Chromatography Forum

I have one doubt regarding my work. I am unable to proceeding forward without clarifying this.
I am getting problem regarding internal standard. I am trying 2 internal standards. one ISTD is giving very much high intensity than analytes. other ISTD is giving very less intensity than analytes. Finally I ve decided to use 2nd ISTD by giving more conc. like
100uL(1000ppm of ISTD) + 100uL(100 ppm of 1st analyte) + 100uL(100 ppm of 2nd analyte)+700uL of solvent.
Can I proceed like this? If possible How can I do validation in invitro study and invivo study. Please suggest me.

You really have not given enough information for a detailed answer (for instance, what kind of detector, what kind of matrix, at what point in the workup are you adding the internal standard, etc., etc,).

However: internal standards work best when the response (peak area or height) of the IS is comparable to that of your analyte(s), since you will be taking a ratio of peak areas. The actual concentration needed to attain that is less important (so long as you don't have to overload!)

thank you Tom,
I am using flourascence detector. I have to do chiral separation of analyte 1 and its metabolite analyte 2 with an ISTD in plasma by using polar organic mode. For this I have taken more conc of ISTD(1000ppm)+less conc of analytes(100ppm)+plasma. Then only I am getting comparable areas of ISTD and analytes. what I want to ask is Can I proceed like this? If possible how can I proceed in invivo study. Please suggest me.

As long as you are getting comparable areas for the analytes and IS, there should be no problem. The internal standard is added during sample preparation, so has nothing to do with the "in vivo" aspect of your analysis.