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### ISD

Posted: Sun Mar 26, 2017 2:28 am
hello every one I really need a help from you Im using a multi level calibration curve with the internal standard method ( adding equal amount of IS in all the unknown samples ) when I analyse the data to get the unknown concentration of my plasma samples I do the following :
1* find the response ratio ( sample peak / internal standard peak )
2* draw the standard calibration curve and finding the slope and the intercept
3* calculate the concentration for all the unknown samples using the equation

(y=mx+b)

where
y =the response ratio ( sample peak / internal standard peak )
m = slope
b = intercept
x = the unknown concentration
then multiply the x by the dilution factor snice my sample used was 7.5ul and the final volume of preparation is 150ul so I multiply by 20 to get my rea concentration in 7.5ul
my internal standard volume was 15ul
but I Don't included it in my calculation is that right ?. because I SAW my lab mate included it in the calculation to get the unknown conc. like he will take the resulted concentration directly after calculating it from the equation (y=mx+b) and multiply it by 15 divided by 7.5

Im confused

and please can you suggest me a good reference to help me in doing such a calculation because Im in japan and they cant communicate in English with me and this is totally new for me

### Re: ISD

Posted: Sun Mar 26, 2017 7:04 pm
Hello Aya

aya wrote:
I SAW my lab mate included it in the calculation to get the unknown conc. like he will take the resulted concentration directly after calculating it from the equation (y=mx+b) and multiply it by 15 divided by 7.5

Didn't you or him miss the zero in 15?

### Re: ISD

Posted: Mon Mar 27, 2017 12:47 am
no I asked him about this 15 he told me is the internal standard volume so he will multiply by the IS volume and then divided by the plasma volume I don't know why

### Re: ISD

Posted: Mon Mar 27, 2017 5:35 am
If you are working from a calibration of analyte peak area / IS peak area vs known analyte concentration then the concentration of the IS makes no difference and correcting for the volume of the IS makes no sense.

Peter

### Re: ISD

Posted: Mon Mar 27, 2017 12:05 pm
aya wrote:
...
and please can you suggest me a good reference to help me in doing such a calculation because Im in japan and they cant communicate in English with me and this is totally new for me

There is probably a teacher there in the school who can explain you differences betweewn external and internal standard calibration.
But start from reading what is calibration in chromatography and how to calculate concentration of the analyte from calibration curve. Dilution of the sample doesn't complicate calculations so much and you can make it later, not by chromatography data system.