Isolation and identification of a major impurity in a new bulk drug candidate by preparative LC, ESI-MS(n), LC-MS-MS, and NMR.

A simple preparative liquid chromatography (LC) method is developed to isolate a major impurity in a new bulk drug candidate, 6-bromo-4-(carbamidinemethyl)-5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H-indole-3-carboxylic acid ethyl ester hydrochloride monohydrate (carmidole). The carmidole solution for preparation is exposed to daylight before isolation. Based on the electrospray ionization (ESI)-mass spectroscopy (MS(n)) spectral data of the impurity fraction and carmidole, the impurity is preliminarily characterized as 6-bromo-4-(carbamidinemethyl)-5-hydroxy-1-methyl-2-methyl-1H-indole-3-carboxylic acid ethyl ester. LC-MS-MS is used to analyze a carmidole sample. The impurity, lyophilate, is obtained from the fraction of preparative LC, and the impurity standard is synthesized. By comparison of the retention times of high-performance liquid chromatography, ESI-MS(n), and (1)H-nuclear magnetic resonance of the impurity lyophilate with impurity standard and carmidole itself, the structure of the impurity is confirmed and its formation is discussed.

Rapid determination of the antiparasitic drugs flubendazole and febantel in feeds by HPLC with ultraviolet detection.

A rapid and very effective analytical procedure for the simultaneous HPLC determination of two anthelmintics, Flubendazole (FLUB) and Febantel (FEBA), in swine and poultry feeds was developed and tested. The ground feed samples were extracted using dimethylformamide. The extracts were directly analyzed, without any purification, on a reversed-phase ODS column (150 mm x 4.6 mm, 5 microm) with acetonitrile-phosphate buffer (pH 3; 0.017 M) as mobile phase. Ultraviolet detection of FLUB and FEBA was carried out at 300 nm. The method was validated for specificity, linearity, accuracy, repeatability, limit of detection and limit of quantification. The limits of detection (LOD) of FLUB and FEBA in feeds, based on a detector signal-to-noise ratio of 3, were 3 mg kg(-1) and the lowest levels tested in feeds by this procedure (limit of quantification, LOQ) were 10 mg kg(-1). The mean recovery of FLUB and FEBA from spiked samples, in a concentration range of 10-200 mg kg(-1), was 98% with a CV% of 4% and 99% with a CV% of 2%, respectively.

Treatment of post transfusion graft-versus-host disease.

BACKGROUND AND OBJECTIVES: In Japan, so many cases of post-transfusion graft-versus-host disease (PT-GVHD) have been reported, and no effective treatment has been reported. RESULTS: Totally 61 cases of PT-GVHD have been collected, and their background were analyzed. A serine protease inhibitor, nafamostat mesilate(N.M.) was examined as a drug for treatment of PT-GVHD using 4 patients. RESULTS: N.M. was confirmed to inhibit cytotoxic activities of established cytotoxic T cell (CTL) clones in vitro. Simultaneously it was confirmed that fever and skin rash disappeared and liver function tests were normalized soon after the administration of N.M. in all four cases. Leukopenia and thrombocytopenia simultaneously improved. CONCLUSION: The effects of N.M. seem to be limited to inhibit cytotoxic activity of CTL. In addition to this drug, how to remove the CTL from the patients will be the essentially important theme for the future.