Anna Gumieniczek

Revisiting thin-layer chromatography as a lipophilicity determination tool—A comparative study on several techniques with a model solute set

The lipophilicity of a compound is a fundamental property related to pharmaceutical and biomedical activity. As many approaches are mixed together in every-day published studies, the subject needs some standardization. The paper presents a comparative study on several approaches of TLC lipophilicity determination: a single TLC run, extrapolation of a retention, principal component analysis of a retention matrix, PARAFAC on a three-way array and a PLS regression. All techniques were applied to 35 model solutes with simple molecules, using nine concentrations of six modifiers: acetonitrile, acetone, dioxane, propan-2-ol, methanol and tetrahydrofurane. The elaborated comparative analysis formed several general recommendations. Methanol and dioxane were the best modifiers, while acetonitrile gave the worst and inacceptable correlation of retention with lipophilicity. Surprisingly, good correlations were obtained for the single TLC runs and this method is underestimated in the literature. The advanced chemometric processing proposed recently, such as PCA, PARAFAC and PLS did not show a visible advantage comparing to classical methods. A need to use a robust regression and robust correlation measures, due to presence of significant outliers, was also noticed and studied.

Changes in antioxidant status of lung tissue in experimental diabetes in rabbits

OBJECTIVES Hyperglycaemia can result in oxidative stress which may affected as cellular tissue damage. DESIGN AND METHODS After 3, 6, 12 and 24 weeks of hyperglycaemia oxidative stress related parameters were measured in lung tissue of diabetic and control rabbits. RESULTS Decreased activities of antioxidative compounds and intensification of lipid peroxidation process were found in diabetic lung. CONCLUSIONS The data obtained suggest that hyperglycaemia induces oxidative stress in lung tissue which may play an important role in pathogenesis of diabetic complications.

Reversed‐Phase Thin‐Layer Chromatography of Three New Oral Antidiabetics and Densitometric Determination of Pioglitazone

Abstract The thin‐layer chromatographic behavior of new oral antidiabetic drugs, pioglitazone, rosiglitazone, and repaglinide has been investigated. For reversed‐phase (RP) chromatography, chemically bonded cyanopropyl plates with mobile phases comprising 1,4‐dioxane with phosphate buffers were used. The influence of the pH on the separation of the drugs was also examined. Then, a simple, rapid, and stability‐indicating high performance thin‐layer chromatographic method has been developed and validated for the quantitative determination of pioglitazone in tablets. Analysis was performed with 1,4‐dioxane–phosphate buffer of pH 4.4 (5:5) as the mobile phase. Detection and quantification were performed by classical densitometry at the wavelength of maximum absorption of pioglitazone, 266 nm. A calibration plot was constructed in the range of 0.4–2.4 µg/10 µL and was linear with a good correlation coefficient (r = 0.9957). Precision was validated by replicate analyses of standard solutions, and accuracy by analysis of fortified samples. The precision of the proposed chromatographic method expressed as mean relative standard deviation (RSD) was 4.99% and 2.57%, respectively, for the lowest and the highest calibration levels. Recovery from the fortified samples ranged from 98.09% to 103.28%. The mean (±SD) recovery from tablets was 99.79% ± 1.57%.

Differences in antioxidant status in skeletal muscle tissue in experimental diabetes.

BACKGROUND It has been suggested that oxidative stress may play an important role in pathogenesis of diabetic complications. The present study was designed to evaluate the oxidative stress-related parameters in alloxan (A)-induced long-term diabetes in rabbits. METHODS After 3, 6 and 12 weeks of diabetes, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and concentrations of ascorbic acid (AA) and free sulfhydryl compounds (SH) were measured in skeletal muscle of diabetic rabbits and the normal control subjects. The products of lipid peroxidation (MDA) were also estimated. RESULTS In our tests, the muscle SOD activity, SH and AA concentrations were significantly reduced. CAT activity increased significantly at all time intervals. GSH-Px activity decreased after 3 weeks and then remained at the control level. GSSG-R activity decreased progressively at 3rd and 6th week and then significantly increased. MDA level increased initially, dropped below baseline after 6 weeks and then remained at the level of the control group. CONCLUSIONS The changes observed in the present experiment suggest a significant imbalance in antioxidative system in the skeletal muscle of rabbits with alloxan-induced diabetes. Such study may lead to therapeutic approaches for limiting the damage from oxidation reactions and preventing the diabetic complications.

Quantitative analysis of repaglinide in tablets by reversed-phase thin-layer chromatography with densitometric UV detection

A simple, rapid, and stability-indicating thin-layer chromatograph-ic method has been developed for quantitative determination of repaglinide in tablets. Analysis was performed on RP-8 TLC plates with acetonitrile—pH 6.0 phosphate buffer, 60 + 40 (% v/v), as mobile phase. Detection and quantification were performed by classical densitometry at the wavelength of maximum absorption of repaglinide, 225 nm. A calibration plot constructed in the range 0.6–3.6 µg/10 µL was linear with a good correlation coefficient (r = 0.998 ± 0.001, mean ± SD, n = 5). Limits of quantitation and detection of repaglinide were 0.27 µg/10 µL and 0.08 µg/10 µL, respectively. Instrumental precision established at three concentrations of the drug ranged from 3.92 to 0.97% for the lowest and highest concentrations of repaglinide, respectively. The mean intra-day and inter-day variability, including three concentrations of re-paglinide, were 1.93 and 2.25% (n = 9), respectively. Recovery from model mixtures, at three levels of addition, ranged from 103.06 to 102.49% for the lowest and highest levels, respectively. Total mean ± SD recovery was 102.71 ± 2.04% (n = 15). The mean ± SD recovery from commercially available tablets was 101.85 ± 1.83% (n = 10). The effect of pH, temperature, and UV light on degradation of repaglinide was also investigated. The analytical method presented was found to be simple, reliable, and convenient for routine pharmaceutical analysis. Its analytical performance fulfilled acceptance criteria established for TLC methods in the official literature.

Normal- and Reversed-Phase Thin-Layer Chromatography of Seven Oral Antidiabetic Agents

The chromatographic behavior of seven oral antidiabetic drugs -chlorpropamide, tolbutamide, glibenclamide, metformin, pioglitazone, rosiglitazone, and repaglinide - has been investigated. Normal-phase chromatography was performed on silica gel and alumina layers with mixtures of chloroform, diethyl ether, and ethyl acetate as mobile phases. For more effective resolution aqueous ammonia or acetic acid was added to the mobile phases. Silica gel enabled better separation than alumina. Reversed-phase chromatography was performed on octadecyl-bonded silica gel (RP-18) with mixtures of acetonitrile or 2-propanol with phosphate buffer as mobile phases. The effect of pH on the separation of the drugs was also examined. For separation of these drugs reversed-phase chromatography was more effective than use of normal-phase mode.

Rapid HPTLC Determination of Rosiglitazone in Pharmaceutical Formulations

Abstract A new, simple, rapid, and stability‐indicating high‐performance thin layer chromatographic (HPTLC) method has been developed and validated for the determination of rosiglitazone in tablets. Analysis was performed on silica gel 60F254 plates in horizontal chambers with chloroform–ethyl acetate–25% ammonium hydroxide (5:5:0.1, v/v) as mobile phase. Detection and quantification were performed by classical densitometry at 240 and 254 nm. The active substance was extracted from tablets with ethanol. Calibration plots were constructed in the range 0.2–1.0 µg/10 µL and were correlated with good correlation coefficients (r 240 = 0.9993; r 254 = 0.9994). Precision was validated by replicate analyses of standard solutions, and accuracy by analysis of fortified samples. The precision of the proposed chromatographic method, expressed as mean RSD was 3.58 and 2.76% for 240 nm, and 8.23 and 6.56% for 254 nm, for the lowest and the highest calibration levels, respectively. The mean recoveries from the fortified samples ranged from 89.48% to 99.38% for 240 nm, and from 89.05% to 100.89% for 254 nm. The mean recoveries from tablets were 101.95% and 103.2% for assays at 240 and 254 nm, respectively.

Modulation of nitrosative/oxidative stress in the lung of hyperglycemic rabbits by two antidiabetics, pioglitazone and repaglinide.

The lungs are involved in diabetes in the cause of the complex phenomena diabetes generates. In the present study, hyperglycemia inhibited pulmonary antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione. These effects were accompanied by significant elevation of lipid peroxidation, total nitrites, and nitrotyrosine levels. The study investigated the effects of 2 oral antidiabetics, pioglitazone and repaglinide, on the mentioned parameters. It is concluded that pioglitazone exerts protective effect in the lung by inhibiting nitrosative stress and normalizing the nitrites and nitrotyrosine levels. Administration of repaglinide prevents oxidative and, to a smaller extent, nitrosative changes.

Synthesis, biological evaluation and molecular modeling of new tetrahydroacridine derivatives as potential multifunctional agents for the treatment of Alzheimer's disease.

A novel series of 9-amino-1,2,3,4-tetrahydroacridine derivatives with 4-dimethylaminobenzoic acid moiety was synthesized and tested towards inhibition of cholinesterases and amyloid β aggregation. Target compounds were designed as dual binding site cholinesterase inhibitors able to bind to both the catalytic and the peripheral site of the enzyme and therefore potentially endowed with other properties. The obtained derivatives were very potent inhibitors of both cholinesterases (EeAChE, EqBChE) with IC50 values ranging from sub-nanomolar to nanomolar range, and the inhibitory potency of the most promising agents was higher than that of the reference drugs (rivastigmine and tacrine). The kinetic studies of the most active compound 3a revealed competitive type of AChE inhibition. Moreover, all target compounds were more potent inhibitors of human AChE than tacrine with the most active compound 3b (IC50 = 19 nM). Compound 3a was also tested and displayed inhibitory potency against AChE-induced Aβ 1-42 aggregation (80.6% and 91.3% at 50 μM and 100 μM screening concentration, respectively). Moreover, cytotoxicity assay performed on A549 cells did not indicate toxicity of this agent. Compound 3a is a promising candidate for further development of novel multi-functional agents in the therapy of AD.

Effects of two oral antidiabetics, pioglitazone and repaglinide, on aconitase inactivation, inflammation and oxidative/nitrosative stress in tissues under alloxan-induced hyperglycemia.

Pathological changes identified in different tissues in hyperglycemic state are undoubtedly connected with increased oxidative/nitrosative stress and inflammation. In our study myeloperoxidase (MPO), nitrotyrosine and lipid peroxidation were enhanced in the heart and lung of alloxan-treated hyperglycemic animals. Additionally, pulmonary aconitase was inhibited. In the testis the changes occurred as an increase of MPO and lipid peroxidation, and as a decrease of aconitase. The effects of two different antidiabetics, the peroxisome proliferator activated receptor gamma (PPARγ) agonist, pioglitazone, and a short acting insulin secretagogue, repaglinide, on the mentioned parameters, were investigated and compared. The insulin deficient alloxan-induced hyperglycemic animals were used to differentiate a direct anti-oxidative effect of the drugs from secondary effects mediated via increased insulin sensitivity or secretion. Pioglitazone acted by normalization of pulmonary and testicular aconitase, normalization of pulmonary and cardiac nitrotyrosine, reduction of pulmonary and testicular MPO, and by reduction of lipid peroxidation in all tissues examined. Repaglinide prevented oxidative changes by normalization of aconitase activity in the lung and testis, and by reduction of lipid peroxidation and nitrotyrosine in the heart and lung. At the same time, no effect of this drug on MPO was observed. Finally, principal component analysis was performed to explore and visualize similarities and differences of the results obtained for the both drugs.