Dear All,Back to the Top
I am desparately in need to know about calculating the
percentage recovery of an analyte from a biological
sample. I am very much confused to see the scanty
information about it in literature. I will be thankful
to know the exact protocol and method to calculate it.
I used radiolabled pure compounds. Knowing dpm in vs. dpm out, one canBack to the Top
determine losses [recovery, degradation, etc.]. Alternately, do UV-vis
standard calibration curve, quantitate amount added in test biological
matrix, & via absorption peak of recovered sample, calculate % loss.
Probably easiest is D-labeled [2H] standard added to mix at earliest
stage [since virtually indistinguishable from 1H of native compound].
Then quantitate recovery for both with dual detection selective mass
detection [if you are fortunate to have an MS discriminating 1 amu].
Let me know if you need more details.
650/281-9399; fax: 707/313-0955
555 Bryant St., #506
Palo Alto, CA 94301-1704
You spike the same amount of drug in buffer say 1:1 ACN/water or MeOHBack to the Top
water and do the same prep for and biological sample.
Compare the area under the drug peak in both cases, ratio of biological
sample to buffer would give the % recovery. ie. area of buffer
considered to be 100% recovery.
Hope this helps you.
Hello Jimmy,Back to the Top
Guidance for Industry: Bioanalytical Method Validation (2001) by US-FDA
(CDER) gives a very clear defination for recovery and its
have copied the same from there:
The recovery of an analyte in an assay is the detector response
from an amount of the analyte added to and extracted from the biological
matrix, compared to the detector response obtained for the true
concentration of the pure authentic standard. Recovery pertains to the
extraction efficiency of an analytical method within the limits of
variability. Recovery of the analyte need not be 100%, but the extent of
recovery of an analyte and of the internal standard should be
precise, and reproducible. Recovery experiments should be performed by
comparing the analytical results for extracted samples at three
concentrations (low, medium, and high) with unextracted standards that
represent 100% recovery.
Vijay V Upreti
Department of Pharmaceutical Sciences
University of Maryland,
20 Penn St., Baltimore, MD 21201
Dear Martin,Back to the Top
Thanks for your answer.But everybody is not fortunate to have
everything radiolabeled. And while talking about calibration curve and
spiking known amount in matrix, are you refering to QC samples? Then how
many QC samples are considered sufficient to calculate recovery?
Are you talking about QC samples for calculation of recovery? How much of
them are needed to be run on HPLC and are they be run everytime you do
An alternative is to put blank matrix (e.g., plasma) through the prepBack to the Top
procedure and then spike a known amount of drug into a measured aliquot
of the blank extract. Compare the peak area to a sample aliquot of
matrix spiked with the same amount of drug. For Mass Spec detection,
matrix components sometimes affect the signal either positively or
Thomas L. Tarnowski, Ph. D.
Project Team Leader
Dept. Head, Drug Metabolism and Pharmacokinetics
Roche Palo Alto
3431 Hillview Avenue, Palo Alto, CA 94304
Dear Jimmy,Back to the Top
Any concentration but make sure you are comparing same concentration
with plasma preparation vs buffer preparation.
During real sample analysis always run your QC's to make sure your
std.curve is valid to back calculate the unknown samples.
Normally QC's are selected from lower, mid and higher ends of your std.
curve and again it depends on whether your research is in preclinical,
clinical, ordevelopement. Based on the stage of the program guidelines
differ for the percentage error of your QC's.
Hope I had given enough info.
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