Atsuhiko Toyama (Presenter)
MS Business Unit, Shimadzu Corporation
Bio: Atsuhiko Toyama earned his Masters Degree in Biochemistry at Cambridge University in 2004 and joined Shimadzu Corporation in the same year to start his career as an analytical chemist. His scientific publication is focused towards developing new analytical techniques, such as for biomarker research and glycopeptide quantitation using mass spectrometry. He studied the Ph.D. course in University of Tokyo along with his Shimadzu work and earned his degree in 2012.
Authorship: Atsuhiko TOYAMA(1), Ichiro HIRANO(1), Yuki NAKAMURA(2), Kumie SATOU(2)
(1) MS Business Unit, Shimadzu Corporation, (2) Advanced Technology Center, Medical Solution Segment, LSI Medience Corporation
| Short Abstract In this study, we demonstrated the analytical performance of two LC/MS/MS methods that quantitated urinary catecholamines and their various metabolites for efficient clinical research. One method involved acid hydrolysis of urine samples followed by solid phase extraction to quantitate deconjugated norepinephrine (NE), epinephrine (E), dopamine (DA), metanephrine (MN) and normetanephrine (NMN). The other method was a dilute-and-shoot method to quantitate free MN, NMN, vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA). Both methods had quantitative range to cover biologically relevant concentrations and analysis time of less than 5 minutes. |
Long Abstract
INTRODUCTION
Catecholamines and their metabolites in circulation are readily excreted to urine, both in free form and in conjugated forms (sulfate or glucoronate), and their levels are higher in urine than in plasma by orders of magnitude. Given this and also the non-invasive nature of sample collection, urinary catecholamines and their metabolites are of growing research target in clinical context. Moreover, analysis for such research is increasingly performed by tandem mass spectrometry (LC/MS/MS) since it can selectively detect free and conjugated metabolites at high sensitivity. Our aim in this study is to accelerate clinical research by providing a fast and robust LC/MS/MS method to meet the requirement to quantitate both total (deconjugated by acid hydrolysis) and free catecholamine metabolites.
METHODS
Described herein are two methods: one method analyzed norepinephrine (NE), epinephrine (E), dopamine (DA), metanephrine (MN) and normetanephrine (NMN) after acid hydrolysis of urine samples followed by WCX-SPE cleanup; the other method was a dilute-and-shoot method to quantitate free MN, NMN, vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA). Both methods were ran on Nexera-MX UHPLC system coupled to triple quadrupole mass spectrometer LCMS-8060 (Shimadzu Corporation) and analysis time was less than 5 minutes.
RESULTS
As a pre-validation study, we evaluated precision and accuracy of a neat standard curve ranging from 0.01 to 200 ng/mL, corresponding to urinary concentration of 1 – 20,000 ng/mL. Next we evaluated the accuracy of the neat standard curve with respect to matrix calibration curve, prepared using fresh urine sample that contained endogenous target compounds. As a result, urinary concentrations determination by the neat standard curve were demonstrated to reproduce the matrix calibration curve at 85-115% accuracy. Finally, our developed method was tested for quantitative consistency with a conventional methodology (independent HPLC system for 3 catecholamines and metanephrine) by measuring the same sample aggregate in a side-by-side fashion.
In conclusion, the described high-throughput methods achieved sufficient sensitivity and linearity to cover biologically relevant concentration range in urine and was demonstrated for accuracy, robustness and consistency with conventional methodology.
References & Acknowledgements:
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