Technical Tip: Optimizing Wash and Elution Protocols in SPE Method Development

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:


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.


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.


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.


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.

More technical support:

Request-a-Method™: Contact our Method Development Team for a tailored solution!
Applications: We have thousands of them! Search by analyte name or structure.
Free Guides and Posters: Download free guides and posters for troubleshooting, method development, and more.
Product Videos: See the technology in action. Watch our product videos and demonstrations.
The Technical Library: Check out our helpful collection of technical notes, posters, webinars, how-to videos, guides, and more.
Interactive Web Tools: Simple, interactive tools to instantly find the right products and solutions.
FAQ Portal: Find answers to popular technical questions here.
Quality and Safety Documents: Download your CQA, C of A, and MSDS.
Product Care and Use: Love your products and treat them right using these handy guides.
Training & On-Site Support: We’ll come to you! Classroom training or in-lab demonstrations—you pick the topic.
Troubleshooting: Did something go wrong? We’ll fix it!
Chat With Us: Our Phenomenex technical experts (Phenoms) are ready to chat with you about method development and technical support.

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

Leave a Reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.