Electromembrane Extraction of Biologically Active Substances – Investigation of Fundamental Aspects

Electromembrane extraction (EME) was introduced in 2006 as a fast and selective microextraction technique that offered good recoveries for basic analytes. Since then, more than 90 publications have been presented on the technique in various applications and technical setups. The principle is based on extraction of analytes across a thin supported liquid membrane (SLM) by the use of an electric field. Several key parameters for an efficient EME setup has been described previously, but systematic knowledge about the extraction process and the importance of the SLM was lacking at the beginning of this PhD project. The main objective of the work with this thesis was to further develop the theoretical understanding of EME on biologically active substances and to build systematic knowledge about the extraction process. Special attention has been given to the SLM, distribution of analytes throughout the EME system over time, stability of the EME system, and how the extraction process was affected by high amounts of either salts or organic solvents in the sample solution.
In paper I, a screening of different SLM compositions for the extraction of eight model peptides with EME was performed. The model peptides were selected to represent a broad range of physical chemical parameters. This paper confirmed previous findings on the importance of combining an organic solvent with a carrier for efficient extractions, as well as identifying several new compositions of carriers and solvents that were effective as SLMs. The effective compositions comprised a mono- or dialkylated phosphate acting as a carrier and a primary alcohol or ketone acting as a solvent. Especially the combination 2-octanone and tridecyl phosphate (9:1 w/w) was shown to give higher extraction recoveries and lower standard deviations than previously reported SLMs.
In paper II, a phenomenological theoretical model for the time dependent distribution of analytes in EME was presented and experimentally verified on several unpolar basic drugs and peptides, representing a broad range of physical chemical properties. Distribution profiles were made, where the amount of analytes in the sample, SLM, and acceptor solution at different extraction times were investigated. The distribution profiles were in good accordance with the theoretical model, but a deviation was seen for some of the peptides where a relatively high amount became trapped in the membrane. The resulting observations demonstrated that the mass transfer across the SLM in EME had elements of both a distributive and electrophoretic process. This can be seen from the theoretical model by the inclusion of a voltage dependent distribution coefficient.
In paper III, EME was performed on samples containing a substantial amount of the organic solvents ethanol, methanol, dimethyl sulfoxide, or acetonitrile together with five unpolar basic drugs as model analytes. The main purpose was to investigate the stability and efficiency of EME when organic solvents were present in the sample. When nitrophenyl octyl ether (NPOE) was used as SLM, stable extractions were achieved from samples containing up to 50 % (v/v) ethanol or methanol, and up to 75 % (v/v) dimethyl sulfoxide. Acetonitrile partially dissolved the SLM solvent, and samples containing acetonitrile were unsuitable for EME. The maximum recovery was unaffected by the presence of organic solvent in the sample, but the time to reach this level increased from 5-10 minutes to 15-25 minutes. A practical example of these discoveries was successfully performed on the highly organic eluate from a commercial dried blood spot card.
In paper IV, a large systematic screening of 61 potential SLM solvents in EME was performed and evaluated according to stability during extractions and their ability to give high extraction recoveries for five unpolar basic model drugs. Several relevant solvent properties were correlated to these parameters through partial least square regression (PLS) analysis. The efficient EME solvents were characterized with a low water solubility (<0.5 g/L), high dipole moments, high proton acceptor properties, and low proton donor properties. Especially some nitroaromatics and ketones belonged to this group, and several efficient solvents that had not been previously described were identified from these criteria. Some solvents were classified as unsuitable because they gave a high extraction current, often combined with an electroosmotic flow of water through the SLM. This was solvents with a low log P value and high water solubility. Finally, some solvents were inefficient and provided no extraction recovery. These were solvents with a high log P value (log P > 4).
In paper V, EME was performed on samples containing different concentrations of NaCl. The presence of NaCl in the sample solution and its effect on extraction recovery, repeatability, and membrane current in EME was thoroughly investigated on 17 unpolar basic drugs with various physical chemical properties. For eight drugs, a substantial reduction in recovery was seen when more than 1 % (w/v) of NaCl was present in the sample solution and NPOE was used in a hollow fiber membrane setup. No correlation was seen between this recovery loss and the physical chemical properties of these analytes. With a NaCl content of5 % (w/v) the repeatability of the extractions was compromised. The reduction in recovery was hypothesized to be caused by ion pairing in the SLM, and a mathematical model was made according to this hypothesis and the experimental data. Changing the SLM solvent from NPOE to 6-undecanone, or reducing the SLM to acceptor solution volume ratio by using a thinner membrane, reduced the observed recovery loss, which was consistent with the ion pair hypothesis