ROBUSTNESS EVALUATION OF HPLC DETERMINATION OF ATORVASTATIN AND LISINOPRIL ON COLUMN PUROSPHER C8 STAR IN PHARMACEUTICALS

Introduction. Innovative pharmaceutical development of various antihypertensive drugs with statins and the creation of domestic fixed-dose combinations of drugs with different effects is an urgent task of modern pharmacy, which will help attract more patients to the treatment and prevention of cardiovascular disease. Pharmaceutical development of atorvastatin and lisinopril by our scientific group proposes for using the ratio of (1/1) for lisinopril (10 mg) and atorvastatin (10 mg). HPLc (High-Performance Liquid chromatography) technique is adopted as it is considered as the most common technique in realm of quality control analysis. The aim of the study – to evaluate the robustness of HPLc (High-Performance Liquid chromatography) method for the quantitation of lisinopril and atorvastatin and determine the analytical parameters that present greater influence in the final results of the analysis. Research Methods. An efficient method to assess the robustness of analytical methods is by Youden’s test, by means of an experiment design which involves seven analytical parameters combined in eight tests. In the recent studies, we assessed the robustness of a chromatographic method to quantify lisinopril and atorvastatin in tablets using Youden’s test. Results and Discussion. By using the criteria of Youden’s test, HPLc method proved to be greatly robust regarding content of lisinopril and atorvastatin, when variations in seven analytical parameters were introduced. The most variation in effects of the analytical parameters in retention time (Rt) for lisinopril and atorvastatin HPLc quantitation was when used column supplier. Purospher c8 STAR (55 mm×4 mm, 5 μm) is based on high purity silica and an almost complete surface coverage. Purospher c8 STAR provides excellent peak symmetry for acidic, basic and even chelating compounds, highest column efficiency in terms of the number of theoretical plates, and exceptional stability from pH 1.5 to 10.5. Conclusion. Youden’s test can be applied successfully for the ro bustness evaluation in validation process of analytical methods and results ontained in our work should be interest to the scientific population dealing with pharmaceutical analytical chemistry.

INTRODUCTION. Innovative pharmaceutical development of various antihypertensive drugs with statins and the creation of domestic fixed-dose combinations of drugs with different effects is an urgent task of modern pharmacy, which will help attract more patients to the treatment and prevention of cardiovascular diseases [1][2][3][4]. Analysis of modern scientific publications in Pubmed and Science-Direct on the creation of pharmaceutical development based on lisinopril and atorvastatin has not yielded any results. Scientists are focused on the study of analysis methods of API in drugs, because SPhU or other pharmacopoeias have monographs on some substances of antihypertensive drugs, but usually for their analysis are used methods that can not be used for analysis of combination drugs [5,6]. Pharmaceutical development of atorvastatin and lisinopril by our scientific group proposes for using the ratio of (1/1) for lisinopril (10 mg) and atorvastatin (10 mg). HPLC (High-Performance Liquid Chromatography) technique is adopted as it is considered as the most common technique in realm of quality control analysis.
The aim of the study was to evaluate the robustness of HPLC method for the quantitation of lisinopril and atorvastatin and determine the analytical parameters that present greater influence in the final results of the analysis.
RESEARCH METHODS. Atorvastatin calcium (purity 99.1 %, as determined by HPLC) and lisi-ISSN 2410-681X. Медична та клінічна хімія. 2021. Т. 23. № 1 nopril (purity 99.3 %, as determined by HPLC) were purchased from Sigma-Aldrich (Switzerland). 10 mg Atorvastatin calcium (standard sample) and 10 mg lisinopril (standard sample) were put in 100 mL measuring flask and dissolved in 50 mL diluent com posed of 50 % v/v methanol and 50 % v/v perchloric acid (0.05 % v/v), ultrasound crushed and treated for 2 minutes and shaked 15 min with shaker. After that measuring flask was filled up to mark with diluent and filtered hrough 0.2 µm RC syringe filter before injection. Final concentrations of 0.1 mg/mL of both analytes. After filtration through the above filter, 10 µL were injected on the working column.
Solvents used in experiments were HPLC gradient grade purchased from Merck Darmstdat, Ger many. Analytical Balance Mettler Toledo MPC227, pH-metter Metrohm 827, deionized water from TKA Micro system, with final conductivity less than 0.05µS/cm. IKA orbital shaker KS4000i was used for sample agitation. The nylon and regenerated cellulose RC 0.45 μm syringe filters were purchased from Agilent Technologies.

Sample preparation
Twelve tablets of each preparation were studied to obtain statistically significant results. The tablets with declared contents of 10 mg atorvastatin and 10 mg of lisinopril were purchased from local drug store, pharmacy. The tablets were put in 100 mL measuring flask and dissolved in 50 mL diluent composed of 50 % v/v methanol and 50 % v/v perchloric acid (0.05 % v/v), ultrasound crushed and treated for 2 minutes and shaked 15min with shaker. After that measuring flask was filled up to mark with diluent and filtered hrough 0.2 µm RC syringe filter before injection. Final concentrations of 0.1 mg/mL of both analytes. After filtration through the above filter, 10 µL were injected on the working column.
RESULTS AND DISCUSSION. The robustness evaluation of HPLC method for lisinopril and atorvastatin quantitation was performed using the method proposed by Youdene Steiner. Seven analytical parameters were selected and small variations were induced in the nominal values of the method. Then, eight runs were performed with an aim to determine the effect of each parameter in the final result. The seven analytical parameters employed, as well as the introduced variations are demonstrated at Table 1. The analytical conditions at the nominal values are represented by capi tal letters and the conditions with the small variation are represented by lowercase letters [7][8][9][10][11][12][13].
The seven parameters and its respective variations were combined in eight assays or chromatographic runs, performed in a random order. Table 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 obtai ned result was t, and so successively.
In each combination, three injections of each sam ple and standard solutions were carried out, at the work concentration. After the alteration of chroma tographic column or mobile phase composition, there was a waiting of 30 min for system stabilization. The evaluated re sults in each combination were peak area, retention time (Rt), tailing factor (T), theoretical plates number (N) and lisinopril and atorvastatin content. For evaluating the effect the following equation was used: Effect C/c=(s+u+w+y)/4-(t+v+x+z)/4Eq. (1). Through the use of robustness elavulation, 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 of quality control fixed combination of lisinopril and atorvastatin.
In our study, main challenges wеre dirеcted to find optimаl chromatographic cоnditions for HPLC determination of lisinopril and atorvastatin in pharmaceuticals. Our objеctivе of the chromatоgraphіc method develоpment wаs to achiеve a peak tailing factor <1.5, retention time up to 3 min, along with very gооd resоlution. In both equipments (Shi mad zu Nexera XR UPLC system and Agilent 1260 Infinity II system), were carried out simultaneously the assays for the robustness evaluation of the chromatographic method. The results obtained in the eight runs to enalapril sample and standard solutions. In Table 3 are presented effects of the parameter variations in the analysis.
By using the criteria of Youden's test, HPLC method proved to be greatly robust regarding content of lisinopril and atorvastatin, when variations in seven analytical parameters were introduced. The most variation in effects of the analytical parameters in retention time (Rt) for lisinopril and atorvastatin HPLC quantitation was when used column supplier. Purospher C 8 STAR (55 mm x 4 mm, 5 μm) is based on high purity silica and an almost complete surface coverage. Purospher C 8 STAR provides excellent peak symmetry for acidic, basic and even chelating compounds, highest column efficiency in terms of the number of theoretical plates, and exceptional stability from pH 1.5 to 10.5.
pH of solution potassium dihydrogen phosphate in mobile phase CONCLUSION. The main target of the work was to evaluate the robustness of HPLC method for the quantitation of lisinopril and atorvastatin and determine the analytical parameters that present greater influence in the final results of the analysis. Youden's test can be applied successfully for the ro bustness evaluation in validation process of analytical methods and results ontained in our work should be interest to the scientific population dea ling with pharmaceutical analytical chemistry.
funding. Authors are grateful to the Ministry of Health of Ukraine Fund for providing scholarship for studies related to solutions for development of original combinations of antihypertensive agents, their analysis and standardization (0120U104201 (No. 509 of February 24, 2020).