Analytical Quality by Design: Achieving Robustness of an LC-CAD Method for the Analysis of Non-Volatile Fatty Acids.

An alternative to the time-consuming and error-prone pharmacopoeial gas chromatography method for the analysis of fatty acids (FAs) is urgently needed. The objective was therefore to propose a robust liquid chromatography method with charged aerosol detection for the analysis of polysorbate 80 (PS80) and magnesium stearate. FAs with different numbers of carbon atoms in the chain necessitated the use of a gradient method with a Hypersil Gold C18 column and acetonitrile as organic modifier. The risk-based Analytical Quality by Design approach was applied to define the Method Operable Design Region (MODR). Formic acid concentration, initial and final percentages of acetonitrile, gradient elution time, column temperature, and mobile phase flow rate were identified as critical method parameters (CMPs). The initial and final percentages of acetonitrile were fixed while the remaining CMPs were fine-tuned using response surface methodology. Critical method attributes included the baseline separation of adjacent peaks (α-linolenic and myristic acid, and oleic and petroselinic acid) and the retention factor of the last compound eluted, stearic acid. The MODR was calculated by Monte Carlo simulations with a probability equal or greater than 90%. Finally, the column temperature was set at 33 °C, the flow rate was 0.575 mL/min, and acetonitrile linearly increased from 70 to 80% (v/v) within 14.2 min.

Optimization of Extraction and HPLC-MS/MS Profiling of Phenolic Compounds from Red Grape Seed Extracts Using Conventional and Deep Eutectic Solvents.

Winemaking generates large quantities of grape waste consisting of seeds, skin and stalks. Given that grape seeds are a rich source of different bioactive compounds, the main goal of this research was to optimize grape seed phenol extraction using a Box-Behnken design. The following conditions were derived from the optimization process: sample:solvent ratio of 1:10 w/v, extraction time of 30 min and extraction temperature of 50 °C. In addition, a sustainable (green) approach for obtaining extracts was developed by comparing choline chloride:citric acid-ChCit (natural deep eutectic solvent (NADES)) and ethanol extraction methods with respect to phenol profiles and antioxidant activity. This study was conducted on seeds from eight different red grape varieties. Phenolic acids, flavan-3-ols and procyanidins were characterized using HPLC-MS/MS, and the concentration of procyanidin B1 was above 1 mg/g of dry weight in all analyzed samples. The contents of all phenol classes and antioxidant activities were found to not differ significantly between the solvents, but NADES was found to offer valuable advantages. Importantly, ChCit showed a strong affinity toward procyanidins and a strong correlation between antioxidant activity and quantified phenolic compounds.

A comprehensive study on retention of selected model substances in ß-cyclodextrin-modified high performance liquid chromatography.

The quantitative structure-retention relationship (QSRR) models are not only employed in retention behaviour prediction, but also in an in-depth understanding of complex chromatographic systems. The goal of the present research is to enable the comprehensive understanding of retention underlying the separation in ß-cyclodextrin (CD) modified reversed-phase high performance liquid chromatography (RP-HPLC) systems, through the development of mixed QSRR models. Moreover, the amount of ß-CD adsorbed on the stationary phase surface (ß-CDA) is added as the model's input in order to evaluate its contribution to both model performances and retention. Nuclear magnetic resonance (NMR) experiments were conducted to confirm the predicted inclusion complex structures and support the application of in silico tools. The most significant descriptors revealed that retention is governed by the steric factors 7.5 Å distant from the geometrical centre of a molecule, 3D arrangement of atoms determining the molecular size and shape, lipophilicity indicated by topological distances, as well as the unbound system's energy, related to the inclusion complex formation. In addition, a notable effect of the pH of the aqueous phase on the retention of ionizable analytes was shown. In the case of pH of the aqueous phase and ß-CDA the change in retention behaviour of the studied analytes was observed only at the highest ß-CDA value (5.17 µM/m2), but it was not related to the ionization state of analytes. When the analytes did not change the ionization form across the investigated studied pH range, and the acetonitrile content in the mobile phase was 25% (v/v), the retention factor had low values regardless of the ß-CDA; under these circumstances the retention is probably acetonitrile driven.

Compounding of a topical drug with prospective natural surfactant-stabilized pharmaceutical bases: physicochemical and in vitro/in vivo characterization--a ketoprofen case study.

Recently, healthcare professionals again began realizing the benefits of preparing customized medications to meet specific patient needs. The objective of this work was to develop and evaluate simple pharmaceutical bases stabilized with natural-origin surfactant of alkyl polyglucoside (APG) type as prospective ready-to-use bases and compare them to widely used pharmacopoeial ones. Additionally, the ability of the formulated bases to sustain isopropyl alcohol was assessed as well as its influence on ketoprofen skin absorption (as a co-solvent and potential penetration enhancer). In order to evaluate the manifold characteristics a topical drug product should possess, a comprehensive characterization was performed using different techniques. Physicochemical characterization demonstrated satisfactory physical stability of APG-stabilized bases upon the addition of alcohol. In vitro release/permeation studies failed to show significant difference in ketoprofen liberation/permeation profiles from different bases. However, the extent of ketoprofen delivery in vivo was clearly increased from APG bases, relative to that obtained from pharmacopoeia quality one, implying a distinct influence of the emulsion systems' colloidal structures. Taking also into account the rheological behavior of APG bases, revealing their ameliorated sensory characteristics, it could be concluded that the investigated APG bases could be considered as preferential option in drug compounding related to the conventional ones.

Validation of a column liquid chromatographic method for the analysis of pramipexole and its five impurities.

In this paper, a previously optimized method for HPLC analysis of pramipexole and its impurities was subjected to method validation in accordance with official regulations. The optimized chromatographic conditions were as follows: mobile phase acetonitrile-water phase [15 + 85, v/v, water phase contained 1% triethylamine (TEA), pH adjusted to 7.0 with orthophosphoric acid]; detection at 262 nm for pramipexole, BI-II 751 xx, BI-I 786 BS, BI-II 820 BS, and 2-aminobenzothiazole and at 326 nm for BI-II 546 CL; column temperature, 25 degrees C; and flow rate, 1 ml/min. Acetonitrile and TEA content, pH of the water phase, flow rate, column temperature, and column type were factors studied in robustness testing. According to the experimental plan defined by a Plackett-Burman design, five dummy variables were added in order to have 12 factors. As output, resolution factor was chosen. Robustness was assessed by graphical (half-normal probability plots and Pareto charts) and statistical (t-test) methods. Also, nonsignificance intervals for significant factors were estimated, and limits for the system suitability test were determined. Finally, linearity, accuracy, and precision of the proposed HPLC method were defined. LOD and LOQ values for analyzed impurities were determined. The method was completely defined by these experiments.

Optimization and validation of an RP-HPLC method for analysis of hydrocortisone acetate and lidocaine in suppositories.

An RP-HPLC method has been optimized and validated for the simultaneous determination of hydrocortisone acetate and of lidocaine in suppositories. For the method optimization, response surface methodology was applied, and the obtained model was tested using analysis of variance. The optimal separations were conducted on a Beckman-Coulter 150 x 4.6 mm, 5 microm particle-size column at 20 degrees C. The mobile phase was methanol-water (65 + 35, v/v), pH adjusted to 2.5 with 85% orthophosphoric acid, with a flow rate of 1.0 ml/min. UV detection was performed at 250 nm. Phenobarbital was used as an internal standard. The method was validated for selectivity, linearity, precision, and robustness.

Computer-assisted optimization and validation of LC analysis of trimetazidine dihydrochloride and its impurities.

Trimetazidine dihydrochloride is an anti-anginal drug, which possesses protective properties against ischemia inducing heart damage. In this paper, a new procedure for liquid chromatographic analysis was successfully developed, optimized, and applied in assessment of trimetazidine dihydrochloride content and its impurities, Y-145, Y-235, and Y-234 at most 1.0%, 0.2%, and 0.2%, respectively, in commercially available pharmaceutical preparation containing 35 mg of trimetazidine dihydrochloride. The retention behavior of trimetazidine dihydrochloride and its impurities is investigated by using several stationary and mobile phases to settle a simple, sensitive, and precise RP-HPLC method. The separation conditions are optimized by DryLab 2000 Plus Chromatography Optimization Software version 3.5.00. Separations are performed on PurospherSTAR RP18 endcapped (150 x 4.6 mm, 5 microm particle size) column at 20 degrees C with UV detection at 210 nm. The mobile phase composition is acetonitrile-aqueous phase (10 mmol/L disodium hydrogenphosphate and 2 mmol/L sodium dihydrogen phosphate, pH 7.6) (30:70 v/v). Afterwards, the method is validated; the important statistical parameters for selectivity/specificity, linearity, precision, limit of detection, and quantitation are defined. The recovery value of the trimetazidine dihydrochloride is 98.06%, and the content of impurities is 0.23% for Y-145, less than 0.02% for Y-235, and less than 0.01% for Y-234. In addition, this method is used for analyzing trimetazidine dihydrochloride and its impurities in pharmaceuticals and bulk drug.

Retention modelling in liquid chromatographic separation of simvastatin and six impurities using a microemulsion as eluent.

A novel and unique approach was used for retention modelling in the separation of simvastatin and six impurities by liquid chromatographic using a microemulsion as mobile phase. A microemulsion is a modification of a micellar system where a lipophilic organic solvent is dissolved in the micelles; for that reason, microemulsions are usually treated as solvent-modified micellar solutions. When microemulsions are used as eluents in HPLC separations, solutes partition between the charged oil droplets and the aqueous buffer phase. The complexity of the composition of the microemulsion permits extensive manipulations to be made during method development in order to achieve acceptable resolution of such a complex mixture of substances. In order to avoid a laborious "trial and error" procedure, a 2(3) full factorial design was applied for choosing an optimal microemulsion composition to obtain good separation in a reasonable run time. Organic solvent, sodium dodecyl sulphate, and n-butanol content were varied within defined experimental domain. Optimal conditions for the separation of simvastatin and its six impurities were obtained using an X Terra 50 x 4.6 mm, 3.5 microm particle size column at 30 degrees C. The mobile phase consisted of 0.9% w/w of diisopropyl ether, 2.2% w/w of sodium dodecylsulphate (SDS), 7.0% w/w of co-surfactant such as n-butanol, and 89.9% w/w of aqueous 25 mM disodium phosphate pH 7.0.