Sample Preparation by Mixed-Mode SPE Using ISOLUTE® HCX

Importance of the Topic

The accurate extraction of basic pharmaceutical compounds from complex biological fluids is critical for pharmacokinetic studies, clinical diagnostics, and therapeutic drug monitoring. Mixed-mode solid-phase extraction (SPE) combines hydrophobic and cation-exchange interactions to selectively retain target analytes while removing matrix interferences, thus improving sensitivity and reproducibility in downstream analyses.

Study Aims and Overview

This technical note evaluates the performance of the ISOLUTE HCX series of mixed-mode SPE sorbents for the isolation of basic drugs from plasma and urine. The study investigates sorbent conditioning, sample loading, interference removal, and final analyte elution to deliver highly purified extracts suitable for low-level detection.

Methodology and Instrumentation

Sample Pretreatment:

• Combine 100 µL of biological fluid (plasma or urine) with 100 µL of 0.05 M ammonium acetate buffer (pH 6.0).

Sorbent Conditioning and Equilibration:

• Condition wells with 1 mL methanol under gravity or a brief vacuum/pressure pulse.
• Equilibrate with 250 µL of 0.05 M ammonium acetate buffer (pH 6.0).

Sample Loading and Interference Elution:

• Load 200 µL of buffered sample using low vacuum (< –5″ Hg) or 1–3 psi pressure pulses.
• Elute acidic and neutral interferences sequentially with 250 µL each of ammonium acetate buffer (0.05 M, pH 6.0), 1 M acetic acid, and methanol, allowing a 1-minute soak before each brief vacuum/pressure pulse.

Analyte Elution:

• Elute basic analytes with two 100 µL aliquots of methanol/ammonium hydroxide (95:5, v/v), permitting a 2–4 minute soak before vacuum-assisted collection.
• Evaporate eluent and reconstitute in a solvent compatible with LC-MS or other analytical platforms.

Instrumentation

• VacMaster –10 or –20 vacuum manifold
• VacMaster-96 vacuum manifold
• Biotage PRESSURE+96 positive pressure manifold
• Biotage Extrahera automated liquid handling system

Main Results and Discussion

The ISOLUTE HCX series (25 mg, C8/C18/C4 chemistries) demonstrated dual retention via hydrophobic and strong cation-exchange mechanisms, enabling rigorous removal of matrix interferences. Tests with 1 mL plasma spiked at 0.1 mg/mL showed no analyte breakthrough and delivered exceptionally clean extracts. The hydrophobic interaction remained robust against variations in matrix ionic strength, ensuring consistent recoveries.

Benefits and Practical Applications

• High-purity extracts with minimal co-extracted matrix components.
• Compatibility with 96-well plate formats for high-throughput workflows.
• Applicability to pharmacokinetic, clinical, and QA/QC analyses of pharmaceuticals and food additives.
• Improved sensitivity and reproducibility in LC-MS and other detection methods.

Future Trends and Applications

Advances may include tailored mixed-mode sorbent chemistries optimized for specific drug classes, integration with fully automated platforms for increased throughput, and coupling with high-resolution mass spectrometry to achieve trace-level detection in complex biological matrices.

Conclusion

ISOLUTE HCX mixed-mode SPE provides a robust and selective method for extracting basic drugs from biological fluids. The dual interaction mechanism delivers clean extracts and high analyte recoveries, supporting accurate, sensitive, and reliable analysis in research and clinical settings.

References

Biotage. Technical Note TN125.V.1. Sample Preparation by Mixed-Mode SPE Using ISOLUTE HCX. 2020.