Ichiro Hirano (Presenter)
Shimadzu Corporation
Bio: March 1994: Graduated faculty of Chemistry, Hiroshima University April 1994 to July 1998: Sales department, Analytical Instrument Division, Shimadzu Corporation July 1998 to November 1998: International Marketing Division, Shimadzu Corporation November 1998 to January 2004: Shimadzu do Brasil Comercio Limitada January 2004 to April 2009: Marketing department, Analytical Instrument Division, Shimadzu Corporation April 2012 to current: Product Manager, Mass Spectrometry Business Unit, Analytical Instrument Division, Shimadzu Corporation
Authorship: Ichiro HIRANO (1), Atsuhiko TOYAMA (1), Kumie SATOU (2), Yuki NAKAMURA (2)
(1) Mass Spectrometry Business Unit, Shimadzu Corporation, Kyoto, Japan, (2) Special Pharmacology Analysis Department, LSI Medience Corporation, Tokyo, Japan
| Short Abstract Plasma levels of catecholamines and their metabolites, namely norepinephrine, epinephrine, dopamine, metanephrine and normetanephrine, are of significant relevance in clinical research in the field of endocrinology and disease screening. Fast, high-sensitivity analytical system is warranted. To this end, we developed an optimized method for simultaneous determination of these compounds using Shimadzu LCMS-8060, achieving complete chromatographic separation and low pg/mL LLOQ in plasma within 5 minute analysis time. Sample preparation was automated by Biotage Extrahera using WCX-SPE in 96-well format. Pre-validation study was performed, including demonstration of quantitative consistency with HPLC-based predicate device. |
Long Abstract
Catecholamine levels in plasma, namely norepinephrine (NE), epinephrine (E) and dopamine (DA), give indication of disease states and are routinely measured for clinical research. Recently, plasma catecholamine metabolites such as metanephrine (MN) and normetanephrine (NMN) are increasingly studied as an alternative of or to complement catecholamine levels. Therefore high-sensitivity and high-throughput analytical system that covers all of these compounds is warranted. We herein demonstrate our optimized LCMS method for simultaneous determination of plasma NE, E, DA, MN and NMN in plasma.
For plasma sample preparation we employed weak cation exchange solid-phase extraction (WCX-SPE) in 96-well format, which was automated by Extrahera system (Biotage AB, Sweden). The eluate of WCX-SPE was subjected directly to LCMS analysis from 96-well plates to maximize total throughput. LCMS analysis was performed using Nexera-MX UHPLC system coupled to triple quadrupole mass spectrometer LCMS-8060 (Shimadzu Corporation, Japan).
Difficulty in catecholamine analysis is that this class of compounds are poorly retained by conventional reversed-phase chemistry, making them susceptible to strong matrix effect. Here we used a unique type of ODS column, MAqC-ODS I, to achieve baseline-separation of all of the five compounds within 4 minutes. MAqC-ODS, whose name comes from Metal Aquo-Ion Conjugated ODS, contains metal particles on the surface of ODS-conjugated silica beads; aquo-ion formation around metal ions results in a net negative charge that exerts ionic interaction in addition to the reversed-phase mode.
As a pre-validation study, we evaluated precision and accuracy of a neat standard curve ranging from 10 to 30,000 pmol/L. Next we evaluated the accuracy of the neat standard curve with respect to matrix calibration curve, prepared using fresh plasma that contains endogenous catecholamines. As a result, plasma 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 predicate device (HPLC-based system for 3 catecholamines) by measuring the same sample aggregate using the two systems in a side-by-side fashion.
In conclusion, the described high-throughput method achieved sufficient sensitivity and linearity to cover biologically relevant concentration range in plasma and was demonstrated for accuracy, robustness and consistency with conventional methodology.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Shimadzu Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Shimadzu Corporation |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |