Determination of Ethylene Oxide (ETO) in Mainstream Cigarette Smoke Using Hydrobromic Acid Derivatization and Gas Chromatography-Mass Spectrometry Method Xinyu LIU, Peter JOZA, Andrew MASTERS, Bill RICKERT 2014 CORESTA CONGRESS October 12-16, 2014 Québec City, Canada LABSTAT INTERNATIONAL ULC. 262 Manitou Drive Kitchener, Ontario, Canada N2C 1L3 Phone: (519) 748-5409 Fax: (519) 748-1654 Web: www.labstat.com 1
Objectives To develop a reliable method for the analysis of ethylene oxide in tobacco smoke To analyze low concentrations of ethylene oxide in new generation products (e.g. e- cigarette aerosols and heat-not-burn emissions) 2
Methods for the Direct Analysis of ETO Authors & Journal Sample matrix Method Gordon et al. 57 th TSRC (2003) Mainstream cigarette smoke GC-MS Dong et al. 62 th TSRC (2008) Masters et al. Coresta (2009) Gillman et al. Coresta 2012 Otte et al. Coresta 2013 Mainstream cigarette smoke Mainstream cigarette smoke Mainstream cigarette smoke Mainstream cigarette smoke GC-MS GC-MS GC-MS GC-MS 3
Physicochemical Properties Appearance: colorless gas Molecular formula: C 2 H 4 O (MW=44.05) BP: 10.8 C MP: -111 C 4
Relative Abundance (%) EI Mass Spectrum Mass library: Wiley, NIST/EPA/NIH, FFNSC 5
Potential Interference Peaks 6
Direct Method : Analytical Conditions Instrument Column Parameters DB-5ms (60 mx0.25 mmx1.0 µm) Injector temperature 220 C Oven temperature Column flow 35 C for 6.5 minutes; 20 C per minute to 210 C; hold 3 minutes. 1.5 ml/min Injection mode Split mode with a split ratio 20:1 Injection volume 1 µl Transfer line temperature 200 C Source temperature 230 C MS quad 150 C Ionization mode Scan 7
Direct Method: Chromatogram (3R4F Smoke ) Methanethiol Match 808, R. Match 962, Prob. 92.7% m/z=47 Ethylene oxide RT time window m/z=44 TIC 2.6 3.6 4.6 5.6 6.6 7.6 min 8
Direct Method: Analytical Conditions Instrument Parameters Column: DB-624 ms (60 mx0.25 mmx1.4 µm) Injector temperature 220 C Column temperature 35 C for 10 minutes; 20 C per minute to 250 C; hold 3 minutes. Column flow 1.5 ml/min Injection method Split mode with split ratio 20:1 Injection volume 1 µl Transfer line temperature 200 C Source temperature 230 C MS quad 150 C Ionization mode Scan 9
Direct Method: Chromatogram (3R4F Smoke ) m/z=44 44 Ethylene oxide? TIC MeOH 3.7 4.7 5.7 6.7 7.7 8.7 min 10
Effects of Interference Peak on ETO Analysis 11
Reaction Sequence: Indirect Method C 2 H 4 O (Ethylene Oxide) + HBr (Hydrobromic Acid) BrC 2 H 4 OH (2-Bromoethanol) Eliminate the interference from common trapping solvent (e.g. methanol) Provide more specific ion m/z=124 for reliable quantitation analysis 12
Experimental Steps Add 1.0 g anhydrous Na 2 SO 4 Accurately transfer 2 ml sample Add 200 µl 48% HBr, wait 5 min Add 0.3 g Na 2 CO 3, wait for 30 min Spike 200 µl ISTD (D 4-2-bromoethanol ) Centrifuge 5min at 2500 rpm Transfer supernatant in vial GC/MS analysis 13
Analytical Conditions Instrument Parameters Column ZB-1 (60 mx0.25 mmx1.0 µm) Injector temperature 250 C Oven temperature 100 C for 10 minutes; 20 C per minute to 250 C; hold 3 minutes Column flow 1mL/min Injection method Split mode with split ratio 10:1 Injection volume 1 µl Transfer line temperature 250 C Source temperature 230 C MS quad 150 C Ionization mode SIM 14
MS Quantization Parameters Component RT Quantifier ion 2-bromoethanol (ETO-HBr) D 4-2-bromoethanol (ISTD) Dwell time Qualifier ion (min) (m/z) (ms) (m/z) 6.373 124 100 107/95 6.329 128 100 NA 15
Response Optimization of Derivatization Time (3R4F smoke) 2850 2800 2750 2700 2650 2600 2550 2500 2450 1 10 40 70 100 130 160 190 220 250 Derivatization time (min) 16
Response Optimization Amount of Derivatization Reagent (3R4F Smoke) 1400 1200 1000 800 600 400 200 0 10 25 50 100 200 300 500 600 Volume (µl) of 48% aqueous HBr 17
Std level Linear Concentration Range 2-Bromoethanol (µg/ml) 1 21.1 2 16.9 3 12.6 4 8.43 5 4.21 6 2.11 7 0.843 8 0.421 9 0.211 10 0.084 Y=0.392x R 2 =0.999 18
Recovery (Accuracy) Method Characteristics Laboratory Reagent Blank (LRB) (ng/cig) 0.00 Laboratory Fortified Blank (LFB) (%) 83.6-92.0 Laboratory Fortified Matrix (LFM) (%) 81.1-94.4 Detection Limit Limit of Detection (LOD) (ng/cig) 33.7 Limit of Quantification (LOQ) (ng/cig) 112 19
Mainstream Smoke Analysis (3R4F) ETO-HBR m/z=124 ISTD m/z=128 2.9 3.4 3.9 4.4 4.9 5.4 5.9 6.4 6.9 7.4 Min 20
E-cigarette Aerosol Analysis ETO-HBR m/z=124 ISTD m/z=128 2.8 3.3 3.8 4.3 4.8 5.3 5.8 6.3 6.8 7.3 Min
Method Comparison (Derivatization vs Direct analysis) Method Sample Sample Replicates Mean Std. Dev. RSD ID Matrix [n] [µg/cig] [µg/cig] [%] Direct analysis* 3R4F MS ISO 70 23.9 6.83 28.6 HBr-derivatization 3R4F MS Intense 57 65.3 12.8 19.6 3R4F MS ISO 19 8.37 1.74 20.8 3R4F MS Intense 112 24.6 4.58 18.6 E-cig** MS Intense 3 0.212 0.022 10.3 *DB-5 column, full scan mode, ion-trap detector was used. ** Example for demonstration. 22
Summary The Hydrobromic acid-derivatization method can be used to effectively solve sample matrix interference The developed method can be applied to analyze ethylene oxide in cigarette smoke and in new generation products ( e.g. e-cigarette aerosols and heat-not-burn emissions) 23
Acknowledgements Labstat International ULC Sample Preparation Technician and Analytical Team (Mr. MingZhong Cui, Mrs. Helena Coetzer, Mrs. Gabriela Pop, Mr. Ali Ahmed) 24