Chiralpedia
The recent development of monolithic chiral stationary phases (CSPs) for liquid chromatography (LC) is mainly focused on reducing backpressure, maximizing flow rates, faster run time, column efficiency, and stability. This review paper emphasizes recent progress in the development of polysaccharide and cyclodextrin-based monolithic CSPs. Further the paper draws attention to competing techniques, like non-porous particle-packed columns, core-shell and monoliths as chromatographic support matrix, available for achieving fast and efficient chromatographic separation. A brief discussion on the three main classes of chiral monolithic stationary phase viz. silica, organic polymer and hybrid-based monolithic stationary phases is also presented. In addition, the paper highlights various studies on the application of monolith chiral CSPs in LC and capillary electrochromrography separation and analysis of chiral compounds.
Chiral inversion of pharmaceutical drugs 
2-Arylpropionic acid nonsteroidal anti-inflammatory drugs (NSAIDs) provide one of the most demonstrated pharmaceutical examples of chiral inversion. Chiral inversion depends on various factors (viz. biological-, solvent-, light-, temperature-induced, etc.) and the energy barrier associated with the stereogenic element present in the chiral molecule. The pharmacological properties of chiral drugs depend on the activity of one enantiomer or both the enantiomers targeting different biological targets. Consequently, chiral inversion can alter the biological activities of the pharmaceutical drug. Hence a better understanding of chiral inversion, factors facilitating such inversion, and the tools employed to determine chiral inversion are of great significance from a pharmacological and toxicological perspective.
Impact of cyclofructan derivatives as efficient chiral selector in chiral analysis: An
overview
The development of chiral selectors for the separation and analysis of chiral molecules has been an evolving process happening over three decades, since the introduction of the first chiral stationary phase (CSP) in 1938. The main impetus for designing new chiral selectors is to get to most promising one which has a broad chiral recognition property, separation capability for a wide range of chiral analytes, and the cost-effective CSP, which is also a major concern. Today, we have more than 100 commercially available CSPs, and these are prepared by coating or immobilizing the classical chiral selectors on to the chromatographic support, normally, silica gel. The purpose of this review is to look at progress and the impact of cyclofructan derivatives, a novel chiral selector introduced recently, for performing chiral analysis.
Enantiopurity Assessment of Chiral Switch of Ondansetron by
Direct Chiral HPLC
A simple, selective, robust, and rapid enantiospecific HPLC method was developed and optimized for simultaneous determination of ondansetron enantiomers. The chiral separation was performed on Chiralpak AS-3R analytical column (150 mm x 4.6 mm i.d., 3 µm). AD-optimal mixture design methodology was employed to evaluate the influence of solvent mixtures on retention factor of first peak (k1), resolution between enantiomers (Rs1,2) and runtime (tR2). Solvent mixtures are delivered at 1.5 mL min-1 flow rate, and enantiomeric peaks were detected at 222 nm. Experiments were carried out, and results were analyzed by the two-component mix plot graph of the design software. The mobile phase containing methanol/water/diethylamine (85/15/0.1% v/v/v) leads to a best possible combination adequate retention (k1 = 1.4), enantiomeric resolution (Rs1,2 = 2.9) in shorter runtime (3.5 min). The proposed method was validated according to ICH guidelines and found to be linear, sensitive, selective, precise, and accurate. Furthermore, the pertinence of this developed method was established by analyzing two commercially available tablets: Emeset-8 (racemic mixture) and Zordil-4 (R-ondansetron). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good % recovery and %CV. The study resulted in a better chromatographic system for chiral impurity profiling of ondansetron chiral switch.
A novel, selective and robust enantiospecific HPLC method was developed for simultaneous determination of amlodipine and atenolol enantiomers. Box-Behnken design was employed to identify the effect of factors (% ethanol, % diethylamine and flow rate) and their interactions on enantioresolution and analysis time. Chromatography was performed using mobile phase comprising acetonitrile, ethanol and DEA (92:8:0.2% v/v/v) delivered at a flow rate of 1.2 mL min-1 on a Lux Cellulose-4 column. The enantiomers were monitored at a wavelength of 240 nm and separation was achieved within 8 min. The method was validated in terms of specificity, linearity, accuracy, precision, limit of detection and quantification. The method was found to be linear (R2 = 0.991), accurate (99.8-101.4%) and precise (%RSD = 3%). Additionally, fractional factorial design was used to evaluate the robustness of the method and non-significant intervals for mixture related factors were established using contour profiling. Furthermore, the pertinence of this validated method was established by analyzing three different commercially available formulations. The obtained results confirmed that the proposed method can be extended for routine enantiopurity assay of amlodipine and atenolol in pharmaceutical formulations.
In this paper, a simple, robust and selective liquid chromatographic method was developed and validated for the analysis of warfarin and its process-related impurities. The chromatographic separation was achieved on Phenomenex Gemini C18 column by employing mobile phase comprising water:acetonitrile:acetic acid-41:59:0.1 % v/v at 1.1 mL min-1 flow rate. Factorial design with multi-criteria decision-making approach was utilized to evaluate the effects of quantitative factors (aqueous content, flow rate and types of acid additive) over selected qualitative responses to achieve a compromise between resolution and analysis time. Finally, the method was validated according to ICH Q2(R1) guidelines and successfully applied for determination of warfarin and its two process-related impurities in commercially available tablets. The robustness of the method was also studied using fractional factorial design and robust domains were established using contour profiling. The proposed method separated warfarin and its related impurities within a short run time of 5 min thereby enabling the developed method cost-effective than the reported methods.
The aim of the study was to develop a new HPLC method for direct chiral separation of Ofloxacin enantiomers using polar non-aqueous mobile phase by application of response surface methodology. Rotatable central composite design (CCD) with eight factorial points, six axial points and six replications in central point was used to evaluate the influence of three independent variables (concentration of methanol, diethylamine and flow rate) on the output responses (capacity factor of first peak, tailing factors of both the enantiomers, resolution between the Ofloxacin enantiomers, retention time of the last peak and chromatographic optimization function). Further, CCD data were combined with multiple response optimization in order to obtain a set of optimal experimental conditions (% methanol/hexane/acetonitrile-43.33/10/46.62 (v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as 1.4 mL min-1) leading to the most desirable compromise between resolution and analysis time. The method demonstrated good correlation between observed and predicted responses. The developed method was validated according to ICH guidelines and applied for quantitative analysis of two commercially available tablets Zenoflox (Ofloxacin) and Glevo (Levofloxacin). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good %recovery and %CV. The study resulted in a better chromatographic system for the determination of Ofloxacin enantiomers.
Warfarin is a well-known anticoagulant agent that occurs in two enantiomers, [(R)-(+)-warfarin and (S)-(-)-warfarin], in which 4-hydroxycoumarin (Imp. A) and benzalacetone (Imp. B) are commonly found as impurities. Due to the lack of analytical reports for the simultaneous estimation on warfarin and its impurities in bulk drug and pharmaceuticals, we aim at the simultaneous estimation and optimization of the chromatographic separation of Warfarin and its related substances employing experimental design. Central composite design was employed to evaluate the influence of two independent variables (concentration of organic modifier and flow rate) on the output responses: capacity factor (k1), resolution of the peak (Rs, 3, 4), and retention time of the last peak (tR5), as well as to model these responses.. Further, the central composite design results were combined in a multicriteria decision- making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.
This present study describes a specific, precise, sensitive and accurate method for simultaneous determination of hydroxyzine, loratadine, terfenadine, rupatadine and their main active metabolites cetirizine, desloratadine and fexofenadine in serum and urine by using meclizine as an internal standard. Solid phase extraction method for sample clean-up and preconcentration of analytes was carried out by using Phenomenex Strata-X-C and Strata X polymeric cartridges. Chromatographic analysis was performed on a Phenomenex cyano (150 mm x 4.6 mm i.d., 5 µm) analytical column. A D-optimal mixture design methodology was used to evaluate the effect of changes in mobile phase compositions on dependent variables and optimization of the response of interest. The mixture design experiments were performed and results were analyzed. The region of ideal mobile phase composition consisting of acetonitrile-methanol-ammonium acetate buffer (40 mM; pH 3.8 adjusted with acetic acid): 18/36/46% v/v/v respectively was identified by a graphical optimization technique using an overlay plot. While using this optimized condition all analytes were baseline resolved in less than 10 min. Solvent mixtures are delivered at 1.5 mL min-1 flow rate and analytes peaks were detected at 222 nm. The proposed bioanalytical method was validated according to USFDA-CDER guidelines. The proposed method was sensitive with detection limits of 0.06-0.15 µg mL-1 in serum and urine samples. %RSD for inter and intra-day precision data was found to be less than 7%. The proposed method may find application in the determination of the selected antihistaminic drugs in biological fluids.
Systematic Robustness Testing of a Liquid Chromatographic Method: A Case Study
Robustness testing of a method plays a crucial role in establishing its reliability. It examines the potential sources of variability in one or more responses of the proposed method. In this study, the robustness testing of a method proposed for simultaneous determination of warfarin and its two process related impurities was evaluated by using two level, fractional factorial design. Factors that are sensitive to a variation during method transfer were selected as independent variables [aqueous content (range: 39-43%, v/v), concentration of acetic acid (range: 0.08-0.12%, v/v), flow rate (range: 0.93-1.33 mL/min), and wavelength (range: 218-222 nm)]. Variables that determine the quality of separation, viz., retention factor of the first peak, resolution between the critical peak pair, tailing factor of warfarin, and total analysis time were selected as responses. Robustness was assessed by graphical (half normal probability and Pareto plots) and statistical (analysis of variance) methods. It was found that, among the studied variables, aqueous content had a significant effect on capacity factor and analysis time. Furthermore, non-significant intervals for significant factors were established by contour profiling. This study demonstrated the significance of experimental design and other statistical tools in understanding the effects of investigating factors of the chromatographic system and in defining their limits.
In this paper, a simple, robust and selective liquid chromatographic method was developed and validated for the analysis of warfarin and its process-related impurities. The chromatographic separation was achieved on Phenomenex Gemini C18 column by employing mobile phase comprising water:acetonitrile:acetic acid-41:59:0.1 % v/v at 1.1 mL min-1 flow rate. Factorial design with multi-criteria decision-making approach was utilized to evaluate the effects of quantitative factors (aqueous content, flow rate and types of acid additive) over selected qualitative responses to achieve a compromise between resolution and analysis time. Finally, the method was validated according to ICH Q2(R1) guidelines and successfully applied for determination of warfarin and its two process-related impurities in commercially available tablets. The robustness of the method was also studied using fractional factorial design and robust domains were established using contour profiling. The proposed method separated warfarin and its related impurities within a short run time of 5 min thereby enabling the developed method cost-effective than the reported methods.
Loxoprofen belongs to a class of Nonsteroidal anti-inflammatory drug acts by inhibiting isoforms of cyclo-oxygenase 1 and 2. In this study an improved RP-HPLC method was developed for the quantification of loxoprofen in pharmaceutical dosage form. For that purpose an experimental design approach was employed. Factors-independent variables (organic modifier, pH of the mobile phase and flow rate) were extracted from the preliminary study and as dependent variables three responses (loxoprofen retention factor, resolution between loxoprofen probenecid and retention time of probenecid) were selected. For the improvement of method development and optimization step, Derringer's desirability function was applied to simultaneously optimize the chosen three responses. The procedure allowed deduction of optimal conditions and the predicted optimum was acetonitrile: water (53:47, v/v), pH of the mobile phase adjusted at to 2.9 with ortho phosphoric acid. The separation was achieved in less than 4minutes. The method was applied in the quality control of commercial tablets. The method showed good agreement between the experimental data and predictive value throughout the studied parameter space. The optimized assay condition was validated according to International conference on harmonisation guidelines to confirm specificity, linearity, accuracy and precision.
Developed and optimized a validated isocratic reverse phase HPLC separation of Rosuvastatin, Telmisartan, Ezetimibe and Atorvastatin in pharmaceutical preparation using response surface methodology. The separation was carried out by using phenomenex C18 column (15 cm x 4.6 mm id, 5 micron particle size) and UV detection at 239 nm. The ranges of the independent variables used for the optimization were MeCN: 33-38%, buffer conc.: 10-20 mM and flow rate: 1-2 ml/min. The influence of these independent variables on the ...
This paper deals with optimization of a liquid-liquid extraction procedure for simultaneous HPLC analysis of domperidone and pantoprazole in human plasma. Central composite design and Derringer's desirability function were used to optimize the concentration of KOH and the volume of ethyl acetate as the main factors affecting the liquid-liquid extraction procedure. After extraction, the analytes were separated quantitatively on a C 18 column with 10 mM pH 7.0 phosphate buffer-methanol-acetonitrile 48.46:20:31.54 ( u / u ) as mobile phase at a flow rate of 1.20 mL min -1 and with UV detection at 285 nm. It was concluded that extraction recovery of both the analytes was affected by KOH concentration and that recovery of pantoprazole was affected by ethyl acetate (extraction solvent) volume. Extraction recovery under optimum extraction conditions was 93.52% for domperidone and 92.72% for pantoprazole. The optimized extraction method was validated. Linearity was established for six levels in the ranges 10-1000 ng mL -1 for pantoprazole and 15-1000 ng mL -1 for domperidone. The lower limit of quantitation (LLOQ) and detection (LOD) were estimated as 9.84 and 5.91 ng mL -1 , respectively, for pantoprazole and 14.56 and 8.79 ng mL -1 for domperidone. The optimized method was linear, specific, accurate, and precise; the high recovery (>92%) and low relative standard deviation (< 2.5%) enable reliable quantification of these analytes in spiked human plasma.
A stability-indicating HPLC method has been developed and subsequently validated for the simultaneous determination of domperidone and pantoprazole in commercial tablets. The proposed HPLC method utilizes Phenomenex ® Gemini C 18 column (150 mm x 4.6 mm id, 5 µm) and mobile phase consisting of methanol-acetonitrile-20 mM dipotassium hydrogen phosphate and phosphoric acid buffer pH 7.0 (20: 33: 47, v/v/v) at a flow rate of 1.19 mL min- 1. Quantitation was achieved with UV detection at 285 nm ...
Statistical experimental design and Derringer's desirability function were applied to develop an improved RP-HPLC method for the simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations. Four independent factors were considered: acetonitrile content in the mobile phase; buffer pH; buffer concentration; and flow rate. The preliminary screening step was carried out, according to a 24-1 fractional factorial design, to identify the significant factors affecting the analysis time response. Then central composite design was applied for a response surface study, in order to examine in depth the effects of the most important factors. Subsequently, Derringer's desirability function was employed to simultaneously optimize the six responses: retention factor of first peak; two resolutions; and three retention times, each having a different target. This procedure allowed deduction of two separate optimum conditions, intended for the analysis of quality control and plasma samples, within the experimental domain. The predicted optimum for the quality control samples was: methanol-acetonitrile-15 mM K2HPO4 buffer (pH 5.33) (10:42.08:47.92, v/v/v) as the mobile phase and 1.12 mL/min as the flow rate. The method using this optimized condition showed higher sensitivity and shorter analysis time than the previously published reports. The optimized assay condition was validated according to International Conference on Harmonization guidelines.
This paper deals with multiple response simultaneous optimization using the Derringer's desirability function for the development of a reversed-phase HPLC method for the simultaneous determination of domperidone and pantoprazole in commercial pharmaceutical preparations. Twenty experiments, taking the retention factor of the first peak, the two resolutions, and three retention times as the responses with three important factors, mobile phase composition, buffer molarity and flow rate, were used to design ...
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous determination of domperidone and pantoprazole in capsules. The compounds were separated on an ODS analytical column with a mixture of methanol, acetonitrile, and triethylamine solution (10 mM, pH 7.0±0.05 adjusted with 85% phosphoric acid) in the ratio 20: 33: 47 (v/v) as mobile phase at a flow rate of 1.0 mL min- 1. UV detection was performed at 285 nm. The method was ...
A reversed-phase high-performance liquid chromatographic method for separation of the enantiomers of ketoprofen in formulations and in plasma matrices has been developed and optimized. A central composite design was used to develop response models and Derringer's desirability function was then used for simultaneous optimization of chiral resolution and analysis time. This procedure enabled discovery of two separate sets of optimum conditions for analysis of quality-control samples and plasma samples within the ...
Summary The present study was undertaken to evaluate the possibility of chiral discrimination in the release of enantiomers of ketoprofen (KT) in tablet form and, in turn, the bioavailability of the individual enantiomers, using the rabbit as a model. The enantiomeric concentration of KT in plasma was determined using a customized chiral HPLC analysis. First the plasma concentration-time profile was established for pure (±) KT in order to assess the extent of chiral discrimination. Subsequently the tablet formulation (Rhofenid-100mg) ...