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Development of Capillary Electrophoresis Methods Coupled to Mass Spectrometry for Biomedical and Pharmaceutical Analysis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices.

Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 804
Keywords [en]
Capillary Electrophoresis, Capillary Surface Modificaions, Electrospray Ionization, Mass Spectrometry, Peptides, Intact Proteins, Basic Pharmaceutical Drug Molecules and Complex Biological Samples
National Category
Other Basic Medicine
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-143814ISBN: 978-91-554-7996-1 (print)OAI: oai:DiVA.org:uu-143814DiVA, id: diva2:391693
Public defence
2011-03-11, C8:301, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 734Available from: 2011-02-18 Created: 2011-01-25 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
Open this publication in new window or tab >>Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
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2008 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 8, p. 1619-1625Article in journal (Refereed) Published
Abstract [en]

A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 X 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).

Keywords
Capillary electrophoresis, M7C4l, Peptides, Proteins, Protein digests, Time-of-flight
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-16217 (URN)10.1002/elps.200700737 (DOI)000255703100005 ()
Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2017-12-08Bibliographically approved
2. Analysis of peptides using N-methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS
Open this publication in new window or tab >>Analysis of peptides using N-methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS
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2010 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 7, p. 1151-1156Article in journal (Refereed) Published
Abstract [en]

In this study, the N-methylpolyvinylpyridinuim polymer has for the first time been used as a silica surface modifier for CE in combination with ESI MS (CE-ESI-MS). The compatibility for ESI-MS was demonstrated by the analysis of peptides and protein digests. The N-methylpolyvinylpyridium surface interacts electrostatically with the ionized silanol groups, giving a cationic surface with a reversed EOF. The surface modifier gave rapid and repeatable separations of peptides, proteins and protein digests at acidic pH for more than 4 h of continuous use. The CE separation yielded peak efficiencies of up to 4.3 x 10(5) plates/m. The surface coating is highly compatible with ESI and facilitates the separation and analysis of complex peptide mixtures as shown by the analysis of BSA digests.

Keywords
CE, ESI, MS, N-methylpolyvinylpyridinuim, peptides
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-125809 (URN)10.1002/elps.200900536 (DOI)000276811000005 ()20209570 (PubMedID)
Available from: 2010-05-28 Created: 2010-05-28 Last updated: 2017-12-12Bibliographically approved
3. The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.
Open this publication in new window or tab >>The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.
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2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 17, p. 3613-3620Article in journal (Refereed) Published
Abstract [en]

The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 x 10(6) apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific.

Keywords
Capillary electrophoresis, alkali salt, intact protein analysis, coated capillary, stacking effect
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-100628 (URN)10.1016/j.chroma.2008.12.037 (DOI)000265467200004 ()19150070 (PubMedID)
Available from: 2009-04-03 Created: 2009-04-03 Last updated: 2017-12-13Bibliographically approved
4. Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries
Open this publication in new window or tab >>Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries
2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 6-7, p. 647-658Article in journal (Refereed) Published
Abstract [en]

In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4×105plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.

Keywords
Capillary electrophoresis, Mass spectrometry, Tricyclic Antidepressant Drugs, Quantification, Human plasma
National Category
Other Basic Medicine
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-143784 (URN)10.1002/elps.201000566 (DOI)000288602000001 ()21341290 (PubMedID)
Available from: 2011-01-25 Created: 2011-01-25 Last updated: 2018-01-12Bibliographically approved
5. A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS
Open this publication in new window or tab >>A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS
2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 13, p. 1778-1785Article in journal (Refereed) Published
Abstract [en]

In this study, the extraction recoveries of an anticancer drug (Imatinib) from human plasma using a common liquid-liquid extraction (LLE) method and a new strong cation exchange (SCX) solid-phase extraction (SPE) column was investigated. The extracts were analyzed with CE coupled on-line to electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) using a monoquaternarized piperazine compound (M7C4I) for capillary coatings. Clean extracts with high and reproducible extraction recoveries ranging between 85 and 91% with % RSD values of 2.5% (n = 3) were obtained using the SCX-SPE columns. This can be compared with the recoveries obtained with the LLE method ranging between 30 and 35%. The CE-ESI-TOF-MS analysis was performed in = 0.997 and % RSD values of 0.5% (n = 3). The intra-day and inter-day assay variations were lower than 8%. The presented CE-ESI-TOF-MS method with the use of SCX-SPE columns yielded rapid, efficient and high extraction recoveries together with high sensitivity (LOD 5 ng/mL), selectivity and good linearity. Accordingly, the method can readily be used for accurate determination and therapeutic monitoring of the Imatinib blood levels for more effective patient treatment. In addition, it can be applied for the extraction, quantification and clinical assessments of metabolites of Imatinib and other basic pharmaceutical drug molecules in biological fluids or pharmaceutical dosage forms.

Keywords
Capillary electrophoresis, Human plasma, Imatinib, Mass spectrometry, Quantification, Theraputic drug monitoring.
National Category
Other Basic Medicine
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-143791 (URN)10.1002/elps.201100121 (DOI)000292971000027 ()
Available from: 2011-01-25 Created: 2011-01-25 Last updated: 2018-01-12Bibliographically approved

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