Development of a capillary zone electrophoretic method to determine six antidepressants in their pharmaceutical preparations. Experimental design for evaluating the ruggedness of method

A capillary zone electrophoresis method is proposed for the separation of six of the antidepressants most used for the treatment of mental illness (clomipramine, paroxetine, fluoxetine, citalopram, fluvoxamine, and trazodone). Optimum conditions for their separation were investigated. A background electrolyte solution consisting of 50 mM phosphate buffer adjusted to pH 2.0, hydrodynamic injection, and 25 kV as separation voltage were used. Relative standard deviations (RSD) were <0.38% and <2.93% for migration time and corrected peak area (n = 24), respectively. Detection limits obtained for the six antidepressants ranged from 0.03 to 0.11 mg L(-1). Stability of the solutions, linear concentration range, accuracy, and precision were examined during validation of the method. A ruggedness test of this method was performed using the fractional factorial model of Plackett-Burman, requiring in our case design of a matrix of 15 experiments, in which the influence of seven factors at three different levels was tested on different electrophoretic results: efficiency; resolution; and corrected peak area. Statistical evaluation of electrophoretic results was achieved by Youden and Steiner's method. The described method is rapid, sensitive, and rugged and it was tested for the pharmaceutical formulation analysis, giving recoveries between 95.6 and 99.1% with respect to the nominal content.     

Comparative study of robustness between micellar electrokinetic capillary chromatography and high-performance liquid chromatography using one-variable-at-a-time and a new multi-variable-at-a-time approach

Micellar electrokinetic capillary chromatography and reverse-phase liquid chromatography methods were developed in order to perform robustness testing to determine the caffeine content in beverages. Both methods were fully validated and two different robustness approaches were applied. One-variable-at-a-time (OVAT) approach at eleven levels (0; ±1; ±2; ±3; ±4; ±5 units) was carried out and compared with multi-variable-at-a-time (MVAT) approach at three levels (±1 unit per investigated parameter). Four analysts in two laboratories on two capillary electrophoresis and two RPLC equipments have tested the samples. Robustness was statistically analyzed using peak area, migration or retention time, symmetry, and resolution of caffeine and sulfacetamide as internal standard, and presented as R.S.D. values. The RPLC method was found to be more sensitive than the MEKC method. Both methods showed acceptable robustness level for OVAT approach, whilst MEKC method was more robust when the determination of real samples coming from different beverages was based on the MVAT approach.     

Method development and validation for the simultaneous determination of five selective serotonin reuptake inhibitors by capillary zone electrophoresis

Summary A capillary zone electrophoresis method is proposed for the separation of five antidepressants. Optimum conditions for the separation were investigated. A background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.5, hydrodynamic injection, and a 28 kV separation voltage were used. Relative standard deviations (RSD) were <0.9% and <1.7% for migration time and corrected peak area (n=21), respectively. The detection limits for the five antidepressants ranged from 0.3 to 0.7 mg L⁻¹. The stability of the solutions, linearity, accuracy, and precision were examined during validation of the method. The method is rapid and sensitive, when it was tested for the analysis of pharmaceutical formulations the recoveries obtained were between 98 and 103% of the nominal content.     

Application of response surface methodologies in capillary electrophoresis

A fast method of determining ascorbic acid and isoascorbic acid by capillary zone electrophoresis with a photodiode array detector was developed. Response surface methodologies based on three-level, three-variable designs, such as the Box-Behnken design, central composite face-centered and full fractional design, were used comparatively for optimization of buffer pH, buffer concentration and operation voltage. Statistical interpretation of the variables concerning different responses, such as resolution and migration time of the last migrated analyte, were performed. The optimum conditions of these variables were predicted using a second-order polynomial model fitted to the results obtained by applying three designs. The response surface plots using three experimental designs revealed a separation optimum with Tris–HCl buffer of pH 8.5, a concentration of 50 mM, and an operation voltage of 30 kV. The significance of the statistical designs were confirmed by the generally good agreement obtained between predicted responses and actual experimental data. We concluded that experimental designs offer a rapid means of optimizing several variables and provide an efficient test for the robustness of the analytical method.     

Development and validation of a cyclodextrin‐modified capillary electrophoresis method for the enantiomeric separation of vildagliptin enantiomers

The enantiomers of vildagliptin, an orally available and selective dipeptidyl‐peptidase‐4 inhibitor used for the treatment of type II diabetes, have been separated by CD‐modified CZE, using uncoated fused‐silica capillary. After screening 13 negatively charged CD derivatives as potential chiral selectors, sulfobutyl‐ether‐α‐CD (SBE‐α‐CD) was selected for the enantioseparation. For the optimization, a factorial analysis study was performed by orthogonal experimental design. Six experimental factors were chosen as variable parameters: temperature, applied voltage, chiral selector and BGE concentrations, pH, and the parameters of the hydrodynamic injection. The optimized system still was not considered final as the second peak (S‐enantiomer) migrated too close to the EOF, resulting in a potential inaccuracy during the determination of the chiral impurity. To fine‐tune the method “one factor at a time” variation approach was applied. The final method (applying 15°C capillary temperature, 40 mbar × 4 s hydrodynamic injection, 25 kV voltage in 75 mM acetate‐Tris buffer [pH 4.75] containing 20 mM SBE‐α‐CD as chiral selector) was validated according to the ICH guideline. RSD percentage of the resolution value, migration times, and corrected peak areas were below 5% during testing repeatability and intermediate precision. LOD and LOQ values were found to be 2.5 and 7.5 μg/mL, respectively. The method is considered linear in the 7.5–180 μg/mL range for the R‐enantiomer. The robustness of the method was justified using Plackett–Burmann statistical experimental design.     

Enantiomeric determination, validation and robustness studies of racemic citalopram in pharmaceutical formulations by capillary electrophoresis

A chiral capillary electrophoresis (CE) method has been developed allowing the enantiomeric separation of racemic citalopram (R-(-) and S-(+) citalopram) using as chiral selector carboxymethyl-gamma-cyclodrextrin (CM-gamma-CD). The influence of chemical and instrumental parameters on the separation such as cyclodextrin (CD) and buffer concentrations, buffer pH, voltage, injection pressure, ..., was investigated. Good chiral separation of the racemic mixture was achieved in less than 4 min using a fused-silica capillary and as background electrolyte (BGE) a phosphate buffer solution (20 mM, pH 7) containing 0.15% (w/v) of CM-gamma-CD as chiral selector. The separation was driven in normal polarity mode at 15 degrees C, 30 kV and hydrodynamic injection. In order to validate the method, the stability of the solutions, precision (repeatability, reproducibility and F-Snedecor test), linearity (Lack of Fit and ANOVA tests) accuracy (98-101%), detection and quantitation limits (0.06 and 0.2 mg L(-1), respectively), on a selected analytical placebo, were examined. Besides, a robustness test was performed using the Plackett-Burman fractional factorial experimental design using a matrix of 15 experiments for seven factors (internal parameters) with a statistical treatment suggested by Youden and Steinner. The proposed method is fast, sensitive, inexpensive and, besides, it has been evaluated by means of an extensive validation study and an exhaustive robustness test. The scope of this validated and robust method has been proved in the analysis of four pharmaceutical formulations; two of them (recently available in Spain), which just contained S-(+)-citalopram (escitalopram) as active principle. Recoveries between 101 and 103%, with regard to their nominal contents were obtained. In the other two pharmaceutical ones, the method provided the separation and quantification of both chiral isomers in the existing racemic mixture.     

Multivariate evaluation of the separation performance in micellar electrokinetic capillary chromatography of peptides: Optimization

Summary A simultaneous optimization of resolution, efficiency and migration times of enkephalin-related peptides in micellar electrokinetic capillary chromatography (MEKC) was performed. Six experimental variables; the surfactant concentration, the percentage of organic modifier, the ionic strength of the buffer, the injected plug length, the applied temperature and the sample solution composition were studied via central composite design (CCD). Large differences in separation performance were observed at a commonly used level of organic modifier in the background electrolyte. Partial least squares regression of the responses revealed that the experimental domain was too large and complicated to be explained by the model. A new CCD model was obtained with improved prediction ability at narrower ranges of the experimental factors, especially of the organic modifier. A conflict between maximum resolution and efficiency within the shortest analysis time was observed. Therefore, constraints were set on maximal resolution and analysis time, while solving for maximum efficiency. Optimal operating conditions were found at 35 mM sodium dodecyl sulfate (SDS), 5% v/v of acetonitrile, 1.9 mm injected plug length, 35°C and sample solution with no added micelles, giving high efficiency and resolution at short analysis time. The value predicted by the model was found to agree very well to the observed values, at the optimal experimental conditions, even on a new capillary.     

Development and validation of a capillary electrophoretic method for the determination of amiodarone and desethylamiodarone

Summary A simple, sensitive and rapid capillary electrophoretic method has been developed for the separation and quantification of amiodarone and its metabolite, desethylamiodarone. The compounds were separated in a capillary of 45 cm effective length and 75 μm i.d., by use of an applied voltage of 25 kV and an electrolyte containing 15mm ADA buffer (pH 7.5), 10mm SDS, and 70% (v/v) acetonitrile. The selectivity, precision, linearity, range, sensitivity, and robustness of the method were good. The applicability of the assay was demonstrated by analyzing these drugs in serum. Electrokinetic injection with field-amplified sample-stacking was used to increase sensitivity. The limit of detection of the serum assay was 6.46 ng mL⁻¹ and the precision 3.7%.     

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.     

Reversed-phase liquid chromatography column testing: robustness study of the test

Choosing the right RPLC column for an actual separation among the more than 600 commercially available ones still represents a real challenge for the analyst particularly when basic solutes are involved. Many tests dedicated to the characterization and the classification of stationary phases have been proposed in the literature and some of them highlighted the need of a better understanding of retention properties to lead to a rational choice of columns. However, unlike classical chromatographic methods, the problem of their robustness evaluation has often been left unaddressed. In the present study, we present a robustness study that was applied to the chromatographic testing procedure we had developed and optimized previously. A design of experiment (DoE) approach was implemented. Four factors, previously identified as potentially influent, were selected and subjected to small controlled variations: solvent fraction, temperature, pH and buffer concentration. As our model comprised quadratic terms instead of a simple linear model, we chose a D-optimal design in order to minimize the experiment number. As a previous batch-to-batch study [K. Le Mapihan, Caractérisation et classification des phases stationnaires utilisées pour l'analyse CPL de produits pharmaceutiques, Ph.D. Thesis, Pierre and Marie Curie University, 2004] had shown a low variability on the selected stationary phase, it was then possible to split the design into two parts, according to the solvent nature, each using one column. Actually, our testing procedure involving assays both with methanol and with acetonitrile as organic modifier, such an approach enabled to avoid a possible bias due to the column ageing considering the number of experiments required (16 + 6 center points). Experimental results were computed thanks to a Partial Least Squares regression procedure, more adapted than the classical regression to handle factors and responses not completely independent. The results showed the behavior of the solutes in relation to their physico-chemical properties and the relevance of the second term degree of our model. Finally, the robust domain of the test has been fairly identified, so that any potential user precisely knows to which extend each experimental parameter must be controlled when our testing procedure is to be implemented.     

Uncertainty assessment from robustness testing applied on an LC assay for R-timolol and other related substances in S-timolol maleate

The robustness testing of a normal-phase liquid chromatographic (LC) method for the determination of R-timolol and other related substances in S-timolol maleate was performed applying a two-level Plackett-Burman design. Two qualitative and five quantitative factors were examined. Two types of responses were considered, qualitative, i.e. chromatographic performance criteria, and quantitative ones. The latter were taken into account to determine if the analytical procedure was robust. The quantitative responses were the contents of R-timolol in two S-timolol maleate samples. Even though some significant factor effects were observed on the qualitative responses, the R-timolol contents were not significantly different from those observed at nominal conditions, which demonstrated the robustness of the procedure.Since the experiments of the Plackett-Burman design can be assimilated to laboratories in an interlaboratory study, uncertainty can be evaluated using the robustness test data. The robustness test was set-up in such a way that the required variances could be estimated. It was shown that the robustness set-up allows estimating the reproducibility uncertainty without performing an interlaboratory study.     

Experimental design methodologies to optimize the CE separation of epinephrine enantiomers

Summary A capillary electrophoretic method using a chiral selector was optimized by experimental design for the enantioresolution of epinephrine enantiomers. Two β-cyclodextrins derivatives, namely heptakis-2,6-di-O-methyl-β-cyclodextrin and carboxy-methyl-β-cyclodextrin, respectively neutral and charged, were used as chiral selectors employing an uncoated capillary. By using a statistical experimental design in which all factors are varied at the same time, it was possible to optimize the method with regard to the resolution between peaks and the two migration times. A fractional factorial design and a central composite design were used. A compromise between conflicting goals, such as maximization of resolution and minimization of analysis time, was found by means of a desirability function D. Balancing these goals against each other, the most acceptable solution to the problem was found and the optimized method gave a fast separation with complete resolution between the adrenaline enantiomers. The response surfaces obtained confirmed the robustness of the method.     

Stacking and separation of coproporphyrin isomers by acetonitrile-salt mixtures in micellar electrokinetic chromatography

The effectiveness of the addition of salt and acetonitrile in the sample matrix to induce narrowing of the analyte zones is demonstrated for the first time in micellar electrokinetic chromatography (MEKC). Using coproporphyrin (CP) I and III isomers as test compounds, the use of sodium cholate (SC) as the micelle in the separation buffer and a high concentration of sodium chloride in the aqueous sample solution (without the presence of an organic solvent) were found to provide enhancement in peak heights for both CP I and III, but yielded very poor resolution of these two positional isomers at sample size of 10% capillary volume or larger. With the addition of acetonitrile as the organic solvent in the aqueous sample solution (acetonitrile-salt mixtures), baseline/partial resolution of CP I and III was obtained even at large injection volumes, along with significant increase in peak heights for both isomers. Possible mechanisms responsible for the narrowing of analyte zones are briefly discussed. The effects of experimental parameters, such as concentrations of salt and acetonitrile, on peak heights and resolution of the test compounds were studied. Importantly, the usefulness of the present method was demonstrated for the MEKC determination of endogenous CP I and III present in normal urine samples with good separation and detection performances.     

Robustness test of a headspace solid-phase microextraction method for the determination of chloroanisoles and chlorophenols related to cork taint in wine using experimental design

A robustness test of a solid-phase microextraction-based method optimised for the simultaneous determination of chloroanisoles and acetyl-chlorophenols implicated in the presence of corky taste in wine has been carried out using a hybrid experimental design. The influence of small changes around the nominal level of four factors (Vs/Vt ratio, extraction temperature, exposure time and sample incubation time) on the measured response were evaluated in order to indicate if the method is robust for the experimental range considered. Moreover, it was also necessary to identify the critical parameters in the validated model in order to keep them under strict control. Experimental design provides an effective approach for robustness testing as a part of the analytical method validation.     

MultiSimplex and experimental design as chemometric tools to optimize a SPE-HPLC-UV method for the determination of eprosartan in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug eprosartan from human plasma samples. MultiSimplex program was used to optimize the HPLC-UV method due to the number of experimental and response variables to be studied. The measured responses were the corrected area, the separation of eprosartan chromatographic peak from plasma interferences peaks and the retention time of the analyte.The use of an Atlantis dC18, 100 mm × 3.9 mm i.d. chromatographic column with a 0.026% trifluoroacetic acid (TFA) in the organic phase and 0.031% TFA in the aqueous phase, an initial composition of 80% aqueous phase in the mobile phase, a stepness of acetonitrile of 3% during the gradient elution mode with a flow rate of 1.25 mL/min and a column temperature of 35 ± 0.2 °C allowed the separation of eprosartan and irbesartan used as internal standard from plasma endogenous compounds. In the solid phase extraction procedure, experimental design was used in order to achieve a maximum recovery percentage. Firstly, the significant variables were chosen by way of fractional factorial design; then, a central composite design was run to obtain the more adequate values of the significant variables. Thus, the extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer pH 2 as conditioning agent, a drying step of 10 min, a washing step with methanol-phosphate buffer (20:80, v/v) and methanol as eluent liquid. The SPE-HPLC-UV developed method allowed the separation and quantitation of eprosartan from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Ruggedness and robustness of conversion factors in method of simultaneous determination of multi-components with single reference standard

Single standard to determine multi-components (SSDMC) is a novel and rational method for quality control of botanical products and traditional Chinese medicines (TCMs). However, it is restricted to wide application due to unknown fluctuation in conversion factors when it is performed in different laboratories. To evaluate the fluctuations of conversion factors, we selected Salvia miltiorrhiza as an example to determine three components of tanshinones by SSDMC method. Then ruggedness and robustness test were adopted to comprehensively investigate three kinds of factors that may influence stability of conversion factors, which were related with environmental parametric variables, operational parametric variables and peak measurement parametric variables. Nested-factorial-design was used to perform ruggedness tests. One-variable-at-a-time (OVAT) procedure and Plackett-Burman (PB) design were both used in robustness test. The results showed that stability of conversion factors was principally related with accuracy of wavelength of UV detector, peak measurement parameters and concentration of standard solution. The acceptable range of conversion factors was obtained from robustness test. Our results showed that conversion factors were inevitable to change, but when key parameters were well controlled, the range of its fluctuation was acceptable and the SSDMC method could be used widely in different laboratories.     

Method development and validation for optimised separation of salicylic, acetyl salicylic and ascorbic acid in pharmaceutical formulations by hydrophilic interaction chromatography and response surface methodology

This paper introduces a design of experiments (DOE) approach for method optimisation in hydrophilic interaction chromatography (HILIC). An optimisation strategy for the separation of acetylsalicylic acid, its major impurity salicylic acid and ascorbic acid in pharmaceutical formulations by HILIC is presented, with the aid of response surface methodology (RSM) and Derringer's desirability function. A Box-Behnken experimental design was used to build the mathematical models and then to choose the significant parameters for the optimisation by simultaneously taking both resolution and retention time as the responses. The refined model had a satisfactory coefficient (R2>0.92, n=27). The four independent variables studied simultaneously were: acetonitrile content of the mobile phase, pH and concentration of buffer and column temperature each at three levels. Of these, the concentration of buffer and its cross-product with pH had a significant, positive influence on all studied responses. For the test compounds, the best separation conditions were: acetonitrile/22 mM ammonium acetate, pH 4.4 (82:18, v/v) as the mobile phase and column temperature of 28°C. The methodology also captured the interaction between variables which enabled exploration of the retention mechanism involved. It would be inferred that the retention is governed by a compromise between hydrophilic partitioning and ionic interaction. The optimised method was further validated according to the ICH guidelines with respect to linearity and range, precision, accuracy, specificity and sensitivity. The robustness of the method was also determined and confirmed by overlying counter plots of responses which were derived from the experimental design utilised for method optimisation.     

Multivariate optimisation and validation of a capillary electrophoresis method for the analysis of resveratrol in a nutraceutical

A rapid and simple method based on capillary electrophoresis was developed for the quality control of nutraceuticals containing resveratrol. Setting the UV detector at 280nm, the optimisation involved the separation of 11 effervescent tablet components, including the active compounds vitamin C, vitamin B(2), flavanones and hydroxycinnamic acids. Flufenamic acid was employed as internal standard. The effects of background electrolyte concentration, acetonitrile percentage and voltage were investigated by means of response surface methodology, considering as responses the critical resolution values and analysis time. The optimum conditions were found by Derringer desirability function. The background electrolyte consisted of 23mM borate buffer, adjusted to pH 10.0 with 1M sodium hydroxide, containing 7% (v/v) acetonitrile. Temperature and voltage were set at 25 degrees C and 26kV, respectively. Applying these conditions, the analysis time was below 7min. The performances of the method were tested in terms of selectivity, robustness, linearity and range, accuracy and precision and system suitability, following ICH guidelines.     

Optimization of the micellar electrokinetic capillary chromatographic determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine using experimental design and radial basis function neural network

Orthogonal design has been applied to the optimization of separation and determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine by micellar electrokinetic capillary chromatography. Operational variables, such as the voltage, micelle concentration, buffer concentration and pH were optimized. Their different effects on peak resolution were studied by the experimental design method. Optimized separation conditions were obtained and successfully applied to the separation and determination of dauricine and daurisoline in real samples. The proposed method allows alkaloids in real samples to be determined within 15 min using a buffer system composed of 25 mM HAc, 25 mM NaAc and 2% polyoxyethylene sorbitan monolaurate (Tween-20) (pH 4.5). In addition, a radial basis function neural network with a “4-18-1” structure was developed based on the experimental results of orthogonal design and uniform design, and applied to the prediction of peak resolution of dauricine and daurisoline under the optimum separation conditions given by orthogonal design. The predicted results were in good agreement with the experimental values, indicating that radial basis function neural network may be a potential method for the selection of separation conditions in capillary electrophoresis.