The Importance of Ion-Exchange Capacity in Mixed-Mode SPE

Reporter EU Volume 11


Most researchers who use SPE to process biological samples rely on traditional single-mode retention mechanisms to recover, clean up, and concentrate their compounds of interest prior to LC or GC analysis. Although single-mode SPE chemistries are often adequate, problems can easily arise. For example, reversed-phase chemistries (e.g., C18) can result in insufficient clean up. Although selectivity can be improved when using ion-exchange phases, the technology is often very sensitive to sample matrix effects. In this report, we will discuss the utility of mixed-mode SPE technology, and how ion-exchange capacity can play a critical role in many bio analytical applications.

back to top Back to Top

How Mixed-Mode SPE Works

Discovery DSC-MCAX (Mixed-Mode Cation Exchange) SPE utilises the dual retention mechanisms of both hydrophobic and electrostatic interaction to retain basic, acidic, neutral, and zwitterionic compounds from aqueous sample matrices such as biological fluids. Its broad affinity for such a wide range of compounds is due to the dual bondings (octyl, C8 & benzene sulphonic acid, SCX) contained within the packed-bed.

Although the technology was designed to drastically improve the selectivity/sample clean up of basic and zwitterionic compounds, DSC-MCAX is useful in other applications as well. Through the careful manipulation of pH and organic strength, the user can also fractionate basic and zwitterionic from acidic and neutral compounds, or adjust the protocol to permanently retain any unwanted basic and zwitterionic interfering components while eluting acidic and neutral compounds of interest. Table 1 summarises the generic protocol recommended for DSC-MCAX use.

Table 1 Recommended Generic Protocol for Discovery DSC-MCAX

back to top Back to Top

The Importance of Ion-Exchange Capacity

Most bioanalytical applications provide analytical support for drug metabolism/ pharmacokinetic studies. In some cases, parent drug compounds can metabolise into very polar compounds that are difficult to retain via reversed-phase SPE. As a result, two different retention mechanisms are required to retain both parent drug and metabolite(s).

In this application, piroxicam and piroxicam’s highly polar metabolite, 2-aminopyridine, were spiked in human urine. Spiked samples were then extracted via Discovery DSC-MCAX using the recommended generic protocol, and analysed via HPLC-UV. The results were then compared against two competitor mixed cation phases run in parallel. Using the DSC-MCAX provided excellent clean up of biological samples such as urine (Figure A). Although greater than 80% recovery was observed for piroxicam on all three mixed-cation phases, recovery suffered for 2 aminopyridine on the two competitor phases. Unlike piroxicam, 2-aminopyridine’s polar nature required cation exchange to be the dominant mode of retention. Discovery DSC-MCAX’s high ion-exchange capacity allowed for excellent retention of both piroxicam and its polar metabolite 2 aminopyridine.

Figure A Competitor Comparison of 2-Aminopyridine and Piroxicam from Human Urine (567516-U)

back to top Back to Top


The dual retention properties of Discovery DSC-MCAX provide a broad affinity for a wide range of compounds. By controlling pH and organic strength, the technology offers superior clean up and selectivity relative to most single-mode SPE phases. DSC-MCAX’s high ion-exchange capacity also allows for the selective recovery of polar basic compounds.

back to top Back to Top


Related Links