Cold EI demonstrated its usefulness in the following aspects:
- All the diglycerides, cholesteryl esters and triglycerides were eluted in 10 min using Cold EI GC-MS. They all exhibited good peak shapes without any ion source tailing, while achieving excellent sensitivity and high ratio of peaks to column bleed.
- Cold EI provided molecular ions information for all lipids. The Cold EI mass spectra of each triglyceride exhibited useful molecular ions with relative abundances of 10-50% to the base peak. Molecular ions are not only important for the elucidation of the structure of the triglycerides but it also helped to resolve co-eluting saturated and unsaturated triglycerides in these triglyceride.
- Cold EI provided informative high mass fragment ions information. As a result, it is possible to more accurately obtain the structure of the diglycerides and triglycerides.
- Cold EI provided useful information about diglycerides that eluted among the cholesteryl esters. The Cold EI mass spectra of diglycerides were characterized by having abundant molecular ions plus a nearby high mass fragment of M-18 in view of loss of water. Cholesteryl esters Cold EI MS were characterized by a dominant m/z=368 fragment ion.
- The analysis time was merely 10 min.
- Caffeine was found at various concentrations in the samples, which indicates the ability to simultaneously monitor drugs in blood using the same method.
- Several free fatty acids were found. The most abundant were palmitic, linoleic, oleic and stearic but arachidonic acid and smaller free fatty acids were also found.
- Vitamin E was found, possessing only one major isomer of MW = 430 (alpha tocopherol).
- 25-Hydroxy Vitamin D3 was found, but we are not sure which isomer.
- Cholestadiene was found and its ratio to cholesterol could be measured.
- Cholesterol was the second biggest peak. Its relative and absolute concentration greatly varied between different samples by more than a factor of 3.
- Several diglycerides were found. In total, they were more abundant than the triglycerides. Chemically, they should be more active than triglycerides.
- Six cholesteryl esters were found. Cholesteryl esters were the most abundant lipids in the serum samples and among them cholesteryl C18 (linoleate, etc.) were the most abundant. We were surprised to find that the cholesteryl esters only consisted of multi unsaturated acids such as linoleic and arachidonic but none with palmitic acid or oleic acid. Probably the saturated CEs had very small molecular ions. CEs were more abundant than cholesterol by about a factor of 3-4 (sample dependent).
- The triglycerides were found with a mixture of acids. Accordingly, the molecular ions of the one with tri C18 consisted of a mixture of 0-8 degrees of unsaturation. The most abundant TAGs were with one C16 and two C18. Only small amount of tripalmitin (tri C16) was present despite palmitic acid being the most abundant free fatty acid in the serum.
|Figure 2. Total ion mass chromatograms of serum zoomed at the elution time of free fatty acids and the Cold EI mass spectrum of linoleic acid.|
|Figure 3. Total ion mass chromatogram of serum zoomed at the elution time of vitamin E and 25-hydroxy vitamin D3 and the Cold EI mass spectrum of vitamin E.|
|Figure 4. Total ion mass chromatogram of serum zoomed at the elution time of diglycerides and the Cold EI mass spectrum of a diglyceride with molecular weight of 592.|
Figure 5. RSIM mass chromatogram with m/z = 368 of cholesteryl esters in serum zoomed at the elution time of cholesteryl esters and the Cold EI mass spectrum of cholesteryl linoleate.
Figure 6. Total ion mass chromatogram of serum zoomed at the elution time of triglycerides and the Cold EI mass spectrum of glycerol tri C18 fatty acids mixture
|Figure 7. Cold EI mass spectrum of a triglyceride GC peak of glycerol with three C18 fatty acids. Note the range of degrees of unsaturation from 0 to 8 (peaking at 4).|