Atmospheric Pressure Ionisation Mass

SPECTROMETRY—III. NON-IONIC SURFACTANTS: LC-MS AND LC-MS-MS OF ALKYLPHENOL ETHOXYLATES AND THEIR DEGRADATION PRODUCTS

Mira Petrovic, Horst Fr. Schröder and Damia Barcelo

2.6.1 Alkylphenol ethoxylates (APEOs)

Liquid chromatography-mass spectrometry (LC-MS) analysis of APEOs has been attempted using both normal-phase (NP) and reversed-phase (RP) systems. Using NP-LC, the APEOs are separated according to the increasing number of ethylene oxide units, while corresponding oligomers with the same number of ethoxy units, but different alkyl substituents; e.g. nonylphenol ethoxylates (NPEOs) and octylphenol ethoxylates (OPEOs), co-elute. RP-LC on C8, C18 silica gel columns, alumina-based C18 or polyethylene-coated alumina allows separation according to the character of the hydrophobic moiety and it is particularly well-suited to separate surfactants containing various hydrophobic moieties (homologue-by-homologue separation). In this case, the length of the ethylene oxide chain does not influence the separation and the various oligomers containing the same hydrophobic moiety elute in one peak. However, concentration of individual oligomers (e.g. NPEO1, NPEO2, NPEO3, etc.) can be readily obtained by extracting total ion chromatograms for characteristic m/z values (Fig. 2.6.1). This approach is suitable for routine determination of OPEOs and NPEOs since the quantification is simplified and eluting all the oligomers into one peak has the advantage of increasing the peak intensity and therefore, increasing the sensitivity of determination.

APEOs can be detected using both electrospray (ESI) and atmospheric pressure chemical ionization (APCI), under positive ionization (PI) conditions. Generally, it was found that ESI offers better sensitivity and specificity for a higher range of oligomers than does APCI [1]. However, it is important to mention that the overall sensitivity of MS detection—as well as the formation of adducts and fragmentation— depends strongly on the source design and can vary significantly depending on the particular instrument used. Specific issues related to

Comprehensive Analytical Chemistry XL

© 2003 Elsevier Science B.V. All rights reserved

Fig. 2.6.1. RP—LC—ESI—MS analysis of flocculation sludge from a Barcelona drinking water treatment plant. Column C18 LiChrolute 250 X 4.6 mm, 5 mm, gradient elution with ACN—water. Upper trace total ion current (TIC), lower traces extracted ion chromato-grams for NPEOs, nEO = 1 -5. Inset: ESI mass spectrum of NPEO oligomeric mixture.

Fig. 2.6.1. RP—LC—ESI—MS analysis of flocculation sludge from a Barcelona drinking water treatment plant. Column C18 LiChrolute 250 X 4.6 mm, 5 mm, gradient elution with ACN—water. Upper trace total ion current (TIC), lower traces extracted ion chromato-grams for NPEOs, nEO = 1 -5. Inset: ESI mass spectrum of NPEO oligomeric mixture.

the formation of distinct adducts with ions originating from the buffer, the sample and/or the introduction system (e.g. H+, Na+, K+, Cl", CH3COO"), the formation of cluster and multiple charged ions, ionisation suppression caused by co-elution of other analytes or natural matrix components or due to the presence of ion-pairing agents or high salt concentrations in the mobile phase buffer used, as well as issues related with the response factors of different oligomers are discussed in detail in Chapter 4.3. (Quantitation in surfactant analysis.)

Several authors have reported the identification and determination of APEOs in industrial blends, wastewaters and environmental samples by APCI-MS using flow injection analysis (FIA) [2,3] or preceded by LC, using both RP [4-9] and NP [10,11] separation.

APCI-MS analysis of OPEO in river water and effluents of wastewater treatment plants (WWTP) applying FIA and ionisation promoted by applying ammonium acetate, resulted in equal spaced (D m/z 44) [M + NH4] + ions starting from m/z 312 and ending at m/z 972 [12]. The oligomeric distribution followed a Gaussian curve with a maximum at nEO = 9. LC-APCI-MS was also applied for the determination of NPEOs in tannery wastewaters [8]. Besides NPEO compounds, biochemical oxidation products of PEGs (carboxylated PEGs), resulting from degradation of non-ionic surfactants were determined.

The alicyclic homologue of Triton X-114, an octylcyclohexylethoxylate ('reduced' octylphenolethoxylate) mixture (C8H17-C6H10-O-(CH2-CH2-O)m-H) and the aromatic OPEOs, were examined. Their APCI-FIA-MS(+) spectra are presented in Fig. 2.6.2(a,b), respectively. APCI-LC-MS(+) on a RP-C18 column was applied to separate and differentiate the constituents of this mixture; UV-DAD spectra were recorded in parallel to distinguish the aromatic and the alicyclic compounds. In Fig. 2.6.2 (6) the reconstituted ion current (RIC) and mass traces of aromatic (2 and 3) and alicyclic (4 and 5) homologues both ionised as [M + NH4] + ions equally spaced with Dm/z 44 u are presented. The UV trace 220 nm is plotted in Fig. 2.6.2 (1). Here the aromatic ring system serving as chromophore can be recognised, whereas the alicyclic compounds without chromophoric groups cannot be observed [13].

ESI-MS methods, combined with NP-LC [14-16] or RP-LC [17-21] have been frequently used for the quantitative analysis of ethoxylates in environmental and wastewater samples.

Tic Chromatogram

Fig. 2.6.2. APCI—LC—MS(+) TIC chromatogram (6) and selected mass traces of (2) and (3) of octylphenolethoxylates (OPEOs) together with (4) and (5) their alicyclic homologues, the octylcyclohexanolpolyglycolether; (1) UV trace 220 nm presenting

OPEO signals [13].

Fig. 2.6.2. APCI—LC—MS(+) TIC chromatogram (6) and selected mass traces of (2) and (3) of octylphenolethoxylates (OPEOs) together with (4) and (5) their alicyclic homologues, the octylcyclohexanolpolyglycolether; (1) UV trace 220 nm presenting

OPEO signals [13].

Normal-phase LC-ESI-MS was used for the quantitative determination of individual NPEOs in complex environmental matrices (e.g. marine sediment) [14]. The best chromatographic separation of NPEO oligomers was achieved under NP-LC conditions using non-polar solvents as mobile phases (e.g. gradient elution with toluene as solvent A and 0.5 mM NaOAc in toluene-methanol-water (10:88:2, v/v) as solvent B). However, such mobile phases are not compatible with ESI, and post-column addition of a polar solvent and a modifier is required to facilitate ionisation of the target analytes and evaporation of the preformed ions into the gas phase via the electrospray process and thus to enhance the signal and system stability. High oligomer NPEOs benefit most from the post-column addition (mobile phase B at a flow-rate ratio of 0.75:1) with over 10-times signal intensity enhancement, whereas low oligomers (NPEO1, NPEO2) show approximately a three times increase in signal intensity compared with results from no post-column addition.

Using reversed-phase LC-ESI-MS, NPEOs (nEO = 1 -20) were detected in samples of raw and treated wastewater, river and drinking (tap) water after their pre-concentration by solid phase extraction (SPE) using GBC material and differential elution [22]. RP-LC separation of the NPEOs and electrospray application for MS coupling allowed sensitive detection of higher ethoxylates (nEO > 3). However, the method gave very low sensitivity for NPEO1 and NPEO2, and they were quantified using fluorescence (FL) detection.

NPEOs and OPEOs (nEO = 3-10) as industrial blends or standard compound (Triton X-100), respectively, were separated together with linear alkylbenzene sulfonates (LASs) on a C1-RP column [10]. The intensive ions that could be observed in the spectra were mono-, di- and tri-sodium adduct ions [M + Na]+ (m/z 581), [M + 2Na]+ (m/z 604) and [M + 3Na]+ (m/z 626) of the EO7 homologue. The intensity of the molecular [M + H]+-ion, however, was small compared with the sodium adduct ions. The compounds had been concentrated prior to separation on C18 and SAX SPE cartridges. Samples from river water were handled in the same way.

Ferguson et al. [18] reported on the application of a mixed-mode HPLC separation, coupled with ESI-MS for the comprehensive analysis of NPEOs and nonylphenol (NP) concentrations and distributions in sediment and sewage samples. The mixed-mode separation, which operates with both size-exclusion and reversed-phase mechanisms, allows the resolution ofNPEO ethoxymers prior to introduction to the MS using a solvent system that is readily compatible with ESI. In this method, elution of NPEOs is reversed relative to normal-phase chromatography, with smaller, less ethoxylated compounds, including NP, eluting last (Fig. 2.6.3). The separation allows all NPEOs and NP to be quantified in a single chromatographic run, while removing the effects of isobaric interferences and co-analyte electrospray competition.

Recently, Shao et al. [23] reported on the complete separation of individual NPEO (nEO = 2-20), which was achieved combining a C18 pre-column with a silica analytical column, using acetonitrile-water as eluent followed by ESI-MS detection. The method used ramped cone voltage from 25 to 70 V, which suppressed the appearance of doubly charged adducts effectively and increased the response of oligomers of relatively high molecular mass.

Several methods, applying MS-MS techniques, for the identification and characterization of APEOs and their metabolites, alkylphenoxy carboxylates (APECs) and alkylphenols (APs) in environmental liquid and solid samples and industrial blends are reported. APCI-MS-MS

0 -I-.-.-.-.-1-1-.-.-,-.-,-.-.-1-.-,-,-,-,-,-,-,-.-1-,-.-1-r-

Time (minutes)

Fig. 2.6.3. Mixed-mode HPLC-ESI-MS summed ion chromatogram of a sediment extract (32-36 cm depth core slice) showing resolution of NPEOs and NPs. Numbered peaks correspond to NPEOs and [13C6]NPEOs with the indicated number of ethoxy groups (0 = NP, [13C6]NP; 1 = NPEO1, [13C6]NPEO1, etc.). Peaks 'A' and 'B' are the internal standards, n-NP and n-NPEO3, respectively. (Note the discontinuity at retention time 25.8 min, corresponding to the shift in MS polarity from positive to negative ion mode.)

Reprinted with permission from Ref. [18] © 2001 Elsevier.

0 -I-.-.-.-.-1-1-.-.-,-.-,-.-.-1-.-,-,-,-,-,-,-,-.-1-,-.-1-r-

Time (minutes)

Fig. 2.6.3. Mixed-mode HPLC-ESI-MS summed ion chromatogram of a sediment extract (32-36 cm depth core slice) showing resolution of NPEOs and NPs. Numbered peaks correspond to NPEOs and [13C6]NPEOs with the indicated number of ethoxy groups (0 = NP, [13C6]NP; 1 = NPEO1, [13C6]NPEO1, etc.). Peaks 'A' and 'B' are the internal standards, n-NP and n-NPEO3, respectively. (Note the discontinuity at retention time 25.8 min, corresponding to the shift in MS polarity from positive to negative ion mode.)

Reprinted with permission from Ref. [18] © 2001 Elsevier.

showed ethoxy chain fragments at m/z 89, 133, 177 and the diagnostic fragment of OPEOs at m/z 277 and 291 for NPEOs, which can be explained as shown in Fig. 2.6.4 [12,24—26]. Mixture analysis by FIA— MS —MS(+ ) was applied for the confirmation using the diagnostic precursor scans of m/z 277 and 291 for the detection and identification of OPEOs and NPEOs, respectively [25].

The precursor ion scanning of m/z 121 and 133 and multiple reaction monitoring (MRM) applying APCI—FIA—MS—MS(+) were used for a rapid screening of NPEOs as contained in the industrial blend Igepal CP-720. The precursor scan (PS) of 121, characteristic for ethoxylates with 1—4 chain units (EO1—EO4), and the PS of 133, characteristic for EO5—EO16, demonstrated a preferential elimination of the EOs—EO^ NPEOs from wastewater samples. The PS alone was not characteristic for these compounds because linear alcohol ethoxylates resulted in the same precursors; monitoring 16 MRM transitions allowed the confirmation of results [27].

For confirmation of low concentrations of NPEO homologues in complex samples from the Elbe river, APCI—LC—MS—MS(+) was applied to record the substance-characteristic ion mass trace of m/z 291. The SPE isolates contained complex mixtures of different surfactants. The presence of NPEOs in these complex samples was confirmed by generating the precursor ion mass spectrum of m/z 291 applying MS— MS in the FIA—APCI(+) mode. This spectrum, showed in Fig. 2.6.5, presents the characteristic series of ions of NPEOs at m/z 458, 502,...,678, all equally spaced with Dm/z 44 u. Besides the NPEOs, small amounts of impurities could be observed because of the very low concentrations of NPEOs in the water sample [25]. In the foam sample, the identity of NPEOs could be easily confirmed by APCI—FIA—MS— MS(+) because of their high concentrations in this matrix. The LC—

Fig. 2.6.4. Substance specific fragments for characterisation of APEOs [13,26].
Isomer C6h10
Fig. 2.6.5. APCI-FIA-MS-MS(+) (CID) parent ion mass spectrum of product ion at m/z 291 ofC18-SPE surface water extract from the river Elbe presenting NPEO characteristic ions at m/z 458, 502, 546, 590, 634 and 678, all equally spaced with Dm/z 44 [25].

MS-MS(+) product ion spectrum of the NPEO homologue [M + NH4]+ ion at m/z 678 with nEO = 10 together with its fragmentation scheme is presented in Fig. 2.6.6.

The alicyclic isomer of Triton X-114, an octylcyclohexylethoxylate (reduced octylphenolethoxylate) mixture (C8H17-C6H10-O-(CH2-CH2-O)n-H) was examined by APCI-FIA-MS-MS(+). The equally spaced ions (Dm/z 44 u) could be fragmented as presented in the fragmentation scheme together with the daughter ion spectrum in Fig. 2.6.7. No fragmentation could be observed in the alkyl chain; however, unlike aromatic homologues, a bond scission took place between the alcoholic oxygen and the alicyclic ring system. PEG ions as base peak ions with the general structure HO-(CH2-CH2-O)x-H were generated according to the PEG chain length of the precursor parent ion while a neutral loss of 193 could be observed in parallel [28].

Methods applying LC-MS-MS for the quantitative determination of APEO were seldom reported. Houde et al. [29] recently described a LC(ESI)-MS-MS method for the determination of NPEO and nonylphenoxy carboxylates (NPEC) in surface and drinking water using a reversed-phase column (C8) with isocratic elution. Transitions from the [M + NH4]+ ions to different product ions (m/z 127, 183, 227, 271, 315, 359 for NPEO nEO = 1 -6 and m/z 291 for nEO = 7-17) were monitored, yielding detection limits from 10 to 50 ng L"1.

Mass Spec Polystyrene

Fig. 2.6.6. APCI-LC-MS-MS(+ ) (CID) daughter ion mass spectrum of [M + NH4]+ ion at m/z 678 generated from Ci8-SPE of foam sample. Compound could be identified as non-ionic surfactant NPEO (CgHig-C6H4-O-(CH2-CH2-O)m-H); (inset)

fragmentation scheme under CID conditions [28].

Fig. 2.6.6. APCI-LC-MS-MS(+ ) (CID) daughter ion mass spectrum of [M + NH4]+ ion at m/z 678 generated from Ci8-SPE of foam sample. Compound could be identified as non-ionic surfactant NPEO (CgHig-C6H4-O-(CH2-CH2-O)m-H); (inset)

fragmentation scheme under CID conditions [28].

Tripalmitin Mas Spectra

Fig. 2.6.7. APCI-FIA-MS-MS(+ ) daughter ion mass spectrum of [M + NH4]+ ion of octylcyclohexylethoxylate homologue at m/z 582 from 'reduced' octylphenolethoxylate mixture (C8H17-C6H10-O-(CH2-CH2-O)m-H); (inset) fragmentation scheme under

CID conditions [28].

Fig. 2.6.7. APCI-FIA-MS-MS(+ ) daughter ion mass spectrum of [M + NH4]+ ion of octylcyclohexylethoxylate homologue at m/z 582 from 'reduced' octylphenolethoxylate mixture (C8H17-C6H10-O-(CH2-CH2-O)m-H); (inset) fragmentation scheme under

CID conditions [28].

2.6.2 Alkylphenols (APs)

Octylphenol (OP) and NP, were detected under negative ionisation (NI) conditions, using both APCI and ESI interfaces. The sensitivity of detection, using an ESI source was approximately 40-50 times higher than that obtained with an APCI source [30]. In contrast to the GC-MS analysis that reveals the presence of 22 isomers of the alkyl chain in the technical mixture of 4-NP, LC-MS analysis yields a single very broad peak. Using an ESI, APs give exclusively [M — H] — ions with m/z 205 for OP and m/z 219 for NP as shown in Fig. 2.6.8.

Using an APCI, at higher voltages, using so-called in-source CID, the spectra show fragmentation that closely resembles that obtained by the MS-MS technique. APs give, in addition to the [M — H]— ions, fragment m/z 133, resulting from the loss of a C5H12 (OP) and C6H14 (NP) group [31]. A similar fragmentation pattern is obtained using a MS-MS [32]. Fragments m/z 147, 133, 119 and 93 result from the progressive fragmentation of the alkyl chain, whereas m/z 117, observed also by Pedersen and Lindholst [31], cannot be explained in such a straightforward way. Therefore, reaction channels m/z 205 ! 133 (for OP) and m/z 219 ! 133 (for NP) and precursor (parent) ion scan of m/z 133 can be used to monitor APs. However, the reaction is not specific, and it can also be used for APECs (Fig. 2.6.9).

2.6.3 Carboxylated degradation products (APECs and CAPECs)

Alkylphenoxy carboxylates (APEnC) were detected in both the NI mode [1,18,33] and PI mode [21]. In the NI mode, using ESI, APECs give two types of ions, one corresponding to the deprotonated molecule [M — H] — and the other to deprotonated alkylphenols [M — CH2COOH]— in the case of APE1Cs and [M — CH2CH2OCH2COOH]— for the APE2Cs. The relative abundance of these two ions depends on the extraction voltage. Using a low voltage, the ESI source is capable of producing deprotonated molecular ions, and the spectra display only signals at m/z 277 and 263 corresponding to NPE1C and OPE1C and m/z 321 and 307 for NPE2C and OPE2C. At higher voltages, using so-called in-source CID, the spectra give fragmentation that closely resembles that obtained by the MS-MS technique [34]. Figure 2.6.9(a) shows a product

Liver Sims Spectrum

Fig. 2.6.8. Full-scan LC-ESI-MS chromatogram (upper trace) of river sediment (Anoia, downstream of WWTP) and extracted chromatogram for m/z 205 and 219 corresponding to OP and NP, respectively. Inset: mass spectra of OP and NP. Reprinted with permission from Ref. [40] © 2001 AOAC International.

Fig. 2.6.8. Full-scan LC-ESI-MS chromatogram (upper trace) of river sediment (Anoia, downstream of WWTP) and extracted chromatogram for m/z 205 and 219 corresponding to OP and NP, respectively. Inset: mass spectra of OP and NP. Reprinted with permission from Ref. [40] © 2001 AOAC International.

Mass Fragmentation Surfactin

Fig. 2.6.9. MS-MS chromatogram (MRM channel m/z 219 ! 133) of raw effluent (river water) treated in a Barcelona drinking water treatment plant. Insets: product ion scan of NPE2C (A) and NP (B), obtained using argon as collision gas at collision energy of

40 eV. Reprinted with permission from Ref. [32] © 2002 Elsevier.

Fig. 2.6.9. MS-MS chromatogram (MRM channel m/z 219 ! 133) of raw effluent (river water) treated in a Barcelona drinking water treatment plant. Insets: product ion scan of NPE2C (A) and NP (B), obtained using argon as collision gas at collision energy of

40 eV. Reprinted with permission from Ref. [32] © 2002 Elsevier.

ion scan of m/z 321 for NPE2C. Intense signals at m/z 219 and 205 are produced after the loss of the carboxylated (ethoxy) chain, while m/z 133 and 147 corresponded to the fragmentation of the alkyl chain, as described above for NP.

The short chain NPE1C, which had been synthesised from branched NPs for standard comparison purposes, was also detected by APCI-FIA-MS in the positive and negative modes. In the presence of ammonium acetate, APCI under PI conditions resulted in [M + NH4]+ ions at m/z 296, whereas NI generated the prominent [M — H] — ion besides negatively charged acetate adduct ions [M — H + acetate]— and the dimeric ions [2M — H]— at m/z 277, 337 or 555, respectively [13]. NPE2C gave [M + NH4]+ ion at m/z 340. This ion, examined by CID(+), Labo generated alkyl fragments at m/z 57, 71 and 85, the [C9H19]+ product ion at m/z 127 and the prominent ion at m/z 103, characteristic for carboxylated PEG chains as presented in the product ion spectrum and fragmentation scheme in Fig. 2.6.10.

Aerobic degradation in laboratory scale experiments was carried out by Di Corcia et al. [167]. They could prove the postulate, which resulted in either NPECs, alkyl chain carboxylated NPEO (CNPEO) or compounds carboxylated in both positions, in the alkyl chain and in the polyether chain (CNPEC). Additionally these compounds could be generated in the mechanical-biological WWTP [21]. Analysis of treated sewages showed that CAPECs were the dominant products of the A9PEO biotransformation, accounting for 66% of all the metabolites leaving the WWTP [18].

The identity of the dicarboxylated breakdown products was confirmed by reversed-phase LC-ESI-MS [18] and LC-ESI-MS-MS [33]. Under NI conditions, at low cone voltage no CID process was possible and the spectrum displayed only signals tentatively assigned to the [M — H]— and [MNa — 2H]—. However, with the cone voltage of 55 V, structural confirmation of these species was achieved by observing different fragment ions (Fig. 2.6.11). Typical total ion current (TIC) chromatogram and ion current profiles of CAPECs obtained using a C18 reversed-phase column (Altima, Alttech, Italy) and gradient elution with acetonitrile and water (both acidified with formic acid, 1 mmol L—1) is shown in Fig. 2.6.12.

At low extraction voltages, the in-source CID process is greatly inhibited and the spectra display intense signals for the protonated molecular ions. By raising the extraction voltage, in-source CID spectra were obtained. Neutral losses of the carboxylated ethoxy chain and

Surfactin Mass Spectrometry

Fig. 2.6.10. APCI-FIA-MS-MS(+ ) (CID) daughter ion mass spectrum of selected [M + NH4]+ parent ion (m/z 340) of potential carboxylated non-ionic surfactant metabolite of precursor NPEO prepared by chemical synthesis; structure of short-chain NPEC: C9H19-C6H4-O-(CH2-CH2-O)-CH2-COOH; fragmentation behaviour under CID presented in the inset [28].

Fig. 2.6.10. APCI-FIA-MS-MS(+ ) (CID) daughter ion mass spectrum of selected [M + NH4]+ parent ion (m/z 340) of potential carboxylated non-ionic surfactant metabolite of precursor NPEO prepared by chemical synthesis; structure of short-chain NPEC: C9H19-C6H4-O-(CH2-CH2-O)-CH2-COOH; fragmentation behaviour under CID presented in the inset [28].

Fig. 2.6.11. NI mass spectra of the dicarboxylated (CAePE2C) metabolite of NPEO taken at two different cone voltages. Reprinted with permission from Ref. [19] © 2000 American

Chemical Society.

Fig. 2.6.11. NI mass spectra of the dicarboxylated (CAePE2C) metabolite of NPEO taken at two different cone voltages. Reprinted with permission from Ref. [19] © 2000 American

Chemical Society.

carboxylated alkyl chain, respectively, and methanol loss followed by formation of acylium ions, were found to be typical fragmentation patterns for methylated CAPECs. In Fig. 2.6.13 the daughter ion spectra of underivatised CNPECs (A) and methylated CNPECs (M) together with their fragmentation behaviour are presented. The alkyl chain branched CNPE1C compounds could be confirmed as extremely recalcitrant intermediates in the biochemical degradation process.

However, using LC-MS, under conditions giving solely molecular ions, identification of dicarboxylated compounds is difficult since CAnPEmCs have the same molecular mass as APECs having one ethoxy unit less and a shorter alkyl chain (An_1PEm_1C). Moreover, since some compounds partially co-elute, the unequivocal assignment of the individual fragments can be accomplished only by using LC-MS-MS. Jonkers et al. [33] studied the aerobic biodegradation of NPEOs in a laboratory scale bioreactor. The identity of the CAPEC metabolites was

Fig. 2.6.12. TIC chromatogram (bottom trace) and extracted chromatograms for CAPECs obtained in NI mode. Reprinted with permission from Ref. [19] © 2000 by American

Chemical Society.

Fig. 2.6.12. TIC chromatogram (bottom trace) and extracted chromatograms for CAPECs obtained in NI mode. Reprinted with permission from Ref. [19] © 2000 by American

Chemical Society.

confirmed by the fragmentation pattern obtained with LC-ES-MS-MS. Of 17 degradation products that were found to accumulate, nine were confirmed to be CAPEC metabolites. In Fig. 2.6.14 MS-MS fragmentation pattern is given for some of the compounds that were positively identified. In addition to the carboxy-alkylphenoxy fragment, typical fragments observed were the carboxy-alkylphenoxy fragment that has additionally lost CO2 or an acetic acid group, which, in the case of CA5PE1-2C, led to fragments of m/z 149 and 133.

2.6.4 Halogenated derivatives of alkylphenolic compounds

APEOs and their acidic and neutral metabolites can be halogenated to produce chlorinated and brominated products. The formation of these compounds has been reported during the chlorination processes at drinking water treatment plants [1,35,36] and after biological wastewater treatment [37].

Chlorination Mass Spectrum

Using an ESI interface, halogenated APEOs like their non-haloge-nated analogues, show a great affinity for alkali metal ions, and they give exclusively evenly spaced sodium adduct peaks [M + Na]+ with no further structurally significant fragmentation. The problem arises from the fact that the chlorinated derivatives (ClAPEnO) have the same molecular mass and they gave the same ions as brominated compounds with one ethoxy group less (BrAPEn—1O, respectively). However, they can be distinguished by their different isotopic profiles. The doublet signal in the mass spectrum of brominated compounds shows the contribution of bromine isotopes of 79Br/81Br = 100:98, while the contribution of chlorine isotopes is 35Cl/37Cl = 100:33. Therefore, chromatographic separation of these two groups of compounds is a pre-requisite of their quantitative determination. Halogenated NPEOs were detected in flocculation sludge from a Barcelona drinking water treatment plant (DWTP) (Figs. 2.6.15 and 2.6.17) [1]. Halogenated OPEOs were also identified. Like halogenated NPEOs, OPEO derivatives gave regularly spaced signals corresponding to [M + Na]+ ions with m/z 395/397-571/573 corresponding to ClOPEOs, nEO = 3- 6 (assigned as ■ in Fig. 2.6.16(c)) and m/z 395/397-615/617 corresponding to BrOPEOs, nEO = 2-7 (assigned as ■ in Fig. 2.6.16(d)).

Halogenated APs and halogenated APECs were analysed in the NI mode using an ESI interface [1,17]. Halogenated NPs (XAPs) gave a characteristic isotope doublet signal of the [M — H]— ions (m/z 297/299 for BrNP and m/z 253/255 for ClNP). Halogenated NPECs (XNPECs) gave two signals, one corresponding to a quasi-molecular ion and another to [M — CH2COOH]— in the case of XNPE1Cs or [M — CH2 CH2OCH2COOH]— for XNPE2Cs. The relative abundance and absolute intensity of these two ions, as compared with quasi-molecular ions, depends largely on the cone voltage. At higher values, the base peak, with high absolute intensity, for ClNPE1C and ClNPE2C is m/z 253/255 and for BrNPE1C and BrNPE2C m/z 297/299.

With gradient elution using methanol-water on a C18 reversed-phase column, APECs, APs, XAPs and XAPECs can be easily separated.

Fig. 2.6.13. Fragmentation behaviour of the acidic NPEO metabolites (CNPEC); (A) ESI-LC-MS(+) of underivatised compound and (M) di-methyl ester of HOOC(CH2)6-C6H4-O-(CH2CH2O)COOH: Source CID conditions, see Ref. [21]. Reprinted with permission from Ref. [21] © 1998 by American Chemical Society.

Ionisation Isomer
Fig. 2.6.14. MS-MS fragmentation patterns of some CAEC (NI mode): (a) CA9PE2C, (b) CA5PE2C, and (c) CA5PE1C. The exact branching of the alkyl chain is unknown; the alky] isomer structures shown are chosen arbitrarily. Reprinted with permission from Ref. [33] © 2001 American Chemical Society.
Cid Fragmentation Esi Mass
Fig. 2.6.15. Total ion LC-ESI-MS chromatogram (bottom trace) and reconstructed chromatograms of halogenated APEOs and APEOs, obtained in PI mode, found in sludge from a Barcelona drinking water treatment plant. Reprinted with permission from Ref. [1] © 2001 American Chemical Society.

However, when analysing real samples containing LAS, ClNPE1C co-elutes with C11LAS, a compound having the same molecular weight (MW = 312) and base ion m/z 311. LASs are the major surfactant class used in detergents throughout the world and high concentrations (up to several mgL"1) are often found in environmental and wastewater samples. All attempts to separate ClNPE1C and C11LAS using gradient elution with standard mobile phases for reversed-phase separation (methanol/water or acetonitrile/water) failed and determination of ClNPE1C was achieved by monitoring a fragment ion at m/z 253/255, which also suffered the isobaric interferences in some real samples.

ESI-MS-MS permitted unambiguous identification and structure elucidation of compounds detected under NI conditions (halogenated NPECs and NPs), while for NPEOs, detected under positive ionisation conditions no fragmentation was obtained and these compounds were analysed using a single stage MS as [M + Na]+ in selected ion

Cid Fragmentation Esi Mass

monitoring (SIM) mode [38,39]. With collision-induced dissociation (CID) under NI conditions, halogenated NPECs and NPs undergo fragmentation with few major pathways, depicted in Fig. 2.6.17. For halogenated NPE1Cs and NPE2Cs, the predominant reaction was loss of CH2COOH and CH2CH2OCH2COOH, respectively, which resulted in intense signals at m/z 253/255 for ClNPECs and m/z 297/299 for BrNPECs. Further fragmentation of [ClNP]— occurred primarily on the alkyl moiety leading to a sequential loss of m/z 14 (CH2 group), with the most abundant fragments at m/z 167 for 35Cl and m/z 169 for 37Cl with the relative ratio of intensities of 3.03. A fragment corresponding to a chlorine ion was produced 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 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 81 corresponding to the [Br]— (ratio of isotopes is 1.02), while the fragmentation of the side chain was suppressed and resulted just in a low-intensity fragment at m/z 211/ 213 produced after the loss of C6H14. Such a difference in the mechanism of fragmentation of chlorinated and brominated compounds is presumably the consequence of the lower energy of a Br-benzene bond compared with a Cl-benzene bond.

The detection limits of the LC-MS-MS method [38] fell down to 1-2ngL—1 for the analysis of halogenated NPECs and NPs in water samples (after SPE pre-concentration) and to 0.5-1.5 ng g—1 in sludge samples (target compounds were extracted using pressurised liquid extraction). A specificity of MRM mode permitted unequivocal identification and quantification of isobaric target compounds (e.g. BrNPE1C and ClNPE2C) and elimination of interference of co-eluting isobaric no-target compounds (e.g. C11LAS), both having a base ion at m/z 311, observed using an LC-MS in SIM mode as shown in Fig. 2.6.18.

Fig. 2.6.16. ESI mass spectra of halogenated APEOs detected in the DWTP sludge (chromatogram shown in Fig. 2.6.17): (a) ClNPEO; (b) BrNPEO; (c) ClOPEO (assigned as B); and (d) BrNPEO (assigned as B). Reprinted with permission from Ref. [1] © 2001

American Chemical Society.

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