ISTD concentration for LC-MS/MS

[limeiluo]

Dear all,

I am developing a method for plasma samples. I have got liear calibration curve, but my QCs at mid-concentration always have high positive bias. I am not sure if it is something to do with my ISTD concentration. For analyte quantitation with LC-MS/MS, how ISTD concentration affects the results (e.g. STDs and QCs accuracy)?

Many thanks to any response.

[sandip.gurav]

Internal Standard Concentration :

First of all an internal standard should be added at the beginning of the sample work-up, typically before the plasma crash or solid phase extraction. The internal standard should be added at the same level in every sample including the standards. An internal standard should give a reliable MS response. Care should be taken that the amount of the internal standard is well above the limit of quantitation but not so high as to suppress the ionization of the analyte. "How much internal standard should I add?", this is an important question. It pays to know roughly how much compound is in your sample. This can be accomplished by making trial analyses of an early, middle and late time point with perhaps one or two standard points. This information will be very valuable when building an appropriate standard curve and in knowing how much internal standard to add. If you were trying to quantify samples in the range of 100 fg to 25 pg and the limit of detection was 100 fg you might add 5 to 10 pg of internal standard to every sample. A good rule of thumb is to target the internal standard to the lower 1/3 of the working standard curve. This is a range that will give a comfortable response without interfering with the ionization of the analyte.


Apart from this if U need any clarification u ca mail on

sandip.gurav@gmail.com

[limeiluo]

Hi, Sandip,

Thanks a lot for your explanation. Normally, I use an internal standard (ISTD)concentration which gives a similar response to the middle level standards of my calibration curve. This practice worked for many methods, but this time when I used the same rule my QCs at low concentrations had high positive bias. So I increased ISTD concentration, then I noticed high positive bias at mid-concentration level. Maybe I should try lower ISTD concentraton.

best regards

Li