Comparison of Single Phase and Biphasic Extraction Protocols for Lipidomic Studies Using Human Plasma

Area of Science:

• Lipidomics
• Biochemistry
• Analytical Chemistry

Background:

• Lipidomic profiling of plasma is crucial for understanding cellular pathways and identifying disease biomarkers.
• Existing lipid extraction methods can be complex and may not optimize the recovery of all lipid classes.
• Plasma lipidomics holds promise for diagnosing and monitoring conditions such as dementia and neuropsychiatric disorders.

Purpose of the Study:

• To evaluate the efficiency of three distinct solvent-based lipid extraction protocols for human plasma.
• To compare the novel single-phase methanol/1-butanol (Alshehry) method against established Folch and Matyash (methanol/tert-butyl methyl ether) methods.
• To assess the reproducibility and suitability of the Alshehry method for plasma lipidomic analysis.

Main Methods:

• Human plasma samples were subjected to lipid extraction using the Folch, Matyash, and Alshehry methods.
• Extracted lipids were analyzed via liquid chromatography-mass spectrometry (LC-MS) in both positive and negative ion modes.
• Quantification involved normalized peak areas, with reproducibility assessed through intra- and inter-assay coefficients of variation (CV).

Main Results:

• Over 500 distinct lipids were identified across all methods and ionization modes.
• The Alshehry method demonstrated comparable extraction efficiency to Folch and Matyash for most lipid classes, excelling in polar lipid recovery.
• High correlations (r² = 0.99 and 0.97) were observed between Alshehry and the other methods; intra-assay CVs were 14.1% (Alshehry), 15.1% (Folch), and 21.8% (Matyash).

Conclusions:

• The Alshehry single-phase extraction method is a viable and effective alternative to traditional biphasic methods for plasma lipidomics.
• This method requires minimal plasma volume (10 μL), is highly reproducible (inter-assay CV 14.4%), safer, and more environmentally friendly (chloroform-free).
• The Alshehry method facilitates comprehensive lipid analysis and holds potential for biomarker discovery in neurological and psychiatric diseases.