Robustness evaluation of the chromatographic determination of nifedipine in pharmaceuticals

Topicality. Robustness tests were originally introduced to avoid problems in interlaboratory studies and to identify the potentially responsible factors. The aim of this study was the rubustness evaluation of the chromatographic determination of nifedipine in medicines using Youden’s test. Materials and methods. Youden’s test is a reliable method to evaluate the robustness of analytical methods, by means of an experiment design which involves seven analytical parameters combined in eight tests. In the present study, we assessed the robustness of a chromatographic method to quantify nifedipine using Youden’s test. Youden’s test showed to be a simple and feasible procedure to evaluate the robustness of chromatographic methods. Results and discussion. Using the criteria of Youden’s test, the chromatographic method showed to be highly robust regarding of nifedipine content, when variations in seven analytical parameters were introduced. The highest variation in nifedipine content was 0.28 %, when the concen tration of trifluoroacetic acid in the mobile phase was altered; a value considerably low and not significant in routine analyses. Conclusions. Youden’s test showed to be a reliable and useful tool for the robustness evaluation of the chromatographic method for assay of nifedipine. Therefore, Youden’s test can be successfully applied for the ro bustness evaluation in validation process of analytical methods by HPLC.

Ключевые слова: нифедипин; валидация; робастность; хроматография; количественный анализ; Юден тест INTRODUCTION Both the ICH and the USP guidelines define the robustness of an analytical procedure as a measure of its capacity to remain unaffected by small but deliberate variations in procedural parameters listed in the documentation, providing an indication of the method's or procedure's suitability and reliability during normal use. But while robustness shows up in both guidelines, interestingly enough, it is not in the list of suggested or typical analytical characteristics used to validate a method (again, this apparent discrepancy is changing in recently proposed revisions to USP chapter 1225. Robustness tests were originally introduced to avoid problems in interlaboratory studies and to identify the potentially responsible factors. This means that a robustness test was performed at a late stage in the method validation since interlaboratory studies are performed in the final stage. Thus the robustness test was considered a part of method validation related to the precision (reproducibility) determination of the method. However, performing a robustness test late in the validation procedure involves the risk that when a method is found not to be robust, it should be redeveloped and optimised. At this stage much effort and money have already been spent in the optimisation and validation, and therefore one wants to avoid this. Therefore the performance of a robustness test has been shifting to earlier points of time in the life of the method [1].
The evaluation of the robustness of chromatographic methods often is complex and laborious, taking into account the large number of analytical parameters that should be considered to carry out the test. Some authors select specific analytical parameters to be evaluated, introducing small variations in the nominal conditions and the statistical interpretation is performed by means of Student's t-test or ANOVA test. Other wider alternative to determine the robustness of analytical methods is the Youden's test. This test allows not only evaluating the method robustness but also pointing out the influence of each analytical parameter in the final results. The basic idea of Youden's test is not to study one alteration at time but to introduce several changes at once, in such a manner that the effects of individual changes can be ascertained [2,3].
The aim of the work was to evaluate the robustness of the chromatographic method for the quantitation of nifedipine, using Youden's test, and determine the analytical parameters that present higher influence in the final results of the analysis.

MATERIALS AND METHODS
The objects of the study were tablets "Fenigidin Zdorovja" (Ukraine). The chromatographic analysis of nifedipine performed on liquid chromatographs Agilent 1290 and HP 1100 systems. The columns used Nucleosil C18 (4.6 × 150 mm with a particle size of 5 microns) and Ascentis Express C18 (column size 4. The seven parameters and its respective variations were combined in eight assays or chromatographic runs, performed in a random order. Tab. 2 demonstrates the factorial combination of the parameters for the Youden's test. The analyses results are shown by letters from s to z. Hence, when combination 1 was assayed, the obtained result was s. When combination 2 was assayed, the obtained result was t, and so successively. In each combination, three injections of each sample and standard solutions were carried out, at the work concentration. After the change of chromatographic column or mobile phase composition, 30 min were awaited for system stabilization. The evaluated re sults in each combination were peak area, retention time (Rt), tailing factor (T), theoretical plates number (N) and verapamil hyrdochloride content.
To determine the influence of variations of each parameter in the final result, the mean of the four values corresponding to the capital letters (nominal conditions) was compared to the mean of the four values correspon ding to the lowercase letters (altered conditions). For example, to evaluate the effect of the column temperature in the final result of the analyses, the following equation was employed:

Effect C/c = (s + u + w + y) / 4 -(t + v + x + z) / 4 Eq. (1)
Thus, the influence of the seven analytical parameters regarding the peak area, retention time (Rt), tailing factor (T), theoretical plates number (N) and nifedipine content were evaluated. By means of Youden's test, it is possible to establish certainly the parameters which present higher influence in the final result of the analyses and perform a more rigorous control in the eventual variations of these parameters that may occur during a routine analysis.

RESuLTS AND DISCuSSION
The assays for the robustness evaluation of the chromatographic method were carried out simultaneously in both equipments, Agilent 1290 and HP1100. The results obtained in the eight runs to nifedipine sample and standard solutions [4,5,6,7].
To evaluate the effect of each parameter, the average of the four values corresponding to altered conditions was subtracted from the average of the four values obtained at the nominal conditions, as demonstrated in Eq. (1). The effects of the parameter variations in the analysis results are presented in Tab. 3.
Using the criteria of Youden's test, the chromato graphic method showed to be highly robust regarding nifedipine content, when variations in seven analytical parameters were introduced. The highest variation in nifedipine content was 0.28 %, when the concen tration of trifluoroacetic acid in the mobile phase was altered; a value considerably low and not significant in routine analyses. The retention time of nifedipine peak was more considerably influenced by three analytical parameters. Some parameters such as column temperature, mobile phase flow rate, column supplier and chromatograph model presented low influence in the evaluated factors of the chromatographic method.

CONCLUSIONS
Youden's test showed to be a reliable and useful tool for the robustness evaluation of the chromatographic method for assay of nifedipine. Therefore, Youden's test can be successfully applied for the ro bustness evaluation in validation process of analytical methods by HPLC.
Conflict of Interests: authors have no conflict of interests to declare.