Darko Ivanović

Reversed-phase ion-pair HPLC determination of some water-soluble vitamins in pharmaceuticals

A reversed-phase ion-pair high-performance liquid chromatografic method (RP-IPC) was developed to assay some water-soluble vitamins in solution dosage forms. Vitamins of the B-group B1, B2, B3, and B6, including vitamin C were determined in Oligovit coated tablets. In Beviplex coated tablets the vitamins B1, B2, B3, B6 and p-aminobenzoic acid were analysed. Hexanesulphonic acid sodium salt and triethanolamine in water methanol were used as mobile phase with adjusting pH to 2.8 with orthophosphoric acid. Phenol was used as an internal standard. For quantitative simultaneous analysis of vitamins in pharmaceutical formulations, the method of internal standard was used. All parameters for the validation of the method are given.

Effect of pH on the retention behaviour of some preservatives-antioxidants in reversed-phase high-performance liquid chromatography

SummaryIn this paper the dependence of eluent pH on the efficiency of separation in RP-HPLC is described. Preservatives-antioxidants, esters ofp-hydroxybenzoic acid (methyl, ethyl, propyl and butyl parabens), sorbic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and L-ascorbic acid (Vitamine C) were investigated by reversed-phase high-performance liquid chromatography, utilising isocratic elution with UV detection. The mobile phase composition (methanol-water v/v containing 1% acetic acid), was varied in order to obtain the best separation. For all substances analysed the pH, pKa and the fitted pKa values were calculated in different mixtures of mobile phases using the graphical method of Charlot and Trémillon [1]. The retention factors (k) and the separation factors (α) were also calculated.

Simultaneous assay of ephedrine hydrochloride, theophylline, papaverine hydrochloride and hydroxyzine hydrochloride in tablets using RP-LC.

The analytical problem was to control the quality of imported antiasthmatic tablets containing ephedrine hydrochloride, theophylline, papaverine hydrochloride and hydroxyzine hydrochloride. The aim of the analytical method for the assay was to separate, identify and quantify all compounds, at the same time. A gradient capable RP-LC system was used, using a commercially packed Nucleosil C18 column connected to a dual channel variable, programmable wavelength detector. The analysis was performed in the gradient program of increasing concentration of acetonitrile in water. The influence of pH of the mobile phase was established. The proposed method is reliable, reproducible, easy to perform and satisfies the aim.

Chemometrical evaluation of ropinirole and its impurity's chromatographic behavior

The aim of this study was the chemometrical evaluation of ropinirole and its impuritys (4-[2-(dipropylamino)ethyl]-1H-indol-2,3-dione) chromatographic behavior in systematic and the most efficient way. For that purpose, as very descriptive, response surface designs are most preferable. Face-centered central composite design (CCD) with 2(3) full factorial design, +/-1 star design and four replication in central point was applied for a response surface study, in order to examine in depth the effects of the most important factors. Factors-independent variables (acetonitrile content, pH of the mobile phase and concentration of sodium heptane sulfonate in water phase) were extracted from the preliminary study and as dependent variables five responses (retention factor of ropinirole, retentin factor of its impurity, resolution, symmetry of ropinirole peak and symmetry of impurity peak) were selected. For the improvement of method development and optimization step, Derringers desirability function was applied to simultaneously optimize the five chosen responses. The procedure allowed deduction of optimal conditions and the predicted optimum was acetonitrile-5mM of sodium heptane sulfonate (21.6:78.4, v/v), pH of the mobile phase adjusted at 2.0 with ortho phosphoric acid. By calculating global desirabilitys determination coefficients (R(D)(2)), as well as by the visual inspection of 3D graphs for global desirability, robustness of the proposed method was also estimated.

Improved chromatographic response function in HILIC analysis: Application to mixture of antidepressants

This paper presents exploration of chromatographic behavior in HILIC system by experimental design and improved chromatographic response function denoted as N(CRF)*. As a model mixture six antidepressants were chosen: selegiline, mianserine, sertraline, moclobemide, fluoxetine and maprotiline. Due to complexity of retention mechanisms in HILIC system, detailed examination of experimental space assessing the influence of important factors (acetonitrile content in the mobile phase, buffer concentration and pH of the mobile phase) and their interactions was done by applying 3(3) experimental design. N(CRF)* is developed and designed to be the only output of the system which simultaneously measures the separation of all the examined substances, the chromatographic run duration and the quality of the obtained peaks shape. It allowed objective estimation of overall chromatogram quality and excluded the arbitrary judgment in ambiguous situations. The applied function highlighted the influence of investigated factors on entire mixture and enabled identification of experimental regions where the chromatographic behavior was satisfactory. Applied experimental design strategy combined with N(CRF)* proved to be valuable assistance in HILIC separation of complex mixtures.

Comparison of Full Factorial Design, Central Composite Design, and Box-Behnken Design in Chromatographic Method Development for the Determination of Fluconazole and Its Impurities

This paper presents the development and optimization of a liquid chromatographic method for the determination of fluconazole and its impurities by experimental design methodology. Four experimental design types were applied: two-level full factorial design, central composite design, Box-Behnken design, and three-level full factorial design. The advantages and drawbacks of each design are described and detailed statistical evaluation of mathematical models was performed. The central composite design and three-level full factorial design created significantly better models comparing to the other methods. As the central composite design requires a smaller number of experiments, its models were used for theoretical examination of experimental space. Multiobjective optimization aiming to achieve maximal separation of all investigated substances and minimal analysis duration was performed by a grid point search. The defined optimal separation was achieved on a C18 (125 mm × 4 mm, 5 µm particle size) column with a mobile phase consisting of acetonitrile and water (5 mM ammonium formate) (15:85, v/v); a column temperature of 25°C; a flow rate of 1.2 mL min−1; and a detection wavelength of 260 nm.

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities.

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.

Monitoring of Impurity Level of Valsartan and Hydrochlorothiazide Employing an RP–HPLC Gradient Mode

Abstract The multi‐component preparation Co–Diovan® is indicated for the hypertension treatment in patients whose blood pressure is not adequately controlled by monotherapy. Its active ingredients are valsartan and hydrochlorothiazide. The reversed–phase high performance liquid chromatographic method (RP–HPLC) for the determination of valsartan and hydrochlorothiazide, as well as their impurities level, was developed and described in this paper. As the investigated substances were structurally different with extremely different lipophilicity and polarity, isocratic elution was not possible, so an optimal gradient mode was settled on. The chromatograms were recorded using the Agilent 1100 Series chromatographic system with DAD detector. Separations were performed on a Hypersil 120–5 ODS column (250 mm×4.6 mm; 5 µm particle size) at 25°C column temperature. The gradient high performance liquid chromatographic system was developed, and the following mobile phases were used: A) mixture acetonitrile–water (10:90 V/V); pH of the mobile phase was adjusted to 2.5 with 85% orthophosphoric acid, and B) mixture acetonitrile–water (90:10 V/V); pH of the mobile phase was adjusted to 2.5 with 85% orthophosphoric acid. Injection volume was 50 µL, flow rate 1 mL min−1 and UV detection was performed at 256 nm. Methyl parahydroxybenzoate was used as an internal standard and acetonitrile–water (40:60 V/V) as a solvent. Afterwards, the developed method was subjected to the method validation. The investigated validation parameters (selectivity, linearity, precision, accuracy, LOQ, and LOD) proved the suitability of the method for the simultaneous determination of valsartan, hydrochlorothiazide, and their impurities in appropriate tablets.

Assessment of β‐lactams retention in hydrophilic interaction chromatography applying Box–Behnken Design

In this paper, the retention prediction models for mixture of β-lactam antibiotics analyzed by hydrophilic interaction chromatography (HILIC) are presented. The aim of the study was to investigate the retention behavior of some organic acids and amphoteric compounds including cephalosporins (cefotaxime, cefalexin, cefaclor, cefuroxime, and cefuroxime axetil) and penicillins (ampicillin and amoxicillin). Retention of substances with acidic functional group in HILIC is considered to be interesting since the majority of publications in literature are related to basic compounds. In the beginning of the study, classical silica columns were chosen for the retention analysis. Then, preliminary study was done and factors with the most significant influence on the retention factors were selected. These factors with the impact on the retention factors were investigated employing Box-Behnken design as a tool. On the basis of the obtained results the mathematical models were created and tested using ANOVA test and finally verified. This approach enables the presentation of chromatographic retention in many ways (three-dimensional (3-D) graphs and simple two-dimensional graphical presentations). All of these gave the possibility to predict the chromatographic retention under different conditions. Furthermore, regarding the structure of the analyzed compounds, the potential retention mechanisms in HILIC were suggested.

Stepwise optimization approach for improving LC-MS/MS analysis of zwitterionic antiepileptic drugs with implementation of experimental design

In this article, a step-by-step optimization procedure for improving analyte response with implementation of experimental design is described. Zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, were chosen as model compounds to undergo chloroformate-mediated derivatization followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Application of a planned stepwise optimization procedure allowed responses of analytes, expressed as areas and signal-to-noise ratios, to be improved, enabling achievement of lower limit of detection values. Results from the current study demonstrate that optimization of parameters such as scan time, geometry of ion source, sheath and auxiliary gas pressure, capillary temperature, collision pressure and mobile phase composition can have a positive impact on sensitivity of LC-MS/MS methods. Optimization of LC and MS parameters led to a total increment of 53.9%, 83.3% and 95.7% in areas of derivatized vigabatrin, pregabalin and gabapentin, respectively, while for signal-to-noise values, an improvement of 140.0%, 93.6% and 124.0% was achieved, compared to autotune settings. After defining the final optimal conditions, a time-segmented method was validated for the determination of mentioned drugs in plasma. The method proved to be accurate and precise with excellent linearity for the tested concentration range (40.0 ng ml(-1)-10.0 × 10(3)  ng ml(-1)).