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LAAN-A-LC-E293

Application Supercritical Fluid Chromatography

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Analysis of Choline and Acetylcholine in
Rat Cerebrospinal Fluid Samples Using the
No. L519 Nexera UC-MS/MS System

Choline, which is a structural element of cell membranes, and
Area
acetylcholine, which is known as a neurotransmitter, are both
familiar compounds in the field of bioanalysis. Since 15000000 Choline
acetylcholine is biosynthesized in the body from choline, it is
possible to estimate the quality of internal activity by monitoring 10000000
both of these compounds. This article focuses on the SFC analysis
of these compounds in a rat cerebrospinal fluid sample by direct
5000000
injection of the cerebrospinal fluid to the Nexera UC SFC system.
Also introduced is automatic extraction and analysis of a r = 0.9993
2
cerebrospinal fluid sample impregnated into filter paper, in 0 0 250 500 750 Conc (μg/L)
consideration of convenience and durability for storage and
transport, using the Nexera UC online SFE-SFC-MS/MS system.
Y. Watabe, T. Iida Area
100000000

„ SFC-MS/MS Analysis Acetylcholine
75000000
A CN column provided favorable separation of choline and
acetylcholine in SFC-MS/MS analysis. Calibration curves were 50000000
created from the peak area values from six times repeated
analyses for each of the three concentrations of 10, 100, and 25000000
1000 μg/L. Good linearity was obtained and the quantitation r = 0.9982
2
limit (LOQ, ASTM method) was 30 μg/L for choline and 10 μg/L 0 500 750
for acetylcholine. Table 1 lists the conditions of SFC-MS/MS 0 250 Conc (μg/L)
analysis. Fig. 1 shows the structural formula of choline and
acetylcholine and Fig. 2 shows the obtained calibration curves. Calibration Curves of Choline and Acetylcholine

The retention time and peak area repeatabilities after six
repetitions at each concentration of 10, 100, and 1000 μg/L
was confirmed at calibration curve creation and the results
2
are summarized in Table 2. The linearity (r ) was 0.9993 for
choline and 0.9982 for acetylcholine. Fig. 3 shows the MRM
Structure of Choline (Left) and chromatograms for 100 μg/L.
Acetylcholine (Right)
Table 2 Repeatabilities of Choline and

Acetylcholine Standards (n = 6)
Table 1 SFC-MS/MS Analytical Conditions
Retention time Peak area
Column : Inertsil CN-3 250 mm L. × 4.6 mm I.D., 5 μm (%RSD) (%RSD)
Mobile phase : A) Supercritical fluid of CO2 Choline 10 μg/L 0.22 7.5
B) Modifier: Methanol containing 20 mmol/L
ammonium formate / water =95/5 (v/v) Choline 100 μg/L 0.05 1.7
Time program : B Conc. 10 % (0 min) → 25 % (10 min) → Choline 1000 μg/L 0.07 2.2
50 % (10.1-12 min) → 10 % (12.1-15 min)
Flow rate : 2.5 mL/min Acetylcholine 10 μg/L 0.07 5.7
Column temp. : 40 °C Acetylcholine 100 μg/L 0.06 4.2
Injection volume : 1 μL Acetylcholine 1000 μg/L 0.07 6.0
BPR pressure : 10 Mpa
BPR temp. : 50 °C
Detector : LCMS-8050 (ESI, MRM mode) (×1,000,000)
Make-up : Methanol 1.5
Make-up flow rate : 0.2 mL/min
MRM transitions : (+) m/z 104.1>60.1 (for choline)
(+) m/z 146. 1>87.1 (for acetylcholine) 1.0

Choline
0.5 Acetylcholine

0.0
0.0 5.0 10.0 min

Choline and Acetylcholine Standards (100 μg/L)