is 15:1 at a total mefloquine concentration of 1.6 mM.

Determination of mefloquine in blood, filter paper-absorbed blood and urine by 9-fluorenylmethyl chloroformate derivatization followed by liquid chromatography with fluorescence detection.

We describe a method for determination of mefloquine (MQ) in 100-microliters samples of urine, whole blood, and capillary blood collected on filter paper; quantification is by liquid chromatography with fluorescence detection at 475 nm of the 9-fluorenylmethyleneoxycarbonyl derivative. Whole blood and urine samples were prepared by extraction of MQ and internal standard from aqueous base with methyl tert.-butyl ether (MTBE), separation and evaporation of the MTBE layer, and derivatization using a solution of 9-fluorenylmethyl chloroformate in acetonitrile. Filter paper spots were immersed for 16 h in 0.1 M hydrochloric acid, followed by extraction with MTBE from aqueous sodium carbonate. The separated and evaporated organic layer was treated with the derivatizing solution. An aliquot was injected onto a high-performance liquid chromatography system using a C18 reversed-phase column and acetonitrile-water (72:28) mobile phase for filter paper spot extracts as for whole blood and urine extracts. The method has a limit of determination in blood, blood spots, and urine of 50 ng/ml with 100 microliters sample size (coefficient of variation = 16%). Linearity and precision (within-day and between-day) for the method are good. The MQ derivative was isolated and characterized spectroscopically. Values for MQ concentrations in filter paper blood spots compared favorably with values found in corresponding whole blood samples analyzed by a published method.