title: A bioanalytical HPLC method for coumestrol quantification in skin permeation tests followed by UPLC-QTOF/HDMS stability-indicating method for identification of degradation products.
abstract: Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07-107.28 μg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products.
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Bianchi
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil.
authors.forename: Helder F
authors.initials: HF
authors.lastname: Teixeira
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil.
authors.forename: Samuel
authors.initials: S
authors.lastname: Kaiser
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil.
authors.forename: George G
authors.initials: GG
authors.lastname: Ortega
authors.affiliation: Programa de Pós-Graduação em Química, Instituto de Química-Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, CEP 91501-970, Porto Alegre, RS, Brazil.
authors.forename: Paulo Henrique
authors.initials: PH
authors.lastname: Schneider
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, CEP 90610-000, Porto Alegre, RS, Brazil. Electronic address: valquiria.bassani@ufrgs.br.
authors.forename: Valquiria L
authors.initials: VL
authors.lastname: Bassani
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003375:Coumestrol / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics*; D004355:Drug Stability; D004423:Ear / Q000502:physiology; D057230:Limit of Detection; D016014:Linear Models; D015203:Reproducibility of Results; D012869:Skin Absorption / Q000502:physiology*; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003375:Coumestrol
keywords: Bioanalytical method; Coumestrol; Skin permeation; Stability-indicating method; UPLC-QTOF/HDMS; Validation