Hi, Can any one explain the spike and recovery calculation for Liquid Liquid Extraction and Solid Phase Extraction using HPLC for urine sample? Is there any protocol to do this recovery study?
Usually I will make 1.0 ng/ml by spiking 40 micro liters of 50 ng/ml to 2 ml of pooled urine. Then do the process for LLE or SPE. Finally I will reconstitute into 250 micro liters and inject into HPLC. Here I need calculation support get the final concentration ~1.0 ng/ml
can any body help me?
Recovery calculation can be done in terms of quantified amount of a given analyst. If you do internal spiking of the sample in question then there would be commensurate rise in peak area for the given analyst corresponding to the added standard in a given amount. The rise in peak area in itself can be correlated with concentration or amount recovered of a given analyst. In external spiking to calculate the recovery you need to compare the peak area of standard with that of the analyte in question. Better you develop calibration curve of peak area v/s concentration for the said cause.
I agree with Shrikaant, the best and only accurate way of doing such a recovery study is by quantitation via calibration curve.
Once you have a concentration from your sample the only thing you need to do is account for the dilution factor. You spike into 2 mL but reconstitute in 0.25 mL. This means, assuming you have calibrated in ng/mL of injected analyte, that you would need to multiply your result by 2.0/0.25 to get the amount recovered from the original 2.0 mL.
You have concentrated a 1.0 ng/mL solution a factor of 8. If you get 100% recovery you will find 8.0 ng/mL in your injected solution. Divide whatever the quantified concentration of your spike is by 8.0 and you will get your percent recovery.
Thank you all ,
I agree with you all and I got clarify in my calculation.
Finally I reached my % recovery .
And also please clarify this small calculation
Can i include 40 micro liters injection volume like
(amount-ng *250 micro lit)/(40 micro liters*2 ml)
to get final concentration ng/ml.
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