Dear,
I'm having some problems implementing the Stewart assay to measure the concentration of phosphate. Can you send Stewart assay protocol for liposomal suspensions?. I have the article but there are parts that are not explained well there.
Calibration curve of lipid mixture:
All glassware’s used in the study were washed with chromic acid solution (5g sodium dichromate was dissolved in 5mL deionised water in a 250mL beaker. 100mL of concentrated sulphuric acid was slowly added with constant stirring and allowed the mixture to cool to room temperature) to avoid the possible contamination from surface active cleansing agents.
A stock solution containing 90mg of lipids mixture (HSPC:35mg; DPPC:35mg; DPPG: 8.6mg; DSPE-mPEG2000: 11.4mg) was prepared in 100ml chloroform (900µg/mL).
The stock solution also contains cholesterol of 13.5mg as one of the component of the final formulation. The 2.5mL of the above stock solution was diluted up to 25mL with chloroform to obtain a concentration of 90µg/mL solution. From this stock solution 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 2mL were pipetted off (9µg to 180µg), added to 3.0mL ammonium ferrothiocyanate solution in a test tube, and enough chloroform was added to make the final chloroform volume 3.0 ml. The biphasic system was then vigorously mixed on a spinex for 3min. The lower chloroform phase was separated with a syringe and the optical density of the chloroform was read at λmax 472nm against chloroform as a blank and the average optical density was plotted against concentration.
Liposomes: A Practical Approach (Practical Approach Series)5 Jun 2003
by Vladimir Torchillin and Volkmar Weissig
New, R. R. C. 1989. Liposomes A Practical Approach. New York: Oxford University Press Inc.
I have attached a technical note I prepared a while back for this.
You must note that if you run a standard curve for say DPPC only, but your liposome formulation has DPPC and DPPG... then you will get the incorrect absorbance. This is demonstrated in the attachment.
The attached technical note references the article below:
1. Stewart, M. Colorimetric Determination of Phospholipids with Ammonium Ferrothiocyanate. 1980. Analy. Biochem. 104.
Thanks Antonio, very good technical note. I still doubt if AF is Anhydrous sodium sulfate. You can confirm this please ?.
"AF" is the combination of ferric chloride hexahydrate and ammonium thiocyanate. The sodium sulfate is only used if the solution is not clear... meaning some precipitate is visible. The solution should be a clear pink/purple from what I remember. If you have access to a LC/MS, I would recommend using that instead.. as the Stewart assay is good, but you may run into some issues. Run lots of controls to avoid any interference.
You can please follow the link below to obtain certain valuable informations:-
http://www.liposomes.org/2009/01/stewart-assay.html
dears,
I have a problem. I did according the protocol but there is no absorption for liposome sample, while I got a fine standard curve. Do you encounter such problem? I guess it happens because liposome does not solve in the chloroform? I am waiting for your precious comments. thanks
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