Publicação
Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms
| Resumo: | Sterols belong to the unsaponifiable fraction of several matrices, where they can be found as free or conjugated structures. In the latter, the 3β-hydroxyl group is esterified with a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty acyl hexose [1]. Ergosterol (an important vitamin D2 precursor), is clearly the main sterol in mushrooms. Typically, the analysis of individual sterols includes the extraction of lipids, saponification (which might include a previous acid hydrolysis), extraction of unsaponifiable matter and separation/partial purification of sterols. The subsequent separation step might be performed by high performance liquid chromatography (HPLC), which is faster than gas chromatography analysis and operates under milder column temperatures and non-destructive detection conditions [2]. Herein, an analytical method for ergosterol determination in cultivated and wild mushrooms was developed using HPLC coupled to ultraviolet detection. The chromatographic separation was achieved in a Inertsil 100A ODS-3 reverse phase column using an isocratic elution with acetonitrile:methanol (70:30, v:v) at a flow rate of 1 mL/min. Different extraction methodologies were tested, using n-hexane, methanol:dichloromethane (75:25, v:v) or chloroform:methanol (20:10, v:v). After studying the linearity (11 levels) for ergosterol (tR = 13.0±0.1 min; coefficient of variation, CV = 0.65%), a seven-level calibration curve (y = 0.6566ϰ + 0.01098; R2 = 0.9996) was made using the peak/area ratio versus concentration of the standard (in μg/mL). The average of triplicate determinations for each level was used. The limit of detection (LOD), calculated as the concentration corresponding to 3.3 times the standard error of the calibration curve divided by the slope, was 0.3498 μg/mL; the limit of quantification (LOQ), calculated using the concentration corresponding to ten times the standard error of the calibration curve divided by the slope, was 1.060 μg/mL. The precision of the equipment was evaluated injecting seven consecutive times the sterols extract of the same mushroom. The optimized method proved to be precise (CV = 1.25%). Repeatability was assessed by applying the extraction procedure three times to the same dried mushroom powder. The method proved also to be repeatable (CV = 1.88%). The method accuracy was evaluated by the standard addition procedure (percentage of recovery). The standard mixture was added to the samples in three concentration levels (25, 50 and 100 % of the peak/area concentration, each one in triplicate) before the extraction. The method showed good recovery (above 90%). Overall, a reproducible and accurate HPLC-UV technique was fully optimized. Chromatographic signals presented good resolution, indicating the suitability of this methodology to assess sterol profiles in future studies. Moreover, the chosen extraction and saponification steps do not require complex conditions, being suitable for routine analysis. |
|---|---|
| Autores principais: | Barreira, João C.M. |
| Outros Autores: | Ferreira, Isabel C.F.R. |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | documento de conferência |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Instituto Politécnico de Bragança |
| Idioma: | inglês |
| Origem: | Biblioteca Digital do IPB |
| _version_ | 1867172977151311872 |
|---|---|
| author | Barreira, João C.M. |
| author2 | Ferreira, Isabel C.F.R. |
| author2_role | author |
| author_facet | Barreira, João C.M. Ferreira, Isabel C.F.R. |
| author_role | author |
| contributor_name_str_mv | Biblioteca Digital do IPB |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Barreira, João C.M.\",\"Person.identifier.orcid\":\"0000-0003-1233-0990\"},{\"Person.name\":\"Ferreira, Isabel C.F.R.\",\"Person.identifier.orcid\":\"0000-0003-4910-4882\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Biblioteca Digital do IPB |
| datacite.creators.creator.creatorName.fl_str_mv | Barreira, João C.M. Ferreira, Isabel C.F.R. |
| datacite.date.Accepted.fl_str_mv | 2013-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2014-03-27T17:44:02Z |
| datacite.date.embargoed.fl_str_mv | 2014-03-27T17:44:02Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.titles.title.fl_str_mv | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| dc.contributor.none.fl_str_mv | Biblioteca Digital do IPB |
| dc.creator.none.fl_str_mv | Barreira, João C.M. Ferreira, Isabel C.F.R. |
| dc.date.Accepted.fl_str_mv | 2013-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2014-03-27T17:44:02Z |
| dc.date.embargoed.fl_str_mv | 2014-03-27T17:44:02Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10198/9400 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.title.fl_str_mv | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_c94f |
| description | Sterols belong to the unsaponifiable fraction of several matrices, where they can be found as free or conjugated structures. In the latter, the 3β-hydroxyl group is esterified with a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty acyl hexose [1]. Ergosterol (an important vitamin D2 precursor), is clearly the main sterol in mushrooms. Typically, the analysis of individual sterols includes the extraction of lipids, saponification (which might include a previous acid hydrolysis), extraction of unsaponifiable matter and separation/partial purification of sterols. The subsequent separation step might be performed by high performance liquid chromatography (HPLC), which is faster than gas chromatography analysis and operates under milder column temperatures and non-destructive detection conditions [2]. Herein, an analytical method for ergosterol determination in cultivated and wild mushrooms was developed using HPLC coupled to ultraviolet detection. The chromatographic separation was achieved in a Inertsil 100A ODS-3 reverse phase column using an isocratic elution with acetonitrile:methanol (70:30, v:v) at a flow rate of 1 mL/min. Different extraction methodologies were tested, using n-hexane, methanol:dichloromethane (75:25, v:v) or chloroform:methanol (20:10, v:v). After studying the linearity (11 levels) for ergosterol (tR = 13.0±0.1 min; coefficient of variation, CV = 0.65%), a seven-level calibration curve (y = 0.6566ϰ + 0.01098; R2 = 0.9996) was made using the peak/area ratio versus concentration of the standard (in μg/mL). The average of triplicate determinations for each level was used. The limit of detection (LOD), calculated as the concentration corresponding to 3.3 times the standard error of the calibration curve divided by the slope, was 0.3498 μg/mL; the limit of quantification (LOQ), calculated using the concentration corresponding to ten times the standard error of the calibration curve divided by the slope, was 1.060 μg/mL. The precision of the equipment was evaluated injecting seven consecutive times the sterols extract of the same mushroom. The optimized method proved to be precise (CV = 1.25%). Repeatability was assessed by applying the extraction procedure three times to the same dried mushroom powder. The method proved also to be repeatable (CV = 1.88%). The method accuracy was evaluated by the standard addition procedure (percentage of recovery). The standard mixture was added to the samples in three concentration levels (25, 50 and 100 % of the peak/area concentration, each one in triplicate) before the extraction. The method showed good recovery (above 90%). Overall, a reproducible and accurate HPLC-UV technique was fully optimized. Chromatographic signals presented good resolution, indicating the suitability of this methodology to assess sterol profiles in future studies. Moreover, the chosen extraction and saponification steps do not require complex conditions, being suitable for routine analysis. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | conferenceObject |
| fulltext.url.fl_str_mv | https://bibliotecadigital.ipb.pt/bitstreams/c76b3fc2-9e59-45e1-bc9b-863e322e1a40/download |
| funding.funder.alternateName_str_mv | FCT FCT FCT |
| funding.funder.identifier_str_mv | http://doi.org/10.13039/501100001871 http://doi.org/10.13039/501100001871 http://doi.org/10.13039/501100001871 |
| funding.funder.name_str_mv | Fundação para a Ciência e a Tecnologia Fundação para a Ciência e a Tecnologia Fundação para a Ciência e a Tecnologia |
| funding.name_str_mv | 5876-PPCDTI 6817 - DCRRNI ID |
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| identifier.url.fl_str_mv | http://hdl.handle.net/10198/9400 |
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| institution | Instituto Politécnico de Bragança |
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| language | eng |
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| oai_identifier_str | oai:bibliotecadigital.ipb.pt:10198/9400 |
| organization_str_mv | urn:organizationAcronym:ipb |
| person_str_mv | Barreira, João C.M. Barreira, João C.M. http://orcid.org/0000-0003-1233-0990 0000-0003-1233-0990 Ferreira, Isabel C.F.R. Ferreira, Isabel C.F.R. https://www.ciencia-id.pt/9418-CF95-9919 9418-CF95-9919 http://orcid.org/0000-0003-4910-4882 0000-0003-4910-4882 |
| publishDate | 2013 |
| reponame_str | Biblioteca Digital do IPB |
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| spelling | engporSterols belong to the unsaponifiable fraction of several matrices, where they can be found as free or conjugated structures. In the latter, the 3β-hydroxyl group is esterified with a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty acyl hexose [1]. Ergosterol (an important vitamin D2 precursor), is clearly the main sterol in mushrooms. Typically, the analysis of individual sterols includes the extraction of lipids, saponification (which might include a previous acid hydrolysis), extraction of unsaponifiable matter and separation/partial purification of sterols. The subsequent separation step might be performed by high performance liquid chromatography (HPLC), which is faster than gas chromatography analysis and operates under milder column temperatures and non-destructive detection conditions [2]. Herein, an analytical method for ergosterol determination in cultivated and wild mushrooms was developed using HPLC coupled to ultraviolet detection. The chromatographic separation was achieved in a Inertsil 100A ODS-3 reverse phase column using an isocratic elution with acetonitrile:methanol (70:30, v:v) at a flow rate of 1 mL/min. Different extraction methodologies were tested, using n-hexane, methanol:dichloromethane (75:25, v:v) or chloroform:methanol (20:10, v:v). After studying the linearity (11 levels) for ergosterol (tR = 13.0±0.1 min; coefficient of variation, CV = 0.65%), a seven-level calibration curve (y = 0.6566ϰ + 0.01098; R2 = 0.9996) was made using the peak/area ratio versus concentration of the standard (in μg/mL). The average of triplicate determinations for each level was used. The limit of detection (LOD), calculated as the concentration corresponding to 3.3 times the standard error of the calibration curve divided by the slope, was 0.3498 μg/mL; the limit of quantification (LOQ), calculated using the concentration corresponding to ten times the standard error of the calibration curve divided by the slope, was 1.060 μg/mL. The precision of the equipment was evaluated injecting seven consecutive times the sterols extract of the same mushroom. The optimized method proved to be precise (CV = 1.25%). Repeatability was assessed by applying the extraction procedure three times to the same dried mushroom powder. The method proved also to be repeatable (CV = 1.88%). The method accuracy was evaluated by the standard addition procedure (percentage of recovery). The standard mixture was added to the samples in three concentration levels (25, 50 and 100 % of the peak/area concentration, each one in triplicate) before the extraction. The method showed good recovery (above 90%). Overall, a reproducible and accurate HPLC-UV technique was fully optimized. Chromatographic signals presented good resolution, indicating the suitability of this methodology to assess sterol profiles in future studies. Moreover, the chosen extraction and saponification steps do not require complex conditions, being suitable for routine analysis.application/pdfporOptimized chromatographic analysis of ergosterol in wild and cultivated mushroomsPersonalBarreira, João C.M.DSpacehttp://dspace.org/items/4629b12c-39b0-4da8-8b8d-6efba5cf2d81DSpacehttp://dspace.org/items/4629b12c-39b0-4da8-8b8d-6efba5cf2d81BarreiraJoão C.M.ORCIDhttp://orcid.org0000-0003-1233-0990Researcher IDhttps://www.researcherid.comD-8269-2013Scopus Author IDhttps://www.scopus.com54895546900PersonalFerreira, Isabel C.F.R.DSpacehttp://dspace.org/items/bd0d1537-2e03-41fb-b27a-140af9c35db8DSpacehttp://dspace.org/items/bd0d1537-2e03-41fb-b27a-140af9c35db8FerreiraIsabel C.F.R.Ciência IDhttps://www.ciencia-id.pt9418-CF95-9919ORCIDhttp://orcid.org0000-0003-4910-4882Researcher IDhttps://www.researcherid.comE-8500-2013Scopus Author IDhttps://www.scopus.com36868826600HostingInstitutionOrganizationalBiblioteca Digital do IPBe-mailmailto:dspace@ipb.ptdspace@ipb.ptISSNIsPartOf978-989-98415-5-02014-03-27T17:44:02Z20132013-01-01T00:00:00ZHandlehttp://hdl.handle.net/10198/9400http://purl.org/coar/access_right/c_abf2open access4671467 bytesFundação para a Ciência e a TecnologiaPortuguese Wild Mushrooms: Chemical characterization and functional study of antiproliferative and proapoptotic properties in cancer cell lines5876-PPCDTICrossref Funder IDhttp://doi.org/10.13039/501100001871Fundação para a Ciência e a TecnologiaStrategic Project - UI 690 - 2011-20126817 - DCRRNI IDCrossref Funder IDhttp://doi.org/10.13039/501100001871Fundação para a Ciência e a TecnologiaBIOACTIVE PROPERTIES & CYTOPROTECTIVE POTENTIAL OF NATURAL EXTRACTS/INDIVIDUAL COMPOUNDS: APPLICATION OF SINGLE CELL GEL ELECTROPHORESIS AND OTHER BIOCHEMICAL, CHEMICAL AND ELECTROCHEMICAL ASSAYSCrossref Funder IDhttp://doi.org/10.13039/501100001871other research producthttp://purl.org/coar/resource_type/c_c94fconference objecthttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://bibliotecadigital.ipb.pt/bitstreams/c76b3fc2-9e59-45e1-bc9b-863e322e1a40/download1st International Symposium on ProfilingCaparica – Portugal |
| spellingShingle | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms Barreira, João C.M. |
| status | SINGLETON |
| title | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| title_full | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| title_fullStr | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| title_full_unstemmed | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| title_short | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| title_sort | Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms |
| url | http://hdl.handle.net/10198/9400 |
| visible | 1 |