Page 32 - Clinical-Medical Solution for Clinical Chemistry

Evaluation of an automated LC-MS/MS system for
analyzing hydrophilic blood metabolites

Introduction

Recently, metabolomics has been developed and applied to biomarkers, a simple and quick automated sample
a variety of research elds, such as the food science, preparation method involving metabolite extraction and
agriculture, engineering, and medical elds. In the medical metabolite measurement should be developed. In this
research eld, metabolomics is used to search for novel study, we assessed whether the plasma levels of
metabolite biomarkers of a variety of diseases and metabolites could be quantitatively measured using a fully
elucidate pathogenic mechanisms, etc., and there have automatic pre-treatment system for LC/MS that can be
been a considerable number of metabolite biomarker connected online to an LC/MS device.
studies. As a step toward the practical use of metabolite

Methods and Materials

Reagents: Acetonitrile (LC/MS grade), formic acid (LC/MS (Amicon Ultra 0.5-mL centrifugal lters, Ultracel-3K). The
grade) and methanol (MeOH; LC/MS grade) were mixture was then centrifuged at 14,000 g for 60 min at
purchased from Wako Pure Chemical Industries (Osaka, 4ºC, and the collected solution was subjected to the
Japan). 2-Morpholinoethanesulfonic acid (MES), which was LC/QqQMS-based analysis of L-valine, L-leucine,
employed as an internal standard of primary metabolites, L-isoleucine, L-tyrosine, and L-phenylalanine.
was purchased from Sigma Aldrich. L-valine, L-leucine,
L-isoleucine, L-tyrosine, and L-phenylalanine were acquired Automatic method: The automatic method used to
from Sigma Aldrich (MO, USA). Isotopically labeled L-valine analyze L-valine, L-leucine, L-isoleucine, L-tyrosine,
(D 8 ), L-leucine ( C 6 ), L-isoleucine (D 10 ), L-tyrosine ( C 9 , N), L-phenylalanine levels was performed using an CLAM-2000
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and L-phenylalanine (D8) were purchased from Cambridge (Shimadzu Corporation, Kyoto, Japan). In the CLAM-2000,
Isotope Laboratories (MA, USA). Commercially available MeOH containing 10 µM isotopically labeled L-valine,
pooled plasma (Kohjin-Bio Co., Saitama, Japan), which was L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine and MES
collected using EDTA-Na as an anticoagulant, was utilized (as internal standards) was added into the solvent
as human plasma, and pooled plasma with the same lot container, and 20 L of plasma (N=5) were also applied
number was used for all experiments. into another tubes. By running the CLAM-2000, 20 L of
plasma were automatically mixed with 230 L of MeOH
Manual method: 20 L of plasma (N=5) were mixed with and the internal standards, before the resultant mixture
230 L of MeOH containing 10 µM isotopically labeled was shaken at 1,900 rpm for 30 min at room temperature.
L-valine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine Then, the mixture was automatically subjected to suction
and MES as internal standards. Next, the mixture was ltration for 90 sec, and the ltered solution was
shaken at 1,200 rpm for 30 min at room temperature, transferred to an SIL-30AC autosampler online, before
before being passed through an ultra ltration lter being subjected to LC/QqQMS analysis.

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