Profiling phospholipid elution in reversed-phase LC-MS/MS bioanalytical methods in order to avoid matrix effects

Area of Science:

• Analytical Chemistry
• Biochemistry
• Pharmacokinetics

Background:

• Endogenous phospholipids cause matrix effects in bioanalytical LC-MS methods.
• Strategies to minimize phospholipid impact include sample processing and chromatographic separation.
• This research focuses on chromatographic separation under reversed-phase conditions.

Purpose of the Study:

• To investigate the chromatographic separation of phospholipids under reversed-phase conditions.
• To understand the retention behavior of glycerophosphocholines (GPCs) in bioanalytical LC-MS.
• To evaluate the impact of mobile phase composition on GPC elution.

Main Methods:

• Reversed-phase liquid chromatography (LC) was employed.
• Mobile phases with varying percentages of organic modifiers (acetonitrile and methanol) were tested.
• The effect of mobile phase pH on retention was analyzed.

Main Results:

• Glycerophosphocholine (GPC) retention showed a 'U-shaped' relationship with organic percentage in acetonitrile-based mobile phases.
• GPC retention was predictable and unaffected by pH in methanol-based mobile phases.
• GPC elution profiles were consistent across different plasma species.

Conclusions:

• The predictable retention of GPCs with methanol in reversed-phase chromatography is a key finding.
• This predictability allows for streamlined and simplified bioanalytical assay development.
• The study offers a more efficient strategy for managing phospholipid matrix effects.