Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls
The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exog...
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| Veröffentlicht in: | Analytica chimica acta 2018-11, Vol.1030, p.105-114 |
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| creator | Putz, Marlen Piper, Thomas Casilli, Alessandro Radler de Aquino Neto, Francisco Pigozzo, Fausto Thevis, Mario |
| description | The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity.
Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure.
Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for |
| doi_str_mv | 10.1016/j.aca.2018.05.016 |
| format | Article |
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Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure.
Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.
[Display omitted]
•A MDGC-C-IRMS was developed as an initial testing method for analyzing endogenous anabolic steroids in urine.•A substantial reduction of the manual workload and an acceleration of the sample preparation was accomplished.•The method was successfully validated according toWADA guidelines and by means of linear mixing models.•A reference population (n = 74) was investigated and reference limits were deduced.•The method was found to be suitable for a routine application in doping control analysis.</description><identifier>ISSN: 0003-2670</identifier><identifier>EISSN: 1873-4324</identifier><identifier>DOI: 10.1016/j.aca.2018.05.016</identifier><identifier>PMID: 30032759</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Acetates ; Acetic acid ; Analytical chemistry ; Androstenedione ; Arylsulfatase ; Capillary pressure ; Carbon isotope ratio ; Carbon isotopes ; Chromatography ; Cutting equipment ; Data processing ; Doping ; Endogenous anabolic steroids ; Excretion ; Flame ionization ; Flame ionization detectors ; Gas chromatography ; Gas Chromatography-Mass Spectrometry ; Healthy Volunteers ; High performance liquid chromatography ; Humans ; Ionization ; Ionization counters ; Isotope ratios ; Isotopes ; Linear Models ; Liquid chromatography ; Liquid-liquid extraction ; Male ; Mass spectrometry ; Mass spectroscopy ; Metabolites ; Online heart-cutting ; Polarity ; Purification ; Purity ; Quadrupoles ; Retention time ; Sample preparation ; Separation ; Solid phases ; Spectroscopy ; Sports drug testing ; Stable isotope analysis ; Steroid hormones ; Steroids ; Steroids - metabolism ; Steroids - urine ; Testosterone ; Windows (intervals)</subject><ispartof>Analytica chimica acta, 2018-11, Vol.1030, p.105-114</ispartof><rights>2018 Elsevier B.V.</rights><rights>Copyright © 2018 Elsevier B.V. All rights reserved.</rights><rights>Copyright Elsevier BV Nov 7, 2018</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c381t-2c2d11533bc6554a745b94c44e3067d863c310be142df71c631711fbbd62659e3</citedby><cites>FETCH-LOGICAL-c381t-2c2d11533bc6554a745b94c44e3067d863c310be142df71c631711fbbd62659e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.aca.2018.05.016$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30032759$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Putz, Marlen</creatorcontrib><creatorcontrib>Piper, Thomas</creatorcontrib><creatorcontrib>Casilli, Alessandro</creatorcontrib><creatorcontrib>Radler de Aquino Neto, Francisco</creatorcontrib><creatorcontrib>Pigozzo, Fausto</creatorcontrib><creatorcontrib>Thevis, Mario</creatorcontrib><title>Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls</title><title>Analytica chimica acta</title><addtitle>Anal Chim Acta</addtitle><description>The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity.
Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure.
Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.
[Display omitted]
•A MDGC-C-IRMS was developed as an initial testing method for analyzing endogenous anabolic steroids in urine.•A substantial reduction of the manual workload and an acceleration of the sample preparation was accomplished.•The method was successfully validated according toWADA guidelines and by means of linear mixing models.•A reference population (n = 74) was investigated and reference limits were deduced.•The method was found to be suitable for a routine application in doping control analysis.</description><subject>Acetates</subject><subject>Acetic acid</subject><subject>Analytical chemistry</subject><subject>Androstenedione</subject><subject>Arylsulfatase</subject><subject>Capillary pressure</subject><subject>Carbon isotope ratio</subject><subject>Carbon isotopes</subject><subject>Chromatography</subject><subject>Cutting equipment</subject><subject>Data processing</subject><subject>Doping</subject><subject>Endogenous anabolic steroids</subject><subject>Excretion</subject><subject>Flame ionization</subject><subject>Flame ionization detectors</subject><subject>Gas chromatography</subject><subject>Gas Chromatography-Mass Spectrometry</subject><subject>Healthy Volunteers</subject><subject>High performance liquid chromatography</subject><subject>Humans</subject><subject>Ionization</subject><subject>Ionization counters</subject><subject>Isotope ratios</subject><subject>Isotopes</subject><subject>Linear Models</subject><subject>Liquid chromatography</subject><subject>Liquid-liquid extraction</subject><subject>Male</subject><subject>Mass spectrometry</subject><subject>Mass spectroscopy</subject><subject>Metabolites</subject><subject>Online heart-cutting</subject><subject>Polarity</subject><subject>Purification</subject><subject>Purity</subject><subject>Quadrupoles</subject><subject>Retention time</subject><subject>Sample preparation</subject><subject>Separation</subject><subject>Solid phases</subject><subject>Spectroscopy</subject><subject>Sports drug testing</subject><subject>Stable isotope analysis</subject><subject>Steroid hormones</subject><subject>Steroids</subject><subject>Steroids - metabolism</subject><subject>Steroids - urine</subject><subject>Testosterone</subject><subject>Windows (intervals)</subject><issn>0003-2670</issn><issn>1873-4324</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc2u0zAQhSME4pYLD8AGWWLDJqnHdpJWrNDlV7oSG1hbjj1pXSVxsJ1K5bF4QibqhQULVpbPfHPGnlMUL4FXwKHZnipjTSU47CpeV6Q8Kjawa2WppFCPiw3nXJaiaflN8SylE10FcPW0uJFUEG293xS_3uMZhzCPOGVmJsfOZvDOZB8mFnpm2LgM2TtP9USaGdjBJGaPMYwmh0M08_GytWHslrT2bH0KOczI4mrBRpMSSzPaTDzmeCk7k9CxjCkzEo7BsT5EGmyGy08_HdgS_WTihaWMMXiXmJ-YC_NasmEimyE9L570Zkj44uG8Lb5__PDt7nN5__XTl7t396WVO8ilsMIB1FJ2tqlrZVpVd3tllULJm9btGmkl8A5BCde3YBsJLUDfda4RTb1HeVu8ufrOMfxY6MV69MniMJgJw5K04K0CIRoOhL7-Bz2FJdKniAIaINbNEwVXysaQUsRez9GP9FsNXK-B6pOmQPUaqOa1JoV6Xj04L92I7m_HnwQJeHsFkFZx9hh1sh4ni85H2rt2wf_H_jdYMbTh</recordid><startdate>20181107</startdate><enddate>20181107</enddate><creator>Putz, Marlen</creator><creator>Piper, Thomas</creator><creator>Casilli, Alessandro</creator><creator>Radler de Aquino Neto, Francisco</creator><creator>Pigozzo, Fausto</creator><creator>Thevis, Mario</creator><general>Elsevier B.V</general><general>Elsevier BV</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QP</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7TK</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20181107</creationdate><title>Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls</title><author>Putz, Marlen ; Piper, Thomas ; Casilli, Alessandro ; Radler de Aquino Neto, Francisco ; Pigozzo, Fausto ; Thevis, Mario</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c381t-2c2d11533bc6554a745b94c44e3067d863c310be142df71c631711fbbd62659e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Acetates</topic><topic>Acetic acid</topic><topic>Analytical chemistry</topic><topic>Androstenedione</topic><topic>Arylsulfatase</topic><topic>Capillary pressure</topic><topic>Carbon isotope ratio</topic><topic>Carbon isotopes</topic><topic>Chromatography</topic><topic>Cutting equipment</topic><topic>Data processing</topic><topic>Doping</topic><topic>Endogenous anabolic steroids</topic><topic>Excretion</topic><topic>Flame ionization</topic><topic>Flame ionization detectors</topic><topic>Gas chromatography</topic><topic>Gas Chromatography-Mass Spectrometry</topic><topic>Healthy Volunteers</topic><topic>High performance liquid chromatography</topic><topic>Humans</topic><topic>Ionization</topic><topic>Ionization counters</topic><topic>Isotope ratios</topic><topic>Isotopes</topic><topic>Linear Models</topic><topic>Liquid chromatography</topic><topic>Liquid-liquid extraction</topic><topic>Male</topic><topic>Mass spectrometry</topic><topic>Mass spectroscopy</topic><topic>Metabolites</topic><topic>Online heart-cutting</topic><topic>Polarity</topic><topic>Purification</topic><topic>Purity</topic><topic>Quadrupoles</topic><topic>Retention time</topic><topic>Sample preparation</topic><topic>Separation</topic><topic>Solid phases</topic><topic>Spectroscopy</topic><topic>Sports drug testing</topic><topic>Stable isotope analysis</topic><topic>Steroid hormones</topic><topic>Steroids</topic><topic>Steroids - metabolism</topic><topic>Steroids - urine</topic><topic>Testosterone</topic><topic>Windows (intervals)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Putz, Marlen</creatorcontrib><creatorcontrib>Piper, Thomas</creatorcontrib><creatorcontrib>Casilli, Alessandro</creatorcontrib><creatorcontrib>Radler de Aquino Neto, Francisco</creatorcontrib><creatorcontrib>Pigozzo, Fausto</creatorcontrib><creatorcontrib>Thevis, Mario</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytica chimica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Putz, Marlen</au><au>Piper, Thomas</au><au>Casilli, Alessandro</au><au>Radler de Aquino Neto, Francisco</au><au>Pigozzo, Fausto</au><au>Thevis, Mario</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls</atitle><jtitle>Analytica chimica acta</jtitle><addtitle>Anal Chim Acta</addtitle><date>2018-11-07</date><risdate>2018</risdate><volume>1030</volume><spage>105</spage><epage>114</epage><pages>105-114</pages><issn>0003-2670</issn><eissn>1873-4324</eissn><abstract>The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity.
Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure.
Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.
[Display omitted]
•A MDGC-C-IRMS was developed as an initial testing method for analyzing endogenous anabolic steroids in urine.•A substantial reduction of the manual workload and an acceleration of the sample preparation was accomplished.•The method was successfully validated according toWADA guidelines and by means of linear mixing models.•A reference population (n = 74) was investigated and reference limits were deduced.•The method was found to be suitable for a routine application in doping control analysis.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>30032759</pmid><doi>10.1016/j.aca.2018.05.016</doi><tpages>10</tpages></addata></record> |
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| subjects | Acetates Acetic acid Analytical chemistry Androstenedione Arylsulfatase Capillary pressure Carbon isotope ratio Carbon isotopes Chromatography Cutting equipment Data processing Doping Endogenous anabolic steroids Excretion Flame ionization Flame ionization detectors Gas chromatography Gas Chromatography-Mass Spectrometry Healthy Volunteers High performance liquid chromatography Humans Ionization Ionization counters Isotope ratios Isotopes Linear Models Liquid chromatography Liquid-liquid extraction Male Mass spectrometry Mass spectroscopy Metabolites Online heart-cutting Polarity Purification Purity Quadrupoles Retention time Sample preparation Separation Solid phases Spectroscopy Sports drug testing Stable isotope analysis Steroid hormones Steroids Steroids - metabolism Steroids - urine Testosterone Windows (intervals) |
| title | Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls |
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