First of all I would like to have a clear view about standard solution. What do you mean by standard solution?? A higher absorbance of drug in supernatant is generally observed when 

1. the centrifugation speed utilized is not sufficient enough to settle down all the drug loaded nanoparticles. This leads to presence of drug loaded nanoparticles in supernatant along with free drug. To confirm wheather all the nanoparticles have settled in form of pellet at particular centrifugation speed, you need to carry out mas balance equation. You have to collect the pellet after centrifugation. dry it and weigh it. now find the entrapment efficiency. Now calculate mass balance.

eg: If you took 100 mg polymer and 50 mg drug so total weight of nanoparticles will be 150 mg in case of 00% entrapment.

Now, if you get entrapment efficiency of 50 %..it means only 25 mg drug got entrapped. so weight of your NP will be 125 mg.. accordingly you can calculate mass balance. If its nt correct then you need to increase speed and time of centrifugation.

2. Second reason is related to interaction of drug with polymer leading to shift in absorbance which may b high or low. In that case u need to study compatibility of drug with polymer in form of solution. Drug polymer and solvent in which you are making nanoparticles. If same absorbance of drug is obatained in all cases then its okk or else you will have to go for derivatization method for estimation of your drug. 

You can cross check both the point by using HPLC for precise result.

i suppose , we should go for prompt dilutions and then go for absorbance determination. Or the higher concentration is may be due to improper drug loading and drug getting adssorbed on the surface of the polymeric particles.

I think, this is an issue of low drug entrapment efficiency. so, there is need to increase the drug entrapment efficiency. This may be due to high amount of entrapped drug in the nanocapsules 

First of all I would like to have a clear view about standard solution. What do you mean by standard solution?? A higher absorbance of drug in supernatant is generally observed when 

1. the centrifugation speed utilized is not sufficient enough to settle down all the drug loaded nanoparticles. This leads to presence of drug loaded nanoparticles in supernatant along with free drug. To confirm wheather all the nanoparticles have settled in form of pellet at particular centrifugation speed, you need to carry out mas balance equation. You have to collect the pellet after centrifugation. dry it and weigh it. now find the entrapment efficiency. Now calculate mass balance.

eg: If you took 100 mg polymer and 50 mg drug so total weight of nanoparticles will be 150 mg in case of 00% entrapment.

Now, if you get entrapment efficiency of 50 %..it means only 25 mg drug got entrapped. so weight of your NP will be 125 mg.. accordingly you can calculate mass balance. If its nt correct then you need to increase speed and time of centrifugation.

2. Second reason is related to interaction of drug with polymer leading to shift in absorbance which may b high or low. In that case u need to study compatibility of drug with polymer in form of solution. Drug polymer and solvent in which you are making nanoparticles. If same absorbance of drug is obatained in all cases then its okk or else you will have to go for derivatization method for estimation of your drug. 

You can cross check both the point by using HPLC for precise result.

Hi, Abhijeet, I fully agree with your clarification.

Thanks everybody. Abhijeet I dissolved drug (30 mg) in water (20ml) to prepare standard. I got absorbance of standard 1.2. While in my nanoparticles formulation I have same amount of drug and same volume of water and its supernatant gave the absorbance of 1.5.

It means that, in supernatant, there is likely some excipients exsiting there. You'd better to check whether excipients used for your nanoparticle preparation have absorbance in your detection wavelength range. If yes, you may need seperate the excipients from your drug. Otherwise, it is impossible to get an accuratre data and therefore EE. 

I got your problem. The increase in absorbance according to your explanation may be due to interaction of ur polymer. If your polymer contains any chromophore or functional group such as carboxylic acid or amino group can lead significant shift in absorbance based on ionic interaction between them. I will suggest you to carry out interaction study. I hope it will work else you can contact me.

Yong Zhang, I think HPMC and ethyl cellulose have no absorbance. Correct me If I am wrong.

Abhijeet, HPMC and ethyl cellulose donot have carboxylic acid or amino group. Now what will you suggest?

If you go by effect of solvent on absorbance of drug then you will come to  know that the functional groups in drug will show variation in absorbance with variation in solution pH and polarity. Also there is chance of hydrogen bond interaction with drug. It has been previously reported that HPMC does interact with metformin and affects its absorbance. Also HPMC may lead to change in polarity. The increase in hydrophilic nature of functional groups of drug such as amine and decrease in hydrophilicity of acids will cause variation in absorbance, So you can have a look on change in pH and polarity of solution after addition of HPMC and cellulose.

In the end suggestions are meant for finding so that it can resolve your problem. Well if it work out its good for you. If not then I will have to study more about the topic.. thats all i would suggest.

Abhijeet, is it possible that it requires zero intercept method to determine correct absorbance?

There is a possibility that the polymers and or surfactants that you are using are interfering in the absorbance. In my research group we found that UV method gives a false result. A more reliable analytical method is chromatography (HPLC or GC) to determine drug concentration. This observation is true for both superntant solutions as well as NP extractions.

You could do a particle size analysis on the supernatant to determine if there are any NP left behind after your ultracentrifugation step.

Hope this helps.

There could be a couple of explanations to your findings.

1. The polymers could be interfering or contributing to the increased absorbance.

2. Check if you are using the correct cuvette type usually quartz cuvettes are more reliable at low absorbance maximas.

3. This could be a predicament of a very low encapsulation efficiency of the polymer you are using.

Recommendation:

You could use alternative characterization methods. You can also try a more direct method by destroying the nanoparticles using a solvent (e.g., acetonitrile) then dertemine how much was encapsulated and see how it correlates to the findings in the supernatant.

I hope this helps.  

I suggest to use HPLC. Considering the interference of nanoparticle materials, you should detect the UV spectrum of free drug and the mixture of free drug and materials.The absorbance might change or shift. Above all, you should make sure that the drug was successfully encapsulated. You need to detect the drug encapsulated at the same time.

You have to consider that the nanoparticles may also contribute to reduced transmission. What are the readings in case of nanoparticles only?

I found that the concentration of your drug solution is 1.5mg/ml, and gave a absorbance value of standard 1.2. If you are using UV spectrascopy, this value is so high that it is not likely to reflect the accurate data corresponding to drug concentration as Lambert–Beer law is not applicable and deviation may happen due to high drug concentration. My suggestionis that you can try some dilution and try to make your absorbance value in the range 0.4-0.8.

Titus you mean reading in case of blank nanoparticles?

Yong thank you. How did you find the concentration of drug? 

yes

Titus, there was just minor difference of readings between blank and formulation containing drug.

Hi, any one if i get less than one value from supernatant so how can i find drug entrappment in nanoparticles?@Giulia Fulias,@Titas Sobisch