Background

Sphingolipids are the second largest group of membrane lipids in living organisms and play an important role in many aspects of cell structure, metabolism, and regulation (Lahiri and Futerman, 2007). At first, it was thought that sphingolipids were a complex family of structurally related molecules, but more and more studies have shown that sphingolipids are involved in numerous cellular processes (Mao and Obeid, 2008). Ceramides (Cer) are essential bioactive lipids implicated in various cell biological processes ranging from cell growth regulation to cell death and senescence (Futerman and Hannun, 2004; Hannun and Obeid, 2008) through influencing of multiple signaling pathways. Although the physiological roles of ceramides are widely reported, few studies have described the extraction, identification, and quantification, thus, analysis of ceramides has gained significant interest in investigating the physiological functions of sphingolipid metabolism in Hemiptera Insects.

Currently, various methods have been described for this purpose, such as Diacylglycerol (DAG) Kinase assay (Preiss et al., 1987), Thin-layer chromatography (TLC) (Gorska et al., 2002), Gas chromatography mass spectrum (GC-MS) (Tserng et al., 2003), High-performance liquid chromatography (HPLC) (Yano et al., 1998; Dobrzyn and Gorski, 2002). In the beginning, DAG kinase assay was commonly used for Cer quantitation, but the specificity has been questioned (Watts et al.,1997). Thin layer chromatography was the method of choice, but the resolution, sensibility, and separation were limited, resulting in inefficient separation of similar molecules (Bielawski et al., 2010). Despite the high sensitivity of chromatographic analysis, this method had some limitations, such as the need for standard substances and derivatization (Dobrzyn et al., 2004). High-performance liquid chromatography (HPLC) was introduced to obtain ceramides separation with higher resolution, but complex samples like tissue extracts, therefore, produced many unspecific signals that did not provide any information concerning the metabolism of molecular species by HPLC (Yano et al., 1998; Bode and Graler, 2012).

Given the ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), it is more efficient, rapid, and sensitive, improving the separation condition of extremely complex samples and reducing matrix interference (Cutignano et al., 2010). Therefore, the current choice method is the analysis of ceramides by Ultra Performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). This protocol provides a relatively rapid and reproducible method. Moreover, this method can be used to profile ceramides from the extraction of the plant sample. It has evolved as the method of choice to detect sphingolipids metabolites due to its high sensitivity and superior specificity. The content of ceramide is determined and a quantitative system of sphingolipids in Hemiptera Insects is established, which lay a foundation for understanding the metabolic process and elucidating the biological function of each component. This method was described and used successfully to extract other sphingolipids in previously published studies (Bielawski et al., 2010; Shi et al., 2018 and 2019).