Vitali Luciano, Della Betta Fabiana, Costa Ana Carolina O, Vaz Fernando Antonio Simas, Oliveira Marcone Augusto Leal, Vistuba Jacqueline Pereira, Fávere Valfredo T, Micke Gustavo A
Department of Science and Food Technology, Federal University of Santa Catarina, Florianópolis, SC, Brazil.
Department of Chemistry, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil.
Talanta. 2014 Jun;123:45-53. doi: 10.1016/j.talanta.2014.01.047. Epub 2014 Jan 30.
The aim of this study was to develop a new multilayer coating with crosslinked quaternary ammonium chitosan (hydroxypropyltrimethyl ammonium chloride chitosan; HACC) and κ-carrageenan for use in capillary electrophoresis. A new semi-permanent multilayer coating was formed using the procedure developed and the method does not require the presence of polymers in the background electrolyte (BGE). The new capillary multilayer coating showed a cathodic electroosmotic flow (EOF) of around 30×10(-9) m(2) V(-1) s(-1) which is pH-independent in the range of pH 2 to 10. The enhanced EOF at low pH obtained contributed significantly to the development of a fast method of separation. The multilayer coating was then applied in the development of a fast separation method to determine betaine and methionine in pharmaceutical formulations by capillary zone electrophoresis (CZE). The BGE used to determine the betaine and methionine concentrations was composed of 10 mmol L(-1) tris(hydroxymethyl) aminomethane, 40 mmol L(-1) phosphoric acid and 10% (v/v) ethanol, at pH 2.1. A fused-silica capillary of 32 cm (50 µm ID×375 µm OD) was used in the experiments and samples and standards were analyzed employing the short-end injection procedure (8.5 cm effective length). The instrumental analysis time of the optimized method was 1.53 min (approx. 39 runs per hour). The validation of the proposed method for the determination of betaine and methionine showed good linearity (R(2)>0.999), adequate limit of detection (LOD <8 mg L(-1)) for the concentration in the samples and inter-day precision values lower than 3.5% (peak area and time migration). The results for the quantification of the amino acids in the samples determined by the CZE-UV method developed were statistically equal to those obtained with the comparative LC-MS/MS method according to the paired t-test with a confidence level of 95%.
本研究的目的是开发一种用于毛细管电泳的新型多层涂层，该涂层由交联季铵壳聚糖（羟丙基三甲基氯化铵壳聚糖；HACC）和κ-卡拉胶组成。采用所开发的程序形成了一种新型半永久性多层涂层，该方法不需要背景电解质（BGE）中存在聚合物。新型毛细管多层涂层显示出约30×10⁻⁹ m² V⁻¹ s⁻¹ 的阴极电渗流（EOF），在pH 2至10范围内与pH无关。在低pH下获得的增强EOF对快速分离方法的开发有显著贡献。然后将多层涂层应用于开发一种快速分离方法，通过毛细管区带电泳（CZE）测定药物制剂中的甜菜碱和蛋氨酸。用于测定甜菜碱和蛋氨酸浓度的BGE由10 mmol L⁻¹ 三（羟甲基）氨基甲烷、40 mmol L⁻¹ 磷酸和10%（v/v）乙醇组成，pH为2.1。实验中使用了一根32 cm（内径50 µm×外径375 µm）的熔融石英毛细管，采用短端进样程序（有效长度8.5 cm）对样品和标准品进行分析。优化方法的仪器分析时间为1.53分钟（约每小时39次运行）。所提出的测定甜菜碱和蛋氨酸方法的验证显示出良好的线性（R²>0.999）、对样品浓度足够的检测限（LOD<8 mg L⁻¹）以及低于3.5%的日间精密度值（峰面积和迁移时间）。根据置信水平为95%的配对t检验，用所开发的CZE-UV方法测定样品中氨基酸的结果与用比较性LC-MS/MS方法获得的结果在统计学上相等。
