LC-MS analysis of lipopolysaccharides and their lipid A components  

Lipopolysaccharides (LPS) are predominant (about 75%) constituents of the outer leaflet membrane of most Gram-negative bacteria (Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae ) and of some marine cyanobacteria.  The physiological activities of LPS are attributed to Lipid A. It is bacterial endotoxin that induces various biological reactions when even at small amounts (nanograms and even picograms) enters the bloodstream.  LPS are quite stable and remain active even after the bacteria die (for example, after heat sterilization). Injection of living or killed gram-negative cells or purified LPS into experimental animals caused such pathophysiological reactions as fever, changes in white blood cell counts, hypotension, shock, and death. During the infection of mammals, LPS are released when bacteria multiply or die and break up causing localized inflammations and if uncontrolled, sepsis.

In this work there were investigated chemical properties of the Lipid A. The analysis of standard compounds purchased for the study is demonstrated below.

Experimental: Lipopolysaccharides were analyzed using the LC-MS system, which consisted of Dionex Ultimate 3000 RS HPLC coupled to a Q Exactive Plus hybrid FT mass spectrometer equipped with a heated electrospray ionization source (Thermo Fisher Scientific Inc.). Chromatographic separation of analytes was carried out on the XSelect CSH C18 column (Waters) using a method developed in TSABAM.

Negative ESI LC-MS chromatogram of "Lipid A" purchased from Sigma-Aldrich (P/N L0774 from Salmonella enterica serotype minnesota Re 595). Lipid A was detected only at low amounts in the commercial product (red EIC chromatogram), while there were found many other similar lipids. Such standards are perhaps used for biological studies or, for calibration, for example, ELISA tests. A rhetorical question,  - I am wondering, what can be the reliability of calibration or research using a regent of such purity...?

A credit must be given to Sigma-Aldrich since the company took responsibility for an impure product and compensated it. 

Further, the study was continued using the Kdo2-Lipid A (KLA) standard  purchased from Avanti Polar Lipids which was chemically pure.