Gas Chromatography - Tandem Techniques - Examples of GC/MS Spectrometry Applications > Analysis of Waxes and Lipid Type Materials > Page 74


It is clear, that the peaks depicted on the single ion monitoring chromatograms represent metabolites in which the D-ring has been preserved. This example illustrates how the GC/MS technique can be a valuable tool for following the metabolic processes associated with the 17a-alkyl anabolic steroids.


Analysis of Waxes and Lipid Type Materials


The metabolism of fatty acids in cell cultures can be very important, particularly with regard to cancer cells. The possible conversion of the fatty acids to peroxides and the role of these compounds in carcinogenesis is still not completely understood. To aid in this type of work Wallace and Coleman (20) developed a procedure for the fatty acid assay of cell tissue employing a GC/MS instrument. According to their publication, they studied two human colon carcinoma lines, HT29/219 and HY115, together with a human breast cancer cell line ZR-75-1. The cells were grown in Dulbeccos's modified Eagles medium, augmented with 10% v/v foetal calf serum (DFC10) or horse serum (DH10). They were seeded at a density of 1.9 x 104 cells per cm2 and maintained at 37ūC in a humidified atmosphere of 5% carbon dioxide and 95% air. The cells were harvested into a phosphate buffered saline at specific intervals up to 120 hours. Lipids were extracted with chloroform/methanol (2+1) containing 2, 6-di-tert-butyl-4-methylphenol as an antioxidant. The extract was separated on a thin layer plate using n-hexane-diethyl ether-acetic acid (70+30+1) as a solvent. The phospholipid, triglyceride and free fatty acid spots were scraped off the plates and the lipids removed by eluting with a chloroform methanol mixture(2+1). The phospholipids and triglycerides were trans-esterified (21) using sodium methoxide in methanol and the free fatty acids were methylated with diazomethane.


It was found that for satisfactory operation the column must have a very low level of bleed. Although the use of polymer coated capillary columns would appear to be ideal for this work, the resolution was inadequate and the best stationary phase was found to be a Carbowax polymer column (polyethylene glycol). The combination of retention data with mass spectrum gave virtually unambiguous fatty acid identification.