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Investigating SPE Recovery Problems

One of the most common problems in SPE is poor or inconsistent recovery.  This quick guide details how to determine if you have a recovery problem and defines step by step procedures which can be used to assess at which point in the SPE process loss of analyte(s) is occurring.

In order to assess any problems with recovery it is important to define the overall recovery of the extraction process.  Recovery of the extraction procedure (RE) is given by Equation 1.1

 

Where:

ResponseExtracted sample = average area count for the analyte in matrix which has been through the extraction (SPE ) process

ResponsePost-extracted spiked sample = average area count for the same quantity of analyte spiked into extracted matrix after the extraction procedure

Furthermore, when techniques such as LC-MS are being used the matrix can cause suppression or enhancement of the analyte signal.  The matrix effect (ME) can be calculated from Equation 2.1

 

Where:

ResponsePost-extracted spiked sample = average area count for the same quantity of analyte spiked into extracted matrix after the extraction procedure

ResponseNon-extracted sample = average area count for the same concentration of analyte in neat solution

When it has been determined that there is a problem with the recovery the source of this problem must be identified.

 

Chromatographic System or SPE Protocol?

First it should be verified that the problem is not being caused by a change in the chromatographic system or detector.  The simplest diagnostic test for this is to inject known standards in the final extract solvent to verify that the response factor of the analytical instrument has not changed since the method was validated.  Following this, absolute recoveries from the extraction procedure of both target analytes and internal standards should be calculated by comparison with the injection of neat standards. 

This data can be compared to the original absolute recoveries calculated when the method was first validated (Figure 1, screen 2).

 

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Figure 1: Assessing source of recovery problems in SPE.

 

Once it has been determined that the recovery problem is with the extraction procedure the next step is to determine where the analytes are being lost i.e. are they eluting from the extraction column and being discarded during sample loading or wash steps, are they remaining on the SPE column during elution or are they lost in the sample pre-treatment steps? 

The best diagnostic to answer these questions is to process standards in pure solvents through the extraction procedure, collect fractions from each protocol step and analyze each fraction to determine where the analytes are being lost (Figure 1, screen 3).

 
 

Analyte Loss in Loading or Wash Steps
If the analytes are breaking through the extraction column during sample loading or wash steps it must be verified that the solvents used in these steps are correct, as well as the solvents used in the conditioning and pre-equilibration steps since these steps influence retention.

If the low recovery is a new behavior that has not been seen previously and all solvents have been verified as correct then this would suggest that the sorbent chemistry may have changed.  At this point a comparison of the lot numbers of the sorbent exhibiting poor retention should be made with the original sorbents used.  It is also a good idea to contact the manufacturer to see if new lots may be obtained (Figure 2, screen 2).

 

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Figure 2: Assessing breakthrough in loading and wash steps.

 

If changes to the method are allowed try altering the sample solvent or the wash step s to enhance retention for the mechanism being used (Figure 2, screen 3 and 4).
In the extreme case where the SPE product has changed irrevocably it may be necessary to change to a more retentive sorbent chemistry or even change the extraction mechanism used (Figure 2, screen 5).

 

Analytes Strongly Retained

If the analytes are being retained on the column (as evidenced by their absence from the load and wash fractions) but are not being eluted, first verify that the solvent is correct.  If so, it again may be an indication that the sorbent chemistry has changed and different lot numbers should be compared (Figure 3, screen 2).

 

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Figure 3: Problems with analyte elution.

 

To help elute the analytes from the column alter the elution solvent to one that has a greater elution strength for the mechanism being used (Figure 3, screen 3).  Also, review the possibility of secondary interactions between the sorbent and the analyte and confirm that the elution solvent addresses these (Figure 3, screen 4).  Finally, it may be necessary to change to a less retentive sorbent (Figure 3, screen 5).

If the SPE protocol seems to be working properly it is likely that the analytes are never reaching the column during the loading step, or are breaking through during loading but are not detectable in the fraction.  The former may be caused by analyte instability in the sample, or if the sample pre-treatment involves protein precipitation and the analytes are protein bound they may be precipitating with the protein fraction. 

Alternatively the protein bound analytes may be passing through the SPE column unretained and are undetectable via the analytical procedure in their bound condition.  In either case, if protein binding is suspected try changing the sample pH, adding chaotropic agents or diluting the sample with mechanism compatible organic solvents to disrupt the protein binding (Figure 3, screen 6).

 

References

  1. Anal.Chem. 2003, 75, 3019-3030.
 
 
 

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