Try the method described in this paperI. F. Uchegbu, J. A. Double, J. A. Turton, and A. T. Florence.

Distribution, metabolism and tumoricidal activity of doxorubicin

administered in sorbitan monostearate (Span 60) niosomes in the

mouse.

Pharm. Res.

121019-1024 (1995)

You might want to separate the free drug and the nano-particle (with entraped drug) first, using either dialysis (e.g. 1 ml sample in a dialysis tube and dialyzed against 100 ml medium) or ultracentrifugation (e.g. 12-14k rpm). Be careful though that in both methods you need to choose a membrane with proper molecular weight cut-off (MWCO) to allow passage of the drug but not the nanoparticles. Usually a rule of thumb is that the MWCO needs to be 100x the MW of your drug molecule to allow drug freely diffuse through. FYI, a 100kD membrane roughly equals to about 10 nm. So generally speaking, a 50kD membrane can be your starting point. After separation, you can use normal analytical methods (UV, HPLC, etc.) to determine the drug concentration.

Another separation technique that might be useful is gel filtration, or if you have access to SEC (size exclusion chromotography).

Hope this helps.

I agree with Dr. Xiaoming Xu in what he mentioned. Millipore company has a wide range of ultracentrifugation tubed mounted with membrane has specific MWCO. The trick is to choose the suitable MWCO and the proper centrifugational speed. The ultracentrifugation method preferred to be conducted at 4 degree Celsius.

I hope it is usefull.

Best luck

Tbx for your answers, but when i centrifuge my samples it will seems clear but when i put it in spectrophotometer the device cant read it. How can i solve this problem?

I would also agree with Dr. Xu concerning the dialysis. This is how I usually "clean" my nanoparticles. Though I don't know how permeable for the encapsulated drug the niosome is, as I've never had a chance to work with it. If it is impermeable, then you can purify well with dialysis, but if it leaks your payload, you should take into account that it will be dialyzed, too, leading to the decrease of total encapsulation efficiency.

What do you mean by device not being able to read your solution? Do you see no signal, or too much signal?

TNX Dear V. Andrés-Guerrero,

I'm not sure but i used centrifuge for more than one step to make sure,i separated my supernatant in each step and centrifuged it for many times,

it seems clear,

my drug in water soluble my solvent in Normal Saline serum, i used Normal Salin serum instead of PBS.

how can i find such solvent that you said?

Hi Adel, what is the size of the niosome that you prepared? If it's in the nano-meter range, traditional centrifugation won't give the clear separation you desired (unless it's done at ultra speed, such as >1million G, and for prolonged time, e.g. hours). The efficiency of the separation is normally dictated by Stoke's law of sedimentation: the smaller the particle the higher speed and longer centriguation time is required (refer to Wikipedia). I would suggest using ultrafiltration (centrifuge with a membrane filter, such as Amicon Ultra-0.5mL) or dialysis as mentioned above to avoid particle interference. To test whether you have particle interference issue, try using a dynamic light scattering instrument to determine the size of the particles. If there are nano particles in your solution, even though they are not visible to the eye, they will definitely scatter light. If that's the case, as you are testing them in spectrophotometer, it will give you artificially higher absorption signal due to the scattering (instrument cannot differentiate whether the light loss is due to UV absorption or due to particle light scattering). That being said, there are methods available to account for particle scattering using traditional UV methods, such as Zero Intercept Method (ZIM).

Good luck!

You can make ultracentrifugation for 30 minute at 25000 rpm

Then take a filterate and determine the free drug in the filterate from which you can obtain the entrapped amount

Try the method described in this paperI. F. Uchegbu, J. A. Double, J. A. Turton, and A. T. Florence.

Distribution, metabolism and tumoricidal activity of doxorubicin

administered in sorbitan monostearate (Span 60) niosomes in the

mouse.

Pharm. Res.

121019-1024 (1995)

Dialysis is the best option for it .i was tried it.

Hi,Adel,I agree with Dr.Xiaoming but if your agent is insoluble in water ,you will have some problem in measurement of free agent such as precipitation .In this case you should pass whole suspension through policarbonat membrane by extruder and calculate ingrediant that percipitat behind of membranes(M.Nikoi et al).

Try dialysis method once.

Even we worked with dialysis method, which is more suitable for calculating entrapment.

how is Entrapment efficiency calculating with dialysis bag? i know how is Release study but not Entrapment efficiency

Let me know the procedure of calculation of Drug entrapment efficiency in nanoemulsion.

hi, try sonication to rupture the vesicles and take the OD after suitable dilutions.