Development and validation of a HPLC method for the determination of benzo(a)pyrene in human breast milk

A simple analytical procedure was developed for the quantitation of benzo(a)pyrene in human breast milk using solid phase extraction (SPE) combined with high performance liquid chromatography. Before the chromatographic process, SPE, including C18 functional groups in silicagel cartridges, was conducted for sample preparation. A C18 column (100×4.6 mm id, 3 μm particle size) was used with acetonitrile:water (80:20) as the mobile phase at a flow rate 1mL/min at 30°C. Fluorimetric detection was performed for excitation and emission at 290 and 406 nm, respectively. It was observed that the calibration curve was linear over the range of 0.5-80 ng/mL. The limit of detection and limit of quantitation were found to be 0.5 and 1.07 ng/mL, respectively. Intraday and interday relative standard deviation values were less than 5.15%. Moreover, the newly developed method provides a fast, simple, cost effective, and sensitive assay to detect an important carcinogen substance, benzo(a)pyrene, in human breast milk.