Recovery calculation

General information

Recovery calculation is used to determine losses that can occur during the sample preparation process. Recovery can also be used to determine the recovery factor of a preparative purification or a chromatographic process. The recovery factor can only be determined for a single component.

A calibration curve is produced using a concentration series of an external standard. The calibration range must cover the amount in both the sample and the spiked sample. Two runs are performed, one with the sample and a second with the sample that was spiked prior to the sample preparation with a known amount of the component of interest. Quantitation of the data from the two sample runs allows the recovery factor of the sample preparation to be calculated.

Note: The recovery is measured as the recovery for the sample preparation, not for the separation during the chromatographic analysis.

The recovery factor

The recovery factor can be used to manually compensate for losses during sample preparation. The apparent amount in a sample is divided by the recovery factor to obtain the corrected amount.

How to perform Recovery calculation

The table below describes briefly how Recovery calculation is performed.

Step

Action

1

Perform a run with each level of the standard.

2

Peak integrate the curves to produce a peak table for each level.

3

Use the data from the peak tables to produce a calibration curve.

Note: This is the same process that is used in the External standard quantitation.

4

Spike a portion of the sample with a known amount of the component of interest prior to the sample preparation.

5

Run both the spiked and an unspiked sample.

6

Peak integrate both samples to produce peak tables for the unspiked sample and the spiked sample.

7

The amounts for unspiked and spiked sample are calculated from the calibration curve. The difference between these amounts provides the apparent amount of the addition.

See illustration below:

8

The ratio of this apparent amount compared to the amount actually added to the sample determines the recovery of the system.

Example: If 2 mg of the component of interest had been added to the sample and quantitation indicated an apparent amount added of 1.6 mg, the recovery factor would then be 0.8.

Reliability

Below are some specific factors that determine if the recovery factor result is reliable:

  • A spike amount that is of the same order of magnitude as the sample must be used to maximize the precision.

  • It is assumed that the recovery is the same for both the sample and the spiked sample. However, if the recovery varies according to the amount of the component of interest, the results are unreliable.

2005-06-15