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Troubleshooting SPE Reproducibility Problems

The first step in troubleshooting irreproducibility is to verify that the analytical system is functioning correctly.  Reproducibility problems can be caused by sample-to-sample carryover, detector problems, or a defective autosampler.  Injection of known standards in the final extract solvent will verify that the response factor of the analytical instrument has not changed.  Repeated injections of pure standards can verify injection reproducibility, while injection of standards of wide-ranging concentration (very high levels followed by very low levels) can diagnose carryover (Figure 1).

The most common causes of reproducibility problems in SPE are a non-rugged method, protein binding problems, or changes to the sorbent from the manufacturer.

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Figure 1: Diagnosing analytical instrument reproducibility problems.
 

During SPE method development and optimization the ruggedness of the method should be examined by testing what effect small changes in parameters such as solvent composition, solvent volume, and flow rate have on the protocol (i.e. do they impact recovery, extract cleanliness etc.).

Another factor which can affect ruggedness is the quality of the SPE sorbent, therefore, different lots should be tested to ensure that the protocol performs well - this takes into account that specific sorbent lots may only be available for a short time.  Provided these factors have been taken into consideration during method development then this should not be the source of any reproducibility problems.

To determine the source of the irreproducibility, first confirm that all of the solvents used in the protocol are correct.  Following this each step of the SPE protocol must be examined to re-test the limitations of the method; this is done by varying the solvent composition and collecting fractions from analyte standards in appropriate pure solvents.  Any changes in method performance exhibited by the standards in comparison to the original method are most likely due to a change in sorbent chemistry (to resolve this problem it may be necessary to contact the manufacturer to obtain different sorbent lots for comparison against the original method).

If the standards perform correctly it may be that the analytes are experiencing sample-matrix related phenomena, the most common of which is protein binding.  If this is the case pre-treatment steps such as, pH modification, denaturation with a chaotropic agent or organic solvent, precipitation with acid or organic solvent, addition of binding site competitive compounds, or the use of restricted access media (RAM) SPE phases should be employed.

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Figure 2: Diagnosing SPE method reproducibility problems.

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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