Reverse phase high performance liquid chromatographic method development based on ultravioletvisible detector for the analysis of 1-hydroxypyrene (PAH biomarker) in human urine

Abstract

Objectives: 1-hydroxypyrene is an important biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs), which appears in the urine of exposed human subjects. In developing countries, where advanced instruments are not available, the importance of this biomarker demands convenient and sensitive methods for determination purposes. This study aimed at developing a methodology to quantify 1-hydroxypyrene (a biomarker of PAHs exposure) based on the UV-visible detector in the reverse phase high pressure liquid chromatography (HPLC). Material and Methods: A 20 μl injection of sample was used for manual injection into the HPLC Shimadzu, equipped with the SPD-20 A UV-visible detector, the LC-20AT pump and the DGU-20A5 degasser. The C-18 column was used for the purpose of the analysis. Results: The method showed a good linearity (the range: R 2 = 0.979-0.989), and high detectability up to the nmol level. The average retention was 6.37, with the accuracy of 2%, and the percentage of recovery remained 108%. The overall performance of this method was comparable (in terms of detection sensitivity) and relatively better than previously reported studies using the HPLC system equipped with the UV-detector. Conclusions: This method is suitable and reliable for the detection/quantification of the 1-OHP in human urine samples, using the UV-detector, however, it is less sensitive as compared to the results of a florescence detector. with 5% HNO 3 , including subsequent washing with distilled water, and oven drying. The standards were prepared in the HPLC-grade acetonitrile (mmol/l). Serial dilutions of standards were carried out using the stock of 1 mmol/l in strength. The stock standard was diluted up to nmol/l. All the dilutions were prepared afresh each day before starting the calibration procedure. The stock standards were kept in a refrigerator in sterile glass vials to avoid solvent evaporation and contamination. Analytical proceduresAll the samples were filtered through the 0.02 μl syringe filters. A 20 μl injection of sample was used for manual injection into the HPLC Shimadzu equipped with the SPD-20 A UV-visible detector, the LC-20AT pump and the DGU-20A5 degasser. The C-18 column was used for the analysis purposes. HPLC optimization for the 1-OHPyr analysisThe HPLC conditions were optimized for the 1-OHPyr. The optimization was carried out by running a series of standard dilutions for a number of times, and the conditions were observed for 3 consecutive days to validate optimization. The optimum conditions for the 1-OHPyr were as follows: 20 μl injection, 250 bars pressure, a flow rate of 0.8 ml/minute, with a run time of 10 min, which was later reduced to 8 min. The mobile phase consisted of acetonitrile and the HPLC gradient grade water of 70:30. The column temperature was set at 30°C. Wavelength was set at 250 nm, and the average elution time was 6.371 min (Table 1). RESULTS AND DISCUSSIONThe analysis of the 1-OHPyr was carried using the UVvisible detector. In published literature studies, the florescence...

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