Zorica Vujić

Effects of rutin and hesperidin and their Al(III) and Cu(II) complexes on in vitro plasma coagulation assays.

Two flavonoids, rutin and hesperidin, were investigated in vitro for anticoagulant activity through coagulation tests: activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT). Only an ethanolic solution of rutin at the concentration of 830 µM prolonged aPTT, while TT and PT were unaffected. In order to evaluate whether the prolongation of aPTT was due to the decrease of coagulation factors, the experiment with deficient plasma was performed, showing the effects on factors VIII and IX. Since pharmacological activity of flavonoids is believed to increase when they are coordinated with metal ions, complexes of these flavonoids with Al(III) and Cu(II) ions were also tested. The results showed that complexes significantly prolonged aPTT and had no effects on PT and TT. Assay with deficient plasma (plasma having the investigated factor at less then 1%) revealed that complexes could bind to the coagulation factors, what may lead to a non-specific inhibition and aPTT prolongation. An effort was made to correlate stability of complexes with their anticoagulant properties.

Radioprotectors – the Evergreen Topic

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation‐induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical‐scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR‐2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical‐scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.

Simultaneous Analysis of Irbesartan and Hydrochlorothiazide: An Improved HPLC Method with the Aid of a Chemometric Protocol

Experimental design method was used for HPLC determination of irbesartan and hydrochlorothiazide in combined dosage forms. The traditional approach for optimization of experiments is time-consuming, involves a large number of runs and does not allow establishing the multiple interacting parameters. The main advantages of the experimental design method include the simultaneous screening of a larger number of factors affecting response and the estimation of possible interactions. On the basis of preliminary experiments, three factors-independent variables were selected as inputs (methanol content, pH of the mobile phase and temperature) and as dependent variables, five responses (resolution, symmetry of irbesartan peak, symmetry of hydrochlorothiazide peak, retention factor of irbesartan and retention factor of hydrochlorothiazide) were chosen. A full 23 factorial design, where factors were examined at two different levels (“low” and “high”) was used to determine which factors had an effect on the studied response. Afterwards, experimental design was used to optimize these influent parameters in the previously selected experimental domain. The novelty of our method lies in the optimization step accomplished by Derringer′s desirability function. After optimizing the experimental conditions a separation was conducted on a Supelcosil C18 (150 mm × 4.6 mm, 5 μm particle size) column with a mobile phase consisting of methanol-tetrahydrofuran-acetate buffer 47:10:43 v/v/v, pH 6.5 and a column temperature of 25 °C. The developed method was successfully applied to the simultaneous separation of these drug-active compounds in their commercial pharmaceutical dosage forms.

Direct separation of the enantiomers of (±)-metoprolol tartrate on impregnated TLC plates with D -(−)-tartaric acid as a chiral selector

A method for chromatographic separation of the enantiomers of (±)-metoprolol tartrate (±MeT) employing a chiral mobile phase additive (CMPA) is described. By using silica gel plates previously impregnated with the mobile phase (ethanol—water, 70 + 30, v/v) containing d-(−)-tartaric acid as chiral selector, direct separation of the enantiomers of ±MeT was achieved. The results of experiments with different concentrations of d-(−)-tartaric acid (5.8, 11.6, and 23 mmol L–1) revealed that the best resolution of the enantiomers of ±MeT was achieved with 11.6 mm d-(−)-tartaric acid in both the mobile phase and the impregnation solution, at 25 ± 2°C. Spot visualization on chromatograms was performed by use of a fixed-wavelength (λ = 254 nm) ultraviolet lamp or an iodine vaporsaturated chamber.

An Improved HPLC Method with the Aid of a Chemometric Protocol: Simultaneous Determination of Atorvastatin and Its Metabolites in Plasma

The aim of the present study was to optimize a chromatographic method for the analysis of atorvastatin (acid and lactone forms), ortho- and para-hydroxyatorvastatin by using an experimental design approach. Optimization experiments were conducted through a process of screening and optimization. The purpose of a screening design is to identify the factors that have significant effects on the selected chromatographic responses, and for this purpose a full 23 factorial design was used. The location of the true optimum was established by applying Derringer’s desirability function, which provides simultaneously optimization of all seven responses. The ranges of the independent variables used for the optimization were content of acetonitrile in mobile phase (60–70%), temperature of column (30–40 °C) and flow rate (0.8–1.2 mL min−1). The influences of these independent variables were evaluated for the output responses: retention time of first peak (p-hydroxyatorvastatin) and of last peak (atorvastatin, lactone form), symmetries of all four peaks and relative retention time of p-hydroxyatorvastatin. The primary goal of this investigation was establishing a new simple and sensitive method that could be used in analysis of biological samples. The method was validated and successfully applied for determination of atorvastatin (acid and lactone forms) and its metabolites in plasma.

UV-Densitometric Determination of Maprotiline, Desipramine and Moclobemide in Pharmaceutical Dosage Forms

SummaryMaprotiline, desipramine and moclobemide are widely used in the treatment of depression. The content of these antidepressants in bulk drug and pharmaceutical formulations were determined by TLC andin situ densitometry using a Camag scanner. In order to achieve the best condition, mathematical statistical model factorial design was chosen. The partition coefficients, calculated by applying commercial drug design software, were correlated with chromatographic behaviour of these substances. After separation on Silica gel GF254 using propanolethanol-ammonium solution (25%) (8∶2∶0.3v/v/v) as the mobile phase, the chromatographic zones corresponding to the spots were scanned at 254 nm. Analysing Mapratilin® tablets, Pertofran® dragees and Auromid® film tablets examined the applicability of the method for samples and dosage forms.

Densitometric determination of metoprolol tartrate in pharmaceutical dosage forms

This paper describes a simple densitometric method for the determination of metroprolol tartrate in tablets and ampoules. After separation on silica gel GF254 plates, using acetone-methanol-triethylamine as the mobile phase for the tablets and acetone-triethylamine for ampoules, the chromatographic zones corresponding to the spots of metoprolol were scanned. Quantitation was performed using a computer-controlled Camag TLC scanner and applying five-point calibration with polynomial regression. The calibration function was established in the ranges 1-28 micrograms for tablets and 1-9 micrograms for ampoules. The results obtained are precise and reproducible, with recovery values of 99.1-99.4%.

A preliminary study of β-cyclodextrin/metoprolol tartrate inclusion complex for potential enantiomeric separation

The inclusion complex formation between Metoprolol tartarata (MeT) and beta-cyclodextrin (beta-CD) has been investigated using hyperchromic shift at lambda(max) 274.4 nm of MeT. Different parameters such as stirring time, solvent composition (aqueous and aqueous/methanol solutions with methanol content up to 50%), pH values 4.0 and 8.0 were established for optimal inclusion complex formation and confirmed two stoichiometric compositions 1:1 and 1:2. Preliminary data on usage of MeT/beta-CD complex in reversed-phase HPLC indicate the potential application of this complex as a kind of pre-column derivatization for enantiomeric separation of beta(1)-blockers.

Determination of fluocortolone pivalate and fluocortolone hexanoate in suppositories using reverse-phase HPLC.

Fluocortolone and its esters are synthetic corticosteroids used topically in the treatment of various skin disorders. A method that can be successfully used for the separation and determination of fluocortolone pivalate and fluocortolone hexanoate in suppositories was developed. This method is based on reverse-phase HPLC on Supelcosil LC-18 (25 cm x 4.6 mm, 5 microns), using methanol-acetonitrile-water-glacial acetic acid (17:46:37:0.4 v/v/v/v) as mobile phase at a flow rate of 3.0 ml/min. Detection was carried out using a UV detector at 238 nm. The method developed was validated, and calibration curves were established dependent on peak area. The validated ranges for fluocortolone pivalate and fluocortolone hexanoate are 15-305 micrograms/ml (r = 0.9995) and 15-315 micrograms/ml (r = 0.9996), respectively. The limits of detection and the limits of quantification for both esters were also determined.

Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories.