Lipidomic analysis of human meibum using HPLC–MSn

Abstract

High-pressure liquid chromatography–mass spectrometry (HPLC–MS) has become a de facto standard analytical tool in lipidomic analyses of complex biological samples. This technique offers the best combination of selectivity and sensitivity among the currently available analytical methods, and provides not only the retention times of analytes, but also their m/z values, from which molecular masses of the compounds can be deduced. Further enhancement of the technique comes from the fact that some of the MS instruments (also known as ion traps, or MSn instruments) are capable of multistage fragmenting of the analytes, thus enabling the researcher to perform their structural elucidation. These capabilities make HPLC–MSn an ideal tool for analyzing small, complex lipid samples such as human meibum. Meibum is a very complex lipid mixture which is secreted onto the ocular surface by Meibomian glands. Meibum plays a critical role in the biochemistry and physiology of the human ocular surface. However, despite all efforts, its (bio)chemical composition remains elusive. In this chapter, several HPLC–MSn methods developed for lipidomic analysis of human meibum will be discussed. Considering the nature of analytes (all of which are hydrophobic compounds poorly soluble in water, and most of which are electroneutral), the only MS technique used in the study will be atmospheric pressure chemical ionization (APCI) MSn in both the positive and the negative ion modes. Electrospray ionization technique, though useful in phospholipid analyses, was found to be inadequate for analyzing less polar compounds, such as wax esters. As the data provided in this chapter will show that meibum is composed predominantly of nonpolar lipids of wax ester, cholesteryl ester, and triacylglycerol families with no appreciable amounts of more polar lipids present, APCI MSn seems to be a method of choice for lipidomic analysis of meibum and similar lipid mixtures.