Sample Preparation by Mixed-Mode SPE Using ISOLUTE® HAX

Significance of the topic

Efficient extraction of acidic drugs from biological matrices is essential for accurate low-level quantification in bioanalysis. Conventional sample preparation methods lack sufficient selectivity, leading to high matrix background and compromised sensitivity. Mixed-mode solid-phase extraction (SPE) addresses these challenges by combining hydrophobic and ionic interactions to achieve high purity extracts.

Objectives and study overview

This technical note evaluates the performance of ISOLUTE HAX mixed-mode SPE sorbent for extracting acidic pharmaceuticals from plasma and urine. The study outlines a robust protocol to maximize analyte recovery while minimizing co-extracted interferences.

Methodology

Samples (100 µL) were acidified with formic acid (2% v/v) and diluted 1:1 with buffer. ISOLUTE HAX cartridges (25 mg) or 96-well plates were conditioned with methanol and equilibrated with acidic aqueous solution. Acidified samples were loaded under vacuum or positive pressure. Interferences were removed by sequential elution with ammonium acetate buffer (0.1 M, pH 7) and methanol/water (50:50). Target analytes were eluted with methanol/acetic acid (98:2) in two 100 µL aliquots. Final extracts were evaporated and reconstituted in LC-MS compatible mobile phase.

Instrumentation used

The procedure employed Biotage VacMaster vacuum manifolds, PRESSURE+96 positive pressure manifolds, and the Extrahera automated liquid handling system. Standard glass and tabless SPE formats were tested to suit throughput needs.

Key results and discussion

ISOLUTE HAX demonstrated dual retention via non-polar and strong anion exchange mechanisms, enabling rigorous wash steps for high matrix removal. Tests with spiked plasma (0.1 µg/mL) showed no breakthrough up to 1 mL sample volumes. The mixed-mode platform yielded extracts with excellent cleanliness, particularly from urine, enhancing downstream LC-MS detection.

Benefits and practical applications

• High selectivity reduces matrix effects and improves sensitivity.
• Robust performance across variable ionic strength conditions.
• Compatible with both low-throughput cartridges and high-throughput 96-well plates.
• Suitable for a wide range of acidic pharmaceuticals in bioanalytical workflows.

Future trends and opportunities

Advancements may include integration with fully automated platforms for high-throughput drug monitoring, development of novel mixed-mode sorbents for extended pH stability, and coupling with hyphenated techniques such as LC-MS/MS and UHPLC to further enhance selectivity and sensitivity.

Conclusion

Mixed-mode SPE using ISOLUTE HAX provides a streamlined, selective approach for extracting acidic drugs from biological fluids. Its dual retention mechanisms allow effective interference removal, delivering clean extracts and reliable analyte recovery, which are crucial for sensitive bioanalytical assays.