Journal of Chromatography B, 723 (1999) 95–104 Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene a, a a a * Marina Buratti , Oronzo Pellegrino , Carla Valla , Silvia Fustinoni , b b Gabriella Brambilla , Antonio Colombi a Istituti Clinici di Perfezionamento, Laboratorio di Tossicologia Professionale, Via Commenda 10, 20122 Milan, Italy b University of Milan, Faculty of Medicine, Institute of Occupational Health, Via S. Barnaba 8, 20112 Milan, Italy Received 21 July 1998; received in revised form 16 October 1998; accepted 16 October 1998 Abstract Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 mm film thickness, b 5320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 1628C, for 14.2 min; ramp by 308C/min to 1908C, for 3.5 min; ramp by 308C/min to 2508C, for 4 min; helium flow rate: 1.7 ml / min; detector and injector temperature: 3008C. The sample (1 ml) was injected with a split injection technique (split ratio 5:1). MHA recovery was .95% in the 0.5–20 mmol / l range; the limit of detection was ,0.25 mmol / l; day-to-day precision, at 2 mmol/l, was Cv,10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban 3 air pollution (airborne xylene ranging from 0.1 to 3.7 mmol/m ). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 mmol/l vs. 8.1 mmol/l, p,0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 mmol/l vs. 6.9 mmol/l, p,0.001), and evidenced a 3 relationship between MHAs ( y, mmol / mmol creatinine) and airborne xylene ( x, mmol/m ) ( y50.08510.34x; r50.82, p,0.001, n556). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.  1999 Elsevier Science B.V. All rights reserved. Keywords: Xylene; Methylhippuric acid 1. Introduction is blended into petrol, the remainder is used in a variety of solvent applications, particularly in the Xylene is an aromatic hydrocarbon which exists in paint and printing ink industries. Recently xylene three isomeric forms: ortho-, meta- and para-. became a ubiquitous environmental pollutant, mainly Technical grade xylene contains a mixture of the diffused in urban air because of motor vehicle three isomers. Approximately 92% of mixed xylenes emissions. It is also present in tobacco smoke, being generated by tobacco pyrolysis processes [1]. In *Corresponding author. humans main metabolic pathway of xylene detoxifi- 0378-4347 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0378-4347(98)00482-4