|dc.description.abstract||Electromembrane extraction (EME) was described for the first time in 2006 and demonstrated electrokinetic migration of charged substances from an aqueous sample solution through a supported liquid membrane (SLM) and into an aqueous acceptor solution. EME was reported as a selective and fast extraction technique providing clean extracts from biological samples that are directly compatible with analytical instrumentation. The intention of this thesis was to investigate fundamental aspects of EME and to further develop technical EME configurations. Major focus was on extraction of small molecular substances from human matrices; extraction at low voltages (<10 V), short extraction times (<10 min), exhaustive extraction, and parallel extractions of multiple samples.
In Paper I a portable EME device was developed, this device was operated under stagnant conditions with a 9 V battery as power supply. Amitriptyline, citalopram, fluoxetine, and fluvoxamine were isolated electrokinetically from 70 μL undiluted human plasma and into 30 μL of 10 mM HCOOH. The final extract was analyzed by liquid chromatography coupled to mass spectrometry (LC-MS). Extraction time, extraction voltage, SLM, and acceptor solution were optimized in terms of extraction recovery. This EME setup provided extraction recoveries in the range 12-22% after 1 min of extraction. Although the extraction recoveries were relatively low, the combination of EME with LC-MS provided lower limit of quantification (LLOQ) below the therapeutic range. The r2 in the therapeutic range (1-1000 ng mL-1) was above 0.998 for all analytes. In addition, the sample throughput was increased by operating three sample compartments in parallel with a single 9 V battery.
In Paper II the extraction recovery with EME was investigated by introducing three separate hollow fibers into a single sample compartment. This setup provided exhaustive EME of citalopram, loperamide, methadone, paroxetine, pethidine, and sertraline from pH adjusted water samples. The three-fiber configuration was also utilized to isolate the same basic analytes from untreated human plasma samples. However, the extraction recoveries were reduced to some extent regarding some of the analytes; probably due to proteinbinding in human plasma and thus limited transportation of the analytes through the SLM. The setup was evaluated regarding linearity, reproducibility, and LLOQ, and the results were considered as acceptable in terms of regulatory guidelines. The three fiber approach in combination with LC-MS was used to isolate citalopram from two patient plasma samples; the reported plasma concentration deviated less than 14% compared to data obtained by a reference laboratory.
In Paper III EME was evaluated with samples of human whole blood spiked with the following drugs of abuse: Cathinone, 2,5-dimethoxy-4-iodoamphetamine (DOI), methamphetamine, 3,4-methylendioxy-amphetamine (MDA), 3,4-methylendioxymetamphetamine (MDMA), and ketamine. Extraction time, SLM composition, acceptor solution, and extraction voltage were all optimized regarding extraction recovery. The final setup isolated the analytes of interest from 80 μL sample, through an SLM consisting of 1-ethyl-4-nitrobenzene (ENB), and further into 10 μL of acetic acid, the applied voltage was 15 V, and the extraction was performed for 5 min. The final EME-configuration with a 15 V battery as power supply was utilized to isolate cathinone, amphetamine, methamphetamine, ketamine, MDA, and MDMA from autenthic forensic samples. Although the extraction recovery was in the range 10-30%, the combination of EME with ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) provided LLOQ below the concentrations associated with drug abuse.
In Paper IV the approach from paper II was combined with solid sampling of biological samples on sheets of alginate- and chitosan foam. Samples of 10 μL whole blood spiked with citalopram, loperamide, methadone, and sertraline were spotted on sheets of either alginateand chitosan foam and thereafter dried for 2 hours. Subsequently, the dried blood spots (DBS) were punched out and disintegrated in 300 μL HCl for 3 min. From this solution the analytes of interest were isolated by EME into 20 μL of 20 mM HCOOH, and provided extracts directly compatible with LC-MS analysis within 10 min. In addition, the novel approach was compared with DBS sampling on well-established sampling media like Whatman FTA DMPK-A and Agilent Bond Elut DMS. The elution recoveries reported with those media was inferior compared to sampling on alginate- and chitosan foam and subsequent extraction with EME. The absence of interfering matrix componentes in the final EME extract was also demonstrated in Paper IV
In Paper V samples of oral fluid was spotted on alginate- and chitosan foam. 10 μL of oral fluid spiked with buprenorphine, methadone, methamphetamine, para-methoxy-amphetamine (PMA), and para-methoxy-metamphetamine (PMMA) was applied to alginate- and chitosan foam; subsequently the dried spots were punched out and disintegrated in 300 μL 1 mM HCl for 5 min. The analytes were subsequently isolated from this solution by EME and into 0.1% trifluoracetic acid (TFA) within 5 min. The experimental conditions were optimized. The final setup was evaluated regarding linearity in the relevant concentration range, RSD, extraction recovery, and storage-stability for 30 days. The evaluation results were considered as acceptable with regards to regulatory guidelines. Post-column infusion experiments demonstrated that the final EME extract was free from interfering matrix components and underlined the sample cleanup provided with EME.
In Paper VI EME was introduced into the multi-well platform with flat membranes to enable parallel extractions with EME; this approach increased the overall sample throughput with EME. The idea of parallel electromembrane extraction (Pa-EME) with the hollow fiber configuration was briefly investigated in Paper I; however this setup was highly challenging to operate. In Paper VI amitriptyline, fluoxetine, quetiapine, and sertraline were isolated from eight plasma samples into eight separate extracts of 70 μL 20 mM HCOOH. The experimental conditions were optimized with a Quality by Design (QbD), and were as follows: Extraction time 8 min, extraction voltage 200 V, sample volume 240 μL, acceptor volume 70 μL, and agitation rate 1040 rpm. The linear calibration curves in the therapeutic range were above 0.9974; this calibration curve was used to quantify plasma concentrations of quetiapine and sertraline in patient samples. Additionally, post-column infusion experiments demonstrated the sample cleanup provided with Pa-EME.
In Paper VII the Pa-EME performance was investigated with different matrices to establish how the Pa-EME was affected by small, but deliberate variations in method parameters during normal use. Urine, human plasma, and pH adjusted water samples were utilized as sample matrices. The Pa-EME system was also stressed by perforating one or several of the wells in order to investigate how this influenced the remaining intact wells in the multi-well plate. The total number of samples processed simultaneously was increased from eight in Paper VI to 68 and finally 96 samples in Paper VII. Paper VII demonstrated for the first time extraction of 96 samples simultaneously with EME. The combination of a robust high-throughput sample preparation technique with UPLC-MS/MS provided a powerful platform for analysis of small molecular drug substances from biological matrices.||en_US
|dc.relation.haspart||Paper IV Smith RL, Åstrand OAH, Nguyen LM, Elvestrand T, Hagelin G, Solberg R, Johansen HT, Rongved P. Synthesis of a novel legumain-cleavable colchicine prodrug with cell-specific toxicity. Bioorganic & Medicinal Chemistry. Volume 22, Issue 13, 1 July 2014, Pages 3309–3315. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1016/j.bmc.2014.04.056||