Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

There is an increasing number of studies suggesting the role of genetic factors in the development and progression of chronic obstructive pulmonary disease and hypertension. Therefore, our study aimed to establish the role of ACE and AGT gene polymorphisms in the mechanisms behind the development of oxidative stress in patients with concomitant chronic obstructive pulmonary disease and hypertension. The study group consisted of 96 patients: Group 1 (individuals with a chronic obstructive pulmonary disease), Group 2 (individuals with arterial hypertension), Group 3 (individuals with a chronic obstructive pulmonary disease and arterial hypertension). The control group consisted of 20 healthy subjects. ACE and AGT gene polymorphisms were determined by polymerase chain reaction amplification. Detection of intracellular reactive oxygen species levels was performed by EPICS XL cytometer (Beckman Coulter, USA) with dichlorodihydrofluorescein diacetate and dihydroethidium. Serum levels of 8-isoprostane were assayed with ELISA, Cayman Chemicals (USA). No significant correlations between ACE and AGT gene polymorphisms and parameters of oxidative stress in a setting of comorbid chronic obstructive pulmonary disease and hypertension were observed. However, the increase in oxidative stress parameters was observed to be the most significant in patients with chronic obstructive pulmonary disease + hypertension and with I/I genotype of the ACE gene, which was due to their lowest values in virtually healthy individuals. This suggests that I/I genotype may be associated with lower levels of reactive oxygen species production compared with other genotypes.

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The aim of the study – to evaluate the rubustness of HPLC determination of valsartan in tablets using Youden’s test. Material and Methods. An efficient method to assess the robustness of analytical methodsis by Youden’s test, by means of an experiment design which involves seven analytical parameters combined in eight tests. In the recent studies, we assessed the robustness of a chromatographic method to quantify valsartan in tablets using Youden’s test. Results. The experimental design approach (design of experiment) is the method of simultaneous investigation of the influence of several factors on robustness of the method using a certain plan (matrix) of experiments. By using the Youden’s test criteria, HPLC method showed to be greatly robust concerning valsartan content, at the introduction of variation in seven analytic parameters. This was done in order to make analytical scientists more successful and businesses more profitable and productive. Conclusion. Youden’s test proved to be an efficient and helpful tool for the robustness evaluation for assay of valsartan by HPLC. Youden’s test can be applied successfully for the robustness evaluation in validation process of analytical methods.