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Advances in analytical methodologies for studies of the platinum metallome in malignant cells exposed to cisplatin
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Förbättrade analytiska metodologier för studier av platina-metallomet i maligna celler exponerade för cisplatin. (Swedish)
Abstract [en]

The scientific progress about the important chemotherapeutic drug substance cisplatin (CDDP) and its function has often been rendered by data difficult to interpret, and still many questions about its mode of action remains to be clarified by the scientific community. However, studies of CDDP possess a high complexity due to; i) low intracellular concentration, ii) many potential biomolecule targets, iii) poor or unknown stability of the intact drug and its biomolecule adducts and iv) complex and varying sample matrices. Metallomic studies, using advanced analytical techniques may contribute to clarify the interactions between CDDP and intracellular biomolecules. For a successful outcome sample preparation conditions as well as separation and detection techniques must be carefully selected and optimized to achieve accurate results and correct interpretation of data.

        This thesis describes some new and improved analytical methodologies for characterizing the Pt metallome in CDDP-exposed malignant cells. The developed methods are based on powerful liquid chromatography (LC) methods hyphenated to sensitive detection by inductively coupled plasma- (ICP) and electrospray ionization mass spectrometry (ESIMS). Consideration has also been taken about sample preparation conditions.

        By selecting “chemically inert” sample preparation (cell lysis by osmosis) and separation (using only nonreactive or no additatives) conditions we could avoid the formation of platinum artifact compounds previously described in the literature (Paper I and II). Using oxygen containing organic solvents with high boiling points (dimethylformamide; DMF, 1,4-dioxane, n-propanol and ethanol) as alternatives to acetonitrile in the LC separations, significant improvements were achieved in ICPMS sensitivity and robustness. When evaluated in combination with chromatographic performance and ESIMS detection the overall best performance was achieved with n-propanol (Paper II, III and IV). From the studies in Paper II we could show that free intact CDDP can be found in malignant cells, as supporting evidence for passive or endocytotic uptake of the drug and further estimate a half-life for intracellular CDDP to about 15 minutes. Such data has not been shown before. In Paper V, the above improved LC methods were used to demonstrate differences in the platinum and cupper metallome from sensitive and resistant T289 melanoma cells exposed to CDDP at near clinical levels.

        In a wider perspective we have shown the potential of using hydrophilic liquid interaction chromatography (HILIC) hyphenated to ICPMS detection as a general approach for analysis of hydrophilic metallo-compounds (Paper II). Taking advantage of the superior ICPMS performance using n-propanol gradients for reversed phase liquid chromatography (RPLC) possess a true alternative and /or complimentary technique to size exclusion chromatography (SEC) commonly applied within metallomic studies of biomolecules (Paper V). Using n-propanol in HILIC as well as in RPLC enables parallel detection by ICP- and ESIMS using only one set of chromatographic parameters (Paper III and IV), something commonly called for by scientists in the field.

Place, publisher, year, edition, pages
Umeå: Kemiska Institutionen, Umeå universitet , 2010. , 8+44 p.
Keyword [en]
Method development, Metallome, Inductively coupled plasma mass spectrometry, Electrospray ionization mass spectrometry, Liquid chromatography, Hyphenation, Organic modifier, Cisplatin, Platinum
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-37400ISBN: 978-91-7459-085-2 (print)OAI: oai:DiVA.org:umu-37400DiVA: diva2:360221
Public defence
2010-11-26, KBC-huset, Stora Hörsalen, KB3A1, Umeå Universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-11-05 Created: 2010-11-02 Last updated: 2010-11-05Bibliographically approved
List of papers
1. Alternative organic solvents for HILIC separation of cisplatin species with on-line ICP-MS detection
Open this publication in new window or tab >>Alternative organic solvents for HILIC separation of cisplatin species with on-line ICP-MS detection
2008 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 31, no 4, 599-603 p.Article in journal (Refereed) Published
Abstract [en]

Several low volatile organic solvents were evaluated as organic modifiers in eluents for HILIC separations of cisplatin species to optimize the on-line coupling of HILIC to inductively coupled plasma MS (ICP-MS). The aim was to identify a solvent giving low solvent vapor loading of the ICP, to maximize analyte sensitivity and minimize carbon depositions on instrumental parts, while retaining chromatographic performance. The best overall performance of the HILIC-ICP-MS system for the analysis of cisplatin was achieved using 1,4-dioxane as eluent, yielding high retention and an HILIC type retention mechanism, at the expense of a 50% drop in column efficiency due to the higher viscosity of 1,4-dioxane compared to the more commonly used HILIC solvent ACN. Using 1,4-dioxane as solvent in HILIC provides the best compromise between carbon deposition and separation efficiency among a series of high-boiling water-miscible solvents tested.

Keyword
Cisplatin, Dimethylformamide (DMF), HILIC, Hydrophilic interaction, ICP-MS
Identifiers
urn:nbn:se:umu:diva-9646 (URN)doi:10.1002/jssc.200700480 (DOI)18307161 (PubMedID)
Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-11-05Bibliographically approved
2. Mobile phase selection for the combined use of liquid chromatography-inductively coupled plasma mass spectrometry and electrospray ionisation mass spectrometry
Open this publication in new window or tab >>Mobile phase selection for the combined use of liquid chromatography-inductively coupled plasma mass spectrometry and electrospray ionisation mass spectrometry
2010 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1217, no 30, 4980-4986 p.Article in journal (Refereed) Published
Abstract [en]

Four different organic solvents: dimethylformamide, 1,4-dioxane, n-propanol and ethanol were evaluated as alternative organic modifiers to acetonitrile for liquid chromatography (LC) separations. The aim was to establish common sets of chromatographic conditions that could be applied for LC hyphenation to inductively coupled plasma mass spectrometry (ICPMS) as well as to electrospray ionization MS (ESIMS). The approach was to evaluate candidate solvents that, compared to acetonitrile, potentially could give improved analytical performance (low solvent vapor loading, maximized analyte sensitivity and minimized carbon depositions on instrumental parts) in ICPMS analysis while retaining chromatographic and ESIMS performances. The study showed that dimethylformamide, 1,4-dioxane, n-propanol and ethanol all can be advantageous chromatographic modifiers for LC-ICPMS analysis, giving superior performance compared to acetonitrile. For the combined use of LC-ICPMS and LC-ESIMS with a common set of chromatographic conditions, n-propanol gave the best overall performance. The 195Pt+ signal in ICPMS was continuously monitored during a 0-60% organic solvent gradient and at 25% of organic modifier, 100% of the signal obtained at the gradient start was preserved for n-propanol compared to only 35% of the signal when using acetonitrile. Platinum detection limits were 5-8 times lower using n-propanol compared with acetonitrile. Signal-to-noise ratio in continuous ESIMS signal measurements was 100, 90 and 110 for a 100 microg/ml solution of leucine-enkephaline using acetonitrile, ethanol and n-propanol, respectively. Chromatographic efficiency in reversed phase separations was preserved for n-propanol compared to acetonitrile for the analysis of the whole protein cytochrome C and the peptide bacitracin on a column with particle and pore sizes of 5 microm and 300 A, but slightly deteriorated for the separation of the peptides leucine-enkephaline and bacitracin on a 3 microm and 90 A column as the peak width at half height for both peptides increased by a factor of two. The performance on the smaller dimensioned column could however be improved by running the separations at 40 degrees C.

Keyword
ICPMS, ESIMS, liquid chromatography, mobile phase, hyphenation
Identifiers
urn:nbn:se:umu:diva-35285 (URN)10.1016/j.chroma.2010.05.062 (DOI)000280019300013 ()20573350 (PubMedID)
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2010-11-05Bibliographically approved
3. Hydrophilic interaction liquid chromatography (HILIC) coupled to inductively coupled plasma mass spectrometry (ICPMS) utilizing a mobile phase with a low-volatile organic modifier for the determination of cisplatin, and its monohydrolyzed metabolite
Open this publication in new window or tab >>Hydrophilic interaction liquid chromatography (HILIC) coupled to inductively coupled plasma mass spectrometry (ICPMS) utilizing a mobile phase with a low-volatile organic modifier for the determination of cisplatin, and its monohydrolyzed metabolite
Show others...
2008 (English)In: Journal of Analytical Atomic Spectrometry, Vol. 23, no 7, 948-54 p.Article in journal (Refereed) Published
Abstract [en]

This study demonstrates the on-line coupling of hydrophilic interaction liquid chromatography (HILIC) to inductively coupled plasma mass spectrometry (ICPMS). A low volatile organic solvent, dimethylformamide (DMF), was used as organic modifier of the mobile phase to minimize the solvent loading of the ICP and thereby improve analyte sensitivity and method robustness. The concept was illustrated by the selective determination of cisplatin (cis-DDP), and its mono-hydrolyzed metabolite (MH-DDP). Species were separated on a 2.1 × 150 mm ZIC-HILIC column and detected on-line by selective platinum (m/z 194 and 195) monitoring, giving a detection limit of 2 pg Pt (0.2 ng ml-1). Compared to the use of the conventional solvent acetonitrile (AcN), with DMF, cis-DDP sensitivity was enhanced as much as 36 times and no addition of oxygen to the plasma was needed to avoid carbon depositions on instrumental parts. Furthermore, several non-identified platinum containing compounds were observed when using AcN as a result of unwanted reactions between this solvent and the analytes. No such species were observed when DMF was used. The molecular structures of eluting compounds were verified by electrospray ionization mass spectrometry. Combined with a simple sample treatment protocol, the HILIC-ICPMS system allowed determination of free intracellular cis-DDP in in-vitro grown T289 human malignant melanoma cells up to 60 min after exposure to 50 g ml-1 cis-DDP for 1 h. This work shows that HILIC-ICPMS is a potent hyphenated technique for the analysis of hydrophilic metal compounds and that the use of chromatographic mobile phases with low-volatile organic solvents may be a generic approach to improve analyte sensitivity and system robustness of HPLC-ICPMS when mobile phases with high amount of organic solvent are used.

Identifiers
urn:nbn:se:umu:diva-10266 (URN)doi:10.1039/b716093c (DOI)
Available from: 2008-08-05 Created: 2008-08-05 Last updated: 2010-11-05Bibliographically approved
4. The platinum and copper metallome from sensitive and resistant T289 melanoma cells exposed to Cisplatin studied by a multi-analytical approach
Open this publication in new window or tab >>The platinum and copper metallome from sensitive and resistant T289 melanoma cells exposed to Cisplatin studied by a multi-analytical approach
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The copper and platinum metallome were evaluated in cisplatin (CDDP) sensitive and resistant melanoma T289 cells after exposure to cisplatin. Determination of the total concentrations of copper and platinum were combined with analysis of the intracellular copper and platinum profiles acquired by size exclusion chromatography (SEC) and reversed phase liquid chromatography (RPLC) coupled to inductively coupled plasma and electrospray ionization mass spectrometry (ICPMS and ESIMS, respectively). The total concentration of platinum in sensitive cells was 50-80% higher than in the resistant cell line and the intracellular copper concentration was decreased by ~30% when exposing the cells to cisplatin. SEC-ICPMS and RPLC-ICPMS profiles showed no obvious differences in the platinum profile between sensitive and resistant cells. However, the copper profiles differed showing a significant increase of some still unidentified copper species with an estimated molecular mass of 25-30 kDa. All together the data support the view that components of the copper homeostatic system are involved in CDDP accumulation and are affected by cisplatin exposure.

Keyword
Metallome, HPLC-ICPMS, HPLC-ESIMS, Platinum, Copper
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-37395 (URN)
Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2015-11-11Bibliographically approved
5. Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells
Open this publication in new window or tab >>Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells
Show others...
2010 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 396, no 1, 76-82 p.Article in journal (Refereed) Published
Abstract [en]

Three cell lysis methods-freeze-thaw, osmosis, and a chemical detergent-based method-were evaluated as sample treatment procedures for platinum metallomic studies of in vitro grown human malignant cells exposed to cisplatin. The lysis methods are relatively mild, resemble those commonly used in proteomic studies, and were selected because of the proven reactivity of platinum drug metabolites and indications that platinum in exposed cells and plasma is mainly associated with proteins. The chemical method gave an absolute lysis efficiency of greater than 80%, whereas the freeze-thaw and osmosis methods gave approximately 30% lower efficiency. The within- and between-batch lysis reproducibilities were, for all methods, better than 20 and 24% relative standard deviations, respectively. Total platinum concentration normalized to lysate protein content was statistically the same for all lysis methods. Reagents in the chemical lysis buffer did, however, react with platinum analyte compounds, making this method unsuitable for analysis of reactive compounds or for metallome profiling encompassing analytes with unknown reactivity. Of the lysis methods evaluated here, osmosis gave the highest cisplatin recovery, likely because this protocol is chemically inert and can be carried out at a constant low temperature. Therefore, it is the recommended cell lysis method for the determination of reactive and unknown intracellular platinum compounds.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Cell lysis, Sample preparation, Human malignant cells, Metallomics, Platinum, Cisplatin
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-29753 (URN)10.1016/j.ab.2009.08.044 (DOI)000272406100011 ()19733145 (PubMedID)
Available from: 2009-11-23 Created: 2009-11-23 Last updated: 2012-03-09Bibliographically approved

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