I am developing method of analysis for the compund which is having a very low dose that it 0.4mg/ capsule .

i have developeled a rs method for this which is isocratic one . containing a phosphate buffer 0.01M, 0.2 %TEA and PH adjusted to 6.0with phosphoric acid .

mobile phase buffer 52% 48% acetonitrile
column is : spherisorb ODS1
Flow rate ; 1.5ml/min
wavelength : 225

so for RS analysis i need to prepare a sample having atleast 1mg/ml of concentration so that impurity of 0.1% has sufficient response.

so i am preparing sample by taking 20mg equivalent of the drug ina 100ml flask (50ul injection volume)
means average wt of capsule is 325mg
and if i take 20mg equivalent of the drug then
weight i am taking is about to be 16mgs of the sample , i am preparing my sample in a 50water:50Acetonitrile mixture , sample is sonicated for 20minutes inorder to get the good recovery.

my probleam is that in above doing i am getting so many peaks up to 5minutes period and my one of impurity is eluting at 3.4minutes which is merging with the placebo peaks . anybody can suggest how to remove all this placebo peaks as this is rs method i am not in position to use spe.

my impurity is very polar and its eluting in void in any reverse phase , it is only eluting in ods1
at 3.4 minutes. ion pair at PH 6.0 IS also not working as i used decane suphonic acid at ph 6.0 i got the same retention of this impurity . (impurity is basic having NH2 GROUP)

SO PLEASE SUGGEST SOME PRACTICAL TIPS TO THIS.

Try and use Zorbax SB Phenyl Column (or any phenyl column) or Discovery RP amide C16 with ID of 250 mm x 4.6mm and alter the mobile phase proportions and check. And how do you conclude that these are placebo peaks, have you injected the placebo of proportional concentration and checked. Another option is to use Tetrabutyl Ammonium Hydrogen Sulfate.

What are the excipiens of your capsules?

If you are getting so much placebo peaks, that isn't normal I think. Have you tried to inject a placebo, to wich was added standard, so you dont have to sonicate your solution? You might be degradate your component by sonicating. If this is the problem, you never get a better result if you don't change your sample preparation method.

I HAVE INJECTED A PLACEBO IN SAME WAYS AS SAMPLE PREPARATION AND CONFIRMED THAT THE PEAKS ARE DUE TO THE PLACEBO NOT DUE TO THE COMPOUND DEGRADATION.
WE REQUIRED SONICATION FOR ANALYSIS OF THE CAPSULES AS CAPSULE CONTAINES MATRIX WHICH IS A SR MATRIX CONTAINING UTRAGIT (POLYMERCOATED)
SO ON SONICATION SLOWLY DRUG WILL COME OUT OF THE MATRIX.

BY SHIRISH

Can you determ what component of the sample matrix is the one that is actualy disturbing?

If you can give some more information, there are possible readers that have the same matrix/problems.

i am in a process of evaluating the placebo components , as actually average weight is 325mg , it seems that even 1mg is going to be the 50mg when we weighe 16gm of placebo. so i have to study each components , i am in process of this.

i am in a process of evaluating the placebo components , as actually average weight is 325mg , it seems that even 1mg is going to be the 50mg when we weighe 16gm of placebo. so i have to study each components , i am in process of this.

Here is a suggestion that might work: if you bring your amine into a non-ionic form at alkaline pH it has much more retention than in the ionic form, which you usually have at pH 6. The difference is often factor of 30 in retention. In order to do so, you need to use a column that is compatible with alkaline pH such as XTerra RP18 or XTerra MS C18.

I Already tried a carbonate bufer at ph 9.0
increase in retention is found but i am dealing with the rs analysis so other impurities also i have to locate which is eluting very late and gradient is giving lots of blank peaks above discussion on gradient is also initiated by me.

Most compounds are still half ionized at pH 9. Go to pH 10 or higher to get more retention.