Detection of recombinant insulins in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry after immunoaffinity purification based on monolithic microcolumns
| Autor: | Francesco Botrè, Marta Senofonte, Filippo Martinelli, Monica Mazzarino, Xavier de la Torre |
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| Rok vydání: | 2019 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Analyte Electrospray ionization 02 engineering and technology Tandem mass spectrometry 01 natural sciences Biochemistry Chromatography Affinity Analytical Chemistry Limit of Detection Tandem Mass Spectrometry Liquid chromatography–mass spectrometry Humans Insulin Sample preparation Detection limit Chromatography Chemistry 010401 analytical chemistry Selected reaction monitoring Reproducibility of Results 021001 nanoscience & nanotechnology Recombinant Proteins 0104 chemical sciences Triple quadrupole mass spectrometer 0210 nano-technology Chromatography Liquid |
| Zdroj: | Analytical and Bioanalytical Chemistry. 411:8153-8162 |
| ISSN: | 1618-2650 1618-2642 |
| Popis: | This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography–electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02–0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. |
| Databáze: | OpenAIRE |
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