A key step in developing solid phase extraction (SPE) methods is the wash. Ideally, SPE methods should be optimized to allow the most aggressive organic wash possible to remove matrix components, such as phospholipids that can otherwise interfere with the subsequent sample analysis (i.e. via LC/UV or LC/MS).
However, overly aggressive washing can also result in a loss of target analytes.
We must find a happy medium.
In this Technical Tip, we will use the development of an SPE method for testosterone as a model for optimizing wash steps in the SPE method development process.
Testosterone is a hydrophobic compound, often subject to abuse by athletes for its performance-enhancing properties. Because of the neutral, non-polar nature of the compound, it’s logical that the SPE would be performed using a silica-based SPE sorbent—such as Strata C18-E, or a hydrophobic, polymeric sorbent like Strata-X.
The 96-wp format of 30 mg Strata-X and 50 mg C18-E were used for this demonstration, with the following protocol:
Conditioning
The sorbent was first conditioned with 1 mL of methanol, followed by 1 mL of water. 500 uL of plasma spiked with testosterone (0.5 ug/mL) was diluted with 1 mL of water and then loaded into multiple wells of the well-plates.
Washing
To determine the ideal washing conditions, we applied a series of washes varying from 10 to 100% methanol at 10% increments, with each well containing a different wash. After the wash step, 1 mL of methanol was applied to elute the retained components.
Elution
After the wash step, 1 mL of methanol was applied to elute the retained components. A curve relating % recovery as a function of % of methanol in the wash step was then reconstructed based upon LC/MS/MS analysis of the elutents. As is clear from the curve, when the % of methanol in the wash step is increased beyond ~40%, we begin to see a decrease in the recovery of testosterone from the Strata C18-E media.
Conclusion
However, when using the polymeric Strata-X, we were able to increase the concentration of methanol in the wash to ~50-60%, which should result in a slightly cleaner final elutent.
Based upon these results, we would conclude that the use of Strata-X with a 50-60% methanol wash step would be optimal for this application.
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Related resources:
• Optimization of Wash/Elution Conditions for Automated SPE of Testosterone from Plasma
• Testosterone (Total) from Human Serum by UHPLC/MS/MS
• Ultra-High Performance on Any LC System
• Improved Recoveries Using SPE vs. Liquid-Liquid Extraction
• Strata Silica-Based Solid Phase Extraction (SPE) Sorbents