Development and optimization of a method for the determination of Cylindrospermopsin from strains of Aphanizomenon cultures: Intra-laboratory assessment of its accuracy by using validation standards
Development and optimization of a method for the determination of Cylindrospermopsin from strains of Aphanizomenon cultures: Intra-laboratory assessment of its accuracy by using validation standards
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DOI：
10.1016/j.talanta.2012.07.087
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发表时间：
2012-10-15
期刊：
影响因子：
6.1
通讯作者：
Camean, Ana M.
中科院分区：
文献类型：
--
作者：
Guzman-Guillen, Remedios;Prieto Ortega, Ana I.;Camean, Ana M.
由于水体逐渐富营养化，在世界许多地区，蓝藻水华在水环境中的出现正在增加。由于柱状精液蛋白(Cyn)等毒素的产生，蓝藻对水的污染是世界范围内一个严重的健康问题。因此，有必要开发和验证分析方法，使我们能够量化真实样本中的半胱氨酸氨基转移酶，以提醒公众这种毒素。在这项工作中，建立了一种分析方法，并对其进行了优化，以测定卵孢无孔菌培养物中的细胞色素N。分析方法采用溶剂提取、石墨化小柱纯化和LC-MS/MS法定量。提取和纯化步骤采用两水平完全析因设计。应用验证标准对该方法的真实性和精确度进行了适当和实用的评估。用冻干铜绿假单胞菌PCC7820(R2>；=0.9999)提取液中的标准品计算回归方程，线性范围为5~500mgCyn/L，相当于0.18~18.00mgCyn/g冻干细胞。方法的检出限和定量限分别为0.04和0.15微克CyN/g，回收率(%)在83~94%之间波动，中精密度(RSD%)在5.6~11.0%之间波动。此外，本方法对于所考虑的三个因素：石墨化碳柱的批次、样品通过石墨盒的流速和固相萃取处理后的最终再溶水量显示出较强的稳健性，从而验证了该方法的有效性。该方法已应用于卵孢霉菌X-001的不同冻干培养物中，以评估Cyn的含量。该方法可用于环境研究中冻干天然水华样品中细胞色素C的测定。(C)2012爱思唯尔B.V.保留所有权利。
The occurrence of cyanobacterial blooms in aquatic environments is increasing in many regions of the world due to progressive eutrophication of water bodies. Because of the production of toxins such as Cylindrospermopsin (CYN), contamination of water with cyanobacteria is a serious health problem around the world. Therefore it is necessary to develop and validate analytical methods that allow us to quantify CYN in real samples in order to alert the public of this toxin. In this work, an analytical method has been developed an optimized for the determination of CYN from Aphanizomenon ovalisporum cultures. The analytical procedure is based on solvent extraction followed by a purification step with graphitized cartridges and CYN quantification by LC-MS/MS. The extraction and purification steps were optimized using a two-level full factorial design with replications. A suitable and practical procedure for assessing the trueness and precision of the proposed method has been applied by using validation standards. The method has been suitably validated: the regression equation was calculated from standards prepared in extracts from lyophilized M. aeruginosa PCC7820 (r2 >= 0.9999) and the linear range covered is from 5 to 500 mu g CYN/L, equivalent to 0.18-18.00 mu g CYN/g dry weight lyophilized cells. Limits of detection and quantification were 0.04 and 0.15 mu g CYN/g, respectively, the recovery range (%) oscillated between 83 and 94% and intermediate precision (RSD %) values from 5.6 to 11.0%. Moreover, the present method showed to be robust for the three factors considered: the batch of the graphitized carbon cartridges, the flow rate of the sample through the cartridge, and the final redissolved water volume after SPE treatment, which permits its validation. The validated method has been applied to different lyophilized cultures of A. ovalisporum (LEGE X-001) to evaluate CYN content. This procedure can be used for determining CYN in lyophilized natural blooms samples in environmental studies. (C) 2012 Elsevier B.V. All rights reserved.