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Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine in haemoglobin
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
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2010 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 27, 2497-2501 p.Article in journal (Refereed) Published
Abstract [en]

A molecularly imprinted polymer, MIP, was prepared and evaluated as SPE sorbent for a cyclicized adduct formed to N-terminal valine (Pyr-Val) in hemoglobin from 1,2:3,4-diepoxybutane (DEB). This metabolite plays an important role in the carcinogenesis of 1,3-butadiene. The hydrazide of Pyr-Val, formed after hydrazinolysis of hemoglobin, as well as necessary standards was synthesized. The MIP was prepared from methacrylic acid with a structure analogue to the investigated adduct as template and the method was developed for aqueous conditions. Selective desorption was achieved when the sample was washed with water after loading in 10% acetonitrile. The primary interaction with the binding sites in the imprints was most likely of ionic character. Quantification of the Pyr-Val adduct was performed with LC/ESI-MS/MS, yielding an instrumental LOD of 150 pg injected amount.

Place, publisher, year, edition, pages
2010. Vol. 878, no 27, 2497-2501 p.
Keyword [en]
Hemoglobin adducts, Diepoxybutane, Molecularly imprinted polymers, Solid-phase extraction
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-30127DOI: 10.1016/j.jchromb.2010.02.012ISI: 000283410200006OAI: oai:DiVA.org:su-30127DiVA: diva2:241639
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2011-11-22Bibliographically approved
In thesis
1. New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane
Open this publication in new window or tab >>New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human exposure to harmful compounds in the environment, from intake via food, occupational exposures or other sources, could have health implications. Exposure to reactive compounds/metabolites can be identified and quantified as hemoglobin (Hb) adducts by mass spectrometry. This thesis aimed at improved synthetic pathways for reference standards, and improved analytical methods for adducts to N-terminal valine in Hb from a range of reactive compounds; isocyanates, aldehydes, methyl vinyl ketone (MVK), and diepoxybutane (DEB).

Isocyanates form urea adducts with N-terminal valine by carbamoylation, which are detachable as hydantoins by hydrolysis. A new synthetic pathway for reference standards of adducts from isocyanates and a method for their analysis by liquid chromatography/mass spectrometry (LC/MS) were developed.

Aldehydes form reversible imines (Schiff bases) with N-termini in Hb. After stabilisation by reduction and detachment by isothiocyanates using modified Edman methods, these adducts could be analysed by gas chromatography/mass spectrometry (GC/MS) or LC/MS. 5-Hydroxymethylfurfural, its metabolites, and other aldehydes related to exposure via food, were studied with regard to analysis by these methods with synthesised standard references. A considerably improved analytical method for imines was developed. Many of the studied adducts are too short-lived in vivo or in vitro to be used for long-term biomonitoring. However, different approaches for the analysis were evaluated.

Through synthesised reference standards, an observed unknown adduct in blood was verified as the adduct from MVK. There exist both natural and anthropogenic sources for MVK.

DEB, metabolite of butadiene, forms a cyclic adduct to valine-N. A new approach using hydrazinolysis of protein and enrichment by molecularly imprinted solid-phase extraction was tested on synthesised reference DEB-adduct and gave promising results.

Synthesised standards were characterized by NMR, LC/MS and GC/MS.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Chemistry, Stockholm University, 2009. 64 p.
Keyword
carbamoylation, modified-Edman procedure, 5-(hydroxymethyl)furfural, MISPE, biomarkers
National Category
Environmental Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-30138 (URN)978-91-7155-934-0 (ISBN)
Public defence
2009-10-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.

Available from: 2009-10-08 Created: 2009-10-05 Last updated: 2014-12-11Bibliographically approved
2. Molecularly Imprinted Solid-Phase Extraction and Liquid Chromatography for Biological Samples
Open this publication in new window or tab >>Molecularly Imprinted Solid-Phase Extraction and Liquid Chromatography for Biological Samples
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on the use of molecularly imprinted polymers as selective sorbents for solid-phase extraction (MISPE). The MISPE methods developed were mainly intended for use with biological samples, such as human urine and blood plasma. These body fluids are complex samples, which often need an effective clean-up step before analysis to reduce the levels of possible interfering substances from the matrix, especially if the analytes are present in trace amounts. Solid-phase extraction (SPE) is a well-established and routinely used method for clean-up and preconcentration of samples from diverse matrices. However, conventional SPE sorbents often lack selectivity, leading to co-extraction of interferences, which negatively affects the following detection method. One of the advantages of MISPE is the built-in selectivity for a target analyte, or class of structurally related analytes, enabling the efficient clean-up that is often required for biological samples. The built-in selectivity of MISPE originates from the preparation of a highly crosslinked copolymer network in the presence of an imprint molecule, i.e. the template. Subsequent removal of this template molecule leads to the creation of defined recognition sites, complementary to the shape and functionality of the template.

In this work, molecularly imprinted polymers were synthesized for the first time for several types of target analytes, including diphosphate esters (Papers I-III) and a protein adduct (Paper IV) and evaluated as sorbents for solid-phase extraction. A MISPE method for extracting local anaesthetic drugs from human plasma was also evaluated (Paper V). The development of appropriate methods for using the prepared polymers to extract target analytes directly from body fluids, and the elucidation of factors that influence their performance, were major foci of all the work underlying this thesis. These are not straightforward tasks, since the recognition mechanism of the material is often based on polar interactions, which are not favoured in aqueous environments. In such cases, non-selective adsorption of the analyte(s) to the polymer surface often occurs. In order to use the MIP sorbent most effectively it is important to suppress this non-selective adsorption, without disrupting the selective adsorption of the target analyte(s) to the imprints. Generally in these studies, this strong analyte-polymer surface interaction could be repressed, and selective adsorption enhanced, by carefully optimising the conditions for washing the sorbent, in terms of organic solvent volumes, solvent polarity and the addition of an ionic modifier. The sample matrix, mainly urine, was found to strongly decrease the capacity of the MIP. Hence, this effect was further investigated. It was found that the presence of NaCl in the sample negatively affected the recovery and repeatability of the method. Furthermore, these parameters could be improved by adjusting the sample pH. It was important to control the pH of the sample, in order both to achieve selective extraction and to increase the extraction recoveries. The selectivity of MISPE for the extraction of diphosphate esters from human urine was demonstrated by comparing its performance with that of a conventional SPE sorbent, a mixed-mode-anion exchanger (MAX). Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Signal suppression from the interfering co-eluting compounds was detected when the MAX extracts were analysed, which was not the case for the MISPE extracts. These findings show the importance of efficient and selective sample preparation, even if a selective detector is used.

Development of LC/ESI-MS methods was also an extensive component of this work (Papers I-IV). Different chromatographic conditions have been evaluated for the optimal separation and detection of the investigated compounds. Use of ion-pairing agents and suitable HPLC columns (Hypercarb and C18 Aquasil) for the acidic, polar analytes, was found to give better retention and separation than use of conventional reversed-phase columns. To improve the selectivity and detectability further, selected ion monitoring (SIM) and selected reaction monitoring (SRM) acquisition modes were used for quantification of the investigated compounds.

In summary, the aim of this work was to contribute to the knowledge of the recognition mechanisms of molecularly imprinted polymers in aqueous matrices, which is important for extending the use of MISPE for several types of bioanalytical applications.

Place, publisher, year, edition, pages
Stockholm: Institutionen för analytisk kemi, 2006. 91 p.
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-1042 (URN)91-7155-234-0 (ISBN)
Public defence
2006-06-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2006-05-09 Created: 2006-05-09 Last updated: 2010-01-08Bibliographically approved

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