Chromatography Forum

I am currently trying to develop a method to quantify tranexamic acid in plasma samples. I'm using L-Norleucine as an internal standard, filtering and dervitizing both the txa in the plasma and istd. I have tried quantifying with the internal standard, external standard curve and also with standard addition, but cannot get the same results from day to day.

I am using Chemstation software by Agilent. I am not very comfortable with the Agilent software and have been trying to do most of my calculations on paper/excel (which I'm sure is not good). I am having difficulty finding what equations to use to quantify it.

The original istd equation I was using was area is/area analyte = F* [is]/[analyte]. But I recenly found this equation: area analyte/[analyte]=F(area istd/[istd]).

Then for the standard addition method I was using this:
[analyte initial]/[analyte+std]=area initial/(area analyte+std)

But I don't know if the standard addition method can be used when I'm also using a istd. When I do the standard addition method, instead of using the areas of the pk, I'm using the area of analyte/area of istd.

If anyone could help me with the math, with tranexamic acid or how to program calibration for Chemstation I would be much obliged. Thank you!

You can. Basically, instead of:

A(analyte) / A(analyte+std)
= C(analyte) / C(analyte+std)

you now have:

[A(analyte)/A(ISTD)] / [A(analyte+std)/A(ISTD)]
= [C(analyte)/C(ISTD)] / [C(analyte+std)/C(ISTD)]

You don't have to do the math on paper/in Excel. When defining the compounds for the quantitative data analysis method, include the ISTD. It has to be entered *before* your compound, regardless the retention times. Don't forget to check the appropriate "ISTD?" checkbox. Afterwards, when you create the calibration table or do the quantitation, ChemStation will not use C(analyte) but C(analyte)/C(ISTD) (and the same for peak areas) automatically.

We use the ChemStation for automated sequence summary reports for both external standard and internal standard assays. I set them up myself, and I'm pretty darn computer illiterate.