Micke Gustavo Amadeu, Fujiya Neide Mitsue, Tonin Fernando Gustavo, de Oliveira Costa Ana Carolina, Tavares Marina Franco Maggi
Institute of Chemistry, University of Sao Paulo, P.O. Box 26077, 05513-970 Sao Paulo, SP, Brazil.
J Pharm Biomed Anal. 2006 Aug 28;41(5):1625-32. doi: 10.1016/j.jpba.2006.03.012. Epub 2006 May 2.
The separation of six soy isoflavones (Glycitein, Daidzein, Genistein, Daidzin, Glycitin and Genistin) was approached by a 3(2) factorial design studying MEKC electrolyte components at the following levels: methanol (MeOH; 0-10%) and sodium dodecylsulfate (SDS; 20-70 mmol L(-1)); sodium tetraborate buffer (STB) concentration was kept constant at 10 mmol L(-1). Nine experiments were performed and the apparent mobility of each isoflavone was computed as a function of the electrolyte composition. A novel response function (RF) was formulated based on the production of the mobility differences, mobility of the first and last eluting peaks and the electrolyte conductance. The inspection of the response surface indicated an optimum electrolyte composition as 10 mmol L(-1) STB (pH 9.3) containing 40 mmol L(-1) SDS and 1% MeOH promoting baseline separation of all isoflavones in less than 7.5 min. The proposed method was applied to the determination of total isoflavones in soy germ capsules from four different pharmaceutical laboratories. A 2h extraction procedure with 80% (v/v) MeOH under vortexing at room temperature was employed. Peak assignment of unknown isoflavones in certain samples was assisted by hydrolysis procedures, migration behavior and UV spectra comparison. Three malonyl isoflavone derivatives were tentatively assigned. A few figures of merit for the proposed method include: repeatability (n=6) better than 0.30% CV (migration time) and 1.7% CV (peak area); intermediate precision (n=18) better than 6.2% CV (concentration); recoveries at two concentration levels, 20 and 50 microg mL(-1), varied from 99.1 to 103.6%. Furthermore, the proposed method exhibited linearity in the concentration range of 1.6-50 microg mL(-1) (r(2)>0.9999) with LOQ varying from 0.67 to 1.2 microg mL(-1). The capsules purity varied from 93.3 to 97.6%.
采用三（二）因子设计研究了六种大豆异黄酮（黄豆黄素、大豆苷元、染料木黄酮、大豆苷、黄豆苷和染料木苷）的分离情况，该设计考察了胶束电动毛细管色谱（MEKC）电解质成分在以下水平的变化：甲醇（MeOH；0 - 10%）和十二烷基硫酸钠（SDS；20 - 70 mmol L⁻¹）；硼酸钠缓冲液（STB）浓度保持恒定在10 mmol L⁻¹。进行了九次实验，并计算了每种异黄酮的表观迁移率作为电解质组成的函数。基于迁移率差异、第一个和最后一个洗脱峰的迁移率以及电解质电导率，构建了一种新型响应函数（RF）。响应面分析表明，最佳电解质组成为含有40 mmol L⁻¹ SDS和1% MeOH的10 mmol L⁻¹ STB（pH 9.3），可在不到7.5分钟内实现所有异黄酮的基线分离。将该方法应用于四个不同制药实验室的大豆胚芽胶囊中总异黄酮的测定。采用在室温下涡旋80%（v/v）MeOH进行2小时的提取程序。通过水解程序、迁移行为和紫外光谱比较辅助确定某些样品中未知异黄酮的峰归属。初步鉴定出三种丙二酰异黄酮衍生物。该方法的一些性能指标包括：重复性（n = 6），迁移时间的变异系数（CV）优于0.30%，峰面积的CV优于1.7%；中间精密度（n = 18），浓度的CV优于6.2%；在20和50 μg mL⁻¹两个浓度水平下的回收率在99.1%至103.6%之间。此外，该方法在1.6 - 50 μg mL⁻¹浓度范围内呈线性（r² > 0.9999），定量限（LOQ）在0.67至1.2 μg mL⁻¹之间。胶囊纯度在从93.3%至97.6%之间。
