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Novel Microextraction Techniques for Bioanalysis of Neurotransmitters and Biomarkers in Biological Fluids
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Sample preparation (sample pre-treatment) is the initial step and an essential part in bioanalysis procedure. The main role of sample preparation is to extract and transfer the analyte(s) of interest from a complex matrix to a purified media such as a pure solvent for analysis and quantification. Biological fluids are complex and contain, in addition to the target analyte(s), many different unwanted compounds from salts to proteins. Thus, the analysis of these samples requires an effective sample preparation method prior to the liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) assays. The aim of the present work was to evaluate micro-extraction by packed sorbent (MEPS) and develop new sample preparation techniques for the extraction of neurotransmitters and biomarkers from biological samples. Moreover, two new sample preparation techniques were developed. The first developed technique is molecular imprinting on polysulfone membrane (MIPM), and the second one is molecularly imprinted polymer in tablet form (MIPT).

MEPS is a well-known sample preparation technique that can be used online with analytical instruments without any modifications. In this thesis, MEPS was used online with liquid chromatography-tandem mass spectrometry (LC/MS/MS) for the quantification of dopamine and serotonin in human urine samples (Study I) and with GC/MS for the analysis of methadone in humane urine (Study II). Polystyrene polymer and silica-C8 were used as sorbent in Study I and Study II, respectively. In both studies, small sample volumes (50 µL) were used and full method validation was performed. In both studies (I and II), MEPS enhanced the limit of detection (LOD) and reduced the extraction time compared to the previously published methods. In Study I, the mean accuracies of quality control (QC) samples of dopamine and serotonin were 99–101% while the precision values (RSD) were 6–11%. In Study II, the accuracy values of methadone were between 97 and 107% while the precisions (RSD) were between 11 and 15%.

MIP-sol-gel on polysulfone membrane (MIPM) was developed and used in combination with MEPS for the extraction of hippuric acid (HA) in plasma and urine samples (Study III). A good selectivity was obtained using plasma and urine samples. The precision of QC samples in plasma and urine samples were 2.2–4.8% and 1.1–6.7%, respectively. The method recovery was above 90%.

In Study IV, a new technique was developed using a tablet form of molecularly imprinted sol-gel (MIPT) for the extraction of methadone from human plasma samples. Methadone-d9 was selected as the template for accurate recovery, and 3-(propyl methacrylate) trimethoxysilane (3PMTMOS) was used as a precursor. The extraction recovery was higher than 80%. The LOD and LLOQ were 1.0 and 5.0 ng mL-1, respectively. The validation showed good selectivity, accuracy and precision.

In Study I, III and IV, LC/MS/MS system was used while GC/MS was used in Study II for the separation and detection of target analytes.

There will always be a high demand for rapid, selective, reliable and sensitive techniques for sample preparation. It is convenient to use molecular dynamics simulations as a theoretical tool for the optimization of molecularly imprinted system. Suitable monomers and cross-linkers are crucial to synthesize the new membrane and tablet molecular imprinted polymer platforms for all kinds of molecules.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University , 2016. , 55 p.
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-135001ISBN: 978-91-7649-581-0ISBN: 978-91-7649-582-7OAI: oai:DiVA.org:su-135001DiVA: diva2:1040891
Public defence
2016-12-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-11-22 Created: 2016-10-29 Last updated: 2016-11-17Bibliographically approved
List of papers
1. Determination of dopamine and serotonin in human urine samples utilizing microextraction online with liquid chromatography/electrospray tandem mas spectrometry
Open this publication in new window or tab >>Determination of dopamine and serotonin in human urine samples utilizing microextraction online with liquid chromatography/electrospray tandem mas spectrometry
2007 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 30, no 3, 421-424 p.Article in journal (Refereed) Published
Abstract [en]

A specific LC-MS-MS method for the determination of dopamine and serotonin (5-hydroxytryptamine; 5HT) in human urine is described. The analytes were extracted from urine and preconcentrated by microextraction in a packed syringe (MEPS). The new method is very promising, very easy to use, fully automated, of low cost, and rapid in comparison to previously used methods. The method was validated and the standard curves were evaluated by means of quadratic regression and weighted by the inverse of the concentration: 1/x for the calibration range 50–4000 μg/L. The MEPS applied polymer (silica-C8) could be used more than 300 times. The extraction recovery was about 50%. The results showed close correlation coefficients (r2 A0.999) for all analytes in the calibration range studied. The accuracy of MEPS-LC-MS-MS was 100–101% for dopamine and 99–100% for 5HT. The interday precision (n = 3 days), expressed as the RSD%, was 6.0–7.7% for dopamine and 6.1–11% for 5HT. MEPS reduced the handling time by 12 times compared to a published method.

Keyword
dopamine, serotonine, LC/MS/MS, microextraction in packed syringe, sample preparation, urine samples
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-134996 (URN)10.1002/jssc.200600369 (DOI)
Available from: 2016-10-29 Created: 2016-10-29 Last updated: 2016-11-15Bibliographically approved
2. Quantitative analysis of methadone in human urine samples by microextraction in packed syringe-gas chromatography-mass spectrometry (MEPS-GC-MS)
Open this publication in new window or tab >>Quantitative analysis of methadone in human urine samples by microextraction in packed syringe-gas chromatography-mass spectrometry (MEPS-GC-MS)
2007 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 30, no 15, 2501-2505 p.Article in journal (Refereed) Published
Abstract [en]

A method for the simultaneous analysis of methadone in urine samples by microextraction in a packed syringe online with GC-MS (MEPS-GC-MS) is described. The new method reduced the sample handling and the detection limit by two- to seven-fold compared to published methods. Using a quantitation method based on the calculation of analyte concentration by comparison to an internal standard, we were able to measure methadone levels consistent with values reported for healthy individuals. The intra-assay precisions (RSD) of the method using quality control (QC) samples at three different concentration levels were about 11-14% (n = 6). The interassay precisions (RSD) were 11–15% for methadone in urine samples (n = 18). The accuracy varied from 89 to 109% for intra-assay (n = 6), and 97 to 107% for inter-assay (n = 18). The regression correlation coefficients (r2) were over 0.99 in all experiments.

Keyword
cyclophosphamide, GC/MS, microextraction in packed syringe, polystyrene sorbent, sample preparation, urine
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-134998 (URN)10.1002/jssc.200700067 (DOI)
Available from: 2016-10-29 Created: 2016-10-29 Last updated: 2016-11-15Bibliographically approved
3. On-line detection of hippuric acid by microextraction with a molecularly-imprinted polysulfone membrane sorbent and liquid chromatography-tandem mass spectrometry
Open this publication in new window or tab >>On-line detection of hippuric acid by microextraction with a molecularly-imprinted polysulfone membrane sorbent and liquid chromatography-tandem mass spectrometry
Show others...
2014 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1372, 55-62 p.Article in journal (Refereed) Published
Abstract [en]

Destruction of sorbents during consecutive extractions using the microextraction by packed sorbent (MEPS) technique is a serious problem. In MEPS the complex matrix such as plasma and blood can affect the sorbent physical properties and the sorbent can be deteriorated after handling of few samples. To overcome this problem, the surface of a polysulfone membrane (PSM) was modified by a molecularly imprinted sol-gel and utilized for online extraction of a lung cancer biomarker, hippuric acid (HA), in biological matrices. The molecularly imprinted polymer membrane provided fast, sensitive, selective and robust sample preparation method for HA in biological fluids. In addition, MIP membrane could be used for up to 50 extractions without a significant change in extraction recovery. To achieve the best results, the parameters that influenced the extraction efficiency were thoroughly investigated. Moreover, for evaluating the performance of the molecularly imprinted sol-gel membrane (MISM), a non-molecularly imprinted sol-gel membrane (NISM) as a blank was prepared. The limits of detection CLOD) and quantification (LOQ) for HA in both plasma and urine samples were 0.30 nmol L-1 and 1.0 nmol L-1, respectively. Standard calibration curves were obtained over the range of 1-1000 nmol L-1 for HA in plasma and urine samples. The coefficients of determination (R2) were ≥ 0.997. The extraction recoveries of HA from human plasma and urine samples were higher than 91%. The precision values for HA in plasma and urine samples were 2.2-4.8% and 1.1-6.7%, respectively.

Keyword
molecularly imprinted, polysulfone membrane, microextraction, hippuric acid, human plasma, urine samples
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-134999 (URN)10.1016/j.chroma.2014.10.061 (DOI)
Available from: 2016-10-29 Created: 2016-10-29 Last updated: 2016-11-15Bibliographically approved
4. Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: I. Determination of methadone in human plasma utilizing liquid chromatography-tandem mass spectrometry
Open this publication in new window or tab >>Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: I. Determination of methadone in human plasma utilizing liquid chromatography-tandem mass spectrometry
2016 (English)In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 936, 116-122 p.Article in journal (Refereed) Published
Abstract [en]

In the present work molecularly imprinted sol-gel tablet (MIP-Tablet) was prepared. The MIP-sol-gel was prepared as a thin layer on polyethylene material in a tablet form. Methadone-d9 was selected as the template and 3-(propylmethacrylate)-trimethoxysilane was used as precursor. MIP-Tablet was applied for micro-solid phase extraction (μ-SPE). The MIP-Tablet was used for the determination of methadone in human plasma samples utilizing liquid chromatography-tandem mass spectrometry; and each tablet could be used twenty times. The extraction time was 10 min while desorption time was 6 min. Factors affecting the extraction efficiency such as desorption solvents, sample pH, salt addition, extraction time, desorption time and adsorption capacity were investigated. The calibration curves were obtained within the range of 5-5000 ng/mL using methadone in human plasma samples. The coefficients of determination (r(2)) values were >= 0.999 for all runs and the extraction recovery was >80%. The accuracy values for quality control samples varied from +3.6 to +9.7% and the inter-day precision (RSD %) values were ranged from 5.0 to 8.0%. The limit of detection was 1.0 ng/mL and the lower limit of quantification was 5 ng/mL utilizing methadone in human plasma samples.

Keyword
molecularly imprinted polymer, sol-gel, tablet, micro-solid phase extraction, methadone, human plasma
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-135000 (URN)10.1016/j.aca.2016.07.001 (DOI)
Available from: 2016-10-29 Created: 2016-10-29 Last updated: 2016-11-15Bibliographically approved

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El Beqqali, Aziza
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