Anal. Chem. 2008, 80, 834-842
Analysis of Nitrosamines in Wastewater: Exploring the Trace Level Quantification Capabilities of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer Martin Krauss* and Juliane Hollender
Eawag, Swiss Federal Institute of Aquatic Science and Technology, Ueberlandstrasse 133, CH-8600 Du¨bendorf, Switzerland
A method was developed to determine nine N-nitrosamines in wastewater on the basis of solid-phase extraction and liquid chromatography mass spectrometry using a linear ion trap-orbitrap hybrid instrument at high mass resolution. Analytes and five deuterated internal standards were preconcentrated by solid-phase extraction. Positive electrospray ionization resulted in protonated molecular ions of all nitrosamines. One to three product ions were formed by collision-induced dissociation or higher energy C-trap dissociation. The signal intensity of the product ions differed up to a factor of 3 between the two techniques. The molecular ions were usually used for quantification, because of the better sensitivity, and the product ions for confirmation. An actual mass resolving power of 25 000-40 000 ensured a sufficient selectivity to distinguish all molecular and product ions from interfering background ions. Only for N-nitrosomorpholine was a coeluting isobaric molecular ion detected in wastewater samples, which, however, formed different product ions. The mass accuracy was between -12 ppm at m/z 55 and 0 ppm at m/z 205 and did not change for more than 5 ppm over a sample sequence of 20 h analysis time. The optimized method allowed quantifying nine N-nitrosamines in drinking water and wastewater samples down to method detection limits of 0.3-3.9 ng/L at instrumental detection limits of 2-14 pg on column. Recoveries over the whole method were between 75 and 125% for six compounds, but considerably lower for three compounds, probably due to strong matrix effects causing a signal suppression of up to 95% in wastewater samples. N-Nitrosodimethylamine and N-nitrosomorpholine were the most abundant compounds (3-22 ng/L) in samples from two wastewater treatment plants, another four nitrosamines (N-nitrosopyrrolidone, -piperidine, -diethylamine, and -dibutylamine) were also detected. Our study demonstrates that the LTQ Orbitrap is a powerful instrument to quantify low molecular weight compounds at the picogram level in complex matrixes with both a high sensitivity and selectivity. The occurrence of N-nitrosamines in water resources is of large health concern, as these compounds are potent carcinogens. In * To whom correspondence should be addressed. Phone: +41 44 823 5482. Fax: +41 44 823 5826. E-mail:
[email protected].
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particular N-nitrosodimethylamine (NDMA) has been recognized as a widespread disinfection byproduct in drinking water with levels ranging up to 180 ng/L.1,2 Other aliphatic and alicyclic nitrosamines have been detected at lower levels.2,3 The main source of NDMA is the use of chloramine disinfection, which promotes the reaction of dichloramine with organic nitrogencontaining precursors.4 Also, chlorine disinfection may lead to NDMA formation if ammonia is present to form chloramines or nitrogen-bearing cationic resins are used for coagulation.5 Although the identity of NDMA precursors apart from dimethylamine is not yet well-known, it is evident that treated wastewater is a major source of these precursors.6,7 Thus, the reuse of sewageimpacted ground and surface water yields a potential of NDMA formation either from disinfection of treated sewage prior to discharge or the disinfection of abstracted drinking water. But also in countries where chlorine or chloramine are not extensively used in wastewater and drinking water treatment, NDMA and other nitrosamines, in particular N-nitrosomorpholine (NMOR), are discharged with industrial and municipal wastewater.8 The need to detect nitrosamines at the low-nanogram per liter range in water samples is challenged by the fact that enrichment of the very polar but uncharged compounds from water and a selective detection of the small molecules are both difficult. In recent years, however, several sensitive methods based on solidphase extraction with carbonaceous adsorbents and gas chromatography-mass spectrometry (GC/MS) have been developed.2,9-11 The use of electron ionization coupled to low-resolution MS lacks in general the selectivity to be applied to complex matrixes, as it results in a low number of rather unspecific fragments. This can be overcome in parts by high-resolution mass spectrometry (1) Mitch, W. A.; Sharp, J. O.; Trussell, R. R.; Valentine, R. L.; Alvarez-Cohen, L.; Sedlak, D. L. Environ. Eng. Sci. 2003, 20, 389-404. (2) Charrois, J. W. A.; Arend, M. W.; Froese, K. L.; Hrudey, S. E. Environ. Sci. Technol. 2004, 38, 4835-4841. (3) Zhao, Y.-Y.; Boyd, J.; Hrudey, S. E.; Li, X.-F. Environ. Sci. Technol. 2006, 40, 7636-7641. (4) Schreiber, I. M.; Mitch, W. A. Environ. Sci. Technol. 2006, 40, 6007-6014. (5) Najm, I.; Trussell, R. R. J. Am. Water Works Assoc. 2001, 93, 92-99. (6) Mitch, W. A.; Sedlak, D. L. Environ. Sci. Technol. 2004, 38, 1445-1454. (7) Pehlivanoglu-Mantas, E.; Sedlak, D. L. Water Res. 2006, 40, 1287-1293. (8) Global Water Research Coalition. Report of the GWRC Research Strategy Workshop, 2007. (9) Munch, J. W.; Bassett, M. V. J. AOAC Int. 2006, 89, 486-497. (10) Taguchi, V.; Jenkins, S. D. W.; Wang, D. T.; Palmentier, J.; Reiner, E. J. Can. J. Appl. Spectrosc. 1994, 39, 87-93. (11) Cheng, R. C.; Hwang, C. J.; Andrews-Tate, C.; Guo, Y. B.; Carr, S.; Suffet, I. H. J. Am. Water Works Assoc. 2006, 98, 82-96. 10.1021/ac701804y CCC: $40.75
© 2008 American Chemical Society Published on Web 01/10/2008
(HRMS).10 The use of positive chemical ionization with ammonia or isobutane as reagent gas results in more selective ionization, less fragmentation, and the formation of higher molecular weight adduct ions besides the molecular ions.2 The use of tandem MS further increases selectivity.9 However, these GC-based methods are not suitable to analyze thermally instable nitrosamines like N-nitrosodiphenylamine (NDPhA). The applicability of liquid chromatography (LC) coupled to MS to detect nitrosamines has so far mainly been shown for tobacco-specific nitrosamines12-14 and for N-nitrosodiethanolamine in cosmetics.15 Only recently, Zhao et al.3 utilized liquid chromatography coupled to a triple-quadrupole tandem MS to detect volatile aliphatic and alicyclic nitrosamines as well as NDPhA in drinking water samples; wastewater samples were not analyzed by LC-MS so far. When starting our own studies on nitrosamines in wastewater, it turned out that LC coupled to low-resolution tandem mass spectrometry was not sufficiently selective for the detection of several of the nitrosamines of interest in this complex matrix. This encouraged us to explore the suitability of highresolution tandem mass spectrometry using the recently developed LTQ Orbitrap MS.16 The performance of this hybrid instrument has so far been studied with regard to compound identification/metabolomics17-19 and proteomics,20 but not to tracelevel quantification. Therefore, the objective of this study was to develop a method for the determination of nitrosamines in wastewater on the basis of solid-phase extraction and LC-HRMS detection. Our main focus was to explore the capabilities of the LTQ Orbitrap for trace level identification and quantification of nitrosamines as a case study for small polar molecules in complex matrixes. EXPERIMENTAL SECTION Reagents. We analyzed nine N-nitrosamines (Figure S1, Supporting Information), N-nitrosodimethylamine (NDMA), -methylethylamine (NMEA), -diethylamine (NDEA), -di-n-propylamine (NDPA), -di-n-butylamine (NDBA), -diphenylamine (NDPhA), -morpholine (NMOR), -piperidine (NPIP), and -pyrrolidine (NPYR), which were obtained as a 2 mg/mL solution in dichloromethane from Sigma-Aldrich (Buchs SG, Switzerland). The deuterated N-nitrosodimethylamine-d6 (NDMA-d6), -morpholined8 (NMOR-d8), -pyrrolidine-d8 (NPYR-d8), -dipropylamine-d14 (NDPAd14), and -diphenylamine-d6 (NDPhA-d6) were used as internal standards and obtained from Cambridge Isotope Laboratories (Andover, MA) or Dr. Ehrenstorfer (Augsburg, Germany). Work(12) Xia, Y.; McGuffey, J. E.; Bhattacharyya, S.; Sellergren, B.; Yimaz, E.; Wang, L. Q.; Bernert, J. T. Anal. Chem. 2005, 77, 7639-7645. (13) Wagner, K. A.; Finkel, N. H.; Fossett, J. E.; Gillman, I. G. Anal. Chem. 2005, 77, 1001-1006. (14) Jansson, C.; Paccou, A.; Osterdahl, B. G. J. Chromatogr.. A 2003, 1008, 135-143. (15) Schothorst, R. C.; Somers, H. H. J. Anal. Bioanal. Chem. 2005, 381, 681685. (16) Makarov, A.; Denisov, E.; Kholomeev, A.; Balschun, W.; Lange, O.; Strupat, K.; Horning, S. Anal. Chem. 2006, 78, 2113-2120. (17) Thevis, M.; Makarov, A. A.; Horning, S.; Scha¨nzer, W. Rapid Commun. Mass Spectrom. 2005, 19, 3369-3378. (18) Peterman, S. M.; Duczak, N., Jr.; Kalgutkar, A. S.; Lame, M. E.; Soglia, J. R. J. Am. Soc. Mass Spectrom. 2006, 17, 363-375. (19) Lim, H.-K.; Chen, J.; Sensenhauser, C.; Cook, K.; Subrahmanyam, V. Rapid Commun. Mass Spectrom. 2007, 21, 1821-1832. (20) Yates, J. R.; Cociorva, D.; Liao, L.; Zabrouskov, V. Anal. Chem. 2006, 78, 493-500.
ing standard solutions of analytes and internal standards were prepared in methanol and used for the preparation of calibration standards and spiking of samples. As nitrosamines are strong carcinogens, pure substances and concentrated solutions should be handled with great care. Methanol and water of HPLC grade purity were obtained from Scharlau (Barcelona, Spain) or Acros Organics (Geel, Belgium). Ethyl acetate, n-pentane, and glacial acetic acid of pro analysi grade and dichloromethane of residue analysis grade purity were purchased from Merck (Darmstadt, Germany). Solid-Phase Extraction (SPE). The principal approach was to combine two cartridges: A reversed-phase sorbent cartridge was used in the top position to enrich the less polar nitrosamines and remove most interfering matrix compounds to keep a carbonaceous sorbent cartridge (bottom position) available for the most polar nitrosamines. The reversed-phase sorbents tested were C18- and phenyl-modified silica gel (500 mg of Chromabond C18 Hydra or Chromabond C6H5, respectively; Macherey-Nagel, Du¨ren, Germany) as well as Oasis HLB (200 mg of a divinylbenzeneN-vinylpyrrolidone copolymer; Waters, Milford, MA). The carbonaceous sorbent was Bakerbond Carbon (1000 mg; MallinckrodtBaker, Philippsburg, NJ), which is an activated spherical carbon of 200-450-µm diameter and a surface area of 1300 m2/g produced by pyrolysis of a polymeric resin. The optimized SPE method was as follows: The 500-mL aliquots of samples were filtered through a glass fiber filter (GF/ F; 0.7 µm, Whatman, Brentford, U.K.) prior to extraction and spiked with internal standard (50 ng of each compound). The Oasis HLB cartridges were conditioned using 2 mL of n-pentane, 2 mL of ethyl acetate, 2 × 5 mL of methanol, and 2 × 5 mL of tap water. Bakerbond Carbon cartridges were conditioned with 5 mL of n-pentane, 5 mL of ethyl acetate, 2 × 5 mL of methanol, and 4 × 5 mL of tap water. Cartridges were connected (HLB on top) and samples passed at a flow rate of ∼3 mL/min. Cartridges were rinsed with 5 mL of HPLC grade water, disconnected, and dried in a vacuum for 1.5 h. The reconnected cartridges were eluted with 5 mL of dichloromethane; then the HLB cartridge was removed and the Carbon cartridge further eluted with 10 mL of dichloromethane into a conical flask. We added 800 µL of a water-methanol mixture 95:5 (v/v) to the flask and removed the dichloromethane completely using a rotary evaporator. The extracts were transferred to 2-mL autosampler vials and the volumes adjusted gravimetrically to 1 mL using water-methanol 95:5 (v/v). In case hydrophobic matrix components precipitated after evaporation of dichloromethane, the extracts were filtered using a 0.2-µm syringe filter. Liquid Chromatography-Mass Spectrometry. The HPLC system consisted of a HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) and a Rheos 2200 quaternary low-pressure mixing pump (Flux Instruments, Basel, Switzerland). Sample aliquots of 30 µL were injected into a 20-µL injection loop. Chromatographic separation of nitrosamines and internal standards was achieved using a reversed-phase column (Waters X-Bridge C18, 100 × 2.1 mm, 3-µm particle size, with precolumn 10 × 2.1 mm of the same type) and a gradient elution with water (A) and methanol (B), each containing 0.4% (v/v) acetic acid. The LC program started at 95% of solvent A for 6 min, increasing solvent B up to 95% in 11 min, held for 3 min, Analytical Chemistry, Vol. 80, No. 3, February 1, 2008
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Table 1. Optimization of LTQ Orbitrap Operating Conditions and Effects on Selectivity, Temporal Resolution, and Sensitivity of the MS Detection effect on selectivity mode of operation (scan/ SIM/HCD/CID) increase mass resolution no. of simultaneous MS expts increase ion injection time
may increase or decrease increases no effect no effect
temporal resolution small
effecta
decreases decreases decreases
sensitivity may increase or decrease decreasesb no effect increases
a The additional isolation step in SIM and the isolation and activation steps in MS/MS mode are ∼50-80 ms and thus much shorter than ion injection time and orbitrap scan time. b Due to collisional loss of ions in the orbitrap.
returning back to 95% of solvent A in 2 min, and re-equilibration for 8 min. The MS system was a LTQ Orbitrap hybrid instrument (Thermo Electron, Bremen, Germany), consisting of a LTQ linear ion trap mass spectrometer21 and an additional orbitrap mass analyzer. Ionization of nitrosamines was achieved using an electrospray probe in positive mode. For a detailed description of the LTQ Orbitrap, see ref 16. In brief, ions stored in the LTQ are delivered into a C-shaped quadrupole trap (C-trap) used to store and collisionally cool ions with nitrogen gas (∼1 mTorr). The trapped ions are orthogonally ejected into the orbitrap, where they are trapped in an electrostatic field and oscillate harmonically around a spindle-shaped inner electrode. The moving ions induce an image current at the outer shell-like electrodes. The m/z of the ions can be determined from the frequencies of oscillation after Fourier transformation. The resolving power of the orbitrap increases with increasing oscillation time and is set at discrete nominal steps of R ) 7500, 15 000, 30 000, 60 000, and 100 000 full width half-maximum (fwhm) referenced to m/z 400. This means that the actual mass resolution is usually higher than the nominal one at m/z 45% in both plants; those of NMOR were ∼50% at Eawag, but only
