The Extraction Rifabutin from Serum Using HyperSep SLE

Significance of the Topic

The quantification of rifabutin in human serum is essential for effective tuberculosis therapy and pharmacokinetic studies. Conventional liquid–liquid extraction (LLE) can be time-consuming, solvent-intensive and prone to emulsification, leading to matrix interferences. Solid-supported liquid–liquid extraction (SLE) addresses these challenges by offering cleaner extracts, reduced solvent consumption and easier automation.

Objectives and Study Overview

The primary aim of this application note is to present a robust SLE protocol using HyperSep SLE cartridges or 96-well plates for extracting rifabutin from serum. The study evaluates the workflow’s efficiency, cleanliness of extracts and compatibility with downstream LC analysis compared to conventional methods.

Methodology and Instrumentation

A sample volume of 50 µL serum is applied to a diatomaceous earth-based HyperSep SLE support. After a 5 min adsorption period under vacuum or positive pressure, analytes are eluted with 1.4 mL of methyl tertiary butyl ether containing 0.5–1 % acetic acid and ethyl acetate (50:50). The combined eluate is dried under nitrogen at 40 °C and reconstituted in 400 µL methanol/water/acetic acid (45:55:0.1).

The following instrumentation is employed:

• HyperSep SLE 200 mg/3 mL cartridge or 96-well plate
• Vacuum or positive pressure SPE manifold
• Finnpipette 1000 µL
• Reacti-Therm heating module and Reacti-Vap evaporator
• LC system with Hypersil GOLD 5 µm 50 × 4.6 mm column and Uniguard Hypersil GOLD guard column

Chromatographic conditions are:

• Mobile phase A: 0.1 % formic acid in water
• Mobile phase B: methanol with 0.1 % formic acid
• Gradient: 10 % B to 100 % B over 20 min
• Flow rate: 0.2 mL/min; injection volume: 5 µL; column temp: 40 °C

Main Results and Discussion

The SLE approach yielded clear, phospholipid-free extracts without emulsification. Recoveries were consistent across replicates, and matrix interferences in LC analysis were significantly reduced. Solvent usage was decreased by over 50 % compared to traditional LLE, and total sample-preparation time was shortened by approximately 30 % due to simplified handling and compatibility with automation platforms.

Benefits and Practical Applications

• High throughput workflow suited for clinical and pharmacokinetic laboratories
• Reduced solvent costs and waste generation
• Minimized matrix effects for reliable quantitation by LC or LC–MS
• Potential integration into automated sample-preparation systems

Future Trends and Applications

Emerging directions include coupling SLE directly with online LC–MS systems for fully automated analysis, exploring greener extraction solvents to improve sustainability, and developing miniaturized SLE plate formats for high-density screening. Advances in sorbent materials may further enhance selectivity and throughput.

Conclusion

The HyperSep SLE protocol delivers an efficient, reproducible and automatable solution for extracting rifabutin from serum. Key advantages include cleaner extracts, reduced solvent consumption and faster sample turnovers, making it a valuable tool for therapeutic monitoring and bioanalytical applications.

Reference

• Aspey S. The Extraction of Rifabutin from Serum Using HyperSep SLE. Thermo Fisher Scientific Application Note ANCCSSLERIFABUT, 2011.