Retention mechanisms of imidazoline and piperazine-related compounds in non-aqueous hydrophilic interaction and supercritical fluid chromatography based on chemometric design and analysis.

The experimental design methodology based on central composite design of experiments was applied to compare the retention mechanisms in supercritical fluid chromatography (SFC) and non-aqueous hydrophilic interaction liquid chromatography (NA-HILIC). The selected set consists of 26 compounds that belong to imidazoline and serotonin receptor ligands. The different chemometric tools (multiple linear regression, principal component analysis, parallel factor analysis) were used to examine the retention, as well as to identify the most significant retention mechanisms. The retention mechanism was investigated on two different stationary phases (diol, and mixed-mode diol). In NA-HILIC, the mobile phase contains acetonitrile as a main component, and methanolic solution of ammonium formate (+ 0.1% of formic acid) as a modifier. The same mobile phase modifier was used in SFC, with a difference in the main component of the mobile phase which was CO2. The retention behaviour differs significantly between HILIC and SFC conditions. The retention pattern in HILIC mode was more partition-like, while in SFC the solute-sorbent interactions allowed retention. The retention mechanism between mixed-mode diol and the diol phases varies depending on the applied chromatographic mode, e.g., in HILIC the type of stationary phase significantly affects the elution order, while in SFC this was not the case. The HILIC retention behaviour was influenced by the number of tertiary amines-aliphatic, and N atom-centred fragments in tested compounds. On the other hand, the number of pyrrole and pyridine rings in the structure of the compound showed correlation with their SFC retention, simultaneously increasing the molecular weight and rapid elution of larger compounds. It was found that temperature surprisingly plays a major role in SFC mode. The increase in temperature reduces the relative contribution of enthalpy factors to total retention, so the separation in SFC was more entropy-controlled. For further pharmaceutical research and optimization, the SFC would be considered more beneficial compared to HILIC since it gives good selectivity in separation of chosen impurities.

Direct spectrophotometric determination of quercetin in the presence of ascorbic acid.

The current research provides a simplified sample preparation procedure for the accurate estimation of quercetin in pure and in the pharmaceutical dosage form. Direct spectrophotometric method for the determination of quercetin in the presence of ascorbic acid was established. The influences of medium, wavelength, pH, temperature and the ionic strengths on quercetin determination were investigated. The best conditions for calibration curve are: 50% ethanol, lambda = 370 nm, pH = 4.2, T = 34 degrees C and I = 7.5 x 10(-5) M. Beer's law is obeyed in the concentration range 1.0-12.0 microg ml(-1) for quercetin. The corresponding detection limit is 0.76 microg ml(-1). The proposed method was verified by analyzing Quercetin + C capsules, Twinlab.

A preliminary study of beta-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.

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%.

Spectrophotometric investigation of metoprolol-benzyl orange reaction and its application to the assay in pharmaceutical dosage forms.

A sensitive spectrophotometric method for the determination of metoprolol tartrate in tablets and ampoules is presented. Using spectrophotometric measurements, it was found that metoprolol tartrate and benzyl orange form a chloroform soluble ion-pair complex with an absorption maximum at 401 nm. The composition of the ion-pair complex was determined by applying Job's method to equimolar solutions of metoprolol tartrate: benzyl orange (1:2); molar absorptivity 7.39 x 10(3) mol-1 cm-1. Extraction of the ion-pair complex in chloroform was accomplished easily at a Britton-Robinson's buffered optimum pH = 5.2, mu = 0.1 mol/dm3. The relative stability constant, calculated according to the method of Sommer and Job's non-equimolar solutions, was log K = 9.72 (avg. value). Beer's law was obeyed up to 3.42 micrograms/ml of metoprolol tartrate (the detection limit was also 3.42 micrograms/ml). The precision of the method was checked at three different concentrations. The RSD (n = 7) varied from 0.51 to 2.03%. Reproducibility was examined by analysing Lopresor tablets and ampoules. Recoveries varied from 99-101%. The reported method, applied to the assay of metoprolol tartrate in tablets and ampoules, gives precise and reproducible results.