Electromembrane extraction of methotrexate and its metabolites
Master thesis
Permanent lenke
https://hdl.handle.net/11250/2761439Utgivelsesdato
2021-05-20Metadata
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- Master theses [30]
Sammendrag
Within pharmaceutical analysis, sample preparation is essential to make a sample compatible with the chosen analytical instrument, prevent contamination and damage of the instrument, and to avoid interference from matrix substances in biological samples. Electromembrane extraction (EME) was developed in the mid-2000s, and has proved to be an efficient sample preparation technique for several analytes. It is based on transfer of electrically charged analytes from an aqueous donor solution, across an organic solvent (SLM), and into an aqueous acceptor solution. There are several advantages with EME, including the possibility of rapid extractions, high sample clean-up and enrichment, high selectivity, low consumption of organic solvents, and pre-concentration of the analyte. To this date, most studies on EME have been performed with non-polar, basic analytes. In the present study, EME was for the first time used for sample preparation of methotrexate (MTX) and its metabolites 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA). These are polar, acidic, and zwitterionic analytes, all physicochemical properties that are little explored with EME. For MTX method development, a range of different conditions were tested and optimized in order to yield high analyte recoveries. The extracted samples were analyzed using HPLC-UV during method development. MTX was extracted as either positively or negatively charged, with subsequent adjustments of pH in the donor/acceptor solutions and composition of the SLM. Due to the polarity of the analyte, an ionic carrier was added to the SLM. The highest recovery (79.6%) was achieved when MTX was extracted as an anion, using a 40 mM phosphate buffer with pH 7.4 as the donor solution, 10 mM NaOH with pH 12 as the acceptor solution, and peppermint oil + 1% aliquat 336 as the SLM. The same method yielded recoveries of 59.0% 7-OH-MTX and 32.4% DAMPA in the acceptor solution. This method could not be applied to a donor solution containing a physiological concentration of Cl-, due to an interaction between the chloride ions and the cationic carrier aliquat 336. Therefore, MTX was extracted as a positively charged analyte from plasma, using an anionic carrier for transport across the SLM. The extracted plasma samples were analyzed using LC-MS/MS, and the method yielded 5.5% recovery of MTX in the acceptor solution. Taken together, MTX should be extracted as an anion to achieve high recovery, and the addition of an ionic carrier is essential for transport across the SLM. Of all the conditions tested, results pointed towards the most optimal donor/acceptor solutions, SLM, ionic carrier, and settings for voltage, time, and agitation. However, further experimental work is required in order to improve EME of biological samples, particularly to identify an ionic carrier which has low interference with anionic electrolytes in plasma.