Ask the Expert: How can I quickly and easily identify different lipid classes?

Seetaramanjaneyulu Gundimeda, Agilent Application Scientist

Lipidomics is a fast growing area of research focused on lipids. Expansion in the field has been largely driven by major advances in technologies such as mass spectrometry (MS) and the discovery of linkages between lipid profiles with certain metabolic diseases, such as obesity, hypertension, stroke, and diabetes.

Lipids can be analyzed via LC/MS in either normal phase or reversed phase. Lipids have two important components in their structure: a polar head and a hydrophobic tail. As explained in an earlier Access Agilent article, lipids formed with a glycerol backbone are called glycerol lipids.

Signature ions in glycerophospholipids aid in their identification

The alcohol groups of the glycerol can be esterified with long chain fatty acids. When one of the alcohol groups is attached to a polar group (ex: phosphate, phosphocholine, phosphoethanolamine, and phosphoserine), they are called glycerophospholipids. Glycerophospholipids demonstrate characteristic ions in the MS/MS spectra that help in their identification. Lipidomic scientists worldwide use these signature ions for the identifications of the lipids. The diagnostic ion/features shown by some of the lipid classes are given in Table 1.

Table 1. Diagnostic features of different phospholipid classes in their MS/MS spectra

Figure 1. A screenshot demonstrating use of fragment mass filter.

Filtering data by fragment mass simplifies lipid analysis

Information about diagnostic features of different phospholipid classes is very useful in identifying lipids when they are in a mixture. Filtering the data from the information in Table 1 with the Agilent MassHunter Qualitative Analysis software tool can simplify lipid analysis in many ways. The ‘Filter results by fragments’ feature is one such method, as it helps to filter the data based on the fragment mass or neutral loss of interest. By clicking the ‘Find compounds by Auto MS/MS’ tab, one can access multiple filtering options. Data simplification of a lipid mixture to identify ceramides is shown in Figure 1. Retention time window and MS threshold parameters should be set after careful examination of the chromatogram. The accuracy of the fragment mass information is critical because mass match tolerance is set by the accuracy of the data and can influence the results a great deal. The same tool can be used to identify Phosphocholine containing lipids also by changing fragment mass filter to 184.073.

Figure 2. A screenshot demonstrating use of neutral loss filter.

Filtering data by neutral loss also helps identify lipids

In a similar fashion, lipids can be identified by their characteristic neutral loss also. Phosphatidyl cholines (PCs) give a dominant ion at m/z 184 in positive mode in their MS/MS spectra. The same species demonstrate a neutral loss of 60 Da from its formate adducts or 74 Da from its acetate adducts in negative mode. Phosphotidyl ethanolamines (PE) show a characteristic ion at m/z 140 and at m/z 196 in negative mode. But in positive mode they show a dominant neutral loss of 141Da from the precursor. Based on the ionization mode used during data acquisition, both filtering by fragment ion and by neutral loss may be necessary to simplify the data. Prostaglandings, another lipid class can be identified by ‘Neutral loss filter’ 100.09Da (Figure 2).

By processing the data with a relevant diagnostic feature, a compound list can be generated. This compound list can then be exported to any spectral library to identify the compounds. To learn more about this application, please read Agilent publication 5991-6959EN.

Agilent provides a wide array of equipment and software to deliver fast, accurate lipid analysis

The importance of lipidomic research grows with each passing day. Agilent delivers a full range of LC/MS instruments, LC columns, and advanced informatics software that can help your busy research lab speed lipidomic analysis, while increasing accuracy and reducing costs. We encourage you to visit our Lipidomics Resources page to sign up for valuable free information (videos, webinars, articles, and more!) on this exciting field of research and Agilent’s related technologies. Contact your Agilent representative today to start putting these time-saving solutions to use in your research lab.

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Article Directory – August 2016