A Case Study in SPE Method Development - Understanding the Dual Interaction Properties of Discovery DSC-SCX SPE Using Verapamil (and Metabolite) from Serum as a Test Example

Reporter EU Volume 10

A strong cation-exchange (SCX) SPE method was developed to selectively recover verapamil and its major metabolite, norverapamil from porcine serum for subsequent HPLC analysis. Initial experimentation using standards revealed that neither an increase in eluant pH (to neutralize the basic amine functional groups exhibited by verapamil and norverapamil) nor organic strength alone were sufficient to elute analytes from the SCX phase. Instead, a combination of the two strategies was required to completely elute the analytes of interest. This information was invaluable towards developing a rugged wash system for removing unwanted endogenous sample interferences co-extracted onto the SCX phase.

The extraction method, coupled with the Discovery C18 HPLC column, offers a powerful analytical approach for the quick and accurate analysis of verapamil and norverapamil from serum.

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Principles of Ion-Exchange SPE

Ion-exchange SPE utilizes electrostatic interaction between the analyte and sorbent functional groups to retain charged molecules from a variety of sample matrices. In order for electrostatic retention to occur, both the analyte and sorbent functional groups must be oppositely charged. Strict control of pH is necessary to manipulate the ionization states of both the analyte and sorbent’s acidic/basic functional groups thereby controlling selectivity (retention and elution) during the SPE process.

Although electrostatic interaction is the primary mode of retention in ion-exchange SPE, secondary reversed-phase or mixed-mode interactions may take place. This is especially true when organic analytes are introduced to the sorbent in the presence of an aqueous sample matrix.

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Sample Characteristics and Phase Selection

Verapamil is an anti-anginal/anti-arrhythmic calcium channel blocker used treat cardiovascular disorders. Both verapamil and its major metabolite, norverapamil, carry secondary and tertiary amines, respectively making these basic compounds good candidates for cation-exchange (Figure A). Both compounds have a pKa of 8.6-8.9.

Figure A. Structure of Verapamil and Norverapamil


Discovery DSC-SCX (Figure B) is a polymerically bonded, benzene sulfonic acid functional group with an H+ counter ion and exhibits a low pKa of <1. At virtually all pH levels, the SCX functional groups remained negatively charged.

Figure B. Structure of Discovery DSC-SCX


As stated earlier, in order for electrostatic retention to occur, both sorbent and analyte functional groups must be oppositely charged. Dropping the sample pH to at least 2 pH units below the analytes’ pKa (pH = 6.6) should effectively ionize the analytes’ amine functional groups.

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Load Optimization & Wash/Elution Profile

In this study, 1ml working standards of both verapamil and norverapamil (5.0μg/ml) in 10mM ammonium formate, pH 3.1 were loaded onto Discovery DSC-SCX cartridges (100mg/1ml) previous conditioned and equilibrated with 1ml methanol and DI H2O. The load flow through eluate was then collected and analyzed via HPLC-UV. A lack of analyte presence in the load eluate indicated that adequate retention was observed under acidified aqueous conditions.

To determine application specific wash/elute parameters, Discovery DSC-SCX cartridges were conditioned and equilibrated with 1ml MeOH and DI H2O, and loaded with 1ml of the 0.5μg/ml verapamil/norverapamil low pH test mix. The respective tubes were washed/eluted with 1ml test solvents ranging from 0-100% methanol in 50mM sodium borate, pH 11.3. The wash/elute eluate was collected and analyzed via HPLC-UV. A graph relating organic strength to recovery was used to identify breakthrough (Figure C).

Figure C. Results of the Wash/Elute Profile for Verapamil and Norverapamil on Discovery DSC-SCX SPE


By profiling the major parameters affecting analyte retention and elution, application specific guidelines were established for defining, optimizing, and troubleshooting an extraction method. To elute verapamil and norverapamil from the SCX phase, disruption of the electrostatic interaction between SCX’s sulfonic and the analytes’ amino functional groups was necessary. By increasing the eluant pH to at least two pH units above the analytes’ pKa, the compounds were effectively neutralized disrupting the ionic interaction retaining the compounds of interest. However, because of secondary hydrophobic interactions between the sorbent’s phenyl group and the analytes’ carbon backbone, pH modification alone was not sufficient at eluting the analytes of interest. An organically modified buffer of appropriate pH was required for analyte elution (50% methanol in 50mM sodium borate, pH 11.3). Also learned from the wash/elute profile were two powerful wash steps. Both neat buffer and methanol can be used separately to wash off any co-retained hydrophilic and lipophilic interferences, respectively prior to analyte elution.

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Incorporation of Serum Sample Matrix

Porcine serum was incorporated into a method defined by data generated from precursory load optimization and wash/elute profile studies (Table 1).

Table 1.


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Simpler SPE Procedure, Cleaner Extracts, Reduced Analytical Run Times, & Excellent Recovery and ReproducibilityReferences

Figure D illustrates and compares chromatograms of both the SPE extract and external standards. Using the SPE method development approach, we were able to identify two powerful wash steps (neat methanol and buffer) to remove any coretained lipophilic and hydrophobic interferences inherent with the serum sample matrix. As a result, the baseline was free of interfering background peaks that may have co-eluted with the two peaks of interest. Also, cleaner extracts allowed for shorter analytical run times (<4 min.). Unlike most reversed-phase protocols which require 100% organic elution that must be evaporated and reconstituted prior to HPLC analysis, a weaker solvent (50% methanol in 50mM sodium borate, pH 11.3) wasemployed for final elution. This allowed for the direct injection/analysis of the final eluate reducing the number of extraction steps and minimizing overall processing time.

Figure D. Example Chromatograms of Extracts Generated from the Systematically Developed Method Using Discovery DSC-SCX SPE


Average absolute recoveries and RSDs for both verapamil and norverapamil were 102.9 ± 1.2 and 95.9 ± 2.3%, respectively.

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Conclusion

When extracting ionized organic compounds from aqueous samples using ion-exchange SPE, retention rarely occurs exclusively via electrostatic interaction. Secondary hydrophobic interactions must be considered. Some researchers consider secondary or multiple interactions problematic often complicating the sample prep system. However, if studies are conducted to profile the major parameters affecting analyte retention and elution, highly selective SPE methods can be developed to specifically target and isolate analytes in very complicated/dirty sample matrices.

By profiling the affects of pH and % organic modifier, we were able to determine powerful wash conditions to remove both hydrophilic and lipophilic interferences. Cleaner extracts resulted in shorter analytical run times. Elution conditions using a more selective weaker solvent of appropriate pH allowed for direct injection thereby shortening overall processing time while maintaining high recoveries and low RSDs.

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