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Development of LC-MS/MS Methods for the Analysis of Chiral and Achiral Pharmaceuticals and Metabolites in Aqueous Environmental Matrices
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam.

The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected.

The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam.

The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam.

The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples.

The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 158
Keyword [en]
Environmental water matrices, chiral separation, LC-MS/MS, trace analysis, method development, active pharmaceutical ingredients, metabolites
National Category
Analytical Chemistry Pharmaceutical Sciences
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-171550ISBN: 978-91-554-8313-5 (print)OAI: oai:DiVA.org:uu-171550DiVA: diva2:511372
Public defence
2012-05-04, B22, BMC, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2012-04-13 Created: 2012-03-21 Last updated: 2018-01-12
List of papers
1. Trace analysis of fluoxetine and its metabolite norfluoxetine: Part I: Development of a chiral liquid chromatography-tandem mass spectrometry method for wastewater samples
Open this publication in new window or tab >>Trace analysis of fluoxetine and its metabolite norfluoxetine: Part I: Development of a chiral liquid chromatography-tandem mass spectrometry method for wastewater samples
2011 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1218, no 33, 5587-5596 p.Article in journal (Refereed) Published
Abstract [en]

An enantioselective method for the determination of fluoxetine (a selective serotonin reuptake inhibitor) and its pharmacologically active metabolite norfluoxetine has been developed for raw and treated wastewater samples. The stable isotope-labeled fluoxetine and norfluoxetine were used in an extended way for extraction recovery calculations at trace level concentrations in wastewater. Wastewater samples were enriched by solid phase extraction (SPE) with Evolute CX-50 extraction cartridges. The obtained extraction recoveries ranged between 65 and 82% in raw and treated wastewater at a trace level concentration of 50 pM (15-16 ng L(-1)). The target compounds were identified by the use of chiral liquid chromatography tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (SRM) mode. The enantiomers were successfully resolved on a chiral alpha(1)-acid glycoprotein column (chiral AGP) with acetonitrile and 10 mM ammonium acetate buffer at pH 4.4 (3/97, v/v) as the mobile phase. The effects of pH, amount of organic modifier and buffer concentration in the mobile phase were investigated on the enantiomeric resolution (R(s)) of the target compounds. Enantiomeric R(s)-values above 2.0 (1.03 RSD%, n = 3) were achieved for the enantiomers of fluoxetine and norfluoxetine in all mobile phases investigated. The method was validated by assessing parameters such as cross-contamination and carryover during SPE and during LC analysis. Cross-talk effects were examined during the detection of the analytes in SRM mode. In addition, the isotopic purity of fluoxetine-d(5) and norfluoxetine-d(5) were assessed to exclude the possibility of self-contamination. The interassay precision of the chromatographic separation was excellent, with relative standard deviations (RSD) equal to or lower than 0.56 and 0.81% in raw and treated wastewaters, respectively. The method detection and quantification limits (respectively, MDL and MQL) were determined by the use of fluoxetine-d5 and norfluoxetine-d5. The MQL for the single enantiomers ranged from 12 to 14 pM (3.6-4.3 ng L(-1)) in raw wastewater and from 3 to 4 pM (0.9-1 ng L(-1)) in treated wastewater. The developed method has been employed for the quantification of (R)-fluoxetine, (S)-fluoxetine and the enantiomers of norfluoxetine in raw and treated wastewater samples to be presented in Part II of this study.

Keyword
Wastewater, Solid phase extraction, Fluoxetine, Norfluoxetine, Isotope-labeled compounds, Chiral LC-MS/MS
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-158308 (URN)10.1016/j.chroma.2011.06.024 (DOI)000294031700004 ()
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
2. Trace analysis of fluoxetine and its metabolite norfluoxetine. Part II: Enantioselective quantification and studies of matrix effects in raw and treated wastewater by solid phase extraction and liquid chromatography-tandem mass spectrometry
Open this publication in new window or tab >>Trace analysis of fluoxetine and its metabolite norfluoxetine. Part II: Enantioselective quantification and studies of matrix effects in raw and treated wastewater by solid phase extraction and liquid chromatography-tandem mass spectrometry
2012 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1227, 105-114 p.Article in journal (Refereed) Published
Abstract [en]

The isotope-labeled compounds fluoxetine-d5 and norfluoxetine-d5 were used to study matrix effects caused by co-eluting compounds originating from raw and treated wastewater samples, collected in Uppsala, Sweden. The matrix effects were investigated by the determination of matrix factors (MF) and by a post-column infusion method. The matrix factors were determined to be 38–47% and 71–86% for the enantiomers of norfluoxetine-d5 and fluoxetine-d5, respectively. The influence of matrix effects when quantifying the enantiomers of the active pharmaceutical ingredient and the metabolite in wastewater samples with LC–MS/MS is discussed and methods to overcome the problem are presented. The enantiomeric concentrations of fluoxetine and its human metabolite norfluoxetine, quantified by a one-point calibration method, were 12–52 pM (3.5–16 ng L−1) in raw wastewater and 4–48 pM (1.2–15 ng L−1) in treated wastewater. Furthermore, the calculated enantiomeric fractions (EF) of the substances were found to be between 0.68 and 0.71 in both matrices. Neither the EF values for fluoxetine nor those for norfluoxetine were significantly different in the raw wastewater compared to the treated wastewater. Interestingly, the concentration of (S)-fluoxetine was found to be higher than the concentration of (R)-fluoxetine in both raw and treated wastewater. These results are different from other results presented in the literature, which shows that the relative concentrations of the enantiomers of a chiral active pharmaceutical ingredient might be significantly different in wastewater samples from different treatment systems. We report, for the first time, the concentrations of the enantiomers of norfluoxetine in wastewater samples. The concentrations of (S)-norfluoxetine were found to be higher than the concentration of (R)-norfluoxetine in the raw as well as in the treated wastewater samples.

National Category
Analytical Chemistry Pharmaceutical Sciences Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-170511 (URN)10.1016/j.chroma.2011.12.084 (DOI)000301563700013 ()22265784 (PubMedID)
Available from: 2012-03-12 Created: 2012-03-12 Last updated: 2018-01-12Bibliographically approved
3. Chiral analysis of metoprolol and two of its metabolites, alpha‑hydroxymetoprolol and deaminated metoprolol, in wastewater using liquid chromatography-tandem mass spectrometry
Open this publication in new window or tab >>Chiral analysis of metoprolol and two of its metabolites, alpha‑hydroxymetoprolol and deaminated metoprolol, in wastewater using liquid chromatography-tandem mass spectrometry
2012 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1269, no SI, 208-217 p.Article in journal (Refereed) Published
Abstract [en]

A LC–MS/MS method for the chiral separation of metoprolol and two of its main metabolites, α-hydroxymetoprolol (α-OH-Met) and deaminated metoprolol (COOH-Met), in environmental water samples has been developed. The target bases, metoprolol and α-OH-Met, as well as the acidic metabolite (COOH-Met) were extracted from water samples by a solid phase extraction method employing Oasis HLB cartridges. The extraction recoveries were ≥73% for all compounds in surface water. Four different types of chiral stationary phases were investigated for the separation of the eight stereoisomers of metoprolol and its metabolites, Chiralcel OD-H, Chirobiotic V, Chiral AGP and Chiral CBH. In the final method, the enantiomers of metoprolol and four stereoisomers of α-OH-Met were separated using Chiral CBH, the enantiomers of COOH-Met were separated employing Chiral AGP. The analytes were detected in SRM mode by triple quadrupole mass spectrometry. The method was applied for the chiral analysis of the analytes in treated wastewater samples from Uppsala, Sweden. The enantiomers and diastereoisomers of α-OH-Met were detected and analyzed in the samples. The concentrations of the three first eluting stereoisomers of α-OH-Met were between 54 and 61 pM. Interestingly, the last eluting stereoisomer was found to be present at a concentration of 151 pM at the same sampling occasion. This is, to the best of the authors’ knowledge, the first time the stereoisomers of α-OH-Met have been detected in wastewater samples. The enantiomers of metoprolol were determined to be 1.77 and 1.86 nM in the same matrix. The enantiomers of COOH-Met were not detected above the method detection limit (42 pM) in treated wastewater samples. The developed LC–MS/MS methods were validated in wastewater samples.

National Category
Pharmaceutical Sciences Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-171547 (URN)10.1016/j.chroma.2012.09.090 (DOI)000312475300016 ()
Available from: 2012-03-21 Created: 2012-03-21 Last updated: 2018-01-12Bibliographically approved
4. A liquid chromatography-tandem mass spectrometry method for the trace analysis of diazepam and nordiazepam in environmental water samples. A comprehensive study of storage conditions and identification of an unknown compound.
Open this publication in new window or tab >>A liquid chromatography-tandem mass spectrometry method for the trace analysis of diazepam and nordiazepam in environmental water samples. A comprehensive study of storage conditions and identification of an unknown compound.
(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-171548 (URN)
Available from: 2012-03-21 Created: 2012-03-21 Last updated: 2018-01-12

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