I would try dissolving the system using temperature and a surfactant. Then run sds-page to separate insulin from gelatin and quantify using the same gel

Hi Abdul, I agree with Omar.

At high temperatures (~40oC), gelatin will dissolve and you can use insulin ELISA kits to determine the insulin concentrations.

Dear Dr.Omar and Steingrimur, Immense thanks for the nice suggestions! 

Dear Omar! I would like to know about the surfactant, i should use in association with temperature? Do you mean SDS which is used in SDS page!

Dear Steingrimur! If i undertood your idea correctly is that, I should first heat the gelatin nanoparticles at high temperature (~40oC),followed by using the ELISA kits to quantify the released insuli? This ELISA kit would detect only insulin and not the gelatin? I would highly appreciate if you attach some standard protocols for ELISA kits involved analysis of insulin!

Yes use sds to favor solubilization. For the other answer you will need to determine that insulin is stable enough to resist the heating process. Elisa is always attractive but expensive when compared to quantification using sds-page

Hi Abdul, gelatin aggregates at specific concentrations at specific temperatures.

Dissolving the gelatin at ~40oC and adding 5-to 10X buffer volume (PBS) will prevent the gelatin from re-forming pellets.

There are a lot of insulin ELISA kits available:

https://www.google.com/search?num=100&site=&source=hp&q=insulin+elisa+kits&oq=insulin+elisa+kits&gs_l=hp.3..0j0i22i30k1l9.1177006.1187526.0.1188275.18.18.0.0.0.0.522.3952.0j8j9j5-1.18.0....0...1.1.64.hp..0.18.3935...0i131k1.JWhg0f3YNFY#spf=1

Dear Steingrimur

I am highly grateful for your prompt reply

Dear Omar Gonzalez-Ortega! 

Immense thanks for your expert opinion!

So, you mean I should treat my gelatin based nanoparticles with the surfactant (SDS} accompanied by heating up to approx. 40o Celsius to facilitate or promote solubilization of gelatin. Consequently, this joint heat and surfactant mediated solubilization will cause the release of entrapped insulin, which will be subsequenltly quantified via SDS-page.

Here, at this point, i would like to add my comment.

I have a validated RP-HPLC method for the analysis of human inuslin. So, instead of using SDS-Page, how it would be if I analyse my sample using this validated chromatographic assay procedure after the above treatment of solubilization !

What is the size of the gelatin? What are the conditions for the rp-hplc analysis?

Dear Dr.Omar

I am following the protocols elaborated regarding the rp-HPLC analysis of insulin in the article attached herewith! Hopefully, this will serve the purpose!

The size of gelatin nanoparticles is in the range of 200-300 nm!

Regarding the molecular weight of gelatin, so i am using type B gelatin having bloom number of 175-225 which is associated to a molecular weight of 40 kDa-50 kDa.

The specifications of gelatin is described in the article attached herewith

My concern is that in general high molecular weight proteins block the entrance pore of smaller molecules and thus separation is affected. The diffusion of large proteins is also slow which promotes wide peaks. Unless you are using a large pore silica particle you could have problems in the separation. Also you have to be sure that gelatin is soluble in the orgamic solvent you are using. 

Dear Dr. Omar

Regarding the solubility of gelatin in organic solvents, being a hydrophilic polymer, it is soluble in any organic solvent!

I mean gerlatin is not soluble in organic solvents!