Sample Preparation by Mixed-Mode SPE Using ISOLUTE® HAX

Importance of the topic

Sample preparation for trace analysis of acidic drugs in biological fluids is critical for accurate quantification and regulatory compliance. Mixed-mode solid-phase extraction (SPE) combining hydrophobic and strong anion-exchange interactions enhances selectivity and extract purity, enabling sensitive detection in complex matrices such as plasma and urine.

Objective and study overview

This technical note evaluates the performance of ISOLUTE HAX mixed-mode SPE sorbent for extracting acidic drugs from biological fluids. The study aims to demonstrate optimized loading, interference removal and analyte elution protocols that deliver high recoveries and minimal co-extraction of endogenous compounds.

Methods and instrumentation

The protocol employs 25 mg ISOLUTE HAX SPE cartridges, plates or tabless columns. Key steps include:

• Pre-treatment: Dilute 100 µL plasma/urine with 2% formic acid (1:1).
• Conditioning: Wet sorbent with methanol, equilibrate with acidified water.
• Sample loading: Apply 200 µL acidified sample under low vacuum or positive pressure.
• Interference elution: Wash non-polar and neutral interferents with ammonium acetate buffer and 50/50 methanol/water.
• Analyte elution: Release acidic analytes with methanol/acetic acid (98:2, v/v) in two 100 µL aliquots.
• Post-processing: Evaporate eluates and reconstitute in LC-MS compatible mobile phase.

Instrumentation

Extractions were performed using Biotage VacMasterTM-10 or ‑20 vacuum manifolds, VacMaster96 (96-well format), PRESSURE+96 positive pressure manifold and Biotage ExtraheraTM automated liquid handling system.

Main results and discussion

The mixed-mode HAX sorbent retained acidic drugs via combined hydrophobic and anion-exchange mechanisms, while endogenous acids and neutrals were effectively washed away. Optimized elution conditions yielded clean extracts with negligible matrix interference and demonstrated capacity for up to 1 mL plasma without analyte breakthrough at 0.1 µg/mL levels.

Benefits and practical applications

• High selectivity and purity for trace acidic drug analysis.
• Compatibility with LC-MS workflows.
• Reduced matrix effects in pharmacokinetic, toxicology, and clinical studies.
• Scalable formats: cartridges, 96-well plates, tabless columns.

Future trends and opportunities

Advances in mixed-mode sorbent chemistries will expand application to broader compound classes and challenging matrices. Integration with automated platforms and on-line SPE-LC-MS systems promises higher throughput and reproducibility. Emerging hybrid materials may enhance capacity and selectivity further.

Conclusion

ISOLUTE HAX mixed-mode SPE delivers a robust, selective approach for extracting acidic drugs from biological fluids. Its dual retention mechanisms enable rigorous interference removal and high-purity extracts suitable for demanding analytical workflows.

Reference

No external literature references were provided in the source document.