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HPLC Method Validation- Accuracy
Posted: Mon Feb 21, 2005 3:39 pm
by Chem1
I would like to know how to calculate the percent recovery of spiked samples in accuracy.
Thanks for any suggestions,
Posted: Mon Feb 21, 2005 9:43 pm
by tom jupille
% recovery = (measured mass)/(added mass) X 100
where "measured mass" is the result you obtained on the spiked sample, and
"added mass" is the amount you spiked. This assumes that the matrix sample you used had no analyte in it to begin with.
Posted: Tue Feb 22, 2005 12:28 pm
by Chem1
Is “measured massâ€
Posted: Tue Feb 22, 2005 6:05 pm
by tom jupille
Yes. To expand a bit:
1. Run your standards and establish your calibration plot
2. Run your spiked sample and determine the amount of analyte using that calibration plot
3. The ratio of the amount of analyte determined in step 2 to the amount you actually added (times 100) is the percent recovery.
Posted: Tue Feb 22, 2005 6:13 pm
by Chem1
Tom, thank you for your help.
Posted: Tue Feb 22, 2005 6:19 pm
by tom jupille
You're welcome!

about recovery
Posted: Tue Feb 22, 2005 10:06 pm
by mdyo
Hi ,
How can I get the recovery if the matrix sample I use has an amount of the analyte in it ?
Posted: Tue Feb 22, 2005 10:32 pm
by tom jupille
It's called "standard additions". You spike at multiple levels and then extrapolate back to zero added to get the amount in the matrix. The whole concept of "recovery" is probably hazy in this situation.
Posted: Tue Feb 22, 2005 11:18 pm
by mdyo
Thanks a lot tom but why do you think it is hazy?
Posted: Tue Feb 22, 2005 11:33 pm
by Consumer Products Guy
Like Tom infers, I would only use standard additions as a last resort. If you're in the business, why can't a placebo matrix be prepared for you, which you could then spike? That's what we do.
Posted: Tue Feb 22, 2005 11:56 pm
by tom jupille
The idea behind "recovery" is to be able to account for any bias in the results (for example, some of the analyte sticking irreversibly to the matrix). Standard addition compensates (hopefully!)for the bias (in effect, your actual sample is included in all the calibration runs), but it doesn't account for it in the sense that you don't know how much occurred. The big drawback is that, in effect, you have to run several calibrators for each sample. As Consumer Products Guy (can we just call you "CPG" for short?) points out, it's really a last resort; used most often when analyzing endogenous compounds for which a "blank", per se, doesn't exist.
Posted: Wed Feb 23, 2005 8:22 am
by mdyo
Thank you all.
It seems to me that I have to use the standard addition method as my sample is a growth medium for a bacterial species which is analysed for the analyte at different periods of time.
Don't you think so?
Posted: Wed Feb 23, 2005 5:49 pm
by tom jupille
Can you make or obtain the same medium without the analyte?
Posted: Wed Feb 23, 2005 6:42 pm
by mdyo
Yes. But the real sample after a period of time will have other compounds produced by the bacteria and some others may be consumed.
Don't you think that this will cause matrix differences?
Posted: Wed Feb 23, 2005 10:10 pm
by tom jupille
Yes, but those differences are what you're trying to measure.
Please don't misinterpret what I was trying to say; there is nothing inherently wrong with using standard additions, but it does represent much more work than the usual "external standardization". In "standard addition", you essentially have to generate a separate calibration plot for each sample. In "external standardization", you generate a single calibration plot which you use for many samples.