{"id":{"repo_id":"calgary","oai_identifier":"oai:ucalgary.scholaris.ca:1880/107756"},"canonical_url":"https://search.dev.ndltd.org/etd/calgary/oai:ucalgary.scholaris.ca:1880/107756","repository":{"repo_id":"calgary","name":"University of Calgary","base_url":"https://ucalgary.scholaris.ca/server/oai/request"},"display":{"title":"Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia","abstract":"Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway within the adrenal glands. CAH is characterized by numerous clinical manifestations resulting from dysregulation of various enzymes in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids, cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, pregnenolone and dehydroepiandrosterone could significantly improve CAH laboratory diagnosis. Although clinical laboratories predominantly use immunoassays to measure some of the above steroids, these assays are hampered by low specificity due to cross reactivity of antibodies to molecules with similar structures and high cost of antibodies used in the system. As CAH is diagnosed in neonates, using dried blood spot (DBS) as specimen of choice can further improve patient care due to the very small sample volume. This study aimed to develop a more specific and rapid assay to measure seven steroids using liquid chromatography mass spectrometry (LC-MS/MS) in DBS. An optimized DBS sample preparation method was employed without the need of derivatization. A LC-MS/MS assay was developed and optimized using reverse phase-ultra performance liquid chromatography (RP-UPLC) system combined with a triple quadrupole mass spectrometry using positive electrospray ionization (+ESI) mode. Each steroid was quantified using its deuterated or isotopically labelled internal standard. Calibration curve and quality control specimens were prepared in saline-washed erythrocytes mixed with steroid free serum to achieve a 55±1% hematocrit level. Prepared specimens were enriched with predetermined concentrations of the seven steroids and spotted onto Whatman 903® filter papers. The assay was validated according to CLSI analytical guidelines, including limit of detection (LOD) and lower limit of quantification (LLOQ), linearity, precision, and method comparison. The analytical measuring range of the method for all steroids was 2.5 were 0.11-1.8 ng/ml, 0.001-6.4 ng/ml, 1.8–11.5%, and 5.3-13.8%, respectively. This robust and inexpensive assay can be readily implemented in clinical laboratories equipped with LC-MS/MS and can provides superior analytical performance over traditional immunoassays for the diagnosis of CAH.","abstract_html":"Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway within the adrenal glands. CAH is characterized by numerous clinical manifestations resulting from dysregulation of various enzymes in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids, cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, pregnenolone and dehydroepiandrosterone could significantly improve CAH laboratory diagnosis. Although clinical laboratories predominantly use immunoassays to measure some of the above steroids, these assays are hampered by low specificity due to cross reactivity of antibodies to molecules with similar structures and high cost of antibodies used in the system. As CAH is diagnosed in neonates, using dried blood spot (DBS) as specimen of choice can further improve patient care due to the very small sample volume. This study aimed to develop a more specific and rapid assay to measure seven steroids using liquid chromatography mass spectrometry (LC-MS/MS) in DBS. An optimized DBS sample preparation method was employed without the need of derivatization. A LC-MS/MS assay was developed and optimized using reverse phase-ultra performance liquid chromatography (RP-UPLC) system combined with a triple quadrupole mass spectrometry using positive electrospray ionization (+ESI) mode. Each steroid was quantified using its deuterated or isotopically labelled internal standard. Calibration curve and quality control specimens were prepared in saline-washed erythrocytes mixed with steroid free serum to achieve a 55±1% hematocrit level. Prepared specimens were enriched with predetermined concentrations of the seven steroids and spotted onto Whatman 903® filter papers. The assay was validated according to CLSI analytical guidelines, including limit of detection (LOD) and lower limit of quantification (LLOQ), linearity, precision, and method comparison. The analytical measuring range of the method for all steroids was 2.5 were 0.11-1.8 ng/ml, 0.001-6.4 ng/ml, 1.8–11.5%, and 5.3-13.8%, respectively. This robust and inexpensive assay can be readily implemented in clinical laboratories equipped with LC-MS/MS and can provides superior analytical performance over traditional immunoassays for the diagnosis of CAH.","abstract_has_math":false,"creators":["Qasrawi, Deema Omar"],"institution":"Cumming School of Medicine","degree_name":"Master of Science (MSc)","degree_level":null,"degree_discipline":"Medical Science","degree_department":null,"school":null,"contributors":[],"advisors":["Sadrzadeh, S. M. Hossein"],"committee_chairs":[],"committee_members":["Khan, Faisal Masood","Naugler, Christopher T.","Lewis, Ian A."],"year":2018,"date_issued":"2018-09-10","date_published":"2018-09-10","updated_at":"2026-07-24T01:30:31Z","subjects":["Endocrinology","Mass spectrometry","Steroids","Congenital adrenal hyperplasia","dried blood spots"],"languages":["eng"],"rights":["University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["http://dx.doi.org/10.11575/PRISM/32932"],"render_values":[{"text":"http://dx.doi.org/10.11575/PRISM/32932","href":"http://dx.doi.org/10.11575/PRISM/32932","code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/1880/107756","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Sadrzadeh, S. M. 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You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["http://dx.doi.org/10.11575/PRISM/32932"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1880/107756"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway within the adrenal glands. CAH is characterized by numerous clinical manifestations resulting from dysregulation of various enzymes in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids, cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, pregnenolone and dehydroepiandrosterone could significantly improve CAH laboratory diagnosis. Although clinical laboratories predominantly use immunoassays to measure some of the above steroids, these assays are hampered by low specificity due to cross reactivity of antibodies to molecules with similar structures and high cost of antibodies used in the system. As CAH is diagnosed in neonates, using dried blood spot (DBS) as specimen of choice can further improve patient care due to the very small sample volume. This study aimed to develop a more specific and rapid assay to measure seven steroids using liquid chromatography mass spectrometry (LC-MS/MS) in DBS. An optimized DBS sample preparation method was employed without the need of derivatization. A LC-MS/MS assay was developed and optimized using reverse phase-ultra performance liquid chromatography (RP-UPLC) system combined with a triple quadrupole mass spectrometry using positive electrospray ionization (+ESI) mode. Each steroid was quantified using its deuterated or isotopically labelled internal standard. Calibration curve and quality control specimens were prepared in saline-washed erythrocytes mixed with steroid free serum to achieve a 55±1% hematocrit level. Prepared specimens were enriched with predetermined concentrations of the seven steroids and spotted onto Whatman 903® filter papers. The assay was validated according to CLSI analytical guidelines, including limit of detection (LOD) and lower limit of quantification (LLOQ), linearity, precision, and method comparison. The analytical measuring range of the method for all steroids was 2.5 were 0.11-1.8 ng/ml, 0.001-6.4 ng/ml, 1.8–11.5%, and 5.3-13.8%, respectively. This robust and inexpensive assay can be readily implemented in clinical laboratories equipped with LC-MS/MS and can provides superior analytical performance over traditional immunoassays for the diagnosis of CAH."]},{"key":"dc:title","label":"Title","values":["Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia"]}]}],"canonical_facts":{"dc:contributor.advisor":["Sadrzadeh, S. M. Hossein"],"dc:contributor.committeemember":["Khan, Faisal Masood","Naugler, Christopher T.","Lewis, Ian A."],"dc:creator":["Qasrawi, Deema Omar"],"dc:date":["2018-11"],"dc:date.accessioned":["2018-09-18T20:07:18Z"],"dc:date.available":["2018-09-18T20:07:18Z"],"dc:date.issued":["2018-09-10"],"dc:description.abstract":["Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway within the adrenal glands. CAH is characterized by numerous clinical manifestations resulting from dysregulation of various enzymes in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids, cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, pregnenolone and dehydroepiandrosterone could significantly improve CAH laboratory diagnosis. Although clinical laboratories predominantly use immunoassays to measure some of the above steroids, these assays are hampered by low specificity due to cross reactivity of antibodies to molecules with similar structures and high cost of antibodies used in the system. As CAH is diagnosed in neonates, using dried blood spot (DBS) as specimen of choice can further improve patient care due to the very small sample volume. This study aimed to develop a more specific and rapid assay to measure seven steroids using liquid chromatography mass spectrometry (LC-MS/MS) in DBS. An optimized DBS sample preparation method was employed without the need of derivatization. A LC-MS/MS assay was developed and optimized using reverse phase-ultra performance liquid chromatography (RP-UPLC) system combined with a triple quadrupole mass spectrometry using positive electrospray ionization (+ESI) mode. Each steroid was quantified using its deuterated or isotopically labelled internal standard. Calibration curve and quality control specimens were prepared in saline-washed erythrocytes mixed with steroid free serum to achieve a 55±1% hematocrit level. Prepared specimens were enriched with predetermined concentrations of the seven steroids and spotted onto Whatman 903® filter papers. The assay was validated according to CLSI analytical guidelines, including limit of detection (LOD) and lower limit of quantification (LLOQ), linearity, precision, and method comparison. The analytical measuring range of the method for all steroids was 2.5 were 0.11-1.8 ng/ml, 0.001-6.4 ng/ml, 1.8–11.5%, and 5.3-13.8%, respectively. This robust and inexpensive assay can be readily implemented in clinical laboratories equipped with LC-MS/MS and can provides superior analytical performance over traditional immunoassays for the diagnosis of CAH."],"dc:identifier.doi":["http://dx.doi.org/10.11575/PRISM/32932"],"dc:identifier.uri":["http://hdl.handle.net/1880/107756"],"dc:language.iso":["eng"],"dc:publisher.institution":["University of Calgary"],"dc:rights":["University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission."],"dc:subject":["Endocrinology","Mass spectrometry","Steroids","Congenital adrenal hyperplasia","dried blood spots"],"dc:title":["Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia"],"dc:type":["master thesis"],"thesis:degree_discipline":["Medical Science"],"thesis:degree_name":["Master of Science (MSc)"],"thesis:institution_name":["University of Calgary"]},"updated_at":"2026-07-24T01:30:31Z"}