non linearity

[pliva]

dear all,
we are doing analytical method development for dicycloverine hcl using lc method. when i inject 20 to 80 microlitre of my stock , i get good linear response but when i make a serial dilution the stock i cant get the lineariyu at all. the funny thing is, when i check with lowest concentration by injecting 10,20, 30 to 80 microlitre it shows linearity. i am wondering what went wrong with me. i check the dilution but it was correct. can anybody find this problem before.

[Kostas Petritis]

It can be a number of factors including bad pipeting (instrument or human error). Have you calibrated your pipets? Can you describe the way that you are making the dilutions?

[prashant.pqa]

dear all,
we are doing analytical method development for dicycloverine hcl using lc method. when i inject 20 to 80 microlitre of my stock , i get good linear response but when i make a serial dilution the stock i cant get the lineariyu at all. the funny thing is, when i check with lowest concentration by injecting 10,20, 30 to 80 microlitre it shows linearity. i am wondering what went wrong with me. i check the dilution but it was correct. can anybody find this problem before.

Dear
I think Kostas is saying correct. Also so many factors such after preparing solution where your storing it or immediatly your keeping in autosampler it is nothing but you have to check stability of your drug in diluent also.

Also concern about the pipette.
All the best

[AdrianF]

On several occasions it has been pointed out that serial dilution should not be used to prepare standards. Any errors are multiplied as you increase the dilution. Dilutions should all be made by taking various volumes from the stock standard.

Serial dilution may be OK for homeopathy but not HPLC!

[mbicking]

We would need more information about what you mean by "non-linearity." Is the response non-linear at high concentrations or low concentrations? Also, injection different volumes in LC can cause linearity problems as well. It is unusual, however, to see a better result with different injection volumes.

I would disagree with the statement that serial dilutions should not be used. If you use "good laboratory technique" and a Class A glass pipet, you can get excellent linearity. I have done this many times. But the other replies are also correct, if your technique is bad or your pipets are not calibrated, then you will have calibration problems. A combination of serial and parallel dilutions is often the best approach.