# Indoor Air Pollution Increases the Risk of Lung Cancer
## 2 Materials and Methods
### Exclusion Criteria
#### 2.2.3 Pleural Fluid Analysis  
Instruments, Reagents, and Standards  
We analyzed the headspace air of the pleural fluid by gas chromatography-mass spectrometry (GC-MS) using the solid-phase microextraction (SPME) technique. GC-MS analysis was performed on a Hewlett-Packard 6890 gas chromatograph equipped with a 5973 mass-selective detector (Agilent Technologies, Santa Clara, CA, USA) as well as a 30 m \(\times\) 0.25 mm i.d., 0.25 \(\upmu\)m thick DB-5 MS column (J&W Scientific, Folsom, CA, USA). In our study, we established a GC-MS program using standard solutions, which included eight PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, and pyrene) at 10 \(\upmu\)g/mL each in acetonitrile, and BTEX (benzene, toluene, ethylbenzene, p-xylene, m-xylene, and o-xylene) at 1 mg/mL each in DMSO. All standard solutions were obtained from Sigma-Aldrich (Milwaukee, WI, USA). Standard solutions were diluted by phosphate-buffered saline (PBS) (Sigma-Aldrich, Milwaukee, WI, USA). A stock solution containing the two classes of compounds (PAHs and BTEX) was prepared using acetone as the solvent for further dilution. Internal standards (ISs) included benzene-d6 (2000 \(\upmu\)g/mL in methanol) and naphthalene-d8 (99 atom % D isotopic purity; 1 g in a glass bottle prepared at a concentration of 2000 \(\upmu\)g/mL in dichloromethane). All stock solutions were kept in vials and stored at 4 \({}^{\circ}\)C. To overcome matrix effects, in which the matrix coextracted with the analytes can invoke a signal response in MS, we added 100 ng/mL of the IS benzene-d6 and naphthalene-d8 during the extraction process [11]. Instrument Analysis  
The thawed pleural fluid was analyzed by GC-MS within 24 h after collection. This study followed a validated procedure to measure PAHs and BTEX simultaneously [12]. We modified the extraction time, desorption time, and mass range based on our pilot study. We used a 65 \(\upmu\)m PDMS/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellaofente, PA, USA). The SPME fiber was inserted into the headspace of a 4 mL vial containing 100 ng/mL of the IS solutions (benzene-d6 and naphthalene) in acetone-d8 and then exposed for 60 min in an 80 \({}^{\circ}\)C oil bath. After extraction, the fiber was inserted into the GC injector for analysis. The thermally desorbed trace components were separated by a capillary column with a helium flow rate of 1.3 mL/min using the splitless mode. The chromatographic analytical column temperature was initially set at 35 \({}^{\circ}\)C, with a 10 min hold, ramped to 80 \({}^{\circ}\)C at a rate of 20 \({}^{\circ}\)C/min, and then ramped to 300 \({}^{\circ}\)C (10 min) at a rate of 6 \({}^{\circ}\)C/min. For the MS measurement, ionization was executed by the electron impact (EI) method at 70 eV while operating in selected ion monitoring (SIM) mode. Optimization of SPME Extraction Conditions  
The isolation process was optimized by selecting the appropriate extraction time to obtain the highest extraction efficiency. The optimum conditions were determined by the sum of the peak areas obtained under different extraction times. Hence, the impact of the extraction time was examined in the test sample at 50 \({}^{\circ}\)C for various time durations, i.e., 10 min, 15 min, 20 min, 25 min, and 30 min. The extraction time profiles for the total peak area indicated that a sampling time of 25 min adsorption resulted in the highest total peak area. Based on these experimental results, the optimum extraction condition was established as 25 min. Quality Assurance and Quality Control  
We conducted blank tests before the analysis of each batch of samples; the blanks included the following:  
(1) Instrument blank-A blank instrument test was performed to confirm that there were no contaminants in the system before performing the sample analysis. Before each batch of samples, we ran the GC-MS instrument without any substances to confirm that there were no contaminants in the device.