Publicação
Validação de um método analítico para a determinação de antidepressivos em amostras de sangue por LC-MS/MS
| Resumo: | The consumption of antidepressants (ADs) in the last decade has been increasing and, therefore, the request for their detection in biological samples in the Forensic Toxicology and Chemistry Service of the National Institute of Legal Medicine and Forensic Sciences is also increasing. For this reason, it is necessary to have a continuous scientific evolution of detection techniques and their validation in order to keep up with this growth. This work aimed to validate an analytical method for the simultaneous determination of 20 ADs (amitriptyline, bupropion, citalopram, clomipramine, dosulepin, duloxetine, fluoxetine, fluvoxamine, imipramine, maprotiline, mianserin, mirtazapine, paroxetine, reboxetine, sertraline, tianeptine, trazodone, trimipramine, venlafaxine and vortioxetine) and 11 of their metabolites. The analytical procedure for the determination of ADs in blood samples (100 μL) involved a protein precipitation (PP) and its subsequent analysis by liquid chromatography coupled to sequential mass spectrometry (LC-MS/MS) using electrospray ionization (ESI) as a source of ionization and detection by multiple reaction monitoring (MRM). In the chromatographic separation, an Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 μm) was used and the mobile phase consisted of an aqueous solution of 2 mM ammonium formate (with 0.1 % formic acid) and methanol, in gradient mode, with a flow rate of 0.4 mL/min and an injection volume of 10 μL. The method was validated according to the Standard Practices for Method Validation in Forensic Toxicology by the American Academy of Forensic Science (AAFS) Standards Board (ASB) guidelines. Validation parameters studied included: study of interferences, ion suppresion, linearity, carryover, precision and accuracy, limits of detection and quantification, dilution integrity and stability. The method demonstrated the absence of interfering substances from other substances and from the internal standard, with the exception of maprotiline and nortriptyline, which proved to interfere with each other, particularly in the second ionic transition. The range of work chosen proved to be linear, considering a weighting factor of 1/x, always with a correlation coefficient greater than 0.99. No relevant carryover nor matrix effect was detected. The detection and quantification limits obtained are adequate taking into account the range of work studied. In the study of precision (intermediate precision and repeatability) and accuracy, the results obtained from the correlation coefficient (CV) were <20.0 %. For these substances, it was verified stability up to 72 h at room temperature and 1 week after a freezing process. The dilution integrity study demonstrated precision and accuracy at 1:5 (v/v) dilution. The analytical method described met all the necessary parameters according to the guidelines followed, so it can be concluded that it has been validated and allows the simultaneous determination and quantification of 20 ADs and 11 of its metabolites in blood samples by LC-MS/MS. |
|---|---|
| Autores principais: | Coelho, Maria João Vieira |
| Assunto: | Antidepressants LC-MS/MS Protein precipitation Blood Validation Antidepressivos LC-MS/MS Precipitação proteica Sangue Validação |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | português |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | The consumption of antidepressants (ADs) in the last decade has been increasing and, therefore, the request for their detection in biological samples in the Forensic Toxicology and Chemistry Service of the National Institute of Legal Medicine and Forensic Sciences is also increasing. For this reason, it is necessary to have a continuous scientific evolution of detection techniques and their validation in order to keep up with this growth. This work aimed to validate an analytical method for the simultaneous determination of 20 ADs (amitriptyline, bupropion, citalopram, clomipramine, dosulepin, duloxetine, fluoxetine, fluvoxamine, imipramine, maprotiline, mianserin, mirtazapine, paroxetine, reboxetine, sertraline, tianeptine, trazodone, trimipramine, venlafaxine and vortioxetine) and 11 of their metabolites. The analytical procedure for the determination of ADs in blood samples (100 μL) involved a protein precipitation (PP) and its subsequent analysis by liquid chromatography coupled to sequential mass spectrometry (LC-MS/MS) using electrospray ionization (ESI) as a source of ionization and detection by multiple reaction monitoring (MRM). In the chromatographic separation, an Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 μm) was used and the mobile phase consisted of an aqueous solution of 2 mM ammonium formate (with 0.1 % formic acid) and methanol, in gradient mode, with a flow rate of 0.4 mL/min and an injection volume of 10 μL. The method was validated according to the Standard Practices for Method Validation in Forensic Toxicology by the American Academy of Forensic Science (AAFS) Standards Board (ASB) guidelines. Validation parameters studied included: study of interferences, ion suppresion, linearity, carryover, precision and accuracy, limits of detection and quantification, dilution integrity and stability. The method demonstrated the absence of interfering substances from other substances and from the internal standard, with the exception of maprotiline and nortriptyline, which proved to interfere with each other, particularly in the second ionic transition. The range of work chosen proved to be linear, considering a weighting factor of 1/x, always with a correlation coefficient greater than 0.99. No relevant carryover nor matrix effect was detected. The detection and quantification limits obtained are adequate taking into account the range of work studied. In the study of precision (intermediate precision and repeatability) and accuracy, the results obtained from the correlation coefficient (CV) were <20.0 %. For these substances, it was verified stability up to 72 h at room temperature and 1 week after a freezing process. The dilution integrity study demonstrated precision and accuracy at 1:5 (v/v) dilution. The analytical method described met all the necessary parameters according to the guidelines followed, so it can be concluded that it has been validated and allows the simultaneous determination and quantification of 20 ADs and 11 of its metabolites in blood samples by LC-MS/MS. |
|---|
Atividades financiadas
Não foram encontrados projetos financiados.