Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend^®) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min⁻¹. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and p^H; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and p^H have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control.     

Validation of an in-line Raman spectroscopic method for continuous active pharmaceutical ingredient quantification during pharmaceutical hot-melt extrusion

A calibration model for in-line API determination was developed based on Raman spectra collected during hot-melt extrusion. This predictive model was validated by calculating the accuracy profile based on the analysis results of validation experiments. Furthermore, based on the data of the accuracy profile, the measurement uncertainty was determined. Finally, the robustness of the model was evaluated.     

The application of experimental design to the robustness testing of a method for the determination of drug-related impurities by capillary electrophoresis

Summary Robustness testing of capillary electrophoresis methods is an important part of method validation. Appropriate use of experimental designs can be employed in this robustness testing. In this study experimental designs were used in the assessment of a capillary electrophoresis method used to determine drug related impurities. Initially a fractional factorial screening design was used to identify the critical parameters which were found to be pH and voltage. A central composite design was then performed to evaluate the response surfaces for pH and voltage which showed operation at the optimum pH and voltage values.     

Rational experimental design for bioanalytical methods validation illustration using an assay method for total captopril in plasma

Generally, bioanalytical chromatographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess the specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control /dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/ dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/ dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.     

Factor analysis of experimental design in chromatography

Summary Experimental design and factor analysis have been applied to the optimization of the separation of two series of compounds. The behavior of the nonhomogeneous series of test compounds appears to be ideal whereas the second series of congeneric substituted naphthalenes exhibit non-ideal behavior. Factor analysis maps exhibit the projection of the experiment design, on the first factorial plan, as a ternary graph for the ideal model. In this instance the linear combination of the eluents is preserved through the factor analysis projection. For the non-ideal model, the projections of the experiment design do not restore the ternary graph. Competitive interactions with ternary or quaternary eluents are clearly apparent. The simultaneous application of experimental design and factor analysis improve the optimization procedure. With the ideal model, the separation optimum can be reached directly through factor analysis maps. With the non-ideal model, the projection of eluents and their contribution to the inertia explained by the factorial axes can define a new experimental domain where maximum selectivity is obtained.Part XIV of the series Factor Analysis and Experimental Design in Chromatography. For part XIII see J. R. Chrétien, M. Righezza, A. Hassani, B. Y. Meklati,J. Chromatography,609, 261 (1992).     

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.     

Optimization and validation of a capillary zone electrophoretic method for the simultaneous analysis of four atypical antipsychotics

A capillary zone electrophoretic method has been developed and optimized for separation of four atypical antipsychotics (AAPs): clothiapine (cT), clozapine (cZ), olanzapine (O), and quetiapine (Q). A three-level full-factorial design was applied to study the effect of the pH and molarity of the running buffer on separation. Combination of the studied parameters permitted the separation of the four AAPs, which was best carried out using 80 mM sodium phosphate buffer (pH 3.5). The same system can also be applied for the quantitative determination of these compounds. The method was then validated regarding linearity, precision, and accuracy. Especially, the possibility of simultaneous quantification and identification of the active ingredient in the finished product is very attractive.     

Experimental design-based development and single laboratory validation of a capillary zone electrophoresis method for the determination of the artificial sweetener sucralose in food matrices

A capillary zone electrophoresis (CZE) method, optimised chemometrically, underwent a complete in-house validation protocol for the qualification and quantification of sucralose in various foodstuffs. Separation from matrix components was obtained in a dinitrobenzoic acid (3 mM)/sodium hydroxide (20 mM) background electrolyte with a pH of 12.1, a potential of 0.11 kV cm^–1 and a temperature of 22 °C. Detection was achieved at 238 nm by indirect UV. Screening, optimisation and robustness testing were all carried out with the aid of experimental design. Using standard addition calibration, the CZE method has been applied to still, carbonated and alcoholic beverages, yoghurts and hard-boiled candy. The method allows the detection of sucralose at >30 mg kg^–1, with a linearity range of 50–500 mg kg^–1, making it suitable for implementation of the recently amended Sweeteners for use in foodstuffs Directive (European Parliament and Council (2003) Off J L237:3–12), which set maximum usable doses of sucralose for many foodstuffs, most ranging from 200 mg kg^–1 to 450 mg kg^–1.     

Interference modelling, experimental design and pre-concentration steps in validation of the Fenton's reagent for pesticides determination

The determination of atrazine in real samples (commercial pesticide preparations and water matrices) shows how the Fenton's reagent can be used with analytical purposes when kinetic methodology and multivariate calibration methods are applied. Also, binary mixtures of atrazine-alachlor and atrazine-bentazone in pesticide preparations have been resolved. The work shows the way in which interferences and the matrix effect can be modelled. Experimental design has been used to optimize experimental conditions, including the effect of solvent (methanol) used for extraction of atrazine from the sample. The determination of pesticides in commercial preparations was accomplished without any pre-treatment of sample apart from evaporation of solvent; the calibration model was developed for concentration ranges between 0.46 and 11.6 × 10⁻⁵ mol L⁻¹ with mean relative errors under 4%. Solid-phase extraction is used for pre-concentration of atrazine in water samples through C₁₈ disks, and the concentration range for determination was established between 4 and 115 μg L⁻¹approximately. Satisfactory results for recuperation of atrazine were always obtained.     

Development and Validation of a LC Method for the Determination of Pramipexole Using an Experimental Design

A rapid and sensitive RP-HPLC method with UV detection for routine control of pramipexole in tablets was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile/phosphate buffer (60/40; v/v) with a flow rate of 0.8 mL min⁻¹. Quantitation was accomplished with the internal standard method; the procedure was validated by linearity (correlation coefficient = 0.99892), accuracy, robustness and intermediate precision. Limit of quantitation and limit of detection were found to be 4.5 μg and 1.4 μg respectively, which indicates the method is highly sensitive. Experimental design was used during validation to calculate method robustness and intermediate precision, for robustness test three factors were considered; percentage v/v of acetonitrile, flow rate and pH; an increase in the flow rate results in a decrease of concentration found of the drug, while the percentage of organic modifier and temperature have no important effect on the response. For intermediate precision measure the considered variables were: analyst, equipment, days and obtained RSD value (0.56%, n=24) which indicated a good precision of the analytical method. The method was found to be applicable for determination of the drug in tablet formulations and the results of the developed method were compared with those of the UV spectrophotometric method to access the active pramipexole content. Revised: 13 March and 25 April 2006     

Optimum extraction process of polyphenols from Bridelia grandis stem bark using experimental design

Euphorbiaceae barks are known to contain an appreciable amount of polyphenolic compounds responsible for several biological activities. Preliminary extraction from Bridelia grandis stem bark afforded high content of polyphenols, determined by spectrophotometric methods such as Folin–Ciocalteu (for total phenols, TP) and n‐butanol‐HCl (for condensed tannins, CT). A preliminary Plackett–Burman screening design was used to identify the key factors that influence the TP and CT extraction. Between all the variables known to influence the extraction from vegetable matrixes, six were selected; maceration was chosen as traditional extraction methodology. To investigate the effect of solvents and extraction method, methanol, acetone 70% (v/v in water), centrifugation and ultrasound were chosen. A full factorial design 2³ was applied to optimize the extraction procedure. The responses were obtained analyzing the extracts for their TP and CT contents determined by the above‐mentioned spectrophotometric methods. The results confirm that, within the explored domain, the optimum solvent is methanol and the optimum method is one‐cycle centrifugation. Finally, it was also compared with the effect of maceration on the considered responses. It has never given results better than centrifugation, whereas in the case of CT it represents an advantage to employ a three‐cycle centrifugation instead of one.     

Optimization of in situ derivatization SPME by experimental design for GC-MS multi-residue analysis of pharmaceutical drugs in wastewater

This paper presents the development of a procedure, which enables the analysis of nine pharmaceutical drugs in wastewater using gas chromatography‐mass spectrometry (GC‐MS) associated with solid‐phase microextraction (SPME) for the sample preparation. Experimental design was applied to optimize the in situ derivatization and the SPME extraction conditions. Ethyl chloroformate (ECF) was employed as derivatizing agent and polydimethylsiloxane‐divinylbenzene (PDMS‐DVB) as the SPME fiber coating. A fractional factorial design was used to evaluate the main factors for the in situ derivatization and SPME extraction. Thereafter, a Doehlert matrix design was applied to find out the best experimental conditions. The method presented a linear range from 0.5 to 10 μg/L, and the intraday and interday precision were lower than 16%. Applicability of the method was verified from real influent and effluent samples of a wastewater treatment plant, as well as from samples of an industry wastewater and a river.     

Automated ion-selective measurement of lithium in serum. A practical approach to result-level verification in a two-way method validation

 The Quality Assurance Department of Medix Diacor Labservice evaluated a two-way method validation procedure for serum lithium quantification in therapeutic drug monitoring In the process of a company fusion and rationalization of two considerably large production lines, three independent ion-selective electrode (ISE) methods were surveyed, among many others. While tailoring the new medical laboratory production, subcontracting from a collaborating company was discontinued. Likewise, modernization of the ISE instrumentation was unavoidable to increase throughput and effectiveness. It was important that the new result levels should be comparable both with the former subcontractor's levels and with the levels reported from the previously existing instrumentation. The aim of this study was to evaluate the most crucial performance characteristics of a novel lithium method in comparison to the two ISE test methods being withdrawn. The standardized lithium test method was inspected in terms of linear measurement range, analytical variation, bias, past and on-going proficiency testing, in addition to method comparison, to achieve the desired analytical goals. Fulfilling the accreditation requirements in terms of the introduced method validation parameters is discussed. Received: 19 April 2000 / Accepted: 26 July 2000     

Determination of pharmaceuticals in drinking water by CD-modified MEKC: separation optimization using experimental design

A suite of 12 widely used pharmaceuticals (ibuprofen, diclofenac, naproxen, bezafibrate, gemfibrozil, ofloxacin, norfloxacin, carbamazepine, primidone, sulphamethazine, sulphadimethoxine and sulphamethoxazole) commonly found in environmental waters were separated by highly sulphated CD-modified MEKC (CD-MEKC) with UV detection. An experimental design method, face-centred composite design, was employed to minimize run time without sacrificing resolution. Using an optimized BGE composed of 10 mM ammonium hydrogen phosphate, pH 11.5, 69 mM SDS, 6 mg/mL sulphated beta-CD and 8.5% v/v isopropanol, a separation voltage of 30 kV and a 48.5 cm x 50 microm id bare silica capillary at 30 degrees C allowed baseline separation of the 12 analytes in a total analysis time of 6.7 min. Instrument LODs in the low milligram per litre range were obtained, and when combined with offline preconcentration by SPE, LODs were between 4 and 30 microg/L.     

Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 microm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.     

Rapid and quantitative evaluation of the effect of process variables on the kinetics of photocatalytic degradation of phenol using experimental design techniques and parallel factor (PARAFAC) analysis

A 2³ factorial design has been used to analyze the effect of pH, the nature of the catalyst, and the concentration of the substrate on the rate constant of the photodegradation reaction of phenol. The main effects of the considered variables and their interaction are discussed. The significance of the effects has been corroborated using an ANOVA test. The values of phenol concentrations, used to calculate the rate constant, and the concentrations of intermediates were obtained by applying parallel factor (PARAFAC) analysis to the data obtained from monitoring the process by means of excitation–emission fluorescence (EEM). The proposed methodology, which combines experimental design and multivariate techniques, is a rapid alternative for study of chemical kinetics.     

Optimization of a liquid chromatography ion mobility-mass spectrometry method for untargeted metabolomics using experimental design and multivariate data analysis

High-resolution mass spectrometry coupled with pattern recognition techniques is an established tool to perform comprehensive metabolite profiling of biological datasets. This paves the way for new, powerful and innovative diagnostic approaches in the post-genomic era and molecular medicine. However, interpreting untargeted metabolomic data requires robust, reproducible and reliable analytical methods to translate results into biologically relevant and actionable knowledge. The analyses of biological samples were developed based on ultra-high performance liquid chromatography (UHPLC) coupled to ion mobility - mass spectrometry (IM-MS). A strategy for optimizing the analytical conditions for untargeted UHPLC-IM-MS methods is proposed using an experimental design approach. Optimization experiments were conducted through a screening process designed to identify the factors that have significant effects on the selected responses (total number of peaks and number of reliable peaks). For this purpose, full and fractional factorial designs were used while partial least squares regression was used for experimental design modeling and optimization of parameter values. The total number of peaks yielded the best predictive model and is used for optimization of parameters setting.     

Development and validation of a generic high‐performance liquid chromatography for the simultaneous separation and determination of six cough ingredients: Robustness study on core–shell particles

A generally applicable high‐performance liquid chromatographic method for the qualitative and quantitative determination of pharmaceutical preparations containing phenylephrine hydrochloride, paracetamol, ephedrine hydrochloride, guaifenesin, doxylamine succinate, and dextromethorphan hydrobromide is developed. Optimization of chromatographic conditions was performed for the gradient elution using different buffer pH values, flow rates and two C₁₈ stationary phases. The method was developed using a Kinetex® C₁₈ column as a core–shell stationary phase with a gradient profile using buffer pH 5.0 and acetonitrile at 2.0 mL/min flow rate. Detection was carried out at 220 nm and linear calibrations were obtained for all components within the studied ranges. The method was fully validated in agreement with ICH guidelines. The proposed method is specific, accurate and precise (RSD% < 3%). Limits of detection are lower than 2.0 μg/mL. Qualitative and quantitative responses were evaluated using experimental design to assist the method robustness. The method was proved to be highly robust against 10% change in buffer pH and flow rate (RSD% < 10%), however, the flow rate may significantly influence the quantitative responses of phenylephrine, paracetamol, and doxylamine (RSD% > 10%). Satisfactory results were obtained for commercial combinations analyses. Statistical comparison between the proposed chromatographic and official methods revealed no significant difference.