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Simultaneous quantitation of the highly lipophilic atovaquone and hydrophilic strong basic proguanil and its metabolites using a new mixed-mode SPE approach and steep-gradient LC
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Dalarna University College, 781 88 Borlänge; and Uppsala University, Department of Analytical Chemistry, 751 21 Uppsala, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Dalarna University College, 781 88 Borlänge; and Uppsala University, Department of Analytical Chemistry, 751 21 Uppsala, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Dalarna University College, 781 88 Borlänge; and Uppsala University, Department of Analytical Chemistry, 751 21 Uppsala, Sweden.
2005 (English)In: Journal of Chromatographic Science, ISSN 0021-9665, E-ISSN 1945-239X, Vol. 43, no 5, 259-266 p.Article in journal (Refereed) Published
Abstract [en]

A bioanalytical method is described for the simultaneous quantitative analysis of the highly lipophilic atovaquone and the strong basic proguanil with metabolites in plasma. The drugs are extracted from protein precipitated plasma samples on a novel mixed-mode solid-phase extraction (SPE) column containing carboxypropyl and octyl silica as functional groups. The analytes are further separated and quantitated using a steep-gradient liquid chromatographic method on a Zorbax SB-CN column with UV detection at 245 nm. Two different internal standards (IS) are used in the method to compensate for both types of analytes. A structurally similar IS to atovaquone is added with acetonitrile to precipitate proteins from plasma. A structurally similar IS to proguanil and its metabolites is added with phosphate buffer before samples are loaded onto the SPE columns. A single elution step is sufficient to elute all analytes. The method is validated according to published guidelines and shows excellent performance. The within-day precisions, expressed as relative standard deviation, are lower than 5% for all analytes at three tested concentrations within the calibration range. The between-day precisions are lower than 13% for all analytes at the same tested concentrations. The limit of quantitation is 25 nM for the basic substances and 50 nM for atovaquone. Several considerations regarding development and optimization of a method for determination of analytes with such a difference in physiochemical properties are discussed.

Place, publisher, year, edition, pages
2005. Vol. 43, no 5, 259-266 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-108745ISI: 000229224600007PubMedID: 15975245OAI: oai:DiVA.org:uu-108745DiVA: diva2:240694
Available from: 2009-09-29 Created: 2009-09-29 Last updated: 2013-06-20Bibliographically approved
In thesis
1. Development of Analytical Methods for the Determination of Antimalarials in Biological Fluids
Open this publication in new window or tab >>Development of Analytical Methods for the Determination of Antimalarials in Biological Fluids
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis was to develop analytical methods for measuring antimalarial drugs in biological fluids. Solid phase extraction (SPE) was used for the enrichment and purification of the drugs. Automatic extraction procedures using a SPE robot were developed to reduce the workload for the analyst and to minimize variations in the extraction procedure. Liquid chromatography (LC) with either UV or mass spectrometric (MS) detection was used to determine sample concentrations.

Determination of Pyronaridine in whole blood utilised a weak cation exchanger to extract Pyronaridine from blood. To improve LC separation between Pyronaridine and the internal standard, ion-pairing was utilized.

For the simultaneous quantification of the highly lipophilic Atovaquone and the strong basic drug Proguanil with metabolites, a novel mixed mode solid phase extraction column was used. It combines the properties of a carboxylic acid (CBA) column and a non-polar octyl-silica (C8) column to extract the compounds from plasma; it also required a gradient LC separation.

Stability is an important factor when developing new methods. A new approach was used to evaluate the stability of Amodiaquine in blood and plasma. This included the use of a stability marker, a stable compound which was added together with Amodiaquine when preparing the stability samples. This eliminated between-run variations and variations associated with preparation of new stock solutions.

Lumefantrine (LF) is one of the active components in a new drug combination recommended by the World Health Organization as a replacement for older drugs which have lost their effect. The first of the two methods described for this compound is the determination of LF and a possible metabolite in plasma with a calibration range suitable for pharmacokinetic studies. In the second method, a capillary sampling technique is used where the blood is dried on a sampling paper and sent to the laboratory where the extraction and determination of LF concentrations take place. This method facilitates sample collection and will enable drug efficacy studies conducted in rural settings.

To monitor a current change in treatment policy and self medication, a screening assay was developed. Its purpose is to be a complement to interviewing patients about their previous medication (in the previous few weeks) and to detect some of the more common drugs which might have been used.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 676
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-108767 (URN)978-91-554-7620-5 (ISBN)
Public defence
2009-11-12, Clas Ohlson room, Humanistgatan 2, Tenoren, Högskolan Dalarna, Borlänge, 13:00 (Swedish)
Opponent
Supervisors
Note
Paper 6. as ManuscriptAvailable from: 2009-10-22 Created: 2009-09-29 Last updated: 2009-10-22Bibliographically approved

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