A few comments:

Before you can begin quantifying samples, you will need to develop as many of the actual methods as required for each group of compounds you wish to quantify. Select a technique to use (e.g. GC or HPLC), a detector (e.g. FID, MS, DAD, RI....) which can "see" the compounds you are interested in and a method to resolve them. Once you have that information, get help from someone at your University whom can help you learn how to create a method, collect data and run samples.

Once you have all of the methods developed and verified, you will also need to determine what the expected concentration ranges will be for each compound. Once you have that information, you will be able to prepare a range of HPLC stds to create a calibration table that covers the expected ranges. Use at least 5 levels of std for each compound. More is better as you want to cover the expected range so you can interpolate all results (NOT extrapolate!). This is simple math, but it helps to have good software which can perform the curve fits for you for each std.

Remember to use the same injection volume for ALL samples and standards. Don't inject different amounts of the same sample to "create" different stds. Dissolve all final samples and stds in the mobile phase (for HPLC).

Dear Bill Letter

Thanks a lot for your valuable suggestions. I am able to develop the test program to run sample. For your options; I have to run the stds  with various concentration.

I am using Dinex ultimate 3000 HPLC, and ACN, Acetic acid (1%) is my mobile phase. So for preparing std solution what is better to mix with mobile phase or methanol?

Thanks again.

Yousuf

dear yousuf,

it would be better to use mobile phase for sample preparation if it is possible in HPLC.

Yousef: Please see my answer above. "Dissolve all final samples and stds in the mobile phase (for HPLC)."

Dear Bill Letter

Thank you for your kind information. Today I will try it...........

I used Acetonitrile and Acetic acid (1%); which is better to use for standard? I look forward to hearing from you.

"Dissolve all final samples and stds in the mobile phase (for HPLC)."

If your mobile phase is ACN with acetic acid then use that solution.

1. Use the following formula for calculation.

Concentration (mg/kg)= 

   Sample peak area x nanogram of standard injected x Total Volume in milliliter

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Standard peak area x volume of standard injected in microliter x wt. of sample in gram

2. For preparation of standard solution of the compounds first make a 200 ppm (mg/l) stock solution and then by serial dilution go up to 1 ppm level.

Dear Subir Kumar Nag

Thanks a lot, wish your helping hand if I will face further difficulties.

For making standard solutions, first we have to make single stock solution for each compound, then as a second step prepare mixed working solution (based on your system sensitivity you can adjust  concentration by further dilution, expressed as ug/mL ).  Final result can be calculated as below:

 (Peak Area of Sample/Peak Area of Standard )x Concentration of Mixed Standard Solution , ug/mLx (Dilution volume of sample, mL /Sample Weight, g ).