Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents.
Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.
Shen
Chun Fang
CF
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Que., Canada H4P 2R2.
Hawari
Jalal
J
Kamen
Amine
A
D000818:Animals; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000032:analysis*; D004952:Esters / Q000032:analysis / Q000138:chemical synthesis; D005227:Fatty Acids / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry; D007202:Indicators and Reagents; D007313:Insecta; D008055:Lipids / Q000032:analysis*; D008322:Mammals; D011136:Polysorbates; D012015:Reference Standards; D012996:Solutions; D012997:Solvents
D016895:Culture Media, Serum-Free; D004952:Esters; D005227:Fatty Acids; D007202:Indicators and Reagents; D008055:Lipids; D011136:Polysorbates; D012996:Solutions; D012997:Solvents