RESULTS and DISCUSSION Analysis of nine N-nitrosamines using Liquid Chromatography-Accurate Mass High Resolution-Mass Spectrometry on a Q-Exactive instrument Arnaud Djintchui Ngonganga, Sung Vo Duya and Sébastien Sauvéa* aDepartment of Chemistry, Université de Montréal, Montreal, QC, Canada, Phone: +1 514 343-6749. Fax: +1 514 343-7586. E-mail: sebastien.sauve@umontreal.ca. RESULTS and DISCUSSION OVERVIEW Objectives To develop and optimize a selective, sensitive and robust SPE-LC-MS for the determination and the quantification of N-nitrosamines in drinking water and wastewater matrices using an accurate mass spectrometer Q-Exactive instrument and assess the efficiency of our method by quantifying the nine target nitrosamines even at trace concentration levels (ng/L) in different samples. Method validation A selective and robust methodology for the analysis of nine N-nitrosamines (NA) was developed and validated in drinking water and wastewater. Analyte detection was performed by high resolution mass spectrometry using a Q-Exactive Orbitrap using heated electrospray ionization (HESI) in positive ionization mode. Environmentally relevant method detection limits were obtained, falling in the low ng L-1 range. The method was applied to environmental samples i.e. two drinking waters (Montreal and Laval) and a wastewater from the sewage treatment plants located in Repentigny, three cities in the province of Quebec (CAN) to assess the efficiency of the developed method. Figure 4. Chromatogram of N-nitrosamines analyzed on a LC-HRMS Q-Exactive instrument in HPLC grade water; Sample conc. 20 µg/L ; Injection volume 5 µL. NDMA NMor NDPA NDBA NDPhA NDEA NPip NMEA NPyr MATERIALS and METHODS INTRODUCTION Sample preparation and Q-Exactive system Analyte detection was performed by high resolution mass spectrometry (Q-Exactive, Figure 2). The acquisition mode used was: Full Scan MS (FS, R=70000 at m/z < 200). An off-line solid phase extraction (SPE, Figure 2) procedure was used for sample pre-concentration and clean-up. A coconut charcoal SPE cartridge was chosen for the N-nitroamines extraction according to EPA Method 521. Figure 2. Q-Exactive system (left) and off-line manual SPE (right) setup used for the analysis. N-nitrosamines compounds are usually produced by industrial activities such as food (meats) and cosmetics processing, dye and rubber manufacturing, leather tanning and metal casting. Despite the treatment done by industries and water treatment plants, these compounds can still be found in the air, wastewater as well as in drinking water. N-nitrosamines such as NDMA are reported as by-products formed after the disinfection of wastewater effluent by chlorine and the drinking water chlorination and chloramination processes in the presence of nitrogen-containing organic matter. N-nitrosamines might equally pose a risk to water resources and given their potential adverse effects on human health, the presence of these compounds is of more concern in drinking water than in wastewater. NDMA NMor NMEA NPyr NPip NDEA NDPA NDBA NDPhA Table 1. Physico-chemical properties of the studied N-Nitrosamines. * C. Hansch, A. Leo, D. Hoekman, 1995. Exploring QSAR: Hydrophobic, electronic, and steric constants. American Chemical Society, Washington, DC. ** The mass error ranged from 0.0 to 2.0 ppm. This error was calculated using the mean of three injections. There was no drift in the mass error for all compounds during a sequence run. Figure 5. Chromatogram of N-nitrosamines analyzed on a LC-HRMS Q-Exactive instrument in drinking water; Sample conc. 120 µg/L ; Injection volume 5 µL. Compound   Formula Molecular Mass Theoretical Precursor (M+H)+ Experimental Precursor (M+H)+ ∆(M+H)+ in ppm** logKow (*) NDMA C2H6N2O 74.04801 75.05584 75.05599 2.0 -0.57 NMEA C3H8N2O 88.06366 89.07149 89.07150 0.1 0.04 NPyr C4H8N2O 100.06366 101.07149 101.07137 -1.2 -0.19 NDEA C4H10N2O 102.07931 103.08714 0.0 0.48 NPip C5H10N2O 114.07931 115.08714 115.08697 -1.5 0.36 NMor C4H8N2O2 116.05858 117.06640 117.06612 -2.4 -0.44 NDPA C6H14N2O 130.11061 131.11844 131.11830 -1.1 1.36 NDBA C8H18N2O 158.14191 159.14974 159.14928 -2.9 2.63 NDPhA C12H10N2O 198.07931 199.08714 199.08698 -0.8 3.13 NDPhA NDBA NDPA NPip NDEA NDMA NMor NPyr NMEA Figure 6. Chromatogram of N-nitrosamines analyzed on a LC-HRMS a Q-Exactive instrument in wastewater matrix; Sample conc. 120 ng/L in methylene chloride; Injection volume 5 µL. 1. Sample Preparation Drinking water (DW)-no filtration (500 mL) Filtration of wastewater (WW) at 1.2 µm and 0.3 µm (250 mL) 2. SOLID PHASE EXTRACTION SPE (US EPA METHOD 521) i) Coconut charcoal (2g, 6 mL) cartridges conditioning 2 x 3 mL CH2Cl2, 2 x 2 x 3 mL MeOH and 5 x 3 mL H2O ii) Sample extraction iii) Cartridges air-dried under vacuum iv) Elution (12 mL CH2Cl2) 3. EVAPORATION Sample concentrated to 1 mL under a high purity and moderate nitrogen stream at room temperature Concentration factors of 250-fold (WW) and 500-fold (DW)   5. DETECTION-HRMS Q-Exactive HESI Nine N-nitrosamines Full Scan R = 70 000 @ m/z < 200 4. Liquid chromatography conditions Selected Mobile Phase: A: H2O + 0.1% HCOOH and B: MeOH + 0.1% HCOOH Flow Rate: 500 µL/min Autosampler temperature: 8°C Column: Hypersil GOLD C18, 100 x 2.1 mm, 1.9 µm. Column Temperature: 40°C Gradient Duration (min) 0.0 1.0 1.5 4.50 4.51 6.50 6.51 %A 95 90 10 5 100 %B Concentration level in ng/L Method application Figure 7. Analytical results of N-nitrosodiphenylamine (NDPhA), the only N-nitrosamines quantified at a concentration level of 1.2 ng/L which was above the limit of detection (0.4 ng/L). This compound was found in the drinking water sample collected in the city of Trois-Rivières, Quebec Canada. Figure 3. Different steps from sample preparation Solid phase extraction SPE) to the mass spectrometry detection and analysis LC-HRMS). RESULTS and DISCUSSION Regulations about the maximum contaminant level for NDMA at other N-nitrosamines in drinking water has not yet been established in North America, but some limits have been already set in jurisdictions such as in California (USA) at 10 ng/L[1] and in Ontario (Canada) at 9 ng/L [2]. Drinking water samples from Laval and Montréal and wastewater sample WWTP of Repentigny were also analyzed, but none of the studied N-nitrosamines was found above these concentrations. High resolution (R═70000) coupled with the selected full scan acquisition mode was used for the analysis of the samples on the accurate mass spectrometry Q-Exactive device. The target precursor ions of all the studied N-nitrosamines were identified with excellent specificity and sensitivity. Interesting experimental results were also obtained in the complex wastewater samples as shown in Figure 6. The method was optimized and validated in HPLC grade water, drinking water and wastewater matrices with satisfactory results. NDMA-d6 and NDPA-d14 were used as internal standards. The extraction recoveries in real matrices ranged from 68-83% for eight of the nine target nitrosamines, except for NDPhA with values of 22-31%. The detection limits ranged from 0.4 to 12 ng/L (LODs were determined using the error on the y-intercept and slope from the calibration curves). Calibration curves were obtained by the standard addition method. The calibration curves showed fair linearity with R2 > 0.992 for all the selected compounds. CONCLUSION A selective and robust SPE-LC-MS method was developed and optimized for the analysis of N-nitrosamines in drinking water and in wastewater matrices. The sensitivity of our method was comparable with that of published GC/MS and LC-MS/MS based methods. The use of the accurate mass high resolution-mass spectrometer (Q-Exactive) helps for the identification and quantification of the target NA without any ambiguity. Although GC-MS can provide better sensitivity for N-nitrosamines analysis, the advantage of the use of LC-MS is the significant time savings given the longer retention times recorded in GC-MS. Moreover, both GC-detectable and GC-undetectable such as NDPhA can be detected. First report of N-nitrosamines analysis using SPE-LC-MS using HRMS on Q-Exactive. References [1]CDHS. (2005) Notification levels overview [online]. Available from http://www.dhs.ca.gov/ps/ddwem/chemicals/AL/notificationoverview.pdf [cited 20th November 2014]. [2]OMEO (2003) Ontario drinking-water quality standards; O. Reg. 169/03, Sched. 2; O. Reg. 268/03, s. 1; O. Reg. 248/06, s. 2; O. Reg. 242/07, s. 1. [online] Available from http://www.e-laws.gov.on.ca/DBLaws/Regs/English/030169-e.htm [cited 20th November 2014]. ACKNOWLEDGMENTS The authors will like to thank Natural Sciences and Engineering Research Council of Canada for financial support and the Canadian Foundation for Innovation (equipment). Figure 1. Chemical structures and exact masses of the nine N-Nitrosamines studied.