Development and validation of a routine method for the determination of 3-hydroxybenzo[a]pyrene in human urine by GC-MS/MS
Mainstream smoke is a complex mixture containing thousands of unique compounds. Among these are polycyclic aromatic hydrocarbons, which includes benzo[a]pyrene (BaP), a Group 1 carcinogen. 3-Hydroxybenzo[a]pyrene (3OHBaP), a metabolite of BaP, is commonly used as a biomarker of exposure for BaP. Urinary levels of 3OHBaP are expected to be low as this is not the primary route of excretion. Analytical methods developed for the analysis of 3OHBaP must be capable of achieving detection limits appropriate to account for the expected low urinary concentrations.
Here, we report the development and validation of a new method for the analysis of 3-hydroxybenzo[a]pyrene using gas chromatography with triple quadrupole mass spectrometry (GC-MS/MS). During early development, several analytical detection methods were evaluated, including LC-MS/MS and LC-FLR, but these did not achieve the required sensitivity. GC-MS was also evaluated and was sensitive enough for analyte detection, but lacked selectivity from interfering compounds. By using GC-MS/MS, both selectivity and sensitivity were optimized and deemed acceptable for the analysis of 3OHBaP.
Urinary samples were hydrolyzed enzymatically overnight followed by solid phase extraction clean up using a polymeric strong anion exchange column. Sample extracts were derivatized using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and injected on an Agilent 8890 GC with a 7010 MS triple quadrupole (QQQ) mass spectrometer. This method was fully validated and has a LLOQ of 25 pg/mL which equates to 125 fg/mL in sample. Sample extract concentrations were found to vary between non-detected (ND) and 387 pg/mL with an average concentration of 67 pg/mL. Intermediate precision was 8.8 (%RSD) and the average accuracy ranged from 110 % to 122 % across all fortification levels.