Anal. Chem. 2005, 77, 3083-3089
Liquid Chromatography-Tandem Mass Spectrometry Determination of Nonionic Organophosphorus Flame Retardants and Plasticizers in Wastewater Samples Rosario Rodil,† Jose´ Benito Quintana,†,‡ and Thorsten Reemtsma*,‡
Departamento de Quı´mica Analı´tica, Nutricio´ n e Bromatoloxı´a, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain, and Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany
A comprehensive method for the determination of nonionic organophosphorus flame retardants/plasticizers in wastewater samples by solid-phase extraction (SPE) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is presented. It allows the determination of 11 organophosphorus compounds, trialkyl and trichloralkyl phosphates, triaryl phosphate, and biphosphates together with triphenylphosphine oxide (TPPO). Limits of quantification after SPE of 100 mL of water are between 3 and 80 ng/L, which are adequate for most aqueous samples. The sensitivity of the LC-ESI-MS approach allows direct injection of aqueous sample without preceding extraction for concentrations in the low µg/L range. The method was finally applied to municipal wastewater samples, showing the occurrence of six phosphoric acid triesters and TPPO in both raw and treated municipal wastewaters. Flame retardants are important industrial chemicals that are present in many products in quantities ranging from a few to about 50 wt % to reduce flammability of plastics, textiles, and other materials. Among the flame retardants are inorganic compounds such as aluminum trihydrate or antimony oxides and also organic compounds. Of these organic flame retardants, brominated compounds such as polybrominated diphenyl ethers (PBDEs) have attracted much attention in recent years because of their global distribution1 and accumulation in the food chain, leading to exponentially increasing concentrations in the environment and in humans.1 Furthermore, they have been recognized as endocrine disruptors,2 proposed for replacement by the World Health Organization,3 and banned (penta- and octabromodiphenyl ether) by the European Union from August 2004.4 Another important group are organophosphorus flame retardants (OPFR) with an annual consumption of some 80 000 tons/ * Corresponding author. Tel.: +49-30-31426429; fax: +49-30-31423850; e-mail:
[email protected]. † Universidade de Santiago de Compostela. ‡ Technical University of Berlin. (1) Hites, R. Environ. Sci. Technol. 2004, 38, 945-956. (2) Legler, J.; Brouwer, A. Environ. Int. 2003, 29, 879-885. (3) World Health Organization. Environmental Health Criteria 205: Polybrominated dibenzo-p-dioxins and dibenzofurans; Geneva, Switzerland, 1998. (4) Directive 2003/11/EC. Off. J. Eur. Communities L 42/45-46 (15.2.2003). 10.1021/ac048247s CCC: $30.25 Published on Web 03/30/2005
© 2005 American Chemical Society
year and a market share of 20% in Western Europe.5 Because of the ban on PBDEs in the European Union, the importance of OPFR is expected to grow, and new products are reaching the market. However, some OPFR give themselves rise to environmental concern as triphenyl phosphate and tri-n-butyl phosphate are suspected neurotoxic6,7 while chlorinated alkyl phosphates are suspected carcinogenic to animals8,9 and resistant to degradation.10 As a consequence, some OPFRs are undergoing risk assessment by the EU.11 Besides air,12,13 water appears to be an important medium for the distribution of OPFR in the environment, as several compounds have already been found in wastewater,14-17 surface water,15,16,18,19 and groundwater15,16 in concentrations ranging from ng/L up to several µg/L. Present analytical methods for the determination of OPFR from water are based on solid-phase extraction (SPE) and GC-NPD20 or GC-MS determination.13,15,21 However, some of these compounds lead to tailing peaks, like triphenylphosphine oxide (TPPO), when analyzed by GC. (5) European Flame Retardant Association. http://www.cefic-efra.com (accessed Nov 2004). (6) World Health Organization. Environmental Health Criteria 111: triphenyl phosphate, Geneva, Switzerland, 1991. (7) World Health Organization. Environmental Health Criteria 112: tri-n-butyl phosphate, Geneva, Switzerland, 1991. (8) European Commission, European Chemicals Bureau. IUCLID, dataset tris(2chloromethyl) phosphate, EINECS No. 204-118-5, 2001. http://ecb.jrc.it/ESIS. (9) Matthews, H.; Dixon, D.; Herr, D.; Tilson, H. Toxicol. Ind. Health 1990, 6, 1-15. (10) Kawagoshi, Y.; Nakamura, S.; Fukunaga, I. Chemosphere 2002, 48, 219225. (11) European Commission Regulation (EC) No. 2364/2000. Off. J. Eur. Communities L 273/5-7 (26.10.2000). (12) Carlsson, H.; Nilsson, U.; Becker, G.; O ¨ stman, C. Environ. Sci. Technol. 1997, 31, 2931-2936. (13) Toda, H.; Sako, K.; Yagome, Y.; Nakamura, T. Anal. Chim. Acta 2004, 519, 213-218. (14) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Environ. Sci. Technol. 2002, 36, 1202-1211 (15) Fries, E.; Pu ¨ ttman, W. J. Environ. Monit. 2001, 3, 621-626. (16) Fries, E.; Pu ¨ ttman, W. J. Environ. Monit. 2003, 5, 346-352. (17) Meyer, J.; Bester, K. J. Environ. Monit. 2004, 6, 599-605. (18) Knepper, T. P.; Sacher, F.; Lange, F. T.; Brauch, H. J.; Karrenbrock, F.; Roerden, O.; Lindner, K. Waste Manage. 1999, 19, 77-99. (19) Dsikowitzky, L.; Schwarzbauer, J.; Littke, R. Chemosphere 2004, 57, 12891300. (20) Jonsson, O. B.; Nilsson, U. L. J. Sep. Sci. 2003, 26, 886-892. (21) Lebel, G. L.; Williams, D. T.; Benoit, F. M. J. Assoc. Off. Anal. Chem. 1981, 64, 991-998.
Analytical Chemistry, Vol. 77, No. 10, May 15, 2005 3083
Table 1. The Analytes and Their Specific LC-ESI-MS/MS Parameters for MRM Detection in the Positive Ion Mode compound
ret. time (min)
transitionsa
proposed product ion
Tr.1/Tr.2b
1
TCEP
tris(2-chloroethyl) phosphate
6.3
285(25) > 63(14) 285(25) > 161(22)
[C2H4Cl]+ [(M + H) - 2x(C2H3Cl)]+
1.4
2
TPPO
triphenylphosphine oxide
8.7
279(50) > 201(28) 279(50) > 77(44)
[(M + H) - benzene]+ [C6H5]+
2.5
3
TCPP
tris(chloro 2-propyl) phosphate
9.6
327(25) > 99(20) 329(25) > 99(20)
[H4PO4]+ [H4PO4]+
1.0
IS
TPrP (IS)
tri-n-propyl phosphate
9.7
225(20) > 99(16)
[H4PO4]+
4
TDCP
tris(1,3-dichloro-2-propyl)phosphate
11.5
431(25) > 99(25) 433(25) > 99(25)
[H4PO4]+ [H4PO4]+
1.2
5
TPhP
triphenyl phosphate
11.7
327(35) > 215(26) 327(35) > 153(30)
[(PhO)2PO - H2O]+ [C12H9]+
1.7
6
TiBP
tris-iso-butyl phosphate
12.8
267(35) > 99(22) 267(35) > 155(10)
[H4PO4]+ [(M + H) - 2xC4H8]+
2.0
7
TnBP
tri-n-butyl phosphate
13.0
267(35) > 99(22) 267(35) > 155(10)
[H4PO4]+ [(M + H) - 2xC4H8]+
2.0
8
TBEP
tris(2-butoxyethyl) phosphate
13.6
399(30) > 199(12) 399(30) > 299(12)
[(M + H) - 2xC6H12O]+ [(M + H) - C6H12O]+
1.0
9
RDP
resorcinol bis(diphenyl phosphate)
15.2
575(50) > 575(25) 575(50) > 419(35)
[M + H]+ [(M + H) - PO3Ph]+
17
10
BDP
bisphenol A bis(diphenyl phosphate)
17.5
693(45) > 367(36) 693(45) > 327(32)
[(M + H) - OP(OPh)3]+ [HOP(OPh)3]+
13
11
TEHP
tris(2-ethylhexyl) phosphate
21.4
435(45) > 99(14) 435(45) > 113(12)
[H4PO4]+ [C8H17]+
a
2.7
Precursor (cone voltage (V)) > product (collision energy (eV)). b Intensity ratio of the first to the second transition.
Only one LC-MS method has been published for the determination of trialkyl and triaryl phosphates in human blood plasma samples.22 However, TPPO and many important chlorinated trialkyl phosphates were not considered in that work and, moreover, for new and heavier OPFRs such as resorcinol-bi(diphenyl phosphate) (RDP) or bisphenol A-bi(diphenyl phosphate) (BDP), which are not amenable to GC determination, no analytical method is available yet. In this work, a comprehensive analytical method for the determination of organophosphorus flame retardants and plasticizers in wastewater and surface waters by SPE and LC-MS/MS is presented. It covers three chlorinated alkyl phosphates, four nonchlorinated alkyl phosphates, one aryl phosphate (triphenyl phosphate), two bisphosphates (RDP and BDP), and also the synthetic intermediate triphenylphosphine oxide (TPPO). This method improves the determination of many of these compounds from water and facilitates studies on the fate of these nonionic organophosphorus compounds in the aquatic environment. EXPERIMENTAL SECTION Reagents and Chemicals. TCEP, TPPO, TPrP (internal standard), TPhP, TnBP, TBEP, and TEHP were obtained from Aldrich (Steinheim, Switzerland). TDCP and TiBP were bought from Fluka (Steinheim, Switzerland). TCPP (technical mixture of three isomers) was kindly provided by K. Bester (University of Essen), and Fyroflex RDP (technical mixture) and Fyroflex BDP (technical mixture) were kindly supplied by Akzo Nobel (Amersfoort, The Netherlands). Full names of all analytes are given in Table 1. (22) Amini, N.; Crescenzi, C. J. Chromatogr., B 2003, 795, 245-256.
3084 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
Ultrapure water was obtained by an ELGA Maxima HPLC ultrapure water system (ELGA, Ubstadt-Weiher, Germany). Methanol and acetonitrile were supplied by J. T. Baker (Deventer, The Netherlands) and formic acid was purchased from Fluka (Steinheim, Switzerland). Individual stock solutions of 2000 mg/L were prepared in acetonitrile. Mixed stock solutions contained all the analytes except RDP and BDP, which were prepared in methanol/water (1/1) in concentrations of 5 mg/L. A second stock solution was prepared from RDP and BDP and used independently, as these technical products contain impurities of other phosphates. Samples. Twenty-four hour composite samples of the influent, primary effluent, and the tertiary effluent (treated wastewater) of a municipal wastewater treatment plant (WWTP) were collected in August 2004. Tertiary effluent samples were taken with a 24-h delay to account for the hydraulic retention time in the activated sludge treatment. All samples were filtered through 0.45-µm membrane filters (cellulose acetate; Sartorius, Goettingen, Germany) and immediately analyzed. Instrumentation. An HP1100 (Agilent Technologies, San Jose, CA) liquid chromatographic system consisting of a membrane degasser, binary high-pressure gradient pump, autosampler, and column thermostat was used. The system was interfaced to a Quattro LC triple-stage quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with a Z-spray electrospray interface. Nitrogen was provided by a nitrogen generator (Model 75-72, Whatman, Haberville, U.S.) and used as drying and nebulizing gas. Argon (99.999%) was used as collision gas. The system was controlled with Masslynx 3.3 software.
LC-MS/MS Analysis. Optimization of the formic acid concentration in the mobile phase to attain highest sensitivity was performed by flow injection analysis (FIA), injecting 20 µL of individual standard solutions (ca. 100 ng/mL) into a stream of MeOH/water (1/1) containing variable concentration of acid (02%) at a flow rate of 0.25 mL/min. The LC separation with optimized formic acid concentration was carried out on a 150 × 3 mm Luna 3 µm C-18 (2) column (Phenomenex, Eschborn, Germany) at a flow rate of 0.25 mL/ min and column temperature of 45 °C. Eluent A consisted of MeOH/water (20/80) and eluent B pure MeOH, both containing 0.2% formic acid. The gradient was as follows: 0 min (55% B), 0.5 min (70% B), 11 min (100% B), 16 min (100% B), 17 min (55% B), and 27 min (55% B). Analytes were detected by electrospray ionization tandem mass spectrometry operated in the positive ion mode with a capillary voltage of 3.5 kV, a source temperature of 120 °C, a desolvation temperature of 200 °C, a nebulizer gas flow of 100 L/h, and a drying gas flow of 700 L/h. Argon pressure in the collision cell was kept at 1.0 × 10-3 mbar for MS/MS measurements. Quantification of all compounds was made by multiple reaction monitoring (MRM) recording two to four transitions simultaneously. Individual MS/MS parameters for each compound are shown in Table 1. Samples subjected to SPE were quantified by standard addition procedure to the extracts: 50 µL of a standard solution containing increasing concentration of analytes were added to 200-µL aliquots of each extract. TPrP was selected as internal standard and various raw and treated wastewater samples were checked to be free of it. Solid-Phase Extraction. Three commercially available cartridges were tested: Oasis HLB cartridges (3 mL, 60 mg; Waters, Milford, MA), Lichrolut EN (200 mg), and Lichrolut RP-18 (500 mg; both from Merck, Darmstadt, Germany). SPE recovery and matrix effects were determined as in a previous work23 but using treated wastewater as sample matrix. Stardards were spiked at three concentration levels, 0, 0.5, and 2.0 µg/L referred to sample. Solid-phase extraction (SPE) was performed with an Autotrace SPE Workstation (Zymark, Hopkinton, MA). Cartridges were sequentially conditioned with 5 mL MeOH and 5 mL ultrapure water. Samples (100 mL) were then passed through at a flow rate of 10 mL/min, the cartridges were rinsed with 2.5 mL of ultrapure water, dried for 30 min, and finally eluted with the appropriate volume of MeOH. The combined extracts were finally concentrated in a Turbovap II nitrogen concentrator (Zymark) down to ca. 0.3 mL, spiked with 40 µL of IS solution (5 µg/mL), and diluted to a final volume of 1 mL with ultrapure water.
Figure 1. Structures of analytes and internal standard and LCESI-MS/MS chromatogram of a 50 µg/L standard, MRM detection. Peaks of IS and 5 were slightly offset for clarity of presentation; they almost coelute with 3 and 4, respectively. For MRM transitions and retention times, refer to Table 1.
RESULTS AND DISCUSSION Chromatographic Separation. Chromatographic separation of the 11 analytes was obtained by reversed-phase chromatography using a methanol/water gradient with 0.2% of formic acid as organic modifier. Maximum signal intensity for most analytes was obtained at formic acid concentrations between 0.05 and 0.2%. The higher end of this optimum was selected because it provides a higher buffering capacity and was, hence, expected to result in a more robust separation and ionization. The chromatography of the OPFR was hardly influenced by the concentration of formic
acid and a good chromatographic separation is obtained for all analytes under these conditions (Figure 1). MS Fragmentation and Detection. The collision-induced dissociation of the analytes was studied by flow injection to establish the most sensitive transitions to be used for the quantitative determination in the multiple reaction monitoring (MRM) mode. The fragmentation of trialkyl- and trichloralkyl phosphates is dominated by three consecutive McLafferty rearrangements, by which the alkyl substituents are lost as neutral alkenes and protonated phosphoric acid (m/z 99) is obtained as the final cation.22,24 For some of these phosphates, namely, TCEP, TEHP, TBEP, TiBP, and TnBP, the McLafferty rearrangement is accompanied by a charge migration process. In these cases, the positive charge is transferred to one of the alkyl groups that appears as alkyl cation after splitting of the C-O bond (m/z 63 in Figure 2a), leaving phosphoric acid or a mono- or diester behind as neutral molecules. This fragmentation was most prominent for TCEP, whose spectrum (Figure 3a) shows also the protonated phosphoric acid monoethenylester (m/z 125 in Figure 2a), and for TBEP, where the third McLafferty rearrangement is not observed but the formation of the butoxyethyl cation at m/z 101 is observed. Many of the CID spectra obtained from the molecular
(23) Kloepfer, A.; Jekel, M.; Reemtsma, T. J. Chromatogr., A 2004, 1058, 8188.
(24) Lamouroux, C.; Virelizier, H.; Moulin, C.; Tabet, J. C.; Jankowski, C. K. Anal. Chem. 2000, 72, 1186-1191.
Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
3085
Figure 2. Proposed fragmentation pathways of the molecular cations of (a) TCEP, (b) TPhP, and (c) BDP under MS/MS conditions.
cations are very similar to those previously obtained by electron impact ionization (EI) and are stored in spectral libraries. For the aryl phosphate TPhP (Figure 2b), the McLafferty rearrangement is less favorable and only the first one is visible in the spectrum (m/z 251, Figure 3b). Two consecutive losses of water may result in m/z 233 and 215. A precursor ion scan proved that m/z 233 can also be generated from the molecular cation (m/z 327) by expulsion of phenol, as previously reported for some other phosphate esters by EI-MS/MS.25 The biphenyl cation [C12H9]+ (m/z 153) may be formed by a rearrangement from the molecular cation following a similar mechanism as described for dialkyl aryl phosphates.25 Additionally, an intense peak at m/z 77, corresponding to the aryl cation, is observed (Figure 3b) that may be formed from any of the previous fragment ions. Of the two bisphosphates, RDP turned out to be very stable, and fragmentation occurs only at high-collision energies generating low-intensity fragments. This high stability of the molecular cation can be attributed to its high degree of conjugation. The product ions recorded correspond to the loss of phenol (m/z 481) and of phosphoric acid monophenylester (m/z 419). On the contrary, BDP shows two clear fragments (Figure 3c) that are formed by a fission of the bond between the central quarternary carbon and one of the aromtic rings with a parallel hydrogen shift. This fragmentation yields the resonantly stabilized m/z 367 and 327 (Figure 2c). For TPPO, rupture of one phosphorus-carbon bond occurs with the positive charge occurring on either side, yielding m/z 201 (elimination of benzene) and the aryl cation [C6H5]+ (m/z 77). For analyte detection in LC-MS using the MRM mode, the two most intense transitions were selected from these fragmenta(25) Zeller, L. C.; Farrel, J. T., Jr.; Kentta¨maa, H. I. J. Am. Soc. Mass Spectrom. 1993, 4, 125-134.
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Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
tions, except for the internal standard TPrP where one transition was considered sufficient. In case of the very stable RDP, a pseudoMRM transition was used,26,27 in which chemical background is eliminated by increasing the collision energy whereas the molecular anion of the analyte remained largely intact. In this particular example, the highest intensity for the molecular cation (m/z 575) was obtained at low-collision energies (10 eV), but the best signal-to-noise (S/N) ratio is obtained at 20 eV (Figure 4). A low-intensity transition (m/z 575 > m/z 419) was used for confirmation. Solid-Phase Extraction. Three SPE sorbents were tested for elution volume, recovery, and matrix effects. First, Lichrolut EN was excluded as it took more than 8 mL of MeOH to elute the analytes from this sorbent, whereas less than 6 mL was sufficient for the other two phases. To distinguish between the true SPE recovery and matrix effects occurring during the electrospray ionization process, three different response factors (R) were experimentally determined for each set of experiments: external calibration with standards in ultrapure water (R1), standard addition into SPE extracts (R2), and standard addition into a sample prior to SPE (R3). Then, the ratio R3/R2 accounts for the SPE recovery whereas R2/R1 accounts for the matrix effects.23 Recoveries were better with Oasis HLB than with the C-18 material (Table 2), while matrix effects (in this case signal enhancement) were of similar extent. Therefore, Oasis HLB was selected for final method elaboration. The recovery of the late eluting more hydrophobic compounds (Table 2) could be improved by rinsing the sample bottle and the (26) Sancho, J. V.; Pozo, O. J.; Herna´ndez, F. Rapid Commun. Mass Spectrom. 2000, 14, 1485-1490. (27) Andreoli, R.; Manini, P.; Bergamashi, E.; Mutti, A.; Franchini, J.; Niessen, W. M. A. J. Chromatogr., A 1999, 847, 9-17.
Figure 4. LC-ESI-MS/MS of a treated wastewater extract spiked with RDP at the 50 ng/mL level. Transitions: (a) pseudo MRM 575 > 575 (10 eV collision energy), (b) pseudo MRM 575 > 575 (20 eV collision energy), and (c) MRM 575 > 419. Table 2. Comparison of the SPE Recovery (R3 × 100/ R2) and Matrix Effects (R2 × 100/R1) in a Treated Wastewater Sample for Lichrolut RP-18 and Oasis HLB Materiala recovery (%)
Figure 3. Product ion spectra of (a) TCEP, (b) TPhP, and (c) BDP obtained by ESI-MS/MS analysis in the positive ion mode.
tubings of the extraction apparatus twice with 5 mL of methanol. These methanolic solutions were transferred to the SPE cartridge and used for elution of the analytes. The final recoveries obtained, by the standard addition method to the extracts, with this improved procedure are shown in Table 3 and can be considered acceptable for most analytes, except for the most hydrophobic TEHP with 50-70% recovery only. Also, recoveries for RDP and BDP at the lowest level were not considered, as these compounds show less sensitivity and no pure standards were available. The recoveries obtained in this study are in the range of those reported in the literature for a polymeric cartridge (Bond Elute PPL)15 for TnBP, TCEP, and TBEP and a DVB polymeric disc17 for six of the trialkyl and trichloroalkyl phosphates and TPhP. Analytical Performance. The method validation data are summarized in Table 3. A good precision (RSD e 13%) and linearity (R2 ) 0.9865-0.9992) is obtained for aqueous solutions of analytes in the concentration range from 2 to 500 µg/L. Estimated limits of quantification (S/N g 10 for the most intense transition) after SPE of 100 mL of pure water spiked at the 0.1 µg/L level range from 3 ng/L for TPPO to 80 ng/L for BDP. Such experiments could not be done with real wastewater as most analyte concentrations were too high (see below).
matrix effects (%)
comp.
RP-18
Oasis HLB
RP-18
Oasis HLB
TCEP TPPO TCPP TDCP TPhP TiBP TnBP TBEP TEHP
73 69 60 60 43 49 43 47 21
82 90 83 75 65 66 69 71 28
109 138 160 141 174 187 160 176 203
112 131 166 139 160 189 208 153 131
a
Samples spiked at 0, 0.5, and 2.0 µg/L (referred to sample).
These LOQs are adequate for the determination of such compounds in wastewater samples and even for surface water samples. Also, they are better than those reported by LLE and GC-MS for the same volume of sample10 but are slightly higher than those reported for a set of six phosphoric acid triesters obtained by SPE of an unknown sample volume and GC-MS detection in the SIR mode.17 A very low LOQ of 3 ng/L is obtained for TPPO; here, LC-MS has the further advantage that no peak tailing occurs as in GC. The LOQs for RDP and BDP are in the range of 80 ng/L, which are the highest in this study. Even at that concentration level, confirmation may be difficult as the second MRM transition had a very low intensity (Table 1). Anyhow, this method is the first that allows the determination of these bisphosphates together with eight other OPFR and TPPO from water samples. For none of the analytes did breakthrough occur with volumes of 100 mL of pure water or treated wastewater. Thus, the sample volume for extraction could be increased if higher sensitivity is required. Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
3087
Table 3. Performance of the SPE-LC-MS/MS Method. Linearity and Precision of LC-MS Detection and Recoveryg from Different Sample Matrices at Different Spike Levels and Limits of Quantification LOQsc (ng/L)
mean recoveries (RSD)
TCEP TPPO TCPP TDCP TPhP TiBP TnBP TBEP RDP BDP TEHP
lineara/ R2
precisionb/
0.9960 0.9969 0.9992 0.9954 0.9986 0.9975 0.9850 0.9992 0.9940 0.9938 0.9865
10 5 9 5 8 9 12 4 4 4 13
RSD
pure water (0.1 µg/L)
pure water (1 µg/L)
treated wastewat. (4 µg/ L)
raw wastewat. (4 µg/L)
LC-MS/ MSd
SPE-LC-MS/ MSe
74 (7) 78 (8) 74 (8) 76 (8) 63 (8) 75 (12) 65 (11) 76 (4)
89 (2) 88 (2) 87 (3) 89 (1) 71 (2) 60 (2) 69 (4) 75 (3) 89 (6) 108 (11) 70 (1)
106 (2) 107 (2) 103 (4) 107 (3) 101 (3) 62 (3) 66 (1) 104 (4) 128 (5) 103 (10) 67 (16)
95 (5) 91 (4) 97 (5) 85 (1) 95 (4) 106 (7) 90 (2) f 100 (23) 105 (15) 50 (3)
1583 127 874 986 1472 1455 908 1273 3336 3681 1717
35 3 19 22 32 32 20 28 73 81 38
51 (4)
a Six-point calibration (2-500 µg/L, pure water standards). b Fifty microliter injection (n ) 5) of a 50 µg/L pure aqueous standard (2.5 ng on-column). c S/N g 10 (most intense transition). d Direct injection of 100 µL of treated wastewater. e Fifty microliter injection of an SPE extract of 100 mL of pure water. f No recovery determined because sample concentration exceeded added concentration by a factor of 3. g Determined by standard addition, %, mean of three experiments, with relative standard deviation, RSD.
Table 4. Concentration of the Analytes Found in 24-h Composite Samples of a Municipal Wastewater Treatment Planta compound TCEP TPPO TCPP TDCP TPhP TiBPc TnBP TBEP RDP BDP TEHP
Figure 5. Matrix effects occurring in LC-ESI-MS/MS (positive ion mode) of the analytes. Response of compounds added to wastewater extracts (R2) relative to the response in aqueous solution (R1). (Matrix effects for TBEP in raw wastewater could not be calculated, as its concentration in the sample exceeded added concentration by a factor of 3.)
A further advantage of LC-MS/MS over GC-MS is the possibility of directly injecting aqueous samples, without need for SPE, and quantification when the concentration level in the sample exceeds the LOQs that range between 0.13 µg/L for TPPO to 3.7 µg/L for BDP (Table 3). Thus, LC-MS/MS offers a rapid screening method for OPFR, as many of them are found at these levels in wastewater samples. Matrix Effects. Matrix effects that alter the response of analytes in sample matrix as compared to analytes in pure solution can severely compromise quantitation by LC-MS if they are neither compensated nor eliminated.28,29 Matrix effects were quantified as described above by determining the R2/R1 ratio.28 For treated wastewater, the matrix effects range from 0 to 49% and remain below 20% for six of the analytes (Figure 5). They (28) Quintana, J. B.; Reemtsma, T. Rapid Commun. Mass Spectrom. 2004, 18, 765-774. (29) Stueber, M.; Reemtsma, T. Anal. Bioanal. Chem. 2004, 378, 910-916.
3088 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
raw wastewater µg/L (SD)b
primary effluent µg/L (SD)
tertiary effluent µg/L (SD)
0.33 (0.01) 0.026 (0.004) 3.1 (0.1) 0.21 (0.01) nd
0.39 (0.03) 0.025 (0.001) 2.4 (0.2) 0.18 (0.01) nd
0.35 (0.02) 0.046 (0.001) 2.6 (0.1) 0.13 (0.01) nd
0.59 (0.01) 12 (1) nd nd nd
0.69 (0.01) 11 (1) nd nd nd
0.13 (0.01) 0.067 (0.013) nd nd nd
a Average and standard deviation (SD) of three independent analyses of each sample. b SD ) standard deviation; nd ) below LODs c TiBP could not be quantified because of a laboratory contamination.
were stronger in raw wastewater (11-74%). Figure 5 also shows that matrix effects differ strongly for the different analytes within and between samples and cannot be predicted. For example, signal enhancement (+49%) was observed for RDP in treated wastewater, whereas strong suppression occurred in raw wastewater (-74%). This problem has been widely reported in the literature for different classes of compounds,28-31 including TCEP, where a 65% suppression was observed in an extract of surface water.31 Thus, the use of one internal standard (TPrP) cannot compensate for these matrix effects and, therefore, quantification of samples has to be done by the standard addition procedure. Indeed, the standard addition procedure increases the analysis time and introduces a further source of potential error. However, it has demonstrated to be the best approach when no isotopically labeled reference compounds are available,29 as it is the case for most of the analytes. Application to Real Samples. This analytical method was used to determine OPFR in samples from different stages of a (30) Benijts, T.; Dams, R.; Lambert, W.; De Leenheer, A. J. Chromatogr., A 2004, 1029, 153-159. (31) Vanderford, B. J.; Pearson, R. A.; Rexing, D. J.; Snyder, S. A. Anal. Chem. 2003, 75, 6265-6274.
or TPPO (Table 4). This agrees with previous investigations of trichloroalkyl phosphates in wastewater treatment17 and also with the occurrence of trichloroalkyl phosphates and TPPO in surface waters and bank filtrate.18 The novel bisphosphates RDP and BDP could not be detected in the wastewater, which may reflect their limited market share. Lack of occurrence of TEHP in the water phase may be ascribed to its strong tendency to sorb onto particulate organic matter that can be deduced from its octanol-water partition coefficient (log Kow) varying between 4.2 (measured32) and 9.5 (calculated33).
Figure 6. Chromatogram of a (nonspiked) treated municipal wastewater: (a) after SPE of 100 mL of sample (50 µL extract injected) and (b) by direct injection of the original sample (100 µL) without SPE. Concentrations ranged from 46 ng/L (TPPO) to 2.6 µg/L (TCPP). For MRM transitions, refer to Table 1.
municipal wastewater treatment plant (Table 4). Figure 6a shows the chromatogram of an extract from a tertiary treated effluent, in which seven of the analytes are detected. The same sample analyzed by direct injection (no SPE) is shown in Figure 6b. Though some of the analytes are below the limits of quantification, most of them are still detectable (except TnBP and TiBP), demonstrating the potential of direct injection LC-MS/MS without extraction as a fast screening method. These seven analytes were detected in all three untreated wastewater samples in concentrations ranging from 25 ng/L (TPPO) up to 12 µg/L (TBEP). A marked concentration decrease in the activated sludge treatment was visible for TBEP and TnBP, whereas this was not discernible for the trichloroalkyl phosphates
CONCLUSIONS A method for the determination of 11 organophosphorus flame retardants and plasticizers in water samples has been developed. After optimization, the combination of SPE and LC-ESI-MS/ MS allowed the determination of these compounds at the low ng/L level (LOQs ) 3-80 ng/L) for a sample volume of 100 mL. The LC-MS/MS determination offers the advantage that aqueous samples can be directly injected without previous enrichment by SPE if target analytes occur at low µg/L concentrations, thus providing a fast screening procedure for many wastewater samples. Six of the phosphoric acid triesters and TPPO were found in concentrations as high as 12 µg/L of TBEP in raw wastewater and 2.6 µg/L of TCPP in treated wastewater. This points to the need of a more detailed evaluation of the fate of these compounds in the environment, including particulate matter as well as possible metabolites. ACKNOWLEDGMENT We are grateful to Akzo Nobel (Amersfoort, The Netherlands) for kindly supplying us with samples of Fyroflex RDP and Fyroflex BDP. R. Rodil and J.B. Quintana acknowledge Xunta de Galicia and Ministerio de Educacio´n y Ciencia, respectively, for their grants. T. Reemtsma acknowledges funding by European Union for the project Removal of Persistent Polar Pollutants Through Improved Treatment of Wastewater Effluents (P-THREE; EVK1CT-2002-00116). Received for review November 26, 2004. Accepted February 17, 2005. AC048247S (32) Saeger, V. W.; Hicks, O.; Kaley, R. G.; Michael, P. R.; Mieure, J. P.; Tucker, E. S. Environ. Sci. Technol. 1979, 13, 840-844. (33) European Commission, European Chemicals Bureau. IUCLID, dataset tris(2ethylhexyl)phosphate, EINECS No. 201-116-6, 2001. http://ecb.jrc.it/ESIS.
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