Abstract
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
Highlights
Normetanephrine (NMN) and metanephrine (MN), O-methylated metabolites of norepinephrine and epinephrine respectively, are produced by the actions of catechol-O-methyltransferase (COMT) (EC 2.1.1.6), an enzyme largely confined to extraneuronal tissues [1], that needs accurate and selective measurements, for clinical diagnosis and for pathological studies of several diseases
Liquid chromatography coupled with electrochemical detection (LC-ECD) has provided a new tool to evaluate the levels of these compounds in urine [10], plasma [11] and COMT assays [12] and is considered a reliable technique for catecholamine assay [13]
The methodology presented in this work concerns the development and validation of an HPLC method with coulometric detection for assessment of MN, allowing the determination of the biological activity of recombinant COMT as well as native COMT extracted from animal tissues or cell lines
Summary
Normetanephrine (NMN) and metanephrine (MN), O-methylated metabolites of norepinephrine and epinephrine respectively, are produced by the actions of catechol-O-methyltransferase (COMT) (EC 2.1.1.6), an enzyme largely confined to extraneuronal tissues [1], that needs accurate and selective measurements, for clinical diagnosis and for pathological studies of several diseases. We describe a novel method using HPLC with coulometric detection, with adequate sensitivity for reliable measurement of MN in biological bacteria lysate extracts for the first time, by calibration procedures, including intra- and interday precision and stability. This method was fully validated in a wide concentration range with lower limit of quantification. The standard samples were agitated by rotation/inversion movements for 2 min and injected into the HPLC-ECD system according to the experimental conditions
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