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Chapter 20
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Analysis of Halogenated Alkylphenolic Compounds in Environmental Samples by LC/MS and LC/MS/MS Mira Petrovic and Damià Barceló Department of Environmental Chemistry, IIQAB-CSIC, c/Jordi Girona 18-26, 08034 Barcelona, Spain
The state-of-the-art in the LC-MS and LC-MS-MS analyses of halogenated alkylphenolic compounds formed during wastewater and drinking water chlorination is reviewed. Single stage LC-MS with ESI ionization showed limitations in the analysis of real-world samples, resulting from the presence of isobaric interferences when only deprotonated molecules are monitored. With collision-induced dissociation halogenated NPs and NPECs gave specific fragments formed by the cleavage of the alkyl moiety or by the loss of (ethoxy)carboxylic group, respectively. Characteristic pattern of isotopic doublet signals of these specific transitions coupled with formation of [Br] and [C1] permitted unequivocal identification of halogenated alkylphenolic compounds in environmental samples. Limits of detection by LC-MS and LC-MS-MS were in the range of 10 and 1 ng/1, respectively. -
-
Alkylphenol ethoxylates (AP EOs, w=number of ethoxy units) are widely used nonionic surfactants. The findings on toxicity and weak estrogenicity of some of APEOs persistent metabolites have raised concern over their n
338
© 2003 American Chemical Society
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
339 environmental and health effects (1-4). As shown in a simplified way in Figure 1 APEOs may be transformed in wastewater treatment plants (WWTP) or in the environment by: (i) shortening of the polyethoxy chain producing mainly ΑΡ,ΕΟ, AP EO and fully deethoxylated alkylphenols (AP); and (ii) carboxylation of the tenninal EO units or/and the alkyl side chain resulting in the formation of alkylphenol carboxylates (APEC) and carboxyalkyl phenol carboxylates (CAPEC), respectively. These metabolites are susceptible to orthohalogen substitution and they can be further transformed into halogenated by products during chlorination in water treatment works. While the occurrence of non-halogenated metabolites in the environment and their ability to mimic the endogenous hormone, 17P-estradiol, is well documented (1,2), only little data exist on the occurrence and ecotoxicology of brominated and chlorinated alkylphenolic compounds. One of the reasons for this is the low relative abundance of these compounds (generally less than 10% of the total pool of alkylphenolic compounds) and lack of appropriate analytical methods for their unequivocal identification and quantification. Early methods included fast atom bombardment-mass spectrometry (FABMS) (5-9), which proved to be a reliable tool for the identification of halogenated metabolites in raw and drinking water, but not for their quantification. Recently, several authors reported the analysis of halogenated alkylphenolic compounds by gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography with electrospray mass spectrometry (LC-ESI-MS) or tandem mass spectrometry (LC-ESI-MS-MS) (Table I).
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Table I. Survey of analytical methods for quantitative determination of halogenated alkylphenolic compounds Compound XAP, XAPEC
Matrix Water Sludge
Sample preparation Clean-up Extraction SPE-Cis PLE
Analytical Ref method LC-MS-MS (10)
SPE-Cis
(MeOH-Acetone, 1:1)
XAP, XAPEC, Water XAPEO Sludge Sediment XNP Sediment
SPE-Ci Sonication (MeOH-DCM, 7:3) 8
Continuous-flow sonication (MeOH)
SPE-Ci
LC-MS
(11)
LC-MS
(12)
8
SPE-NH2
RP-HPLC
XNPEC XNPEO
Water
SPE-Cis
derivatization to methyl-esters
GC-MS
(13)
BrNP, BrNPEO
Water
SPME
-
GC-MS
(14)
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
r
Short alkylphenol ethoxylates R = octyl (OPEO) R = nonyl (NPEO)
Alkylphenol ethoxylates R » octyl (OPEO) R • nonyl (NPEO)
ι n=1-2 Chlorination
Aerobic biodégradation
eo
n (average)=9-10
OH
Halogenated alkylphenoxycarboxylates R = octyl (BrOPEC, CIOPEC) R = nonyl (BrNPEC, CINPEC)
OCHoCOOH
Halogenated alkylphenol ethoxylates R = octyl (BrOPEO, CIOPEO) R • nonyl (BrNPEO. CINPEO)
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
OH
\Anaerobic biodégradation
2
OCH COOH " η =0-1
Halogenated alkylphenols R = octyl (BrOP, OOP) R = nonyl (BrNP, CINP)
OH
Figure 1. Biodégradation pathway and by-products formation of alkylphenol ethoxylates
Alkylphenols R = octyl (OP) R = nonyl (NP)
Alkylphenoxy carboxylates R = octyl (OPEC) R = nonyl (NPEC) Γ
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342 This review will focus on the current state-of-the-art in the LC-MS and LCMS-MS analyses of halogenated alkylphenolic compounds. It also summarizes knowledge on the occurrence and ecotoxicity of this group of contaminants and addresses future research needs in this area.
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Analysis of halogenated alkylphenolic compounds
LC-MS Owing to the presence of chlorine and bromine atoms, respectively in the molecules, halogenated alkylphenolic derivatives yield a characteristic pattern of isotopic doublet signals. This provides a highly diagnostic fingerprint for this group of compounds. Halogenated APEOs were detected using an ESI-MS under positive ionization (PI) conditions (11). Like their non-halogenated analogs, halogenated APEOs show a great affinity for alkali metal ions, and they produce exclusively evenly-spaced (Δ44 Da) sodium adduct peaks [M+Na] with no further structurally significantfragmentation.Problem arise from the fact that the chlorinated derivatives (ClAP EO) have the same molecular mass and they gave the same ions as brominated compounds with one ethoxy group less (BrAP^EO) and chromatographic separation of these two groups of compounds, which is quite difficult to obtain, is a prerequisite for their quantitative determination. However, the characteristic doublet signal of brominated and chlorinated compounds, respectively due to different contribution of their isotopes ( Br: Br=100:98 and C1: C1=100:33, respectively) provides additional confirmation of their presence. An example of the identification of halogenated NPEOs by LC-ESI-MS is shown in Fig. 2. A similar ion series at m/z from 409/411 to 851/853 revealed the presence of CINPEOs (« =3-13) and BrNPEOs (% =2-12), respectively. In addition halogenated OPEOs were also identified. Several authors reported determination of halogenated NPs (11,12) and NPECs (11) by LC-MS under negative ionization (NI) conditions. Using an ESI interface, halogenated NPs gave a doublet signal of the [M-H]~ ions at m/z 297/299 for BrNP and at m/z 253/255 for C1NP (Fig. 3A and B). Halogenated NPECs gave two signals, one corresponding to deprotonated molecule and another to [M-H-CH COOH]~ in the case of X N P ^ C s or [M-HCH CH OCH COOHr for XNP Ecs (Fig. 3C-F). +
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
343 However, real application showed that with "soft ionization" LC-MS, giving solely deprotonated molecule or very limited number of fragments, the identification of halogenated compounds is quite difficult. Using a single stage of mass selectivity in the analysis of real-world samples, CINPjEC was obstructed by a severe isobaric interference of linear alkyl benzene sulfonate (C LAS), which is often found in environmental and wastewater samples in concentrations several orders of magnitude higher than those of halogenated alkylphenolic compounds (11). All attempts to separate ClNPjEC and C L A S using gradient elution with standard mobile phases for reversed-phase separation (methanol/water or acetonitrile/water) failed. Detennination of CINPjEC was achieved by monitoring fragment ion at m/z 253/255, which also suffered the isobaric interferences in some real samples. n
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LC-MS-MS Driven by the estrogenic potency of some compounds and their low environmental concentrations, the detection limits required for the monitoring of endocrine disruptors are being pushed from the microgram to the nanogram or even to below nanogram per liter range. Currently, a breakthrough is occurring for analytical methods based on LC-MS-MS. However, although considered as one of the most powerful techniques for structure interpretation and quantification, LC-MS-MS has been seldom used in the analysis of acidic and neutral NPEO metabolites and currently only a single paper describes the application of ESI-MS-MS in the analysis of their halogenated derivatives (10). ESI-MS-MS permitted unambiguous identification and structure elucidation under negative ionization conditions (halogenated NPECs and NPs produced signal), while NPEOs under positive ionization conditions produced no fragmentation. As a result these compounds were analyzed using a single stage MS monitoring [M+Na] adduct ion in SIM mode. With collision-induced dissociation (CID) under NI conditions, halogenated NPECs and NPs undergofragmentationwith few major pathways depicted in Fig. 4. For halogenated NPjECs and NP ECs the predominant reaction was loss of CH COOH and CH CH OCH COOH, respectively that resulted in intense signals at m/z 253/255 for CINPECs and m/z 297/299 for BrNPECs. Further fragmentation of [C1NP]~ occurred primarily on the alkyl moiety leading to a sequential loss of m/z 14 (CH group), with the most abundant fragments at m/z 167 for C1 and m/z 169 for C1 with the relative ratio of intensities of 3.03. Fragment corresponding to the [Cl]~ ion was observed only when sufficient collision energy was applied. The intensity of this ion was not very pronounced, but nevertheless remained useful for the identification of chlorinated NP. The +
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. f
Figure 2. ESI mass spectra of halogenated APEOs detected in the flocculation sludge from drinking water treatment plant of Barcelona. A, CINPEO; B BrNPEO; C, ClOPEO (assigned as β ) ; D, BrOPEO (assigned as M). Reproducedfrom(11) ©2001 American Chemical Society
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. Ν»
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Figure 3. ESI mass spectra of standards of halogenated alkylphenolic compounds detected under NI conditions. A, CINP; B, BrNP; C, CINPjEC; D, BrNPjEC; E, CINP2EC; F, BrNP2EC. Reproduced from (11)© 2001 American Chemical Society
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348 brominated compounds showed a markedly different fragmentation pathway. Even at low collision energy [BrNP]~ (m/z 297/299) yielded intense signals at m/z 79 and m/z 81 corresponding to the [Br]~ (ratio of isotopes 1.02), while the fragmentation of the side chain was suppressed and resulted just in a lowintensity fragment at m/z 211/213 resulting from loss of C H . Such difference in the mechanism offragmentationof chlorinated and brominated compounds is presumably the consequence of the lower energy of a B r - C H (benzene) bond compared to a C1-C H bond. 6
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Figure 4. Tentativefragmentationmechanisms for halogenated alkylphenolic compounds in the ESI-MS-MS
A summary of all the ions formed under CID-ESI conditions is shown in Table II. From the observed ion fragmentation pathways a reliable and sensitive quantification method, that overcomes the main drawbacks on existing LC-MS methods, was developed. For each compound, two specific multiple reaction
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
349
monitoring (MRM) channels were chosen for quantitation purposes. Generally, [M-H]" ions where chosen as precursor ions, with the exception of halogenated NP ECs. Even at low cone voltages (5-10 V), in-source CID was obtained for these compounds and their spectra displayed low abundance of the deprotonated molecules. Thus, for these compounds the most intense M R M channels were those monitoring the fragmentation of "the first generation" products (e.g. 297/299 -> 79/81 for BrNP EC and 253/255 -> 167/169 for C1NP EC). 2
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2
2
Table Π. Fragments formed with CID in ESI-MS-MS Compound Precursor ion (m/z)
Product ions (m/z)
BrNP
297/299 [M-H]'
79/81 (100) ; 211/213 (10)
BrNP,EC
355/357 [ M - H f
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BrNP EC
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C1NP
253/255 [ M - H f
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C1NP,EC
311/313 [ M - H f
253/255 (100); 167/169 (45); 181/183 (15);
C1NP EC
355/357 [M-H]-
253/255 (100); 167/169 (45); 181/183 (20); 195/197 (15)
2
2
a
relative abundance at collision energy of 30 eV using argon as collision gas
The high selectivity of the MS-MS detection of halogenated nonylphenolic compounds is shown in Figures 5 and 6, which display extracted M R M chromatograms for halogenated NPECs and NPs in chlorinated river water and in the DWTP flocculation sludge, respectively. The specificity of M R M mode permitted unequivocal identification and quantification of isobaric target compounds (e.g. BrNP^C and C1NP EC) and eliminated interference from coeluting isobaric no-target compounds (e.g. C L A S ) observed using a LC-MS in SIM mode. Positive identification detection criteria were based on: a) L C retention time of the analyte compared to that of a standard (± 2%), and b) the ratio of abundances of two specific precursor ion -» product ion transitions (within 10% of the ratios obtained for the standard). For halogenated compounds a highly diagnostic criteria was the abundance ratio of characteristic isotope ratios ( Br : 2
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Br=1.02; C1 : C1=3.03), and for each transition reaction two specific channels, corresponding to the two isotopes of interest, were monitored and the ratio of their abundances calculated. The detection limits of the LC-MS-MS method (10) fell down to 1-2 ng/L for the analysis of halogenated NPECs and NPs in water samples (after SPE preconcentration) and to 0.5-1.5 ng/g in sludge samples (target compounds were extracted using pressurized liquid extraction), which is significant improvement in comparison to LODs reported previously for GC-MS (14) and LC-MS (11) methods. 253>167 3.16e3
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10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time Figure 5. LC-MS-MS analysis of halogenated alkylphenolic compounds in chlorinated river water (the Llobregat river, Spain). MRM mode: A) 253 ->167 for detection of CI nonylphenolic compounds; Β ) 297 -> 79 for BrNP; B) 355 -> 297 for ^BrNPjEC; D) 399 -> 297for BrNP2EC 35
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In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Figure 6. LC-MS-MS analysis of halogenated nonylphenols in the flocculation sludge from the DWTP Sont Joan Despi in Barcelona (Spain). MRM mode: 297 -> 79 and 299-* 81 for BrNP; 253 ->167 and 255 -> 169 for CINP
Occurrence in the Environment Studies to date have largely focused on short chain NPEOs, NPECs and NP, and only few reports have included halogenated metabolites. Table III lists the reported data on the levels of halogenated alkylphenolic compounds in environmental and wastewater samples. Fujita et al. (16) detected halogenated NPEOs and NPECs in the secondary effluents and in final effluents of 25 out of 40 WWTP studied across Japan. They were produced via chlorination at concentrations up to 52.4 μg/l, for the most abundant BrNPECs, accounting for up to about 10% of nonylphenolic compounds. In another study (12,15), halogenated NPs were found in a sewageimpacted urban estuary sediments (Jamaica Bay, USA). The authors concluded that CINP and BrNP are likely not of major toxicological concerns due to their
In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
352
Table III. Reported levels of halogenated alkylphenolic compounds in environmental and wastewater samples Matrix
Compound
Estuary sediment (Jamaica bay, CINP NY, USA) BrNP
