Matrix effect explained by unexpected formation of peptide in acidified plasma

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Bioanalytical methods using mixed-mode SPE, hydrophilic interaction chromatography (HILIC), and electrospray ionization mass spectrometry (ESI-MS) are crucial for analyzing biological samples.
• Matrix effects, such as signal enhancement or suppression, can significantly impact the accuracy and reliability of these analyses, particularly in complex biological matrices like plasma.

Purpose of the Study:

• To investigate the cause of peak distortion and strong signal enhancement observed in acidified rabbit plasma samples analyzed by a specific bioanalytical method.
• To identify the specific peptide responsible for the matrix effect and understand its formation mechanism.

Main Methods:

• Application of a bioanalytical method involving mixed-mode solid-phase extraction (SPE), hydrophilic interaction chromatography (HILIC), and electrospray ionization mass spectrometry (ESI-MS).
• High-resolution ESI-MS and N-terminal peptide sequencing were employed for peptide identification.
• Hydrogen-deuterium (H/D) exchange ESI-MS was used for confirmation of the identified peptide.

Main Results:

• A peptide, identified as NFQNAL, was found to be responsible for the observed matrix effect.
• This peptide was formed through enzymatic degradation of serum albumin under acidic conditions (pH 3), requiring albumin, acidification, and other plasma constituents.
• Signal enhancement correlated with NFQNAL levels, reaching a maximum enhancement factor of 3.

Conclusions:

• The matrix effect observed in the bioanalytical method was attributed to the peptide NFQNAL, a product of albumin degradation.
• The formation of NFQNAL is dependent on specific conditions including pH, presence of albumin, and other plasma components.
• The interference caused by NFQNAL was successfully mitigated by employing an alternative HILIC column.