Sample Preparation Techniques in Lipidomics Analysis

Mass spectrometry (MS) has become one of the most effective technical tools in lipidomics research. There are several MS-based analytical methods: 1) shotgun method: using MS for direct injection and detection, 2) coupled with other separation techniques such as liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE), and 3) mass spectrometry imaging (MSI) method that enables the spatial distribution of samples. Lipidomics studies are usually performed on biological samples with complex matrices, such as serum, plasma, urine, and tissues. In addition to MSI methods, enrichment and extraction of samples are often required prior to analysis.

The lipidomics workflow (Züllig et al.,2020).

Biological samples are usually frozen directly after collection using liquid nitrogen and then stored at low temperatures (e.g., -80 °C) to minimize alteration of the various substances in the sample. Prior to analysis, liquid samples are re-dissolved at 4 °C and then mixed thoroughly. Solid samples are mixed with the aid of a mortar and pestle or homogenizer by adding the appropriate solvent.

The commonly used techniques for lipid extraction include liquid-liquid extraction (LLE) and solid-phase extraction (SPE). LLE is suitable for non-targeted whole lipid analysis because it can extract a more comprehensive range of lipid molecules. SPE is more suitable for targeted metabolomics analysis of one or more classes of lipid molecules, as the sample undergoes separation and enrichment steps to further remove interfering substances and increase the concentration of the analyte.

Lipids extracted from samples may face the following challenges in the detection process:

1. The variety of lipid molecules and the differences in chemical properties make the ionization efficiency of different lipid molecules vary significantly during MS detection.

2. The dynamic distribution range of lipid molecules is wide, and the ionization process is the ion suppression effect.

3. The sensitivity of the instrument is not sufficient for the detection of low concentration substances. The internal standard molecules of lipids for quantification are not easy to obtain.

4. The existence of a large number of tautomers in lipid molecules makes qualitative quantification difficult, etc.

The chemical derivatization of lipid samples may be one of the effective means to solve these problems. The selection of a suitable derivatization method in lipidomics analysis is beneficial to improve ionization efficiency, increase detection sensitivity, increase compound structural stability, and even distinguish isomers.

Derivatization reactions usually require the selection of suitable derivatization reagents according to the purpose of the study and the reactive groups on the analytes to be measured. To improve the accuracy of quantification, the stable isotope labeling derivatization (SILD) method has been developed. The principle is to react the light and heavy isotope-labeled derivatization reagents with the sample and standard respectively, mix them in a certain ratio and then inject the sample. The relative quantification of the corresponding components is based on the ratio of the light and heavy derivatives in the result. Compared with the conventional derivatization method, SILD can help to solve the problems such as the serious interference of sample matrix and the difficulty to obtain the internal standard, thus improving the quantitative accuracy.

Different types of lipids and their expression are closely related to many diseases. Excellent sample pre-processing techniques provide important technical support for studies to improve the quality of lipidomics data, study the physiological functions of lipids, and discover lipid-related disease markers.

Reference:

1. Züllig, T., Trötzmüller, M., & Köfeler, H. C. (2020). Lipidomics from sample preparation to data analysis: a primer. Analytical and bioanalytical chemistry, 412(10), 2191-2209.