alexa An Improved HPLC Method for Quantification of Metanephr
ISSN: 2157-7064

Journal of Chromatography & Separation Techniques
Open Access

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Research Article

An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection

Pedro AQ1#, Soares RF1#, Oppolzer D1, Santos FM1, Rocha LA1, Gonçalves AM1, Bonifacio MJ2, Queiroz JA1, Gallardo E1 and Passarinha LA1*

1CICS-UBI-Centro de Investigacaoem Ciencias da Saude, Universidade da Beira Interior, 6201-506 Covilha, Portugal

2Departamento de Investigacao e Desenvolvimento, BIAL 4745-457 Sao Mamededo Coronado, Portugal

#Authors contributed equally to this work

*Corresponding Author:
Passarinha LA
Health Sciences Research Centre-University of Beira Interior (CICS-UBI)
Av. Infante D. Henrique, 6200-506, Covilha, Portugal
Tel: +351 275 329 069
Fax: +351 275 329 099
E-mail: [email protected]

Received date: March 12, 2014; Accepted date: April 14, 2014; Published date: April 18, 2014

Citation: Pedro AQ, Soares RF, Oppolzer D, Santos FM, Rocha LA, et al. (2014) An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection. J Chromatograph Separat Techniq 5:217. doi:10.4172/2157-7064.1000217

Copyright: © 2014 Pedro AQ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.



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


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