Biljana Jančić Stojanović

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.

Chaotropic agents in liquid chromatographic method development for the simultaneous analysis of levodopa, carbidopa, entacapone and their impurities.

The simultaneous pharmaceutical analysis of multi-component drugs represents a challenge due to a large total number of analytes present in the sample. These analytes are not only the active pharmaceutical ingredients, but also the impurities that might follow the active substances. The aim of this study was to develop an efficient reversed-phase LC method for the simultaneous analysis of antiparkinsonian drugs levodopa, carbidopa and entacapone along with their six related impurities. For the achievement of desirable separation, different acids with anions possessing different properties according to Hofmeister classification (ortho-phosphoric, trifluoroacetic and perchloric acid) were tested. Finally, in order to draw the unbiased conclusions when optimizing the analytical method, for the final tuning of the gradient program, Box-Behnken experimental design and Derringers desirability function were used. The experiments were performed on Zorbax Extend C18, 150 mm × 4.6 mm, 5 μm particle size column with the UV detection at 280 nm and mobile phase flow rate of 1 mL/min. The optimal mobile phase consisted of methanol and 20mM trifluoroacetic acid (pH 2.0 adjusted with NaOH), while their ratio is changed according to previously defined gradient program. The method was tested for selectivity, sensitivity, linearity, accuracy and precision, and proved to be suitable for routine qualitative and quantitative analysis of levodopa, carbidopa, entacapone and their impurities in their mixture.

Thorough investigation of the retention mechanisms and retention behavior of amides and sulfonamides on amino column in hydrophilic interaction liquid chromatography.

In this paper detailed analysis of a mixture of four amides (tropicamide, nicotinamide, tiracetam, and piracetam) and six sulfonamides (sulfanilamide, sulfacetamide, sulfamethoxazole, sulfafurazole, furosemide, and bumetanide) on aminopropyl column in hydrophilic interaction chromatography (HILIC) was carried out. Since, there are no papers on the topic of the assessment of the contribution of ion-exchange retention mechanism involved in the separation of the acidic compounds on aminopropyl column in HILIC mode, the authors utilized the retention data of the acidic sulfonamides for this purpose. Next, broad range of the aqueous buffer concentrations in the mobile phase was examined providing the separation under either HILIC or RP conditions. Turning points between these two mechanisms were determined and then the fitting of the experimental data in the localized and non-localized adsorption models in both RP and HILIC regions was assessed. Since not many papers in the literature were dealing with the estimation of factor influence on the retention behavior of neutral and acidic compounds on aminopropyl column in HILIC, Box-Behnken design and Response Surface Methodology were applied. On the basis of the obtained data, ten quadratic models were proposed and their adequacy was confirmed using ANOVA test. Furthermore, retention data was graphically evaluated by the construction of 3D response surface plots. Finally, good predictive ability of the suggested models was proved with five additional verification experiments.

Quality by Design approach in the development of hydrophilic interaction liquid chromatographic method for the analysis of iohexol and its impurities

This study presents the development of hydrophilic interaction liquid chromatographic method for the analysis of iohexol, its endo-isomer and three impurities following Quality by Design (QbD) approach. The main objective of the method was to identify the conditions where adequate separation quality in minimal analysis duration could be achieved within a robust region that guarantees the stability of method performance. The relationship between critical process parameters (acetonitrile content in the mobile phase, pH of the water phase and ammonium acetate concentration in the water phase) and critical quality attributes is created applying design of experiments methodology. The defined mathematical models and Monte Carlo simulation are used to evaluate the risk of uncertainty in models prediction and incertitude in adjusting the process parameters and to identify the design space. The borders of the design space are experimentally verified and confirmed that the quality of the method is preserved in this region. Moreover, Plackett-Burman design is applied for experimental robustness testing and method is fully validated to verify the adequacy of selected optimal conditions: the analytical column ZIC HILIC (100 mm × 4.6 mm, 5 μm particle size); mobile phase consisted of acetonitrile-water phase (72 mM ammonium acetate, pH adjusted to 6.5 with glacial acetic acid) (86.7:13.3) v/v; column temperature 25 °C, mobile phase flow rate 1 mL min(-1), wavelength of detection 254 nm.

THEORETICAL AND EMPIRICAL MODELS IN HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

□ Hydrophilic interaction liquid chromatography (HILIC) has found application in the determination of polar molecules. This alternative type of chromatography has become increasingly popular in the last five years and a considerable number of articles regarding the elucidation of its retention mechanisms and the development of methods are available. In order to achieve these goals, various types of theoretical and empirical mathematical models were utilized. However, the knowledge obtained from these models was the subject of various interpretations. The goal of this article is to list and discuss the papers in which these models were used in HILIC and give proper theoretical background of the models. In general, theoretical models in HILIC were applied in order to acquire the knowledge of dominant processes in the retention mechanism, retention prediction, and quantitative description of the influence of mobile phase-related factors on the retention and selectivity of the analytes on different types of stationary phases. In addition, empirical models were used for detailed assessment of the retention behavior in terms of both single factor and factor interactions influence on analyte retention and selectivity. Furthermore, they have also shown a great potential in the optimization of HILIC methods in the method development process.

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)).

Factorial-Based Designs in Liquid Chromatography

In this paper, the review on application of factorial-based designs in liquid chromatography (LC) is given. The most useful and applicable full factorial design and reduced forms of full factorial design (fractional factorial design and Plackett–Burman design) applied in LC are presented. Literature survey shows that experimental design presents very often used tool in screening, optimization and robustness testing of LC methods.

Five different columns in the analysis of basic drugs in hydrophilic interaction liquid chromatography

AbstractFive different columns (two silica, two cyanopropyl and one diol) were investigated in hydrophilic interaction liquid chromatography (HILIC). For the assessment of columns behavior in HILIC mode, six basic drugs (lamotrigine, thioridazine, clozapine, chlorpheniramine, pheniramine and sulpiride) were chosen. The assessment of the influence of the concentration of organic modifier on analytes’ retention on each column was provided by fitting the retention data into theoretical models. Utilizing the statistical analysis, the selection of the model that provides better prediction of the retention behavior was enabled. Dual RP-HILIC mechanism was suggested on cyanopropyl and diol columns, therefore the transition points between the two mechanisms on these columns were calculated. Furthermore, in order to investigate the impact of three factors simultaneously on the retention behavior of the analyzed substances on Betasil Silica column, chemometrically-aided empirical models were built. The experiments were conducted according to the matrix of Box-Behnken design and on the basis of the retention data, six quadratic models were obtained and their adequacy was confirmed using ANOVA test. The obtained coefficients of quadratic models enabled the elucidation of both single factor and factor interactions influence. This was also graphically presented in 3D response surface plots.

Chemometrically assissted optimization and validation of RP-HPLC method for the analysis of itraconazole and its impurities

This paper presents the chemometrically assisted optimization and validation of the RP-HPLC method intended for the quantitative analysis of itraconazole and its impurities in pharmaceutical dosage forms. To reach the desired chromatographic resolution with a limited number of experiments in a minimum amount of time, Box- -Behnken design was used to simultaneously optimize some important chromatographic parameters, such as the acetonitrile content in the mobile phase, pH of the aqueous phase and the column temperature. Separation between itraconazole and impurity F was identified as critical and selected as a response during the optimization. The set optimal mobile phase composition was acetonitrile/ water pH 2.5 adjusted with o-phosphoric acid (50:50, V/V). Separations were performed on a Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 μm particle size column with the flow rate 1 mL min-1, column temperature set at 30 °C and UV detection at 256 nm. The established method was then subjected to method validation and the required validation parameters were tested. For the robustness evaluation, fractional factorial 24-1 design was utilized and factors that might significantly affect the system performance were defined. As other validation parameters were also found to be suitable, the possibility to apply the proposed method for the determination of itraconazole, its impurities B and F in any laboratory under different circumstances has been proven.

Avoiding the False Negative Results in LC Method Robustness Testing by Modifications of the Algorithm of Dong and Dummy Factor Effects Approach

The algorithm of Dong and error estimation based on a priori declared negligible effects (dummy factor effects approach) were evaluated for testing robustness, in case they provide contradictory results. Robustness of LC method for separation of ropinirole and its impurity was determined. Plackett–Burman design was applied to unravel the significant/influential factors. Four selected responses (retention factor of ropinirole and impurity, selectivity and resolution) were analyzed by the standard algorithm of Dong and dummy factor effects approach. The interpretation of the results for retention factor of ropinirole and resolution was inconclusive, since the two applied approaches identified different significant factors. The potential false negative or false positive results were avoided reanalyzing the results by the modified approaches. Modification of the dummy factor effects approach included omitting the large dummy effects values, and modification of the algorithm of Dong included calculating the margin of error values for all different number of factors included, and then selecting the lowest one to be the critical effect. The applied reanalysis allowed final important factor identification and non-significant intervals for significant effects calculation.