Use of response surface methodology for development of new microwell-based spectrophotometric method for determination of atrovastatin calcium in tablets
© Wani et al.; licensee Chemistry Central Ltd. 2012
Received: 9 September 2012
Accepted: 6 November 2012
Published: 12 November 2012
Response surface methodology by Box–Behnken design employing the multivariate approach enables substantial improvement in the method development using fewer experiments, without wastage of large volumes of organic solvents, which leads to high analysis cost. This methodology has not been employed for development of a method for analysis of atorvastatin calcium (ATR-Ca).
The present research study describes the use of in optimization and validation of a new microwell-based UV-Visible spectrophotometric method of for determination of ATR-Ca in its tablets. By the use of quadratic regression analysis, equations were developed to describe the behavior of the response as simultaneous functions of the selected independent variables. Accordingly, the optimum conditions were determined which included concentration of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), time of reaction and temperature. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The method was validated, in accordance with ICH guidelines for accuracy, precision, selectivity and linearity (r² = 0.9993) over the concentration range of 20–200 μg/ml. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision.
The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, environmentally friendly "Green" approach) and reduction in the analysis cost by 50-fold.
KeywordsResponse surface methodology Atorvastatin Validation Optimization Tablets
Response surface methodology (RSM) is a statistical technique used for the development and optimization of complex processes [3, 4]. It was selected and used to find the optimum spectrophotometric conditions for analysis of atorvastatin. The technique has several advantages over conventional experimental or optimization methods in which one variable at a time is used. RSM provides a large amount of information and is more economical approach because a small number of experiments are performed for monitoring the interaction of the independent variables on the response. In conventional optimization, the increase in the number of experiments necessary to carry out the research, leads to an increase in time and expenses as well as an increase in the utilization of reagents and materials for experiments. The equation of the model easily clarifies the effects for binary combinations of the independent variables. Many types of response surface designs are used for optimization like Central composite, Doehlert, and Box–Behnken. Box–Behnken design is preferable to the Central composite and Doehlert designs because it requires fewer test runs and is rotatable. A design is rotatable only when the experiments are roughly situated on a (hyper) sphere. By adequate selection of the number of centre points, it is possible to arrange that the precision of the response of a predicted design is similar over the whole domain. Such a design is said to have uniform precision.
Box–Behnken design is advantageous because it does not contain any points at the extremes of the cubic region created by the two-level factorial level combinations [3–5]. In the present investigation the Box Behnken design was selected and used to optimize, validate and analyze the atorvastatin spectrophotometrically, because the design provides three levels for each factor and requires fewer runs in the three-factor case than Central composite and Doehlert design.
In general, UV-visible spectrophotometry is the most widely used technique in pharmaceutical analysis because of its inherent simplicity and wide availability in most quality control laboratories [6–10]. However, the UV-visible spectrophotometric methods that have been reported for determination of ATR-Ca in its pharmaceutical formulations [11–17] suffer from major drawbacks. These drawbacks include decreased selectivity due to measuring the native light absorption of ATR-Ca in the blue-shifted ultraviolet region, which might be subjected to interferences , employment of multiple-steps of non-selective oxidation reactions [14–16], and tedious liquid-liquid extraction procedures using large volumes of organic solvents in the methods based on formation of ion-pair associates . Microwell based automated spectrophotometric method for determination of ATR-Ca using colored charge-transfer (CT) complex has been developed as alternative for the conventional spectrophotometric technique . Moreover, the conventional methods suffer from the consumption of large volumes of organic solvents, which leads to high analysis cost, and more importantly, the incidence of exposure of the analysts to the toxic effects of the organic solvents [19–23]. These days, there is an urge in scientific community for the improvement in existing analytical methods in such a way that the developed approach includes both the positive traits (selectivity and sensitivity) of the traditional methods as well as the environmentally friendly "Green" approach, in which the amount of hazardous reagents used and chemical waste generated during analysis should be minimal.
Considering the variability of spectrophotometric conditions used in analysis of atorvastatin by CT reaction, need for cost reductions and need for environmentally friendly "Green" approach in routine pharmaceutical analysis, the objective of this research was to optimize and validate CT based reaction of ATR-Ca, and its employment in the optimization of simple and reliable automated microwell based spectrophotometric assay with high throughput analysis. A multivariate approach was adopted as an analytical tool to study the effect of different parameters and to improve spectrophotometric conditions for the analysis of ATR-Ca.
Microwell-plate absorbance reader (ELx 808, Bio-Tek Instruments Inc. Winooski, USA) was used for all the measurements in 96-microwell plates. 96-Microwell plates were a product of Corning/Costar Inc. (Cambridge, USA). Finnpipette adjustable 8–channel-pipette was obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Chemicals and dosage forms
ATR-Ca was obtained from Pfizer Inc. (New York, USA). DDQ was obtained from (Merck, Germany). Lipitor tablets (Parke Davis, Germany) and Lipicure-10 tablets (INTAS Pharmaceuticals, India) labeled to contain 10 mg ATR-Ca were obtained from the local market and from India.
Preparation of standard and tablet solutions
Preparation of stock standard solutions
Into a 5-ml calibrated flask, 10 mg of ATR-Ca was accurately weighed, dissolved in 2 ml methanol, and completed to volume with the same solvent. This stock solution was diluted with methanol to obtain the suitable concentrations that lie in the linear range of the assay.
Preparation of DDQ solutions
Into a 5-ml calibrated flask, 8 mg of DDQ was accurately weighed, dissolved in 2 ml methanol, and completed to volume with the same solvent.
Preparation of tablet sample solutions
Twenty tablets were weighed and finely powdered. A quantity of the powder equivalent to 20 mg of ATR-Ca was transferred into a 10-ml calibrated flask, dissolved in 4 ml methanol, swirled and sonicated for 5 min, completed to volume with the methanol, shaken well for 15 min, and filtered. The first portion of the filtrate was rejected, and a measured volume of the filtrate was diluted quantitatively with methanol to yield the suitable concentrations that lie in the linear range of the assay.
Accurately measured aliquots (100 μl) of the standard or sample solution containing varying amounts of ATR-Ca (20–200 μg) were transferred into wells of 96-microwell assay plates. One hundred microliters of DDQ solution (0.16%, w/v) was added, and the reaction was allowed to proceed at (31 ± 1°C) for 3.5 min. The absorbances of the resulting solutions were measured at 460 nm by the microwell-plate reader. Blank wells were treated similarly except 100 μl of methanol was used instead of sample, and the absorbances of the blank wells were subtracted from those of the other wells.
Optimization of experimental conditions
Box–Behnken experimental design
Levels of tested parameter for Box-Behnken design
Results and discussion
Design of the proposed assay and strategy for its development
The proposed assay was designed to employ 96-microwell assay plate as the CT reaction was carried out in microwells of the assay plate (200-μl reaction volume) instead of the conventional volumetric flasks (10,000-μl volume). The solutions were dispensed by 8-channel pipette, and the absorbances of the colored CT complex were measured by microwell-plate absorbance reader instead of the conventional spectrophotometer.
In the present study, ATR-Ca was selected based on its therapeutic importance, clinical success, and the expected electron-donating ability. This selection was supported by a previous study made by Darwish IA , which demonstrated the excellent electron-donating property of alkali salts of carboxylic acid pharmaceutical compounds. Previous studies involving CT reactions with polyhalo-/polycyanoquinone electron n-acceptors revealed that DDQ is one of the most efficient reagents in terms of its reactivity [24, 25]. Furthermore, its CT reaction with electron-donating analytes is instantaneous [24, 26]. For these reasons, DDQ was used as electron acceptor in the development of the proposed assay. The 96-microwell design of the proposed assay was based on the previous success of Darwish et al. in the utility of this design for determination of some other pharmaceuticals.
Reaction and spectral characteristics
Further support of this assignment was provided by the absorption maxima with those of DDQ radical anion produced by the iodide reduction assay . The dissociation of the (D-A) complex was promoted by the high ionizing power of the polar solvent and the resulting peaks in the absorption spectra of drug-acceptor reaction mixtures were similar to the maxima of the radical anions of the acceptors obtained by the iodide reduction assay .
Response surface optimization
The Box–Behnken design matrix of three variables
Analysis of variance of calculated model for atorvastatin absorbance
9.417 × 10-003
Regression coefficients and their significance in the quadratic model
where wi is the weight of the response, n the number of responses, and di the individual desirability function for each response. In the actual study all with values were set equal to 1.
Validation of the proposed assay under optimized conditions
Linearity and sensitivity
Quantitative parameters for the analysis of ATR-Ca by the proposed assay
LOD (μg/well) a
LOQ (μg/well) a
Accuracy and precision
Accuracy of the proposed assay was assessed by analytical recovery studies. Recovery was determined by the standard addition method. Known amounts of ATR-Ca were added to pre-determined drug-containing pharmaceutical formulation, and then determined by the proposed assay. The mean analytical recovery was calculated using five determinations and was found to be 98.74 ± 0.7% to 100.22 ± 1.24% indicating the accuracy of the proposed assay.
Precision and accuracy of the proposed assay at different ATR-Ca concentrations
Relative standard deviation
80.60 ± 1.28
80.75 ± 0.99
120.5 ± 0.85
120.34 ± 1.11
181.04 ± 1.93
180.42 ± 1.37
Application of the proposed assay in the analysis of pharmaceutical formulations
Analysis of ATR-Ca in its tablets by the reported and proposed methods
Content (% ± SD)a
- Reduction in the consumption of organic solvents (environmentally friendly "Green" approach) in the CT-based UV-visible spectrophotometric analysis, accordingly reduction in the exposures of the analysts to the toxic effects of organic solvents.
- Reduction in the analysis cost by 50-folds which can be reflected on the price for the finished dosage forms, thus it can reduce the expenses for the medications.
- Providing a high throughput analytical methodology that can facilitate the processing of large number of samples in a relatively short time. This property was attributed to the use of multi-channel pipettes for efficient dispensing of the solutions, carrying out the analytical reaction in 96-well plates (as reaction vessels), and measuring the color signals in the 96 wells at ~ 30 seconds by the plate reader.
- The advantages of the proposed assay and in addition to automation could be reached by application of Flow Injection Analysis.
- Although the proposed assay was developed and validated for ATR-Ca, however, it is also anticipated that the same methodology could be used for essentially any analyte that can exhibit CT reaction.
Limit of detection
Limit of quantification
Relative standard deviation
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-VPP-203.
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