Try using an assay kit for phosphatidylcholine. The kit contains a phospholipase that release the choline and a reagent for detecting the choline. The liposomes should be dissolved in detergent before adding the reagents.

Quantitative determination of phospholipids

Colorimetric determination through an acidic digestion method. One milliliter of the oil sample should be digested by heating with 0.65 mL of 70% perchloric acid until the yellow colour disappeared alongside the standard (0.10 to 0.90 μg/mL KH2PO4). The digests should be diluted with 3.5 mL of distilled water followed by 0.5 mL of ammonium molybdate solution and 0.5 mL of ascorbic acid solution. The content should be shaked very well and heated in a boiling water for 30 min for colour development. The absorbances of cool samples (including the standards) are read at 800 mm. (Rouser et al., 1970). Try this. Good luck

I imagine that if you label your SUVs with the FM1-43 dye, you may use FCS (fluorescence correlation spectroscopy) to calculate the vesicle concentration (together with their sizes). Finding the proper dye concentration doesn't seem to be trivial though.

Article Molecular Anatomy of a Trafficking Organelle

 Stewart Assay was successful for POPC quantification 

Dear Morris and all,

Stewart Assay (1980) is the easy and quick method to quantify phospholipid concentration. You can find the method in my paper, i have recently published which is:

"Proliposome powders prepared using a slurry method for the generation of beclometasone dipropionate liposomes"

or you can find it on research gate

https://www.researchgate.net/publication/282812596_Proliposome_Powders_Prepared_Using_A_Slurry_Method_For_The_Generation_of_Beclometasone_Dipropionate_Liposomes

you can find the same method over internet too.

Article Proliposome Powders Prepared Using A Slurry Method For The G...

Which detergent is recommended for determination of PL content in liposome using Stewart protocol?

I was also wondering about the detergent.

The Stewart Assay

The original Stewart assay protocol was published by J.C.M Stewart in Journal of Analytical Biochemistry in 1980:

Stewart, J.C.M. (1980). Anal Biochem, 104, 10. http://www.ncbi.nlm.nih.gov/pubmed/6892980

Stewart assay is based on the ability of phospholipids to form a complex with ammonium ferrothiocyanate.

The advantage of this method is that unlike Bartlett assay, the presence of inorganic phosphate does not interfere with this assay and therefore PBS buffer or other phosphate based buffers can be used.

A simple conversion factor is used to translate the absorbance values (from the spectrophotometer) into milligrams of phospholipid. Keep in mind that this conversion factor is different for phospholipids with different head groups, therefore this method is not applicable to samples where mixtures of unknown phospholipids may be present.

One of the drawbacks of this method is its inability to quantify phosphatidylglycerol(PG).

Equipment and reagents

1) 10 ml centrifuge tubes 2) Centrifuge 3) Cuvettes 4) Spectrophotometer

Preparation of reagents and standards

Ferrothiocyanate Reagent: Dissolve 27.03 gram of ferric chloride hexahydrate (Sigma Aldrich Cat. no. F2877) and 30.4 gram of ammonium thiocyanate (Sigma Aldrich Cat. no. 221988) in double distilled water and volumized it to 1 liter. The solution is stable at room temperature for several months.

Standard: Make up 10 ml of solution of phospholipid in chloroform at concentration of 0.1 mg/ml

Method

1) Pipette reagents and standard into the centrifuge tubes as follows:

Prepare triplicates of each tube. Perform the same procedure with the test samples.

2) Vortex each tube for 20 seconds.

3) Spin each tube for 10 minutes at 1000 r.p.m (approx 300 g) in centrifuge and remove the lower layer using a Pasteur pipet.

4) Read the optical density of standards and samples at 485 nm.

5) Find the concentration in the test sample by comparing it with the standard curve.

For a simple quantification of known PLs I would do a TLC run and then stain the plate with either primulin or molybdenum blue spray reagent (or both consecutively). Silica plates can be developed in chloroform/methanol/28% ammonium hydroxide, 6:4:1 (v/v/v) solvent system (Rf for POPC is about 0.4). The whole procedure with quantification takes about 2 h. Best: Karoly