Yeast lipids classified: Charged aerosol detector covers many classes
- Published: Jun 3, 2013
- Author: Steve Down
- Channels: HPLC
Charged aerosol detector
Ever since yeasts began to be exploited as a source of lipids for biofuels, interest in the lipid content and metabolism has grown. The neutral yeasts comprising steryl esters and triacylglycerols (TAG) are stored in structures known as cytoplasmic droplets and they are enclosed by a monolayer of various phospholipids and proteins.
Phospholipids belong to several different classes. Cardiolipids, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidylcholine each have a central core with different fatty acids attached. The most abundant of these in yeast are palmitoleic and oleic acid but fatty acids like stearic, palmitic and myristic acid are also prevalent.
One of the less expensive ways to analyse lipids involves HPLC. It has been linked to various detectors like mass spectrometry, which is expensive to run, and UV detection, which requires the presence of a chromophore on the lipids, a condition that is not always fulfilled. Another detector is the charged aerosol detector (CAD), which was developed recently and gives almost universal response for any nonvolatile analyte regardless of its chemical structure.
In CAD, droplets are produced from the HPLC eluate and passed through a reaction chamber where they collide with positively charged gaseous ions. The charged analytes are passed to a sensitive electrometer which measures the amount of charge. This value is proportional to the droplet size, which, in turn, is proportional to the analyte concentration. However, even in published methods for analysing lipids by HPLC-CAD, two runs are required for the polar and nonpolar lipids.
Now, scientists in Sweden have developed a single procedure that can analyse all lipid classes by HPLC-CAD. Jens Nielsen and colleagues from Chalmers University of Technology in Gothenborg and Gothenburg University applied the procedure to the lipids that are produced in cultures of yeast cells.
The lipids were extracted by a newly developed microwave-assisted extraction procedure that accomplished complete extraction in one step within 10 minutes, compared with about 6 hours by conventional solvent extraction. Freeze-dried cells were mixed with a mixture of chloroform and methanol and irradiated to 60°C. At higher temperatures, the yields of some lipids began to fall, presumably due to degradation.
This temperature was also the best compromise between extraction efficiency and the tendency of the lipids to esterify any free or bound fatty acids present in the extract, which would reduce the lipid yield. The recoveries of the lipids were high, typically 95% for TAG.
The lipid mixtures were separated on a HILIC column using normal-phase elution conditions at an optimised column temperature of 35°C. The nonpolar and polar lipids were well separated at this temperature, which was chosen to give the best compromise between peak separation, retention time, peak shape and analyte intensity.
Each lipid was identified by matching its retention time to that of a standard that was chromatographed under identical conditions, supported by fraction collection and mass spectrometric analysis. The lipid concentrations were calculated from calibration curves that were established using standard compounds. The relationship between peak height (or peak area) and lipid concentration was not linear, so the researchers resorted to a log-log plot, which gave better linearity.
Lipid classes analysed in one run
The HPLC-CAD system using a HILIC column was able to identify eleven classes of lipids from a single injection. The detection and quantification limits were 1.09-1.91 and 1.34-8.63 µg/mL, respectively, and these values applied to both polar and nonpolar lipids.
One lipid class that the method failed to detect was the ceramides, which are normally present in trace amounts in yeast cells. They eluted within a large noise peak, so could not be analysed. An initial pre-separation of the neutral and polar lipids might prove to be more successful.
Despite the success of the method, Nielsen pointed out that it is effectively a low-resolution procedure, compared with LC/MS or direct mass spectrometry in a shotgun approach. A CAD cannot provide structural information, and has a smaller dynamic range than mass spectrometry.
Nevertheless, HPLC-CAD provides an accurate and consistent way of lipid classification in biofuels and other bioengineered products prepared from yeast, with the great advantage of speed. There is no reason to assume that it cannot be extended to the analysis of lipids on other organisms.
Analytical Chemistry 2013, 85, 4912-4919: "Rapid quantification of yeast lipid using microwave-assisted total lipid extraction and HPLC-CAD"
Article by Steve Down
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.