An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection
An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection
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一种改进的 HPLC 方法，通过库仑检测定量变肾上腺素
DOI：
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发表时间：
2014
期刊：
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通讯作者：
L. Passarinha
中科院分区：
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作者：
A. Q. Pedro;Soares Rf;D. Oppolzer;F. M. Santos;Rocha La;A. M. Gonçalves;M. Bonifácio;J. Queiroz;E. Gallardo;L. Passarinha
建立了一种基于反相高效液相色谱（RP-HPLC）和库仑法的快速、简便的分析方法，用于儿茶酚-甲基转移酶酶测定中o -甲基化产物肾上腺素的定量分析。以0.1 M磷酸二氢钠、0.024 M一水柠檬酸、0.5 mM硫酸辛酯钠和9%乙腈（%v/v）为流动相，在反相柱上进行等密度分离。结果表明，该方法在0.25 ~ 15 nmol/mL范围内呈线性，测定系数为0.9997。日内、日间精密度和准确度符合生物分析方法验证标准，定量限和定量限均为0.25 nmol/mL。该方法的主要重点是获得较低的定量限，这对COMT活性测定的实验室研究具有重要意义，特别是对从天然或重组提取物中获得的蛋氨酸108/158变体。本方法的另一个主要优点是在自动色谱系统上运行时间较短，可以在短时间内分析多个样品。此外，肾上腺素在室温下至少24小时稳定，在HPLC系统注射器中至少24小时稳定，至少三次冷冻/解冻循环稳定。与已有方法相比，该方法具有更高的灵敏度、精密度、准确度、稳定性和线性度。最后是儿茶酚- o -甲基转移酶
A rapid and straightforward analytical method, based on the use of RP-HPLC with coulometric detection, was developed and validated for the quantification of metanephrine, an O-methylated product in catechol-Omethyltransferase enzymatic assays. The isocratic separation was achieved on a reverse column with a mobile phase consisting of 0.1 M sodium dihydrogen phosphate, 0.024 M citric acid monohydrate, 0.5 mM sodium octyl sulphate and 9% acetonitrile (%v/v). The method was found to be linear between 0.25 and 15 nmol/mL with a determination coefficient of 0.9997 for metanephrine. Intra-and interday precision and accuracy were in conformity with the criteria accepted in bioanalytical method validation and the LOD and LLOQ were 0.25 nmol/mL. The main focus of the developed method is the lower LLOQ achieved that can have important implications in laboratory research for COMT activity determinations, in particular for the methionine 108/158 variant obtained either from native or recombinant extracts. Another major advantage of the present method is the shorter run times on automated chromatographic systems that allow the analysis of several samples in a short time. In addition, metanephrine was stable in the samples for at least 24 h at room temperature, for at least 24 h in HPLC system injector and for at least three freeze/thaw cycles. The developed method demonstrated higher sensitivity, precision, accuracy, stability, and linearity when compared with the methods previously described. Finally, a catechol-O-methyltransferase