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Sample Preparation

Liquid/Liquid Extraction Techniques

Aims and Objectives

Aims and Objectives Aims

To describe the basic principles of liquid/liquid extraction To give an overview of limitations and drawbacks of liquid/liquid extraction

protocols and give specific advice for various problems that are routinely encountered

To give an overview of emulsions and how to deal with them To present support-assisted liquid/liquid extraction (SALL) as a useful variation

on standard liquid/liquid extraction Objectives At the end of this Section you should be able to:

Maximise analyte recovery by optimising liquid/liquid extraction conditions Maximise extract cleanliness by optimising liquid/liquid extraction conditions Minimise the impact of some common problems in liquid/liquid extraction Recognise practical aspects of support-assisted liquid/liquid extraction

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Content Liquid / liquid Extraction I 3 Liquid / liquid Extraction II 5 Drawbacks of Liquid/liquid Extraction 6 Emulsions 7 Support-Assisted Liquid / liquid Extraction I 8 Support-Assisted Liquid / liquid Extraction II 9

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Liquid / liquid Extraction I Liquid/liquid extraction is one of the most widely employed and useful techniques in pharmaceutical sample preparation. This is due to a number of characteristics, including simplicity, rapid method development, and reasonable selectivity. Liquid/liquid extraction involves adding a solvent to the sample that is immiscible, followed by selective partitioning of analytes versus contaminants between the two phases. In the interest of extraction completeness, it is necessary to use an adequate amount of extracting solvent to capture all of the analytes from the original sample. In most cases, a volume of extracting solvent equal to or less than the original sample volume is adequate, assuming reasonable analyte solubility in the solvent. This extracting solvent is added to the sample, then the two phases are agitated, by vortexing or shaking, to bring about substantial physical mixing. After agitation, the phases are allowed to separate.

Complete and incomplete analyte extraction After the two phases separate, the phase containing the analytes is removed, either by careful pipetting, or by freeze-pour. Freeze-pour involves placing the samples in a freezer at a sub-zero temperature to freeze the aqueous layer, after which the organic layer may be simply poured off. This approach works best for extracting solvents less dense than water, so the non-frozen organic layer is on top.

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Organic layer isolation Since most pharmaceutical samples are aqueous, the extracting solvents used are typically non-polar organics. Common solvents used for liquid/liquid extraction are ethyl acetate, methyl tertiary-butyl ether (MTBE), methylene chloride, hexane, and mixtures thereof.

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Liquid / liquid Extraction II After collection of the extraction solvent, the solvent may be dried down and reconstituted, in most cases with a different solvent. Since most LC-MS mobile phases are aqueous in nature, and therefore incompatible with most extracting solvents, dry down and reconstitution allows transfer of the purified analytes into a more compatible solvent. In many cases, the reconstitution solvent used is the LC mobile phase.

If the analytes are not yet adequately pure, a second extraction of the organic solvent using an aqueous phase is sometimes used (referred to as a back-extraction). This approach works best with basic compounds, which can be neutralized (making them more organic-soluble) by raising the pH of the original sample, extracting into an organic solvent, then back-extracting into an aqueous phase using an acidic buffer (ionizing the analytes, thus making them more water-soluble). Another approach to liquid/liquid extraction for highly water-soluble analytes is extraction of non-polar, organic-soluble interferences from the sample, leaving the analytes behind in the aqueous phase. This approach has the specific drawback that the low-volatility aqueous phase is not readily concentrated, as is an organic solvent.

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Drawbacks of Liquid/liquid Extraction Liquid/liquid extraction has a number of drawbacks that limit its use as a sample preparation technique. These include limited selectivity, difficulty of automation, and emulsions. Liquid/liquid extraction works via solubility characteristics of the analytes, and relies on differential solubility of the analytes versus other sample matrix components. Since the solvents used are typically non-polar organics, hydrophobic analytes are extracted into the organic layer, but other non-polar interferences (for example, serum lipids) are often co-extracted. Not only does this give a less pure extract than desired, but the co-extractants may accumulate on the analytical column, typically a reversed phase column. For highly water-soluble analytes, in which the organic phase extracts the contaminants versus the analytes, other water-soluble matrix components may stay in the aqueous phase with the analytes, notably peptides and proteins. Again, proteins may accumulate on the analytical column over time, causing back-pressure and other analytical difficulties.

Liquid/liquid extraction drawbacks

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Another drawback of liquid/liquid extraction for large numbers of samples is that it is not well-suited to automation. Other approaches, SPE in particular, are much easier to automate. Emulsions One major drawback of liquid/liquid extraction is the formation of emulsions. Emulsions occur when the sample contains a high level of surfactant-like compounds (notably phospholipids), that prevent clean separation of the two phases. With emulsions, a mid-zone between the two phases is created, having intermediate solubility in each of the two phases, and making it difficult to quantitatively collect one phase or another.

Emulsion formation Emulsions often occur with samples where the animal (or human) diet is high in fats. Thus, emulsions sometimes appear when passing from pre-clinical trials, with animals on low-fat, controlled diets, to clinical trials, with humans who may be on high-fat diets. This characteristic problem makes liquid/liquid extraction a less dependable procedure if it is expected that the same extraction protocol will be used for both pre-clinical and clinical samples. If this problem is anticipated, it is worth trying high-fat samples during method development in addition to the standard test matrices. Emulsions may sometimes be disrupted, or broken-up, by the addition of salt to the emulsion. The salt changes the capacity of the aqueous phase to accommodate the marginally-soluble components in the system, thus driving them into the organic phase.

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Support-Assisted Liquid / liquid Extraction I As discussed (please refer to the Primary Sample Preparation Techniques module from the Sample Preparation Channel), support-assisted liquid/liquid extraction (SALL) is a useful variation on standard liquid/liquid extraction. The chemistry of this technique is virtually identical to liquid/liquid extraction, but the physical nature of the technique offers distinctive benefits unique to the approach. In SALL, an aqueous sample is applied to a high-surface area matrix such that the sample is dispersed over the surface of the matrix, creating a potential interface for extraction. This is most often executed in a syringe barrel-type column format, similar to those used in SPE. The most common matrix employed for sample dispersion is purified diatomaceous earth. A water-immiscible organic solvent is subsequently passed over the matrix holding the aqueous layer, and analytes of interest partition into the organic phase. The amount of matrix required is directly proportional to the volume of aqueous sample to be extracted. Therefore, most commercial suppliers of SALL products specify products based on the sample size for which they are to be used.

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Support-Assisted Liquid / liquid Extraction III Some commercial SALL products are available pre-buffered. This allows for the pH of the sample to be changed simply by applying the sample to the column. The approach is useful for analytes where pH can improve extraction into the organic phase (either basic-buffered for amines, or acidic-buffered for acids).

The organic solvents most commonly used are the same solvents used in conventional liquid/liquid extraction; ethyl acetate, methyl tert-butyl ether (MTBE), methylene chloride, hexane, and mixtures thereof. Another advantage of SALL is that the nature of the column technique allows for superior automation as compared to conventional liquid/liquid extraction.

Finally, the SALL approach is essentially free of the emulsion problems common to conventional liquid/liquid extraction. In pharmaceutical applications, in particular, this allows for the use of the technique across the range of samples from pre-clinical to clinical, with reduced concern for method ruggedness.

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