Page 33 - Clinical-Medical Solution for Clinical Chemistry

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

Prepare blood
collection tubes
Place blood collection Prepared samples are
tubes and reagents
automatically transferred to

Serum/ LC/MS/MS.
Plasma
Extraction of plasma/serum metabolites

Automated sample preparation by CLAM-2000
Mixing at room
Addition of 230 L Filtration

MeOH containing Addition of 20 L temperature • PTFE membrane

• 1900 rpm
plasma
internal standards • 90 sec
• 30 min

Figure 1. Work ow for analysis of metabolites
using fully automated sample preparation LC/MS/MS system

HPLC conditions
Column : Discovery HS F5 2.1 mm × 150 mm, 3.0 µm
Mobile phase A : 0.1% Formic acid/Water
Mobile phase B : 0.1% Formic acid/Acetonitrile
Time program : B conc. 25%(5 min) - 35%(11 min) - 95%(15 min) - 95%(20 min) - 0%(20.01-25 min)
Injection vol. : 1 L
Flow rate : 0.25 mL/min.
Column temperature : 40ºC
MS conditions (LCMS-8040)

Ionization : ESI (Positive/Negative)
Nebulizing Gas Flow : 2.0 L/min.
Drying Gas Flow : 15.0 L/min.
DL temperature : 250ºC
Block Heater Temperature : 400ºC

For the analysis of primary metabolites except L-valine, An amino acid analysis of plasma samples with the same
L-leucine, L-isoleucine, L-tyrosine, and L-phenylalanine, lot number was also performed by SRL (Tokyo Japan), and
LC/MS/MS Method Package for Primary Metabolites the plasma concentrations of L-valine, L-leucine,
(Shimadzu Corporation, Kyoto, Japan) was used. The L-isoleucine, L-tyrosine, and L-phenylalanine (µM) were
multiple reaction monitoring (MRM) transitions of the measured. The Fischer ratio was calculated based on the
native and stable isotopes of L-valine, L-leucine, quantitative results. The resultant data are shown in the
L-isoleucine, L-tyrosine, and L-phenylalanine are shown in ‘Reference concentration ( M)’ (Table 2).
Table 1.

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