Amino Acid Calibration by INTERNAL STANDARD METHOD

[avinashsinha]

Hello Everyone & Merry Christmas,

I am working on amino acid quantification by GCMS. We first derivatize Amino acids to be analyzed on GCMS.

I am interested in quantifying amino acids by INTERNAL STANDARD CALIBRATION. For this purpose I will be using L-Norvaline as the Internal Stnadard.

Kindly guide me in detail how go about sample preparation for this method of calibration. Also how to plot the calibration curve?

Should I prepare Standards of a given amino acid of different concentrations based on mass/volume or on molar basis. Because same concentration (g/L) of different amino acids will be different on molar basis.

Kindly help me in this regard
Thanks

[tom jupille]

Kindly guide me in detail how go about sample preparation for this method of calibration.

Exactly the same as the sample preparation for external standard calibration, except for the addition of the internal standard prior to derivatization.

Also how to plot the calibration curve?

Again, very similar to that of external standard calibration. The only difference is that you plot area ratio (instead of area) versus concentration.

Should I prepare Standards of a given amino acid of different concentrations based on mass/volume or on molar basis. Because same concentration (g/L) of different amino acids will be different on molar basis.

It doesn't matter. Because you know the molecular weights of your analytes you can always convert mass to moles or vice versa, so simply use the one that is more convenient for your purposes.

[avinashsinha]

Thanks a lot Tom,

But I want some further clarification on my first point:-

Lets say I am interested in quantitation of Amino acid L-alanine with L-Norvaline as the Internal standard. Now I describe the procedure for you, 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 50uL (micro Litre) of 10000 ppm stock of L-Norvaline is added and then the volume is made upto 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 to 10 mL/2 mL of my sample (UNKNOWN).

[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 on X-axis and ration of areas of STANDARD/Internal Standard on Y-axis.

[6] Concentration of the unknown can simply be calculated by the slope of the calibration curve.

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

I was also reading a book and it says the we have to find the response factor. Where response factor = ((Area of Standard Signal/concentration of Standard)/(Area of Internal Standard Signal/ Concentration of Internal Standard)).

I understand the utility of Response factor but how do we use it in Internal Standard Calibration curve.

Please guide me all the above.

THANKS

[avinashsinha]

Dear All,

My doubts are not fully clear with the reply posted above. I am working on amino acid quantification by GCMS. We first derivatize Amino acids to be analyzed on GCMS.

I am interested in quantifying amino acids by INTERNAL STANDARD CALIBRATION. For this purpose I will be using L-Norvaline as the Internal Stnadard.

Lets say I am interested in quantitation 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 50uL (micro Litre) of 10000 ppm stock of L-Norvaline is added and then the volume is made upto 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 to 10 mL/2 mL of my sample (UNKNOWN).

[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 on X-axis and ration of areas of STANDARD/Internal Standard on Y-axis.

[6] Concentration of the unknown can simply be calculated by the slope of the calibration curve.

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

Thanks