# Low-Dose Benzene Exposure Monitoring of Oil Refinery Workers: Inhalation and Biomarkers
## 2 Materials and Methods
U-B analysis was performed by headspace (HS) Solid Phase MicroExtraction (SPME) and was analyzed with a Shimadzu QP2010 GC-MS in Selective Ion Monitoring (SIM) mode, equipped with an autosampler with a Multi-Fiber eXchange (MFX) [24]. Extraction was performed via a 20-mL vial, with an added 2 g of NaCl, and filled with an 8 mL urine aliquot and 15 \(\upmu\)L of fluorotoluene (5 \(\upmu\)g/mL, Merck KGaA); it was kept in temperature-controlled agitation at 60 \({}^{\circ}\)C for 20 min. Subsequently, a Fast Fit Assemblies (FFA) Carbosen/PDMS SPME fiber (Merck KgaA) was immersed for 20 min in HS and then desorbed in the GC injector. Separation was performed through a Varian Factor FOUR VF-5 MS with a 10 m EZ-GUARD (J&W Agilent Technologies). U-B was quantified by the \(m/z\) 78 ion. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Monitoring Type** & **Analytes** & **Injection** & **Instrument** & **Autosampler** & **Column** & **Oven Temperature** \\ \hline \multirow{4}{*}{Air Monitoring} & Benzene n-hexane split/splits 30:1 & Shimadzu QP2010 & AOC-5000 Plus with Multi Tools & \ \begin{tabular}{c} Varian Factor FOUR \\ V-5 MS, 60 m, with \\ a 0.25 mm internal \\ diameter, and \\ 1.0 \(\mu\)m film \\ thickness \\ \end{tabular}  &
\begin{tabular}{c} 1 min @ 45 °C \\ rate of 10 °C/min to 320 °C \\ rate of 20 °C \\ rate of 20 °C \\ rate  
SPMA analysis was performed by on-sample derivatization and direct immersion (DI) SPME with a Varian 3900/MS 320 triple quadrupole (TQ) GC-MS/MS, using 1% isobutane in an argon gas mixture for negative chemical impact; the GC-MS/MS system was equipped with a Flex x,y,z autosampler (EST Analytical, Fairfield, Ohio) equipped with a six-position MFX and devices as previously described [25; 26]. The aliquot of 1 mL of sample was transferred into a 20-mL vial, added with 10-\(\upmu\)L of internal standard solution (\({}^{13}\)C\({}_{6}\)-SPMA, Giotto Biotech, Prato, Italy), and then placed into the autosampler. The routine procedure is acidifying the urine to pH \(<\) 1 to convert pre-SPMA to SPMA [27]. After agitation, 215 \(\upmu\)L of 0.1 M KOH (Merck KGaA) solution was added to adjust the pH to 8. Ultrasound-assisted derivatization of the SPMA with 2,3,4,5,6-pentafluorobenzyl bromide solution (Merck KGaA) was carried out [25]. Finally, the 20-mL sample vial was filled with 18 mL of water, and the DI-SPME extraction was carried out using a 65-\(\upmu\)m FFA-PDMS/DVB (Merck KGaA) temperature-controlled agitation at 60 \({}^{\circ}\)C for 20 min. The GC was provided with a Varian Factor FOUR VF-5 MS column. The SPMA derivative was quantified in Q1 \(>\) Q3 mode, \(m/z\) 238 \(>\) 108.8, while the \({}^{13}\)C\({}_{6}\)-SPMA derivative was quantified with \(m/z\) 244 \(>\) 115. Free 2,5-HD was determined with on-sample derivatization; HS-SPME analysis was carried out with a Varian CP 3800/Saturn 2200 Ion Trap, equipped with a CTC CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) and an MFX. Acquisition operated in SIM mode [28]. In a 20-mL vial, 2 mL of urine was supplemented with 100 \(\upmu\)L of O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA, Merck KGaA) water solution at 20 mg/mL and 30 \(\upmu\)L of internal standard (2 \(\upmu\)g/L cyclohexanone, Merck KGaA). Derivatization at 20 \({}^{\circ}\)C lasted 16 h before HS-SPME extraction was performed at temperature-controlled agitation at 60 \({}^{\circ}\)C for 20 min with 30-\(\upmu\)m FFA-PDMS (Merck KGaA). Separation was optimized with a Varian Factor FOUR VF-5 MS. 2,5-HD/bis-PFB-oxime was quantified by \(m/z\) 292, \(m/z\) 181, and \(m/z\) 323 ions; the internal standard was quantified at \(m/z\) 276. The \(t\),\(t\)-SA was analyzed with on-sample derivatization; HS-SPME analysis was performed with a Varian CP3800/Saturn 2200 Ion Trap, mentioned above. An amount of 200 \(\upmu\)L of urine was added to 1.8 mL of water in a 20-mL vial, and then 40 \(\upmu\)L of internal standard (120 \(\upmu\)g/mL cyclopentanecarboxylic acid-CPC, Merck KGaA) was inserted.