Chromatography Forum

I am doing a method validation for the accuracy. the first run give me good linearity and accuracy. When I try to run "between run accuracy", however, the sample accuracy go out of the range, all the accuracy number go out of lower range, say < 15%(20% for LLOQ). I had though it is integeration problem. After adjusting the integeration several time, still the same. Could any body tell what is the reason and what to do, from both experimentally and data processing? THANKS!!!!!!

You said you were trying to check the accuracy.

Were you using a QC plasma sample or just one of your plasma calibrators used to prepare the calibration curve?

James, thank you very much. I simply use three of my plasma calibrators (low mid high) as my QC plasma samples. I has thought they could be same, am I wrong? I am looking fokrward to your further opinion.

I usually use a different group of solutions.

I prepare the calibration standards by adding 25 ul of the appropriate concentration of analyte in 2:1 methanol:water to 100 ul of plasma. That doesn't precipitate the plasma proteins. I then add about 250 ul of the internal standard solution prepared in pure acetonitrile to precipitate the plasma.

I prepare the medium, low, and high QC samples by dissovling 0.25 ul of the appropriate solution of analyte in 2:1 methanol:water in 5 ml of plasma. Then agitate vigorously with vortexer to make sure homogeneous.

I figure this simulates the actual environment of the drug plasma. The calibration is convenient to use, since I can prepare it fresh everyday.

How the QA sample same or not same as the caliberator related to the accuracy check. Thanks for the information.

I think diluting the drug into the plasma at low concentrations of organic is better way to do QC.

If you prepared the standard the same way, would be no difference in my opinion. However, in my previous note I prepare the standard a different way because it wastes less plasma and can prepare fresh whenever I want.

I got that approach from the literature. Don't have the reference with me and will not be back to the office for a week to cite.

Would like to have others opinions..

did you also check your injection medium stability?

the needle rinse properly? maybe require more rinse to preven the bulidup?

injector malfunction?