A Simplified Solid Phase Extraction (SPE) Protocol for Bioanalysis Using Oasis HLB

Importance of the Topic

Solid phase extraction (SPE) is widely regarded as one of the most efficient sample preparation techniques in bioanalysis, offering high sample cleanliness, enrichment of trace analytes, and reduced matrix interferences. Despite these advantages, traditional SPE protocols are often avoided due to the perceived complexity, cost, and time requirements. A streamlined, robust SPE approach can greatly enhance throughput and cost-effectiveness for laboratories in pharmaceutical research, clinical testing, and quality control.

Objectives and Study Overview

This study aimed to develop and evaluate a simplified, three-step SPE protocol using Waters Oasis HLB sorbent in both µElution plate and 1 cc cartridge formats. Key goals included demonstrating equivalent or improved analyte recovery and reproducibility compared to a standard five-step SPE method, reducing solvent consumption and processing time, and assessing long-term lot-to-lot consistency across multiple sorbent production batches.

Methodology

A set of nine pharmaceutical compounds—spanning acidic, neutral, and basic chemistries such as azidothymidine, naproxen, and alprazolam—were spiked into rat plasma and human urine. The simplified protocol omitted conditioning and equilibration, relying on the water-wettable nature of Oasis HLB to retain analytes directly from aqueous samples. The three steps comprised:

• Load: sample diluted 1:1 with 4% phosphoric acid
• Wash: 5% methanol in water
• Elute: two aliquots of 25 µL methanol

Analytes were quantified by UPLC–MS/MS using an ACQUITY UPLC I-Class system with a CSH C18 column, coupled to a Xevo TQ-S mass spectrometer operating in positive ESI mode. Performance metrics included percent recovery, matrix factor, variability, and throughput comparisons.

Used Instrumentation

• Oasis HLB µElution Plate (30 mg) and 1 cc Oasis HLB Cartridges (30 mg)
• ACQUITY UPLC I-Class System with CSH C18 Column (2.1 × 100 mm, 1.7 µm)
• Xevo TQ-S Tandem Quadrupole Mass Spectrometer (ESI+)

Main Results and Discussion

The three-step method achieved average recoveries above 80% with standard deviations below 5% across both matrices and formats. Matrix factors of 1.00 ± 0.15 indicated minimal ion suppression or enhancement. Compared with silica C18 or competitive polymer sorbents, only Oasis HLB maintained high recoveries under the simplified workflow. Sample throughput improved by 40%, allowing 13 µElution plates per day versus 8 with the standard five-step protocol, while solvent usage dropped by 70%. Volume loading experiments confirmed consistent recoveries for plasma inputs from 25 to 375 µL. Long-term reproducibility was verified across four sorbent batches produced over eight years, with recoveries between 80% and 100% and variability below 6%.

Benefits and Practical Applications

• Significant reduction in processing time and solvent consumption
• High analyte recovery and low matrix effects for diverse compound classes
• Robust lot-to-lot consistency supporting long-term studies
• Flexible formats accommodating limited sample volumes or individual cartridges

Future Trends and Possibilities

Further developments may include automation of the simplified SPE workflow for ultra-high-throughput screening, extension to additional biological matrices (e.g., tissues or cerebrospinal fluid), and adaptation to emerging sorbent chemistries. Integration with microfluidic platforms or online SPE-LC-MS systems could further improve efficiency and data quality in bioanalytical laboratories.

Conclusion

The streamlined three-step SPE protocol using water-wettable Oasis HLB sorbent delivers equivalent or superior performance to traditional methods while dramatically reducing time and solvent usage. Its robust recoveries, low variability, and compatibility with both µElution plates and cartridges make it a versatile solution for routine bioanalysis.