A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS
Abstract A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of cotinine and its metabolite, trans-3´-hydroxycotinine, in human serum. Liquid–liquid extraction (LLE) using dichloromethane (DCM) and eth...
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SpringerOpen
2025-05-01
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| Series: | Journal of Analytical Science and Technology |
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| Online Access: | https://doi.org/10.1186/s40543-025-00488-y |
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| author | Xiujiao Joanne Ye Amy M. MacDonald Melissa J. Bennett David W. Kinniburgh |
| author_facet | Xiujiao Joanne Ye Amy M. MacDonald Melissa J. Bennett David W. Kinniburgh |
| author_sort | Xiujiao Joanne Ye |
| collection | DOAJ |
| description | Abstract A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of cotinine and its metabolite, trans-3´-hydroxycotinine, in human serum. Liquid–liquid extraction (LLE) using dichloromethane (DCM) and ethyl acetate (ETAC) (4:1 v/v) was employed for serum sample extractions. Chromatographic separation was achieved on an Agilent Eclipse Plus Phenyl-Hexyl column (4.6 mm × 100 mm, 5 µm) with a gradient elution of 0.05% (v/v) formic acid/5 mM ammonium formate (1:1) in water and methanol at an initial flow rate of 1 mL/min. Tandem mass spectrometry detection used positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode for the determination of cotinine, trans-3´-hydroxycotinine, and their deuterated internal standards. The limits of detection (LOD) for cotinine and trans-3´-hydroxycotinine were 0.005 and 0.006 ng/mL, respectively. The limit of quantification (LOQ) was 0.025 ng/mL for both analytes. The accuracy ranged from 96.1 to 104.9% for cotinine and 98.9 to 102.9% for trans-3´-hydroxycotinine. The linear range for both metabolites was 0.025–10 ng/mL. Extraction recovery ranged from 100.0 to 106.4% for cotinine and 89.6 to 102.5% for trans-3´-hydroxycotinine, respectively. The intra- and interday precision (% CV) ranged from 0.9 to 8.0% for cotinine and 1 to 9.6% for trans-3´-hydroxycotinine. Neither carryover nor endogenous or exogenous interference was observed. The method was successfully applied to pooled serum samples from a biomonitoring study of pregnant women in Alberta, Canada. |
| format | Article |
| id | doaj-art-ea331dadff5444b6b707bd9d2882b921 |
| institution | DOAJ |
| issn | 2093-3371 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | SpringerOpen |
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| series | Journal of Analytical Science and Technology |
| spelling | doaj-art-ea331dadff5444b6b707bd9d2882b9212025-08-20T03:09:19ZengSpringerOpenJournal of Analytical Science and Technology2093-33712025-05-0116111610.1186/s40543-025-00488-yA simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MSXiujiao Joanne Ye0Amy M. MacDonald1Melissa J. Bennett2David W. Kinniburgh3Alberta Centre for Toxicology, University of CalgaryAlberta Centre for Toxicology, University of CalgaryAlberta Centre for Toxicology, University of CalgaryAlberta Centre for Toxicology, University of CalgaryAbstract A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of cotinine and its metabolite, trans-3´-hydroxycotinine, in human serum. Liquid–liquid extraction (LLE) using dichloromethane (DCM) and ethyl acetate (ETAC) (4:1 v/v) was employed for serum sample extractions. Chromatographic separation was achieved on an Agilent Eclipse Plus Phenyl-Hexyl column (4.6 mm × 100 mm, 5 µm) with a gradient elution of 0.05% (v/v) formic acid/5 mM ammonium formate (1:1) in water and methanol at an initial flow rate of 1 mL/min. Tandem mass spectrometry detection used positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode for the determination of cotinine, trans-3´-hydroxycotinine, and their deuterated internal standards. The limits of detection (LOD) for cotinine and trans-3´-hydroxycotinine were 0.005 and 0.006 ng/mL, respectively. The limit of quantification (LOQ) was 0.025 ng/mL for both analytes. The accuracy ranged from 96.1 to 104.9% for cotinine and 98.9 to 102.9% for trans-3´-hydroxycotinine. The linear range for both metabolites was 0.025–10 ng/mL. Extraction recovery ranged from 100.0 to 106.4% for cotinine and 89.6 to 102.5% for trans-3´-hydroxycotinine, respectively. The intra- and interday precision (% CV) ranged from 0.9 to 8.0% for cotinine and 1 to 9.6% for trans-3´-hydroxycotinine. Neither carryover nor endogenous or exogenous interference was observed. The method was successfully applied to pooled serum samples from a biomonitoring study of pregnant women in Alberta, Canada.https://doi.org/10.1186/s40543-025-00488-yCotinineTrans-3´-hydroxycotinineLC-MS/MSHuman serumBiomonitoring |
| spellingShingle | Xiujiao Joanne Ye Amy M. MacDonald Melissa J. Bennett David W. Kinniburgh A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS Journal of Analytical Science and Technology Cotinine Trans-3´-hydroxycotinine LC-MS/MS Human serum Biomonitoring |
| title | A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS |
| title_full | A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS |
| title_fullStr | A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS |
| title_full_unstemmed | A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS |
| title_short | A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS |
| title_sort | simple sensitive method for the simultaneous determination of cotinine and trans 3´ hydroxycotinine in human serum by lc ms ms |
| topic | Cotinine Trans-3´-hydroxycotinine LC-MS/MS Human serum Biomonitoring |
| url | https://doi.org/10.1186/s40543-025-00488-y |
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