Development and Validation of a DLLME-GC-MS/MS Method for the Determination of Benzotriazole UV Stabilizer UV-327 and its Metabolites in Human Blood.

2-(5-chloro-benzotriazol-2-yl)-4,6-di-(tert-butyl)phenol (UV-327) is used as an ultraviolet (UV) absorber in plastic materials and coatings. To investigate its metabolism and to assess human exposure, analytical methods are necessary for the determination of UV-327 and its metabolites in human biological specimens. The method thus presented targets the determination of UV-327 and several of its predicted metabolites in blood using protein precipitation, dispersive liquid-liquid microextraction (DLLME), and derivatization. The trimethylsilylated analytes and internal standards are separated by gas chromatography and analyzed with tandem mass spectrometry (GC-MS/MS). The DLLME procedure was optimized with respect to the type and volume of disperser and extraction solvents, the pH value of the sample solution, and the addition of salt. During method development, an effective ex vivo lactone/hydroxyl carboxylic acid interconversion was observed for two metabolites, each containing a carboxyl group adjacent to the phenolic hydroxyl group. The analytes resulting from interconversion enabled a more sensitive and reliable determination of the metabolites compared to their native structures. Method validation revealed limits of detection (LODs) between 0.02 and 0.36 µg/l. The mean relative recovery rates ranged from 91 to 118%. Precision and repeatability were demonstrated by relative standard deviations in the range of 0.6-14.2% and 1.1-13.7%, respectively. The presently described procedure enables the sensitive and robust analysis of UV-327 and its metabolites in human blood and allows the elucidation of the human UV-327 metabolism as well as the assessment of exposure in potentially exposed individuals.