ANALYTICAL SCIENCES OCTOBER 2007, VOL.

23 2007 The Japan Society for Analytical Chemistry

1221

Determination of 1-Hydroxypyrene in Human Urine by Acid Hydrolysis Coupled to Solid-Phase Microextraction and Semi-microcolumn Liquid Chromatography
Hong-Wen CHEN
Departmant of Environmental Engineering and Health, Yuanpei University of Science and Technology, 306 Yuanpei Street, Hsinchu 300, Taiwan, R. O. C.

This study developed an acid hydrolysis coupled to a solid-phase microextraction method employing a semi-microcolumn liquid chromatography system, instead of enzyme hydrolysis with solid-phase extraction for the pretreatment of human urine samples, to detect urinary 1-hydroxypyrene (1-OHP). The complete separation and detection of urinary 1-hydroxyprene was performed using a high-performance liquid-chromatography fluorescence detection system with an analytical C18 semi-microcolumn, 60% (v/v) aqueous acetonitrile elution, and a ex/em = 348/388 nm pair detection wavelength. Calibration graphs were linear with very good correlation coefficients (r = 0.9997), and the detection limit was 1.0 ng/L. These important parameters for acid hydrolysis and solid-phase microextraction were investigated. The total recovery was above 83% in acid hydrolysis with solid-phase microextraction. The proposed method provided a relatively simple, convenient, and practical procedure to determine the level of urinary 1-hydroxypyrene in biological samples, and was successfully applied to detect the urine of students. (Received October 10, 2006; Accepted May 31, 2007; Published October 10, 2007)

Introduction
Many classes of chemicals are considered to be carcinogenic compounds. Among these, polycyclic aromatic hydrocarbons (PAHs) are one of the most significant pollutants, based upon the amounts of PAHs produced by the combustion of biogenic or anthropogenic materials.1,2 The International Agency of Research on Cancer (IARC) has listed 11 PAHs as being carcinogenic substances, based on animal studies.2 The most prevalent source of PAHs is the combustion of organic materials.3 Human exposure to PAH is generally caused by airborne particulates, dietary consumption of food prepared by grilling, cigarette smoking, and in occupational exposure, such as in the coke-oven industry.4,5 Biomarkers showing human exposure to PAHs are necessary for the accurate determination of exposure and body burden from the above-mentioned sources. By measuring the metabolites of PAH in urine, shortterm exposure may be evaluated. Urinary 1-hydroxypyrene (1-OHP) is a primary metabolite of pyrene in PAHs. Jongeneelen and many other researchers and IARC have used 1-OHP in their studies as a biological marker of environmental and professional PAHs exposure, because of the representativeness and relevancy of such data.2,6,7 Over the past two decades, an enormous amount of research has gone into the development of methods to precisely and accurately analyze urinary PAH metabolites or 1-OHP. Among these, several methods have been published, which employ conventional liquid chromatography8 or gas chromatography9 with different derivations or sample pretreatment techniques, such as liquidliquid extraction, enzymatic hydrolysis, and E-mail: hwchen@mail.ypu.edu.tw

solid-phase extraction.7,1012 The most widely adopted method for the quantification of urinary 1-OHP was published by Their protocol includes enzymatic Jongeneelen et al.7 hydrolysis of the conjugated metabolites, solid-phase extraction (SPE) with C18 media, separation with high-performance liquid chromatography (HPLC), and detection by fluorescence emission. This method was applied to determine the levels of urinary 1-OHP of PAHs in workers for exposure assessment.1315 Solid-phase microextraction (SPME) is a relatively simple and inexpensive method that uses a coated silica fiber to extract organic compounds from aqueous or gaseous samples. Pawliszyn and his group first invented the SPME technique.16 The SPME method has subsequently been applied to detect trace organic micro-pollutants, such as volatile organic compounds (VOCs), PAHs and biological samples.1719 Moreover, when SPME is used for biological samples, it needs a pre-hydrolysis process. Acid hydrolysis is a rapid hydrolysis method used for biological samples, which have been effectively applied for -cellulose, benzo[a]pyrene tetrol, and DNA-adduct pretreatments in past studies.20,21 The objective of the present study is to pave the way for the development of a fast, simple, and straightforward method for the analysis of urinary 1-OHP. The method uses acid hydrolysis coupled to SPME for a pretreatment extraction of 1-OHP. Acid hydrolysis is a rapid method for hydrolysis of the urinary conjugated form 1-OHP. SPME is a solvent-free, rapid, and inexpensive extraction technique for removing 1-OHP from aqueous urinary matrices. A semi-microcolumn liquid chromatographic technique for extracting the detection sensitivity is used to determine urinary 1-OHP after acid hydrolysis coupled to SPME of the sample. In addition, we applied this method for the routine biological monitoring of 1-OHP in the urine of high school students.

1222

ANALYTICAL SCIENCES OCTOBER 2007, VOL. 23

Experimental
Reagents and chemicals Chemicals used in this study were all of HPLC grade. 1-OHP was obtained from Aldrich (Milwaukee, WI, USA). Hydrochloric acid and sodium hydroxide were obtained from E. Merck (Darmstadt, Germany). 1-Hydroxypyrene glucuronide (1-OHPGluc) and 1-hydroxypyrene sulfate (1-OHP-Sulf) were obtained with Singh study.22 Acetonitrile and 2-propanol were obtained from Fisher (Springfield, MO, USA). Creatinine and -glucuronidase/sulfatase (97000 U) were obtained from Sigma (England). Sodium chloride (NaCl), sodium acetate and sodium chloroacetate were purchased from Fluka (Gallen, Switzerland). Using the Millipore 60 system (Bedford, MA, USA), distilled deionized water (conductivity > 18 M/cm) was used for all aqueous solutions. A stock solution of 1-OHP was prepared in 2-propanol at a concentration of 0.05 mg/ml. All solutions were stored in brown bottles and kept in a refrigerator at 20C. The mobile phase for 1-OHP contained 60% aqueous acetonitrile plus a 40% 0.01 M sodium acetate solution. Acid hydrolysis and solid-phase microextraction For acid hydrolysis, urine samples (50 ml) were placed in amber glass vials with 6 M HCl, 100C, 800 revolutions per minute (rpm) stirred and heated for 1 h. After acid hydrolysis, urine samples were processed by an extraction procedure using SPME for clean up and concentration purposes. The SPME selected in this study were coated with an 85 m polyacrylate (85-PA) commercial coating fiber. The samples were saturated with 1.0 M sodium chloroacetate, and then heated at 100C while being stirred with a magnetic stirrer bar for 25 min. The speed was constant at 800 rpm. The analytes were desorbed from the fiber and then injected into the HPLC system using the commercial SPME-HPLC interface. Apparatus Using a fluorescence detector (FLD) (RF-5000, Kyoto, Japan) as the detector, the high HPLC system was equipped with two HPLC gradient pumps, Models LC-10AD and SCL-10A (Shimadzu, Kyoto, Japan), and a reversed-phased semimicrocolumn (Kaseisorb LC ODS-60-5, 150 2.0 mm i.d., Tokyo, Japan). The temperature of the analytical column was maintained at 30C with a thermostated oven (Model CTO-6A, Shimadzu) at a flow-rate of 0.3 ml/min. The analytical data were recorded on a microprocessor integrator using the Chem Win 1.0 system (Taipei, Taiwan). Creatinine in urine was determined photometrically according to the Honglan methods.23 The SPME apparatus (Model 5-7730) and 85 m polyacrylate fiber were purchased from Supelco (Bellefonte, PA, USA). The concentrated urinary 1-OHP was detected using a fluorescence spectrophotometer. Both the excitation and emission slits were set to 10 nm. The fluorescence wavelengths were set to ex = 348 nm and em = 388 nm for the determinations of urinary 1-OHP. Real samples collection We selected a high school, located within 1 km of Hsinchu Semiconductor Industry Park (HSIP) in Hsinchu, Taiwan. The HSIP has not only played a decisive role in the development of Taiwans economy, but has also established an international reputation in semiconductor and related information industries. One hundred first-year high school students (50 boys and 50 girls) were selected from this school, providing a total of 100 non-smoking high-school students for the study. Spot urine

Fig. 1 Effects of time and temperature on urinary 1-OHP-Gluc and 1-OHP-Sulf on acid hydrolysis. The hydrolysis acidity used was 6 M HCl. The urine pool contained 120 mol/L 1-OHP-Gluc and 1-OHPSulf.

samples were collected between 4:30 5:00 p.m. at the students school. An aliquot was taken from each fresh urine sample for creatinine determination and adjusting the 1-OHP concentration. The urine samples were portioned in 50-ml tubes and kept at 20C until analysis. Calibration and recovery tests The urine samples from these non-smoking students were pooled and served as blank urine. These samples were sequentially spiked to 120 mol/L of free form 1-OHP and conjugated form (1-OHP-Gluc, 1-OHP-Sulf) standards for a recovery study. Aliquots (1 ml) of 3 different standard solutions were added to each blank urine sample. The calibration range was 0.07 51.00 ng/ml. These calibrated and spiked samples were treated and analyzed using the previously described procedure. All spike tests were measured in triplicate. Recovery yields were calculated based on the regression line for standard solutions in the same concentration range.

Results and Discussion

Acid hydrolysis efficiency Acid hydrolysis is often used a pretreatment because it can be adapted to a wide variety of biological samples including cellulose, benzo[a]pyrene tetrol, and DNA-adducts.20,21 In the case of strong hydrochloric acid hydrolysis, it is carried out at evaluated temperatures (60 vs. 100C) for various lengths of time (10 120 min). The results are summarized in a plot for 1OHP-Gluc and 1-OHP-Sulf, as shown in Fig. 1. The hydrolysis time is an important parameter. From an analytical point of view, it is desirable to obtain the maximum hydrolysis efficiency in the minimum time. When the exposure time increases, the equilibrium of the reaction will be reached. The results found that an acid hydrolysis time of 60 min at 100C has a sufficiently high hydrolysis efficiency (90%) for the two major 1-OHP conjugates. Increasing the hydrolysis time from 60 to 120 min at 100C had a negligible effect on the hydrolysis efficiency. No significant difference occurred at 100C in the hydrolysis efficiency compared to a much longer incubation time (120 min) and 60 min. The temperature effect was found to have a positive correlation to the hydrolysis of 1-OHP conjugates. Compared with the 100 and 60C hydrolysis conditions for a 60 min incubation, the hydrolysis efficiency is increased by 1.5 2.0 fold. Moreover, the acidity effect (HCl

ANALYTICAL SCIENCES OCTOBER 2007, VOL. 23

1223

Fig. 2 Acidity effect on the urinary conjugated form 1-OHP acid hydrolysis by HPLC/FLD detection. 1-OHP-Gluc and 1-OHP-Sulf were spiked with 120 mol/L into pooled urine samples.

Fig. 4 Effect of the matrix pH on the detection response of 1-OHP. 1-OHP was spiked with 120 mol/L into pooled urine samples.

Fig. 3 Effect of the ion species and the strength on detection response of 1-OHP. Ionic strength = 1/2CiZi2, Ci is the concentration of i in moles per liter (M) and Zi is the charge on species i. 1-OHP was spiked with 120 mol/L into pooled urine samples.

Fig. 5 Adsorption duration effect of 1-OHP at saturation on SPME fiber, using 85-PA, 1.0 M sodium chloroacetate ionic strength, at 100C, pH 4.0, for 120 mol/L 1-OHP.

concentration) depended on the hydrolysis efficiency, and was evaluated at 0.1 6 M HCl (Fig. 2). First-order kinetics can be observed in Fig. 2. The hydrolysis efficiency increased with the HCl concentration and reached 90% yield at 6.0 M HCl. Ionic strength and pH effect on SPME The ionic strength and pH effect are two important factors in SPME efficiency. The ionic strength has been reported for several analytes, along with enhancements of their absorption on the fiber. A composite effect of the ionic strength in solution has been defined as follows: where Ci is the concentration of i in moles per liter and Zi is the charge on species i. Our past studies indicated that organic sodium chloroacetate can increase the SPME efficiency for an aqueous PAHs pretreatment.18 In the present work, we tried to test inorganic and organic salts with ionic strengths of 0.01 1.0 M. Furthermore, a salting-out test was performed using sodium chloroacetate and sodium acetate, and an organic salts buffer system for SPME was developed. The effect of the ionic strength on the detection of 1-OHP by SPME was checked by comparing fluorescence signals when salt was added to the sample, and when salt was not added to the sample. Figure 3 clearly shows that 1.0 M sodium acetate produces the maximum ionic strength effect on the extraction yield (peak area). One potential reason is that sodium acetate improves the 1-OHP dissociation from an aqueous solution and subsequent adsorption into the SPME fiber. Sodium acetate is expected to have a high activity

coefficient for the ion species of urinary 1-OHP, and thus to improve the 1-OHP dissociation. However, when excessive salt was added to the samples (>1.0 M), a decrease in extraction was found. A study by Pawliszyn16 agreed with this phenomenon and our results. On the other hand, changes in the sample matrix yielded significant differences in the signal intensity of analytes for a varying structure in the SPME method. The effect of matrix pH significantly affects the extraction efficiency by providing better selectivity in the SPME process. Adjusting the optimal pH and buffer system of sample matrixes will change K for a dissociable species and the extraction efficiency.24 The effect of the matrix pH on the extraction efficiency of 1-OHP in urine was examined using different pH buffers including sodium acetate/acetate and sodium chloroacetate/acetate buffer systems. The sodium acetate/acetate buffer system is a better buffer system. Figure 4 indicates that a higher extraction efficiency occurs at the pH 4.0 buffer, which is an optimal point for decreasing at pH 3.0 and pH 5 7 values. The value of pH 4.0 is optimal due to fiber erosion at a higher acidity (< pH 4.0). Time profile on SPME Figure 5 shows the adsorption duration test of free-type 1-OHP at saturation on an SPME fiber, using 85 m polyacrylate fiber to adsorb in a solution of 1.0 M sodium chloroacetate ionic strength, at 100C, pH 4.0, for 120 mol/L 1-OHP, with the process repeated six times. According to the theory of SPME, the extent of the saturation for the analyte adsorption of the fiber

1224

ANALYTICAL SCIENCES OCTOBER 2007, VOL. 23

Table 1 Pretreatment characteristics of urinary 1-OHP using two methods (n = 10) Acid SPME hydrolysis Enzymatic hydrolysis SPE

Fig. 6 Desorption of solvent and time profiles for 1-OHP. 1-OHP was spiked with 120 mol/L into pooled urine samples.

Hydrolysis time/min 60 120 Hydrolysis efficiency, % 1-OHP-Gluc 95.2 2.1 95.3 3.1 1-OHP-Sulf 91.5 4.7 90.0 6.8 Extraction time/min 25 60 Extraction efficiency, % 1-OHP-Gluc 90.7 2.7 88.0 5.7 1-OHP-Sulf 90.4 3.8 87.6 4.9 1-OHP 92.6 4.2 88.9 7.2 Total recovery, % 83 88 79 85 1-OHP-Gluc, 1-OHP-Sulf and 1-OHP were spiked with 120 mol/L into pooled urine samples.

The optimal conditions of the system were obtained as the following program: 0.3 ml/min eluent with 60% (v/v) aqueous acetonitrile plus a 40% 0.01 M sodium acetate solution for 7 min; column oven temperature, 30C; ex/em = 348/388 nm pair wavelength on a fluorescence detector. Figures 7(A) and (B) are a pair of standard chromatograms that were sequentially obtained by using a universal use conventional column (4.6 mm i.d.) and semi-microcolumn (2.0 mm i.d.). Comparison of the two methods revealed that the semi-microcolumn method significantly provided a 2.0-fold sensitivity in the detection of urinary 1-OHP. Efficiency of pretreatment and clean-up The metabolites of 1-OHP can be free type or conjugated type. In his toxicokinetic study of pyrene exposure, Namdari25 found free and conjugated-type urinary 1-OHP in fish, at 46 and 54%, respectively. Bouchard26 in his study of urinary excretion kinetics of pyrene exposure found conjugated-type urinary 1-OHP in rat, at around 35%. It can not be ignored that conjugated-type urinary 1-OHP becomes noticeably higher with pyrene exposure. This study aims to reduce the conjugated type 1-OHP to the free type by means of acid hydrolysis before proceeding with measuring the total 1-OHP, so that the measurement obtained can be more representative. Acid hydrolysis with SPME and enzymatic hydrolysis with SPE were compared by using 1 ml of standard materials of a 120 mol/L mixture of 1-OHP-Gluc, 1-OHP-Sulf and 1-OHP, spiked into a blank urine specimen. A consistent trend of high efficiency (> 90%) was obtained in two hydrolysis methods (Table 1). The efficiency of the latter extraction process in SPME was slightly higher than in SPE. The extraction efficiencies were 90.7, 90.4, and 92.6% for 1-OHP-Gluc, 1-OHP-Sulf and 1-OHP, respectively. The total pretreatment efficiency of acid hydrolysis with SPME ranged over 83 88%. It is similar to the efficiency of enzymatic hydrolysis with SPE. However, the advantage of acid hydrolysis with SPME saves even more time than enzymatic hydrolysis with SPE in a whole extraction procedure. In addition, acid hydrolysis with SPME provides a greater clean-up effect in removing impurity compounds. Figures 7(C) and (D) showed the respective HPLC chromatograms of authentic samples using the acid hydrolysis with SPME and enzymatic hydrolysis with SPE. Under the already mentioned conditions, the sample chromatogram of acid hydrolysis with SPME was clear with no residue present, and had a good resolution and a steady baseline. However, the other chromatogram of enzymatic hydrolysis with SPE displayed fluctuations in the baseline with obvious interferences by other species. This proposed method, which is

Fig. 7 HPLC-FLD chromatograms of standard samples and authentic samples. (A) Chromatogram obtained by using the 4.6 mm (i.d.) C18 analytical column and 0.63 ng/ml 1-OHP solution; (B) chromatogram obtained by using the 2.0 mm (i.d.) C18 semimicrocolumn; (C) chromatogram obtained by using acid hydrolysis SPME and semi-microcolumn HPLC analysis (0.18 ng/ml 1-OHP); (D) chromatogram obtained by using enzymatic hydrolysis SPE and semi-microcolumn HPLC analysis in the same urine sample.

will affect the extraction rate of the pretreatment. The experiment of this study compared the results of adsorption with 25 and 50 min duration, but found no significant difference, indicating that the adsorption balance was reached at 25 min duration. The 1-OHP desorption profiles were obtained by evaluating different desorption times (2 30 min) when the fiber was desorbed with four solvents (acetonitrile, methanol, diethyl ether, and 2-propanol). As can be seen in Fig. 6, the desorption efficiency was higher in acetonitrile than in the other three solvents. Five minutes were long enough to guarantee 1-OHP desorption, and reach 92.3%. The fiber was immediately desorbed again at the same conditions to determine carry-over; no peak appeared in the resulting HPLC chromatogram, confirming that PAHs were completely removed and acetonitrile was a good desorption solvent. Chromatographic conditions and column effect Semi-microcolumn HPLC has many advantages over conventional column HPLC such as higher sensitivity and reduced mobile-phase consumption. The semi-microcolumn is used to determine urinary 1-OHP, because it reduces the amount of eluent and simultaneously improves the analytical sensitivity.

ANALYTICAL SCIENCES OCTOBER 2007, VOL. 23

better selected for the analysis of 1-OHP, is a time-efficient, sensitive, cleaned-up, and reliable process. Linearity, detection limit, and performance The linearity and detection limits were evaluated in order to assess the performance of this method. The linearity of the method was established by spiking blank urine samples with 1-OHP 0.07 51 ng/ml (n = 7). Calibration graphs were linear with very good correlation coefficients (r = 0.9997), and the precision of the assay (RSD) was below 5%. According to IUPAC,27 the limit of detection (LOD) is defined as the smallest concentration or amount of an analyte that can be reliably shown to be present or measured under defined condition, and the limit of detection is the lowest concentration level that can be determined to be statistically different from a blank (99% confidence). The LOD is typically determined to be in the region where the signal-tonoise ratio is greater than 3, and LODs are matrix, method, and analyte specific, while LOQ is set at a higher concentration than the LOD. In the statistical method, it is 10 SD above the mean blank value, thus presenting a greater probability that a value at the LOQ is real, and not just a random fluctuation of the blank reading. In our study, the LOD and LOQ of 1-OHP were measured at 1.0 and 4.0 ng/L, respectively. Field determination The present method was successfully applied to analyze the urine of high-school students. The mean of urinary 1-OHP for students living near the Semiconductor Industry Park was 176.8 and 165.2 (ng/g creatinine) for boys and girls. No significant differences (P > 0.05) of 1-OHP levels were found between male and female students. A comparison of the present results (total mean, 171 ng/g creatinine) to those of other published studies revealed that the urinary 1-OHP concentrations of students were higher than those of the Nantou rural area (110.2 ng/g creatinine, Taiwan) and Cheongiu (102.9 ng/g creatinine, South Korea) but were lower than those of Cophenhagen (230.6 ng/g creatinine, Denmark).12,28,29 This inferred that the emission of PAHs from the Semiconductor Industry product was the major contributor, and higher than other regional pollutants. Nevertheless, long-time monitoring should continue in these areas in order to insure the health of residents.

1225
18, 1617. 2. Internal Agency for Research on Cancer (IARC), Polycyclic Aromatic Compounds: Part 1. Chemical, Environmental and Experimental Data, 1986, Vol. 38. 3. J. P. Buchet, M. Ferreria, J. B. Burrion, T. Leroy, M. K. Volders, P. V. Hummelen, J. Jacques, L. Cupers, J. P. Delavignette, and R. Lauwery, Am. J. Ind. Med., 1995, 27, 523. 4. P. Strickland and D. Kang, Toxicol. Lett., 1999, 108, 191. 5. W. P. Tools, P. B. Show, L. K. Lowry, B. A. Mackenzie, J. F. Deng, and H. L. Markel, Appl. Occup. Environ. Hyg., 1990, 5, 303. 6. B. E. Moen, R. Nilsson, R. Nordlineder, K. Bleie, and A. H. Skorve, Occup. Environ. Med., 1996, 53, 692. 7. F. J. Jongeneelen, R. B. M. Anzion, and P. T. Henderson, J. Chromatogr., B, 1987, 413, 227. 8. R. S. Whiton, C. L. Witherspoon, and T. J. Buckley, J. Chromatogr., B, 1995, 665, 390. 9. C. T. Smith, C. J. Walcott, W. L. Hung, V. Maggio, J. Grainger, and D. G. Patterson, J. Chromatogr., B, 2002, 778, 157. 10. J. Jacob and G. Grimmer, Rev. Anal. Chem., 1987, 9, 49. 11. G. Grimmer, J. Jacob, G. Dettbarn, and K. W. Naujack, Int. Arch. Occup. Environ. Health, 1997, 69, 231. 12. C. T. Kuo, H. W. Chen, and J. L. Chen, J. Chromatogr., B, 2004, 805, 187. 13. D. Frdric, J. M. Haguenoer, D. Zmirou, P. EmpereurBissonnet, F. J. Jongeneelen, V. Nedellec, A. Person, C. Ferguson, and W. Dab, J. Occup. Environ. Med., 2000, 42, 391. 14. B. E. Moen and S. vreb, J. Occup. Environ. Med., 1997, 39, 515. 15. E. Siwinska, D. Mielzynska, E. Smolik, A. Bubak, and J. Kwapulinski, Sci. Total Environ., 1998, 217, 175. 16. J. Pawliszyn, Solid-phase Microextraction Theory and Practice, 1997, Wiley, New York. 17. Y. F. Sha, T. M. Huang, S. Shen, and G. L. Duan, Anal. Sci., 2004, 20, 857. 18. H. W. Chen, Anal. Sci., 2004, 20, 1383. 19. G. Gmeiner, C. Krassnig, E. Schmid, and H. Tausch, J. Chromatogr., B, 1998, 705, 132. 20. G. A. Islaam, T. Greibrokk, R. G. Harvey, and S. vrb, Chem. Biol. Interact., 1999, 123, 133. 21. Q. Xiang, Y. Y. Lee, P. O. Pettersson, and R. W. Torget, Appl. Biochem. Biotechnol., 2003, 105, 505. 22. R. Singh, M. Trbek, K. Maxa, T. Jana, and E. H. Weyand, Carcinogen, 1995, 16, 2909. 23. S. Honglan, M. Yongqin, and M. Yinfa, Anal. Chim. Acta, 1995, 312, 79. 24. X. Yang and T. Peppard, LC-GC, 1995, 882, 13. 25. R. Namdari, Ph. D Thesis, Simon Frase University, 1998, B. C., Canada. 26. M. Bouchard, R. Thuot, G. Carrier, and C. Viau, J. Toxicol. Environ. Health, 2002, 65, 1195. 27. IUPAC Compendium of Chemical Terminology, 2nd ed., 1997. 28. J. W. Kang, S. H. Cho, H. Kim, and C. H. Lee, Arch. Environ. Health, 2002, 57, 377. 29. A. M. Hansen. O. Raaschou-Nielsen, and L. E. Knudsen, Sci. Total Environ., 2005, 347, 98.

Conclusions
We have developed an effective extraction process for the detection of 1-OHP in a urinary sample by acid hydrolysis SPME pretreatment coupled with semi-microcolumn HPLC. The method can be successfully applied in real urine samples. It has some advantages, including being solventless, saving time, better sensitivity and recovery. The LOD and LOQ of 1-OHP were measured at 1.0 and 4.0 ng/L, respectively. The recovery yield from acid hydrolysisSPME was above 83%. The proposed system is practical and reliable, and is particularly attractive for routine analysis. This method is sensitive enough to determine urinary 1-OHP of environmental PAHs exposure.

References
1. M. N. Kayali and S. R. Barroso, J. Liq. Chromatogr., 1995,