Simple and Efficient Solution for Robustness Testing in Gradient Elution Liquid Chromatographic Methods

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.     

DNA binding and antitumor activities of platinum(IV) and zinc(II) complexes with some S-alkyl derivatives of thiosalicylic acid

A series of complexes of platinum(IV) (C1–C5) and zinc(II) (C6–C10) with S-alkyl derivatives of thiosalicylic acid were prepared and characterized. The interactions of the complexes with calf thymus DNA were analyzed by absorption (UV–Vis) and emission spectral studies (ethidium bromide displacement studies). The cytotoxic activities of complexes C1–C10 were determined against mouse B cell lymphocytic leukemia cells (BCL1), human B-prolymphocytic leukemia (JVM-13), mouse mammary carcinoma cells (4T1), and human mammary carcinoma cells (MDA-MB-468) and compared to the activities of the free ligand precursors and cisplatin. The cytotoxicities of the platinum(IV) and zinc(II) complexes toward mouse tumor cell lines were higher compared with their effects on human tumor cell lines. The zinc(II) complex C9 showed the highest antitumor activity toward the tested human cell lines, while the platinum(IV) complex C4 exhibited the highest antitumor activity toward mouse BCL1 and 4T1 cells. Both C4 and C9 have ligands derived from S-propyl thiosalicylic acid.     

Some Considerations in Relation to the Matrix Equation AXB = C

In this paper we represent a new form of condition for the consistency of the matrix equation AXB = C. If the matrix equation AXB = C is consistent, we determine a form of general solution which contains both reproductive and non-reproductive solutions. Also, we consider applications of the concept of reproductivity for obtaining general solutions of some matrix systems which are in relation to the matrix equation AXB = C.     

Antioxidant activities of Melittis melissophyllum L. (Lamiaceae)

Extracts of Melittis melissophyllum leaves in ether, chloroform, ethyl acetate, n-butanol and water were evaporated to dryness and dissolved in 50% ethanol to make 10% (w/v) solutions. The potential protective action of the extracts was assessed by the corresponding in vitro and in vivo tests. In the in vitro experiments extracts were tested as potential scavengers of free radicals (DPPH, O?·?, NO, and OH radicals), as well as inhibitors of liposomal peroxidation (LPx). The results obtained show that all extracts (exept n-BuOH extract) are good scavengers of radicals and reduce LPx intensity in liposomes, which points to their protective (antioxidant) activity. In vivo experiments were concerned with antioxidant systems (activities of GSHPx, GSHR, Px, CAT, XOD, GSH content and intensity of LPx) in liver homogenate and blood-hemolysate of experimental animals after their treatment with extracts of M. melissophyllum leaves, or in combination with CCl?. On the basis of the results obtained it can be concluded that the examined extracts have protective (antioxidative) effect and this antioxidative behaviour is more pronounced in liver than in blood-hemolysate. The reason is probably the fact that liver contains other enzymatic systems, which can also participate in the antioxidative mechanism. Of all the extracts the H?O one showed the highest protective activity.     

Antioxidant activities and polyphenolic contents of three selected Micromeria species from Croatia

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69-13.66%), phenolic acid (5.26-6.84%), flavonoid (0.01-0.09%) and tannin (3.07-6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.     

Antioxidant capacity of Ocimum basilicum L. and Origanum vulgare L. extracts

The antioxidant properties of five different extracts (Et?O, CHCl?, EtOAc, n-BuOH, and H?O) of Ocimum basilicum L. and Origanum vulgare L. were studied. Antioxidant activity was assessed in six different model systems. Free radical scavenging capacity (RSC) was evaluated by measuring the scavenging capacity of extracts on DPPH, NO, O??? and OH radical, as well as on hydrogen peroxide (H?O?). In addition, the protective effects on lipid peroxidation in liposomes (LPx) were evaluated by TBA-assay using the Fe2?/ascorbate induction system. The amount of total phenolic compounds and content of total flavonoids was also determined. EtOAc, n-BuOH and H?O extracts of O. basilicum and O. vulgare expressed very strong scavenger activity. Furthermore, the mentioned extracts showed notable inhibition of LPx. On the other hand, Et?O and CHCl? extracts showed much weaker effect in the neutralization of DPPH, NO and O??? radicals and the neutralization of H?O?. When examining the production of OH radicals and inhibition of LPx, the Et?O and CHCl? extracts showed weak prooxidative properties. The observed differences in antioxidant activity could be partially explained by the levels of phenolics and flavonoids in the investigated O. basilicum and O. vulgare extracts.     

Bioorthogonal chemical tagging of protein cholesterylation in living cells

We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins.     

Artificial neural networks modeling in ultra performance liquid chromatography method optimization of mycophenolate mofetil and its degradation products

The study of experimental design in conjunction with artificial neural networks for optimization of isocratic ultra performance liquid chromatography method for separation of mycophenolate mofetil and its degradation products has been reported. Experimental design showed to be suitable for selection of experimental scheme, while Kennard‐Stone algorithm was used for selection of training data set. The input variables were column temperature and composition of mobile phase including percentage of acetonitrile, concentration of ammonium acetate in buffer, and its pH value. The retention factor of the most retentive component and selectivity factors were used as the dependent variables (outputs). In this way, artificial neural network has been applied as a predictable tool in solving a method optimization problem using small number of experiments. Network architecture and training parameters were optimized to the lowest root‐mean‐square error values, and the network with 5‐4‐4‐4 topology has been selected as the most predictable one. Predicted data were in good agreement with experimental data, and regression statistics confirmed good ability of trained network to predict compounds retention. The optimal chromatographic conditions included column temperature of 40°C, flow rate of 700 µl min⁻¹, 26% of acetonitrile and 9 mM ammonium acetate in mobile phase, and buffer pH of 5.87. The chromatographic analysis has been achieved within 5.2 min. The validation of the proposed method was also performed considering selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification, and the results indicated that the method fulfilled all required criteria. The method was successfully applied to the analysis of commercial dosage form. Copyright © 2014 John Wiley & Sons, Ltd.     

New gold(III) chlorophenyl terpyridine complex: Biomolecular interactions and anticancer activity against human oral squamous cell carcinoma

In this work we synthesized new monofunctional gold(III) complex [Au(Cl-Ph-tpy)Cl]Cl₂ (Cl-Ph-tpy = 4′-[4-chlorophenyl]-2,2′:6′, 2″-terpyridine). This complex was characterized by UV–Vis, NMR, IR, and ESI-MS spectrometry. The kinetic study of the substitution reactions of the Au-Cl-Ph-tpy complex with biomolecules showed that the rate constants depend on the nature of the entering nucleophile. Based on the calculated values of entropy (∆H^≠ > 0) and enthalpy (∆S^≠ < 0) the proposed substitution mechanism is associative. Additionally, the relative stability and thermodynamic properties of Au-Cl-Ph-tpy complex were compared with the analogue Au-tpy complex by the B3LYP/def2-svp method. DNA/BSA binding studies showed that Au-Cl-Ph-tpy complex interacts with CT DNA through the intercalation and moderately quenches the fluorescence of tryptophan residues in serum albumin (BSA). Molecular docking confirmed results obtained by spectroscopic experiments and suggested site I (subdomain IIA) for binding of Au complex to BSA. We demonstrated that the Au chlorophenyl terpyridine complex possessed significant in vitro cytotoxic activity against human oral squamous carcinoma cells (CAL-27), induced apoptosis, inhibited proliferation of CAL-27 cells, and induced cell cycle disturbance. Treatment of CAL-27 cells with the Au complex enhanced expression of cyclin-dependent kinase inhibitors p21 and p27, resulting in cell cycle arrest in the G1 phase, reduced the percentage of CAL-27 cells in S phase and decreased expression of Ki-67. Additionally, Au complex reduced expression of phosphorylated STAT3 and downstream regulated molecules associated with cancer stemness, NANOG, and Sox2 protein.