# Low-Dose Benzene Exposure Monitoring of Oil Refinery Workers: Inhalation and Biomarkers
## 2 Materials and Methods
Derivatization with trimethyloxonium tetrafluoroborate (Merck KGaA) was performed [29]. For extraction by HS-SPME, sodium chloride (0.5 g) was added to the vials, and an 85-\(\upmu\)m FFA-PA (Merck KGaA) was used in temperature-controlled agitation at 60 \({}^{\circ}\)C for 20 min, and then it was desorbed in the GC injector. Separation was optimized with a Varian Factor FOUR VF-5 MS. The \(t\),\(t\)-SA derivative was quantified at \(m/z\) 111, while \(m/z\) 67 and 95 were used as confirmation ions. CPC methyl ester was quantified at \(m/z\) 87. The \(t\),\(t\)-MA was analyzed with an on-line SPE LC-MS/MS method, set up on an Alliance e2695 LC Selector Valve coupled with a Quattro Micro API TQ-MS (Waters, Milford, CT, USA), using negative electrospray ionization (ESI) and collision-induced fragmentation. A urine aliquot of 1 mL was added with 20 \(\upmu\)L of internal standard (30 \(\upmu\)g/mL cis,cis-2,5-dimethylmuconic acid, Merck KGaA), 2 mL of a water/methanol 50:50 solution, and 15 \(\upmu\)L of formic acid. After centrifugation, the samples were filtered with an Acrodisc Syringe Filter GHP (13 mm, 0.2 um) (Waters) and analyzed. On-line SPE purification was performed with an Oasis HLB, 25 \(\upmu\)m with 2.1-mm internal diameter and 20-mm column length (Waters), followed by separation with an Atlantis T3, 3-\(\upmu\)m particles with 2.1-mm internal diameter and 100-mm column length (Waters). Elution was conducted at 25 \({}^{\circ}\)C with a 0.3 mL/min flow and 10 \(\upmu\)L of sample injection. The MS utilized multiple reaction monitoring (MRM). The \(t\),\(t\)-MA was quantified by \(m/z\) 141.018 \(>\)\(m/z\) 52.77 and \(m/z\) 141.018 \(>\)\(m/z\) 96.89 as confirmation ion, while the internal standard acquisition was \(m/z\) 169.149 \(>\)\(m/z\) 125.039. U-Cotinine was determined by LC-MS/MS with the same instrument as \(t\),\(t\)-MA but by using positive ESI. A urine aliquot of 1 mL was added with the internal standard (25-\(\upmu\)g/mL daunorubicin, Merck KGaA), then it was treated and analyzed as the \(t\),\(t\)-MA samples. U-Cwas quantified by \(m/z\) 177.093 \(>m/z\) 79.861, while the internal standard acquisition was \(m/z\) 528.256 \(>m/z\) 321.124. Five replicates for each standard sample were processed to perform the internal standard calibration to quantify all these biological markers. The least-square linear regression was used to estimate the slopes (\(m\)) and intercepts (\(b\)) of the calibration curve. The limit of detection (\(LOD\)) was estimated by the equation:  
\[LOD=\frac{(3\,SEb+b)}{m} \tag{1}\]  
where \(SEb\) is the standard error of the intercept. The lower limit of quantification (LOQ) was calculated using the formula 10 \(SEb\), corresponding to 3.3 \(LOD\). The precision of the methods (as a coefficient of variation, CV%) was calculated both as within-session and as intersession repeatability. In addition, the accuracy for toxic organic compounds was evaluated by ClinChek Controls (Recipe, Munich, Germany) and for SPMA using ClinChek Urine Control for Occupational Medicine, Levels I and II (Recipe, Munich, Germany). U-B, U-C, and SA accuracy were verified from a urine pool that had been previously quantified and then fortified by two different amounts of analyte. These accuracy values were then compared with the requirements of US Food and Drug Administration to validate the proposed analytical methods [30]. The Statistical Package for Social Science (SPSS) was used for the statistical analysis. Relationships between the analytes were assessed by simple linear correlation analysis between the data for environmental benzene paired with U-B and between U-B paired with benzene metabolites.