Stability-Indicating HPLC Method for isoflavones aglycones analysis from Trifolium pratense L. extract

Simony Martiny; Mairique Waszczuk; Samuel Kaiser; Marina Cardoso Nemitz; Valquiria Linck Bassani

doi:10.22456/2527-2616.102027

Authors

Simony Martiny Programa de Pós‐Graduação em Ciências Farmacêuticas, Faculdade de Farmácia - Universidade Federal do Rio Grande do Sul
Mairique Waszczuk Programa de Pós‐Graduação em Ciências Farmacêuticas, Faculdade de Farmácia - Universidade Federal do Rio Grande do Sul
Samuel Kaiser Programa de Pós‐Graduação em Ciências Farmacêuticas, Faculdade de Farmácia - Universidade Federal do Rio Grande do Sul
Marina Cardoso Nemitz Faculdade de Farmácia - Universidade Federal do Rio de Janeiro
Valquiria Linck Bassani Programa de Pós‐Graduação em Ciências Farmacêuticas, Faculdade de Farmácia - Universidade Federal do Rio Grande do Sul

DOI:

https://doi.org/10.22456/2527-2616.102027

Keywords:

Trifolium pratense, isoflavone aglycones, HPLC, stability-indicating method, validation

Abstract

The purpose of this study was to develop and validate a fast HPLC stability-indicating method for simultaneously quantifying the four main isoflavones in Trifolium pratense. Validation procedures followed the ICH requirements for complex matrices. The stability-indicating tests were performed by exposing the isoflavones to conditions of forced degradation and further analysis for verifying the formation of degradation products and their possible interferences in the HPLC analysis. The major isoflavones of Trifolium pratense proved to be stable against acid and oxidative media, thermodegradation, and photodegradation. However, they proved to be unstable in alkaline media, even for short periods of exposure like 2h. In this condition, in addition to the peaks corresponding to isoflavones, the HPLC analysis showed the presence of three additional peaks which were eluted at different retention times to the reference substances, without interfering in the quantification of the four analytes of interest, formononetin, biochanin A, daidzein and genistein. The method was validated following ICH guidelines showing to be specific, linear, precise, accurate, and robust. This first report concerning a stability-indicating method revealed that the proposed HPLC method reliably quantify the isoflavones and separate them from the degradation products in a short time of analysis.

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References

Silva AG. Plantas contendo isoflavonas no tratamento da síndrome da menopausa e nos distúrbios do climatério. Nat. Line 2007;5:25–9.

Barnes J, Anderson L, Phillipson J. Fitoterápicos. Porto Alefre: 2012.

Kaurinovic B, Popovic M, Vlaisavljevic S, Schwartsova H, Vojinovic-Miloradov M. Antioxidant profile of Trifolium pratense L. Molecules 2012;17:11156–72.

Ramos GP, Apel MA, Morais CB de, Ceolato PC, Schapoval EES, Dall’Agnol M, et al. In vivo and in vitro anti-inflammatory activity of red clover Trifolium pratense dry extract. Rev. Bras. Farmacogn. 2012;22:176–80.

Kolodziejczyk-Czepas J. Trifolium species-derived substances and extracts—Biological activity and prospects for medicinal applications. J. Ethnopharmacol. 2012;143:14–23.

Chen Q, Li P, Li B, Xiulou, Zhu J, Chen F. Simultaneous determination of formononetin, biochanin A, daidzein, and genistein in Trifolium pratense (red clover) by HPLC. LC GC Eur. 2010;23.

Ramos GP, Dias PMB, Morais CB, Fröehlich PE, Dall’Agnol M, Zuanazzi J a. S. LC determination of four isoflavone aglycones in red clover (Trifolium pratense L.). Chromatographia 2008;67:125–9.

Wu Q, Wang M, Simon JE. Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection. J. Chromatogr. A 2003;1016:195–209.

Oleszek W, Stochmal A, Janda B. Concentration of isoflavones and other phenolics in the aerial parts of Trifolium species. J. Agric. Food Chem. 2007;55:8095– 100.

Lin LZ, He XG, Lindenmaier M, Yang J, Cleary M, Qiu SX, et al. LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense). J. Agric. Food Chem. 2000;48:354–65.

Saviranta NM, Anttonen MJ, von Wright A, Karjalainen RO. Red clover (Trifolium pratense L.) isoflavones: determination of concentrations by plant stage, flower colour, plant part and cultivar. J. Sci. Food Agric. 2008;88:125–32.

ICH. Validation of a analytical Procedures : text and methodology Q2(R1). In: Guidance. 2005. page 17.

FDA F and DA. Guidance for industry: bioanalytical method validation, 2013.

Watanabe E, Kobara Y, Baba K, Eun H. Aqueous acetonitrile extraction for pesticide residue analysis in agricultural products with HPLC-DAD. Food Chem. 2014;154:7–12.

Yatsu FKJ, Pedrazza GPR, Argenta DF, Barreto F, Nemitz MC, Teixeira HF, et al. A new simplified and stability indicating liquid chromatography method for routine analysis of isoflavones aglycones in different complex matrices. Food Anal. Methods 2014;7:1881–90.

Vander Heyden Y, Nijhuis A, Smeyers-Verbeke J, Vandeginste BG, Massart DL. Guidance for robustness/ruggedness tests in method validation. J Pharm Biomed Anal. 2001;24(5-6):723‐753. doi:10.1016/s0731-7085(00)00529-x

de Rijke E, Zafra-Gómez A, Ariese F, Brinkman UAT, Gooijer C. Determination of isoflavone glucoside malonates in Trifolium pratense L. (red clover) extracts: quantification and stability studies. J. Chromatogr. A 2001;932:55–64.

Nemitz MC, Yatsu FKJ, Bidone J, Koester LS, Bassani VL, Garcia C V., et al. A versatile, stability-indicating and high-throughput ultra-fast liquid chromatography method for the determination of isoflavone aglycones in soybeans, topical formulations, and permeation assays. Talanta 2015; 134:183–93.

Niessen WMA, Manini P, Andreoli R. Matrix effects in quantitative pesticide analysis using liquid chromatography–mass spectrometry. Mass Spectrom. Rev. 2006;25:881–99.

Shah B, Jain S, Prajapati K, Mansuri N. Stability indicating HPLC method development: A review. Int. J. Pharm. Sci. Res. 2012;3:2978–88.

Blessy M, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs—A review. J. Pharm. Anal. 2014;4:159–65.

Carr GP, Wahlich JC. A practical approach to method validation in pharmaceutical analysis. J. Pharm. Biomed. Anal. 1990;8:613–8.

Anvisa. RDC N. 166, de 25 de Julho de 2017, Dispõe sobre a validação de métodos analíticos e dá outras providências. Minist. da Saúde 2017;2017:1–21.

Zeaiter M, Roger JM, Bellon-Maurel V, Rutledge DN. Robustness of models developed by multivariate calibration. Part I: The assessment of robustness. TrAC - Trends Anal. Chem.2004;23:157–70.