Taking salt with acetonitrile

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  • Published: Apr 13, 2009
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Taking salt with acetonitrile

Classical liquid-liquid extraction (LLE) is one of the simplest techniques used in the preparation of biological fluids such as urine, plasma and blood. A judicious choice of solvent leads to clean extracts although the technique is not applicable to hydrophilic analytes. It is preferred by many practising scientists due to its speed and simplicity compared with solid-phase extraction and protein precipitation, although its green credentials are questionable.

LLE is accomplished by mixing the sample solution thoroughly with an immiscible solvent in which the target analyte is preferentially soluble. The mixing process creates a large interfacial area between the two liquids to facilitate efficient mass transfer of the target analytes from the sample into the extractant. The two phases are allowed to separate, or are separated by centrifugation, and the extractant is removed for analysis. For biological fluids, a water-immiscible solvent such as ethyl acetate or acetone is generally employed.

There have been some reported cases of LLE with water-miscible solvents but special conditions must be imposed. For instance, acetonitrile is well-known to bioanalysts due to its almost universal application as a deproteinisation agent for biofluids but its use as an extractant it complicated by its water solubility. Freezing an aqueous acetonitrile solution to -20 °C to induce phase separation has been reported, although it is not a technique that is amenable to high throughput.

An alternative to freezing for separating water and acetonitrile is a technique known as salting-out LLE (SALLE), which has had limited application to date. The addition of a salt induced phase separation into two layers within a matter of minutes. This technique has been adopted by scientists in the Department of Drug Analysis at Abbott Laboratories in Abbott Park, Illinois, who have turned it into a high-throughput technique extraction technique that follows good laboratory practice (GLP).

Jun Zhang, Huaiqin Wu, Elaine Kim and Tawakol El-Shourbagy demonstrated the ability of SALLE to enable rapid method development using the extraction of the two active ingredients, ritonavir and lopinavir, from the antiretroviral drug Kaletra which is used to treat HIV/AIDS. All experiments were carried out on a single 96-well plate which was sufficient to evaluate the parameters associated with GLP with reference to the US FDA Guidance for Industry - Bioanalytical Method Validation.

The plate contained 12 sets of calibration standards, each one prepared from 12 matrix lots of plasma from dogs, rabbits and humans. The extraction was carried out by an automated liquid handling system. Solutions of calibration standards, internal standard, the salting agent magnesium sulphate, and acetonitrile were added in turn to each well. The solutions were mixed by 10 aspiration/dispensing cycles before centrifugation for 5 minutes to separate the layers. The upper 100 µL of the upper acetonitrile layer was removed and diluted with an equal volume of water for analysis by liquid chromatography-tandem mass spectrometry.

The accuracy, precision, linearity, carryover, matrix effects, recovery and selectivity were all evaluated in one day, significantly reducing the method development time. No matrix effects were observed and the determination of the analytes was independent of the species. The accuracy and precision were in low, acceptable ranges for both analytes. The recoveries ranged from 62 to 84% across different lots, species and concentration levels, which are comparable to that of conventional LLE.

The use of magnesium sulphate, which has high ionic strength, at the relatively high concentration of 2M, improved the analyte extraction process. However, the researchers cautioned the use of this salt, a strong Lewis acid, in the presence of strong Lewis bases as they would impair the extraction efficiency.

This is the first reported application of SALLE with acetonitrile for biosample preparation but the method has been extended in-house with little modification to the high-throughput extraction of several proprietary Abbott compounds and to other matrices such as urine and blood. The ease of sample preparation, the simple protocol and the ability to automate combined with the rapid development time make this an attractive alternative for bioanalysis.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.


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