Development and validation of a method for the detection and confirmation of biomarkers of exposure in human urine by means of restricted access material-liquid chromatography–tandem mass spectrometry

Abstract

The present article describes the development and validation of a LC–MS/MS method for the determination and confirmation of biomarkers of exposure to different types of xenobiotics in human urine. The method combines the use of a restricted access material (RAM) coupled on-line to a LC–IT-MS system; in this way, a rapid and efficient matrix cleanup was achieved, reducing manual sample preparation to freezing and sample filtration. The ion trap (IT) mass spectrometry detector provided the selectivity, sensitivity and ruggedness needed for confirmatory purposes. The on-line RAM-LC–MS/MS method developed here has been validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The validation steps included the verification of linearity, repeatability, specificity, trueness/recovery, reproducibility, stability and ruggedness in fortified urine samples. Repeatability and within-laboratory reproducibility, measured as intraday and interday precisions, were evaluated at two concentration levels, being 12.7% or below at the concentration corresponding to the quantification limits. Matrix effects and non-targeted qualitative analyses were also evaluated in fortified urine samples. Decision limits (CC α) and detection capabilities (CC β) were in the range of 3.6–16.5 and 6.0–28.1 ng mL −1 respectively. The results of the validation process revealed that the proposed method is suitable for reliable quantification and confirmation of biomarkers of exposure to xenobiotics in human urine at low ng mL −1 levels. In addition, working in Data-Dependent Scan mode the proposed method can be used for the screening of these compounds in urine samples.