Comparison between external and internal standard calibration in the validation of an analytical method for 1-hydroxypyrene in human urine by high-performance liquid chromatography/tandem mass spectrometry

1-Hydroxypyrene is a metabolite of pyrene, a member of the class of polycyclic aromatic hydrocarbons (PAHs) whose toxic properties in some cases include carcinogenicity. The determination of 1-hydroxypyrene in human urine is used as a biological indicator for exposure to PAHs, which is related to the combustion of organic materials, like smoking, living in urban environments, and eating grilled or smoked food. The determination of 1-hydroxypyrene by high-performance liquid chromatography (HPLC) with fluorescence detection has very good sensitivity but it is not highly specific: this can reduce accuracy in the quantitative determination of low levels of analyte in a complex matrix like urine. An HPLC method that uses triple quadrupole mass detection has been validated with the objective both to improve the signal-to-noise (S/N) ratio and to achieve the maximum specificity for the analyte in those urine samples that are richer in possible inteferents. The calibration range for 1-hydroxypyrene is from 0.005-0.1 microg/L in the urine of non-smoking healthy volunteers. After solid-phase extraction, samples were analyzed by HPLC/tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode. In order to obtain reliable results quantitative analysis must be performed by means of the internal standard method (we used deuterium-labelled 1-hydroxypyrene): the method accuracy is not less than 85%. The S/N ratio at a concentration of 0.1 microg/L is about 10, and therefore this can be considered the lowest limit of quantitation. The method performance does not change if urine samples are measured using a calibration curve prepared in methanol, thus reducing the time of analysis and costs.