A capillary electrophoresis-based method with enzymatic reaction inside the capillary for the screening of matrix metalloproteinase (MMP) inhibitors has been developed. MMP-2 and MMP-9, which have been considered as promising targets for cancer therapy, were selected as the model enzymes. The hydrolysis of a fluorogenic substrate catalyzed by MMPs was determined by measuring the increase in fluorescence. For high-throughput screening, the short-end injection was employed. The enzyme, substrate containing inhibitors, and enzyme solutions were injected from the outlet of the capillary via the sandwich mode. They were mixed by alternating the potential at positive and negative polarities. Online hydrolysis, separation, and detection were achieved in 70 s with approximately 0.87 fmol of MMP required for each assay. The applicability of electrophoretically mediated microanalysis (EMMA) with fluorescence detection to estimate the inhibitory mechanism and to determine the IC(50) values was evaluated for two natural inhibitors, epigallocatechin gallate and oleic acid. A few other natural compounds such as resveratrol, quercetin, caffeic acid, glucosamine, and doxycycline were also screened to test their inhibitory potency. The results obtained were compared with those obtained by offline enzyme assay and confirm the effectiveness of the present method. A rapid, cost-effective, and fully automated method for MMP inhibitor screening is proposed.

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A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.