The urine specimens obtained before and after each experimental trial were analyzed for caffeine, paraxanthine, theobromine and theophylline concentrations using an Agilent Technologies HPLC 1200 system (Santa Clara, CA, US) coupled to a triple quadrupole/ion trap mass spectrometer (MS, API 400, US). All the reagents used for these measurements were purchased from Cambridge Isotope Laboratories (Spain). For this measurement, 20 μL of the internal standard theophylline-D6 (2 μg/mL) and 20 μL of the internal standard 13C3 –caffeine (5 μg/mL) were added to 100 μL of urine. A volume of 900 μL of mobile phase (acetic acid 0.1 %) was added to the urine sample and 5 μL of this sample wAS then directly applied to the HPLC–MS system. To calibrate the system, aqueous solutions of caffeine, paraxanthineand theobromine (ranging from 0.1 to 7 μg/mL) and theophylline (from 0.04 to 1 μg/mL) were used before each batch of samples. The correlation coefficients for the calibration of caffeine and its main metabolites were always >0.99. The lower limit for the accurate quantization of these methylxanthines was 0.25 and 0.1 μg/mL for theophylline.
The urine specimens obtained before and after each experimental trial were analyzed for caffeine, paraxanthine, theobromine and theophylline concentrations using an Agilent Technologies HPLC 1200 system (Santa Clara, CA, US) coupled to a triple quadrupole/ion trap mass spectrometer (MS, API 400, US). All the reagents used for these measurements were purchased from Cambridge Isotope Laboratories (Spain). For this measurement, 20 μL of the internal standard theophylline-D6 (2 μg/mL) and 20 μL of the internal standard 13C3 –caffeine (5 μg/mL) were added to 100 μL of urine. A volume of 900 μL of mobile phase (acetic acid 0.1 %) was added to the urine sample and 5 μL of this sample wAS then directly applied to the HPLC–MS system. To calibrate the system, aqueous solutions of caffeine, paraxanthineand theobromine (ranging from 0.1 to 7 μg/mL) and theophylline (from 0.04 to 1 μg/mL) were used before each batch of samples. The correlation coefficients for the calibration of caffeine and its main metabolites were always >0.99. The lower limit for the accurate quantization of these methylxanthines was 0.25 and 0.1 μg/mL for theophylline.
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