MS and Time of Flight

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).


2

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)



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)



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


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.


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). +

n

79

EO

81

35

37

0

2

2

2

2

2


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


U

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 +

2

2

2

2

2

2

35

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50

100

400

409/411

453/455

ι

500

4Θ7/49Θ

600

700 800

m/z

851/853

805/807

761/763

717/719

673/675

629/631

541/543 585/587

50

100

h

4'od

s'od

6'oo

7bd

e'oo

851/853

805/807

761/763

673/675 717/719 629/631 585/587 541/543 497/499 453/455

409/41

Β


m/z

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


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 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


352

Table III. Reported levels of halogenated alkylphenolic compounds in environmental and wastewater samples Matrix

Compound

Estuary sediment (Jamaica bay, CINP NY, USA) BrNP

MS and Time of Flight

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