RESUMO
Objective: We tested the possibility of the use of reverse-phase high performance liquid chromatography (RP-HPLC), a sensitive, simple and cost effective technique, for evaluation of global DNA methylation alteration in stem cells. Methods: We detected genomic methylation in four cell lines of mouse embryonic stem cells and in the cell at various developmental stages, including mouse embryonic fibroblast (n=5) and mouse tissues (n=9), using RP-HPLC. The samples were extracted for single nucleosides and investigated for the concentrations of deoxy-cytidine (dC), deoxy-guanosine (dG) and deoxy-methyl-cytidine (5-dmC) using RP-HPLC. The methylation levels were obtained by a ratio of the entire genomic 5m-C to dC. Results: Our results demonstrated that the RP-HPLC technique can be used for analysis of global methylation on the stem cell genome. It can display the discrepancy of methylation levels among the cells at different degrees of cell differentiation. Conclusion: We present the evidence that the RP-HPLC technique can be used for the entire-genomic methylation detection in stem cells and should be further developed as a strategy for monitoring the global DNA methylation of stem cells in advanced research.
RESUMO
Objective: To develop and validate a high-performance liquid chromatography (HPLC) method for the determination of nevirapine in human plasma. Methods: A plasma sample and an internal standard were extracted with tert.-butyl methyl ether and determined nevirapine concentration by HPLC method. The limit of quantitation (LOQ), accuracy, precision, specificity, stability and recovery were tested for method validation. Results: Standard curve was linear in the range 0.1 ตg/mL to 20 ตg/mL. The limit of quantitation was 0.1 ตg/ml. Coefficients of variation (CV) of intraday and inter-day precision were less than 4 %. Accuracy was range from 97-101%. The extraction recovery was range from 94-112 %. Conclusion: A rapid, sensitive and specific HPLC method was developed and can be used for determination of plasma nevirapine concentration in adult and pediatric patients infected with HIV.