Abstract
Despite the increasing popularity of liquid chromatography−mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids. However, the impact of complex lipid sample complexity and purity on total lipid quantification accuracy has not been investigated. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance (ATR)-FTIR and establish a simple workflow for lipidomics sample quantification. We show that both pure and complex lipids show characteristic FTIR vibrations of CH- and C=O-stretching vibrations, with a quantitative range of 40−3000 ng and a limit of detection of 12 ng, but sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification via CH stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. Taking all tested parameters together, we propose a rapid FTIR workflow for routine lipidomics sample quality and quantity assessment and tested this workflow by comparing to the total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. The uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. With low sample requirement, we anticipate this simple and rapid method will enhance lipidomics workflow by enabling accurate total lipid quantification and normalization of lipid quantity for MS analysis.
Keywords:
FTIR; chemical contaminants; lipids; mass spectrometry; phospholipids; sphingolipids; triglycerides.