Liquid–liquid extraction (LLE) is a commonly used sample preparation technique for the isolation of drugs from biological fluids, such as plasma, prior to analysis by LC-MS-MS. Employing simple methodology, the technique can provide cleaner extracts than alternative solid phase extraction (SPE) or protein precipitation methods, at a fraction of the cost. However, traditional LLE can be labor-intensive and difficult to automate due to manual or off-line steps, and can therefore be unsuitable for high-throughput bioanalytical sample preparation. Good recoveries often require off-line steps such as mixing or centrifuging, which increase the sample preparation time significantly.
The ISOLUTE SLE+ Supported Liquid Extraction Plate (Biotage, Charlottesville, VA) is based on supported liquid extraction (SLE), a technique that eliminates the formation of emulsions and provides complete separation of the organic and aqueous layers, resulting in excellent recoveries of analytes and the reduction of ion suppression/ion enhancement effects in mass spectrometry. Utilizing inert diatomaceous earth, a wide variety of compounds can be extracted by simply transferring an existing method, or by applying a manufacturer-recommended generic method. The 96-well plate format is well suited for automation compatibility and allows scientists to process samples in a fraction of the time it usually takes employing off-line methods. This article explores the use of ISOLUTE SLE+ and the benefits of the technique, giving an example of a common application.
Supported liquid extraction
Traditional liquid–liquid extraction has long been used as part of forensic and toxicological drug determination using individual vials or 96-well collection plates, making the process difficult to automate for high-throughput bioanalytical sample preparation. In order to overcome these challenges, a new format of supported liquid extraction, the ISOLUTE SLE+ 96-well plate, has been developed. Advances in the manufacture and design of diatomaceous earth provide a more rugged support for use in the 96-well plate format. Processing samples with this format requires no manual off-line steps such as capping, mixing, centrifuging, or decapping. With its advanced flow-through format, all steps can be fully automated with no manual intervention.
Figure 1 - ISOLUTE SLE+ procedure.
As part of the ISOLUTE SLE+ procedure, the aqueous biological fluid sample and the immiscible extraction solvent are introduced to the well at different stages, eliminating the problems of contamination and emulsion formation, and maximizing analyte recovery. The technique is carried out using a 96-well plate with an optimized form of diatomaceous earth, providing reproducible flow characteristics from well to well. As the buffered sample is applied to each extraction well, the water is absorbed along with any water-soluble endogenous material, such as phospholipids. The analytes remain on the surface of the diatomaceous earth, forming the interface for the extraction, equivalent to the phase interface in LLE. When the water-immiscible extraction solvent is applied, organic-soluble analytes are efficiently desorbed and eluted into an automation-compatible 96-well plate, as shown in Figure 1.
This simple process provides excellent separation of the two layers, significantly increasing recoveries and sensitivity. By choosing an appropriate extraction solvent, ion suppression and ion enhancement can be drastically reduced to provide cleaner extracts for automated sample preparation.
Experimental: Extraction of drugs from plasma using ISOLUTE SLE+ Supported Liquid Extraction Plates
Extraction efficiency using the ISOLUTE SLE+ plate was investigated and compared to the equivalent LLE procedure (carried out in glass vials). The sample used was 100 μL human plasma containing 10 ng/mL imipramine, trimipramine, and nortriptyline. The sample was diluted with 0.5 M ammonium hydroxide and applied to the ISOLUTE SLE+ plate. The extraction solvent used was hexane: 2-methyl- 1-butanol, 1 mL. The water-immiscible extraction solvent and sample pretreatment conditions used in SLE are the same as those required for LLE, allowing rapid transfer of existing LLE methods, reducing method development time.
Table 1 - Comparison of analyte recovery using the ISOLUTE SLE+ and traditional LLE
In order to effectively prepare the analytes for analysis, the prebuffered sample was applied to the 96-well plate, optimized for the simultaneous extraction of samples. The ISOLUTE SLE+ plate was processed using a vacuum manifold or automated liquid handling system with vacuum capability (to initiate loading and analyte elution). Once the sample was completely absorbed (~5 min), the extraction solvent was applied and allowed to flow under gravity. The vacuum was then applied for 2 min to complete elution. The sample was evaporated to dryness employing the SPE-DRY 96 (Biotage) and reconstituted in mobile phase prior to analysis by LC-MS-MS. Using the ISOLUTE SLE+ plate, analyte recovery is significantly increased, as shown in Table 1, when compared to traditional LLE.
The speed and ease of automation of a typical SLE procedure was also investigated, and the result compared to the equivalent LLE procedure, using the same sample and extraction solvent volumes. The sample used was 200-μL prebuffered human plasma, with 1 mL water-immiscible extraction solvent and a liquid handling instrument equipped with vacuum manifold.
Two hundred microliters aqueous sample (maximum) was dispensed into each well and vacuum was applied to commence loading. Once the sample was absorbed, the water-immiscible extraction solvent was applied to each well and allowed to flow under gravity. The vacuum was applied to facilitate elution, and the extraction solvent was collected in the collection plate.
This simplified process results in a considerable increase in productivity due to full automation. When using LLE, it is necessary to cap the plate, mix and centrifuge, allow the layers to separate prior to evaporation, and uncap the plate. These steps are all off-line and can significantly extend the time for sample preparation. The result with SLE is an easily automated technique, with increased sample throughput by 100% when compared to traditional LLE, as shown in Table 2.
Table 2 - Comparison of SLE+ and traditional LLE methods when investigating ease of automation
Conclusion
LLE is an extremely effective extraction technique across a wide range of applications, but its requirement for off-line preparation renders the method unsuitable for high-throughput bioanalytical sample preparation. By using the completely automated ISOLUTE SLE+ procedure, sample throughput can be increased by 100% when compared to traditional LLE. Analyte recovery and extract cleanliness are significantly improved, and the removal of manual steps decreases extraction time while increasing laboratory productivity.
Mr. Cleeve is Director, Chemistry Consumables Product Management, Discovery Chemistry Group, Biotage, 1725 Discovery Dr., Charlottesville, VA 22911, U.S.A.; tel.: 434-979-2319; fax: 434-979-4743; e-mail: [email protected].