Application of short-end injection capillary electrophoresis mode for assessment of drug stability against human liver microsomes
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| Year of publication | 2010 |
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| Description | Since pharmacological profile of a drug highly depends on its metabolism by cytochromes P450 (CYP), stability against these enzymes belongs to the important drug parameters. During a new drug development, the pharmaceutical companies have to usually decide between analysis of a new drug candidate with chosen CYP isoforms mixture, which has limited evaluation value about processes in a real human body, and laborious and time-consuming analysis of a designed molecule on the liver slices. In this manner, human liver microsomes (HLM) represent a certain trade-off enabling a good simulation of conditions inside the liver and fast analysis. The goal of this study was to introduce a new generic method based on capillary electrophoresis (CE) allowing determination of drug stability against HLM. Work with HLM carries the onus of their high polypeptides and proteins content given by preparation technique nevertheless. These HLM mixture components have a tendency to adsorb onto inner capillary wall. Therefore, we proposed a method based on the short-end injection mode to reduce the time spend by HLM inside the capillary. The second lay-out leading towards higher reproducibility represents utilization of background electrolyte (BGE) containing replaceable polymeric gel. Method optimization and validation was performed with samples containing 0.1 mmol.l-1 bufuralol as the probe drug, 1 mg/ml HLM, 0.1 mmol.l-1 NADPH, 0.1 mmol.l-1 NADP+ and 0.2 mg/ml phtalic acid prepared in 10 mmol.l-1 phosphate buffer pH 7.4. Samples were injected by application of a pressure 0.5 psi to the outlet vial for 3 s. 10 % (v/v) linear polyacrylamide in 50 mmol.l-1 disodium hydrogen phosphate was used as a BGE. An uncoated fused silica capillary with total length of 50 cm (effective length 10 cm) and internal diameter 75 um was thermostated at 37 C. Separations were accomplished by application of 22 kV (positive polarity). All experiments were conducted on the P/ACETM MDQ CE system. As the result we introduced the method based on NADP+ production monitoring. Following summary shows the results obtained by method validation: RSD of 1.77 % for migration time (n = 6), RSD of 2.04 % for relative peak area (n = 6), LOD of 6.5 umol.l-1 (S/N = 3), LOQ of 20 umol.l-1 (S/N = 10) and recovery of 100.63-103.91 % (n = 6). The fast screening of 12 chosen probe drugs was carried out to prove method’s capabilities as the versatile tool for a drug stability determination. |
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