Page 22 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E239

Application High Performance Liquid Chromatography

News Glycerophospholipids Analysis by Comprehensive HPLC

Coupled with a Triple Quadrupole Mass Spectrometer
No.L462

Glycerophospholipids (GPLs) are the major component n Flow Diagram of Comprehensive HPLC
of biological membranes. They can not only act as a Fig. 1 shows the flow diagram of the comprehensive
barrier from the external environment, but can also play HPLC-ESI-MS/MS system. The system comprises 2 flow
a key role in a variety of biological processes including lines: one for the first dimension separation with a
membrane trafficking and signal transduction. Thus, normal phase column and the second dimension
analysis of GPLs is one of the most important studies in separation with a reversed phase column. A mixture of
the metabolomics field. Although reversed phase (RP) GPLs was roughly classified by normal phase
HPLC coupled with electrospray ionization (ESI) MS/MS chromatography in the first dimension. All the eluents
is an effective strategy for lipidomics, there is still room are trapped into two loops alternatively. Then the entire
for further improvement of the analytical methods. One eluents are introduced into second dimensional
drawback to performing determination of GPLs is ion reversed phase UHPLC without any risk of sample-loss.
suppression caused by co-eluting compounds. To obtain The GPLs of interest are separated according to the
reliable results, complete separation of target GPLs by orthogonal retention selectivity and detected with ESI-
comprehensive HPLC with ESI-MS/MS is an effective MS/MS quantitatively.
strategy.

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Fig. 1 Flow Diagram of the Comprehensive HPLC-ESI-MS/MS System

Table 1 Analytical Conditions
1D Column : Nucleosil SIL (150 mm L. × 1.0 mm I.D., 3 µm)
Mobile Phase : A: Isooctane / Acetone / Ethyl Acetate / Acetic acid
= 40/20/20/0.03 (v/v/v/v)
B: Isooctane / 2-propanol / Water / Acetic acid / 28 % Ammonia aq.sol.
= 40/51/9/0.03/0.03 (v/v/v/v/v)
Flowrate : 0.02 mL/min
Time Program : B Conc. 30 % (0 min) → 40 % (25 min) → 100 % (40 min) → 100 % (55 min) → 30 % (55.1 min) → STOP (70 min)
Column Temp. : 40 °C
Injection Vol. : 5 µL
Loop Vol. : 20 µL
2D Column : Phenomenex Kinetex C18 (50 mm L. × 4.6 mm I.D., 2.6 µm)
Mobile Phase : A: Methanol / Water / Acetic acid / 28 % Ammonia aq.sol.
= 90/10/0.05/0.05 (v/v/v/v)
B: 2-propanol / Acetic acid / 28 % Ammonium hydroxide
= 100/0.05/0.05 (V/V/V)
Flowrate : 3.5 mL/min (50 % split to MS)
Time Program : B Conc. 10 % (0 min) → 50 % (0.75 min) → 10 % (0.76 min) → STOP (1 min)
The initial B Conc. has been changed by a stepwise method
Detector : Shimadzu LCMS-8050 (ESI positive, MRM mode)