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Bioanalytical Assay for Neurotransmitters in
Whole Blood by LC-MS/MS
Yang Shi, Catherine Lafontaine, Francois A. Espourteille, Thermo Fisher Scientific, Franklin, MA, USA
Introduction
Taken orally in conjunction with Levodopa (L-DOPA),
Carbidopa (C-DOPA) inhibits the metabolism of L-DOPA
before it reaches the brain so that more is available to be
converted into dopamine in the brain. 3-methoxy-L-tyrosine
(3-OMD) is an important metabolite produced after L-DOPA
administration. The following LC-MS/MS method using
TurboFlow
™
technology for on-line sample extraction using
a Thermo Scientific Aria
™
TLX-1 system coupled with
Thermo Scientific TSQ Quantum Ultra
™
triple quadrupole
mass spectrometer demonstrates its suitability as a
research method for these compounds in human whole
blood.
Goal
To develop a quantitative, fast, automated LC-MS/MS
method for analysis of neurotransmitters in human
whole blood.
Method Information
These analytes were extracted on-line from crashed human
whole blood. Calibration curves were analyzed using an
Aria TLX-1 LC system coupled with a TSQ Quantum
Ultra with heated electrospray ionization (H-ESI) source.
Internal standards used were 4-chloro-L-phenylalanine
and L-DOPA-d
3
.
Experimental Conditions
Sample Preparation
A standard stock solution of 50 µg/mL L-Dopa, C-Dopa
and 3-OMD in methanol was prepared. Methanol-quenched
human whole blood (K
2
EDTA) was centrifuged at
10,000 RPM for 10 minutes. Calibrators were prepared in
the supernatant. Analyte concentration ratio of spiking
solution was 4 to 1 of L-DOPA and 3-OMD to C-DOPA.
Final internal standard concentrations were 90 ng/mL for
4-chloro-L-phenylalanine and 225 ng/mL for L-DOPA-d
3
,
respectively. Injection volumes were 0.010 mL.
Aria TLX-1 System Parameters
Two 0.5 x 50 mm Thermo Scientific Cyclone
™
MAX
TurboFlow columns with a C18 HPLC column
(4.6 x 150 mm, 5 µm particle size).
LC Method Mobile Phases
Loading Pump
Mobile Phase A:
10 mM Ammonium Acetate with 0.2%
Ammonium Hydroxide (aq)
Mobile Phase B:
0.1% Formic Acid (aq)
Mobile Phase C:
50 mM Ammonium Acetate with 10%
Formic Acid (aq)
Mobile Phase D:
50 mM Ammonium Acetate with 10%
Formic Acid in Methanol
Elution Pump
Mobile Phase A:
0.1% Formic Acid (aq)
Mobile Phase B:
0.1% Formic Acid in Acetonitrile
Mass Spectrometer Parameters
Ion Polarity:
Positive ion mode
Vaporizer Temperature:
400 °C
Capillary Temperature:
300 °C
Sheath Gas Pressure (N
2
):
60 units
Auxiliary Gas Pressure (N
2
):
55 units
Scan Type:
Highly-selective reaction monitoring (H-SRM)
Scan Time:
0.050 s
Q1 (FWHM):
0.7
Q3 (FWHM):
0.7
Positive single reaction mode (+SRM) transitions and other
MS parameters for test compounds are shown in Table 1.
The whole experiment was controlled by Aria software.
Key Words
• Aria TLX-1
• TSQ Quantum Ultra
• TurboFlow
Technology
• Parkinson’s
Disease
Application
Note: 436
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