# Urinary Levels of Free 2,5-Hexanedione in Italian Subjects Non-Occupationally Exposed to n-Hexane
## 2 Materials and Methods
### Analytical Method  
Each subject provided one spot fresh urine sample (the second micturition of the morning). The samples were collected in sterile polypropylene containers without the addition of preservatives or stabilizers and stored at -20 \({}^{\circ}\)C until analysis, of which was performed within 2 weeks from collection. Blank samples (water) were analyzed to confirm no contamination from the containers. Free \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Parameter** & **Males** & **Females** & **Total** \\ \hline
**Number** & 52 & 47 & 99 \\ \hline
**Smoking habit** & & & \\ No & 32 & 33 & 65 \\ \textless{}5 cigarettes/day & 9 & 6 & 15 \\ 5–10 cigarettes/day & 3 & 5 & 8 \\ 11–20 cigarettes/day & 8 & 3 & 11 \\ \hline
**Residence** & & & \\ Urban & 37 & 37 & 74 \\ Rural & 15 & 9 & 24 \\ Industrial & - & 1 & 1 \\ \hline
**Traffic intensity** & & & \\ Low & 20 & 14 & 34 \\ Medium & 27 & 21 & 48 \\ High & 5 & 12 & 17 \\ \hline
**Age** & & & \\ \textless{}30 years & 16 & 8 & 24 \\ \textless{}50 years & 25 & 30 & 55 \\ \textgreater{}50 years & 11 & 9 & 20 \\ \hline
**BMI** & & & \\ \textless{}25 kg/m\({}^{2}\) & 30 & 35 & 65 \\ \textless{}30 kg/m\({}^{2}\) & 15 & 8 & 23 \\ \textgreater{}30 kg/m\({}^{2}\) & 7 & 4 & 11 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of the studied population. 2,5-HD urinary concentration was determined by gas chromatography coupled with mass spectroscopy (GC/MS) according to the method of [PERSON] et al. with some modifications [32]. Briefly, after addition of a volume of 50 \(\upmu\)l of a solution of cyclohexanone of 23.7 mg/L, as an internal standard, free 2,5-HD was extracted from the urine samples (5 mL) using diatomaceous earth columns and dichloromethane as solvent (volume of 8 mL, twice). In order to maintain the analyte into the solution and avoid its evaporation, p-Isopropyl-toluene (10 \(\upmu\)L) was added to the organic solution as a _keeper_[33]. The organic solutions were evaporated to reduce their volume until about 500 \(\upmu\)L; the concentrated extract was then transferred to autosampler vials. One microliter of the dichloromethane solution was injected into the GC/MS system to the free 2,5-HD determination. All analyses were performed on a Perkin Elmer AutoSystem XL Gas Chromatograph coupled with a Turbo Mass Upgrade quadrupole mass spectrometer. Chromatographic separation of analytes was carried out through an Agilent Technologies PONA capillary column of the following dimensions: length 50 m, internal diameter 0.2 mm and film thickness 0.5 \(\upmu\)m, at a constant helium flow rate of 1.4 mL/min. The GC oven was programmed from an initial temperature of 50 \({}^{\circ}\)C (held for 1 min) to 80 \({}^{\circ}\)C at 10 \({}^{\circ}\)C/min (held for 5 min) and to 200 \({}^{\circ}\)C at 20 \({}^{\circ}\)C/min (held for 5 min) with a transfer line temperature of 250 \({}^{\circ}\)C. The injection temperature was 250 \({}^{\circ}\)C. The MS detector was operated in EI mode (70 eV). Single ions m/z 55 and 99 Da were registered for the quantification (Dwell time 0.05 sec). The 2,5-HD and cyclohexanone retention time were 11.32 and 10.36 min, respectively. Ten replicates of two standards in urine spiked with concentrations 48.65 \(\upmu\)g/L and 389.2 \(\upmu\)g/L of 2,5-HD independent of the calibration standards, were prepared (50 mL each) and 5 mL were tested the same day in order to assess intra-day precision; the remainder was divided into 5 mL aliquots, stored at \(-\)20 \({}^{\circ}\)C and tested on four subsequent days in order to assess the inter-day precision. For the two concentrations 48.65 \(\upmu\)g/L and 389.2 \(\upmu\)g/L, the intra-assay precisions were 2.98% and 2.20% and the inter-assay precisions were 3.07% and 2.06%, respectively. The accuracy was calculated by analyzing ten aliquots of urine, containing 2,5-HD concentrations at the two levels previously described. The relative percentage error of the calibration curve was found to range between 0.69% and 2.77%.