[avinashsinha]

Dear All,

I am interested in quantifying amino acids by INTERNAL STANDARD CALIBRATION. For this purpose I will be using L-Norvaline as the Internal Standard (IS). I first derivatize Amino acids to be analyzed on GCMS.

Lets say I am interested in quantification of Amino acid, L-alanine, with L-Norvaline as the Internal standard. Please see below a draft of the protocol that I have prepared, kindly check if it is right.


[1] I Prepare 100 ppm stock solution of L-alanine

[2] 10000 ppm STOCK SOLUTION of L-norvaline is prepared.

[3] 10 mL each of 90, 80, 70, 60, 50, 40, 30, 20, 10, 0.0 ppm Standard solution of L-Alanine is prepared from 100 ppm Stock Alanine Standard solution. To each of these Standard solution 50 uL (micro Liter) of 10000 ppm stock of L-Norvaline is added and then the volume is made up to 10 mL. So the final concentration of Norvaline in each Standard would be 50 ppm.

[4] I also add 50 uL/10 uL of Internal standard (IS) to 9.95 mL/1.99 mL of my sample (UNKNOWN). So the final concentration of IS in the unknown sample to be analyzed is also 50 ppm

[4] Then 2 mL each of these different standards and UNKNOWNS are derivatized and finally injected to GCMS.

[5] Now Standard plot is made with Concentration of Standard/Concentration of the IS on X-axis and ratio of areas of STANDARD/Internal Standard (IS) on Y-axis.

[6] Concentration of the unknown can simply be calculated by the slope of the calibration curve and the dilution factor of the unknown based on step [4] .

Is the above procedure right , if NOT please suggest appropriate modifications.

Thanks

Best Regards
Avinash Sinha

[JGK]

If the concentration Of IS is identical in both the STDs and samples, then surely you could just plot:


STD/IS peak area ratio (Y) against STD concentration (X)

Then you can read the sample concentrations directly from the sample peak/IS ratio.

[avinashsinha]

Thanks a lot JGK.

I did get the plot and its quite linear.

Regards
Avinash