Dear Misbah,

I'm guessing you're referring to liquid-liquid extraction method using methanol, chloroform, water and BF3 as solvents for the extraction of FA in serum samples. Is this correct?

In my own experience, the extraction of FA depends greatly on the solvent composition. I've noticed this in LDL (plasma lipoproteins) but I believe this is true no matter the matrix (Reis et al., JLR, 2013, 54, 1812). The extraction of lipids depends on the partition of the lipids across the solvent mixture, so for the particular case of FA profiling I think you might want to choose a solvent mixture with apolar character (either increasing the chloroform content or use hexane in your mixture).

Apart from solvent composition (extraction method) and matrix (serum/plasma), please beware that FA content is also influenced by other factors, such as age-, gender-, and freeze/thaw cycles. These effects are well described in the literature.

Hope this helps,

Ana

We use the following method.

1ml plasma was mixed with internal standard (10-100 µg heptadecanoic acid, Sigma Chemical Co., St Louis, in chloroform depending on the cell number), 2 ml methanol and 3.9 ml chloroform, and then centrifuged (200 0 g, 10 min, 4 0C). The chloroform phase was removed and evaporated to dryness under a stream of nitrogen. Total lipids were saponified with 2% KOH in methanol and the FAs methylated with 14% BF3 in methanol. The resulting FA methyl esters (FAMEs) were extracted with hexane and analysed by capillary gas chromatography (Perkin-Elmer 8420 Capillary Gas Chromatography, Gouda, The Netherlands). Column: 50 x 0.25 mm WCOT fused silica, CP-sil 88; flame-ionization detector (FID) temperature 300 0C; oven temperature programme from 150 to 230 0C at 2 0C min-1; carrier gas N2.  The mass spectra of FAME from representative samples were obtained using a Hewlett-Packard (HP) 6890 capillary GC interfaced with a HP mass selective detector and controlled by a HP Chem. Station. Column: 25 x 0.25 mm ID, QC2 x BP x 70; detector temperature 280 0C; oven temperature programme from 100 0C to 290 0C at 3 0C min-1; carrier gas helium. 

FAMEs were identified by their retention time and compared to those of authentic standards (Sigma Chemical Co., St Louis), and by GC-Mass Spectrometry. The detector response factors were determined by injection equal weights of FAs and internal standard methyl esters on to the column. Their amounts were estimated by calculating the corresponding areas of FA and internal standard.

       The following FAMEs (Sigma Chemical Co., St Louis) were used to determine the total fatty acid profiles: myristic, 14:0; palmitic, 16:0; cis-9 hexadecenoic, 16:1n-9; cis-7 hexadecenoic, 16:1n-7; stearic, 18:0; oleic, 18:1; cis-vaccenic, 18:1; cis-linoleic, 18:2; linolenic, 18:3; 6,9,12,15-octadecatetraenoic, 18:4; arachidic, 20:0; cis-11-eicosanoic, 20:1; 11,14-eicosadienoic, 20:2; cis-11,14,17-eicosatrienoic, 20:3; arachidonic, 20:4; 5,8,11,14,17-eicosapentaenoic, 20:5; behenic, 22:0; erusic, 22:1; cis-13,16-docosadienoic, 22:2; 7,10,13,16-docosatetraenoic, 22:4; 7,10,13,16,19-docosapentaenoic, 22:5; cis-4,7,10,13,16,19-docosahexaenoic,22:6; lignoceric, 24:0 and  nervonic, 24:1.

Reference

Yazıcı Z., Tavares I.A., Soydan A.J., Hollingsworth S.J., Bishai P.M., and Bennett A. Methotrexate alters the fatty acid composition of NC Adenocarcinoma cells in culture. Eur. J. Cancer, 28A:1468-1471, 1992.

Dear Misbah,

In short, there shouldn't be a drastic difference between a blood fatty acid analysis between serum and plasma; unless you're looking for oxylipids or other specific lipids.  I work exclusively with plasma, but have collaborated with labs that often do not alter their protocols when switching between serum and plasma. Obviously there will be some optimization to the method for your work flow, but 2:1 chloroform:methanol works great for lipids. We published a variation of this where we isolated plasma phospholipid from 200uL of plasma (Pickens et al. Plasma phospholipids, non-esterified plasma polyunsaturated fatty acids and oxylipids are associated with BMI. PLEFA. 2015). I've profiled plasma with as little as 50uL of plasma, but in many cases a ratio of 1 part plasma to 3 parts extraction solvent is used.  A great comprehensive paper on profiling of metabolites from serum/plasma/cells is "Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatograph coupled to mass spectrometry" by Dunn et al. Nature. 2011.

Feel free to message me with any troubleshooting or questions.

Best,

Austin

Dear Mishba, 

you can use the same protocol for lipid isolation. However the selection of the procedure is very important depending of your key compounds as Ana Reis and Charles Pikens has commented above. In general is accepted that for fatty acid profiling, isolation using the chloroform:methanol protocols (Folch or Blight and Dyer) would not yield different compositions. In my experience the critical point when using an isolation method is to perform it exactly as it was originally described, taking into account the fat content and water contents in the sample. 

Reading your question, I am not sure if you want to isolate, derivatize and them analyze or you want to perform a single step derivatization. This latter means that you isolate and derivatize in a single step. It is a good option if you want a total profile. On the other hand, if you are willing to analyze different lipid classes, then yes, you need to isolate and them perform some fractionations steps. 

However be very careful when using an acid catalyst to yield FAME: i) acid catalysts are very good as esterification agents (e.g free fatty acids to FAME) but poor for transterification (triacyglicerides to FAME) and moreover ii) when using temperatures they can change your fatty acid profile so you have two options: avoid temperature or use a protectant (DMF or DMSO). Thus, if you want to obtain FAME from TG there you need a basic catalyst but if you want FAME from FFA, then you need an acid catalyst. Finally if you are willing to obtain a total fatty acid composition my suggestion is to combine both catalysts (first basic and them the acid). And please, always include an internal standard for calculations. 

You can use 0.2- 1 ml serum or plasma. Final volume of sample must be 1 ml adding saline solution. 

Yes, you can use the same procedure. You can use 0.3-0.5 ml of blood plasma/serum.

I applied Folch metod for purification of serum and plasma to analyze fatty acid composition (SCFA and LCFA). The results were the same.

Yes, yo can use the same procedure

Yes Luis I need to analyze free fatty acid on GC MS after derivatization to FAME. thank you for you advice, I will be very careful while using acid or base catalyst for  yeilding FAME.

Charles Pickens thank you so much for providing me references of such informative articles. they are indeed of great help.  

THANK YOU everyone for your valuable answers. This made me feel relaxed that I can use same protocol for serum with some prior optimization. 

I used to use  choloform, methanol-BF3 and hexane for derivatization. Its easy and only take 2 hours.  will share the protocol soon.