We want to properly quantify saturated and polyunsaturated fatty acids produced in Yarrowia lipolytica using GC-MS. We have a DB5-HT column (30x250x0.1). Can you recommand extraction and quantification protocols? Do you work with multiple levels of each fatty acid and an internal standard? How do you calculate the final concentration (either in gram FA per gram dry cell weight or liter)?
Thank you for you help.
Michael
If you are looking to just look at total FA and not lipid classes a simple extraction followed by a methylation method works best. We used a one step method. If you send me your e-mail to [email protected] I can send you a copy of the protocol. For internal standard you will want to use something like 17:0 or 21:0 in a concentration of the average expected FA level. Generally ~50 to 100ng/ul is a good start with a small bore column(less than .30 mm ID). To calculate concentration divide the IS conc by peak area to get units of FA/Unit of area. Then just multiple your sample peaks by that factor. You can run a equal wt FA mix to calculate a correction factor for the individual FAMEs. NuCheck prep has some good standard mixes for this purpose. 68a or 462a are good standards for samples that have a wide chain length range of FAMEs. I would go with a more polar column like a BPX70. You will get better separation in a shorter run time than the low polar columns. E-mail me if you have more specific question.
Michael,
you can think about obtaining FAME as suggested Peter or take a look on other alternatives. Generally you need to derivatize them before GC. Check these papers:
Article Sample preparation procedure using extraction and derivatiza...
Article Method for the determination of carboxylic acids in industri...
regards,
G
Great answer Peter, Grzegorz!
I would only add that the selection of derivatization reagent (e.g. BSTFA or MSTFA , with or without TMCS...etc) should be compatible with your analytes for optimum efficency. The type of extraction you are using can give you a hint (e.g. MeOH extractions involve more polar FA than Hexane or Chloroform ones..choose the methyl ester accordingly)
Also, as in articles suggested above, ultrasound-assisted derivatization reactions works better in general (in my experience).
Good luck!
-S
Direct two-stage methylation (sodium methoxide + boron trifluoride in methanol) of partly dried biological material (without preliminary extraction of lipids into organic solvent) is undoubtely the most convenient, quick and correct method. Margaric acid C17:0 is usually used as internal standard. The use of polar polyethylene glycol stationaty phase (such as SUPELCOWAX-10) guaranteers quite efficient separation of fatty acid methyl esters even using rather short columns (15-20 m x 0,25 mm). Nonpolar capillary columns are thought to be undesirable while analyzing FA mixtures - as well as the use of trimethylsilyl FA derivatives (separation efficiency is evidently worse in this case).
Here is the reference, you can read it easily using http://sci-hub.tw method.
Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae, 2010. Lipids 45(11):1053-60
- DOI: 10.1007/s11745-010-3468-2
- We typically separate neutral lipids from phospholipids, and from glycolipids by silicic acid column chromatography using chloroform, methanol, and acetone-methanol (9:1 v/v), respectively. This protocol is described in J. Amino Acids, vol 2013, ID 240537. After samples were dried under N2, we do saponify and methylate fatty acids with BF3 in methanol (14% BF3 in methanol; Sigma) according to Metcalfe et al (Anal Chem 38 (93): 514-515, 1966). We first saponify by adding to each sample 0.5 ml of methanolic-NaOH (2 grams of NaOH/100 ml of methanol and incubate at 100oC for exactly 5 min. Then, 0.62 ml of 14% BF3 in methanol is added and heated at 100oC for exactly 2 min. You can use glass marbles as caps on glass vials. Remove the samples from heat and cool to room temp. To extract FAMEs, add 1.25 of pet-ether to each sample. Remove and save each top pet-ether layer because your FAMEs are in the pet-ether layers.
- After drying under N2, we resuspend in 500 microliters of hexane and add 3 microliters of a stock solution of methylpentadeconaote (MPD; 48.750 micromoles/ 3ml hexane) (Sigma Chemical). This will give you your internal standard of 48.75 nmoles of MPD per sample) and our injection volume is 3 microliters.
- We then separate fatty acid methyl esters (FAMEs) by GC with flame ionization detection using a Supelco Omega-wax capillary GC column (0.53 mm x 30 mm x 0.25 film thickness). Injector temperature is 225o C and flame ionization temperature is 235oC. The split ratio is 3:1 with a helium flow rate of approximately 20 to 25 ml/min. The initial column temperature is 185oC. After a 5 min delay, the column temperature was increased to 205oC at a rate of 2oC per minute. The final temperature (205oC) was held constant for 10 to 15 minutes depending on how many log-chain polyunstaturated fatty acids in your sample. Because we usually work with brain extracts which have high levels of docosahexaenoic acid (22;6,n-3) , and nervonic acid (24;0), our run time ranged from 40 to 45 minutes.
- We quantify how many moles of each biological fatty acid are found by comparing to our internal standard (MPD; 48.75 moles / sample).
- 48.75 nmoles of MPD per area of MPD = (X nmoles) / area of each fatty acid
- We then take the sum of all biological fatty acids in nmoles and then calculate the final %moles of each biological fatty acid with the sum of all biological fatty acids as the denominator.
- While we us GCMS for determining membrane steroid levels, we use GC equipped with FID for membrane fatty acid levels.
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