Egusquiaguirre Susana P, Manguán-García Cristina, Perona Rosario, Pedraz José Luís, Hernández Rosa Maria, Igartua Manuela
NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain.
Instituto de Investigaciones Biomédicas CSIC/UAM, IDIPaz and CIBER de Enfermedades Raras CIBERER, Arturo Duperier, 4, 28029 Madrid, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Dec 1;972:95-101. doi: 10.1016/j.jchromb.2014.09.041. Epub 2014 Oct 5.
In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100μg/ml (R(2)=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.
在本研究中，已开发并验证了一种高效液相色谱（HPLC）方法，用于测定聚乙二醇-聚乳酸-羟基乙酸共聚物（PEI-PLGA）纳米粒中GSE4肽的含量。色谱分离在C18柱上进行，流动相为甲醇和0.1%三氟乙酸（TFA）水溶液，采用梯度洗脱，流速为1ml/min。通过在290nm处的荧光检测对GSE4肽进行鉴定。甲醇:TFA的洗脱最初在（20:80，v/v）保持1分钟，然后在6分钟内梯度变为（80:20，v/v）。然后在此比例下等度洗脱1分钟，再在3分钟内线性变为（20:80，v/v）。所开发的方法根据国际协调会议（ICH）指南进行了验证，具有特异性，在10-100μg/ml范围内呈线性（R(2)=0.9996），精密度良好，日间和日内精密度由相对标准偏差值反映（小于3.88%），准确度高，回收率为100.18±0.95%，当包封在纳米粒中时，在5°C或室温下48小时内稳定。该方法简单、快速，成功用于测定负载GSE4的PEI-PLGA纳米粒中的肽含量。
