MS and Time of Flight

Chapter 21

Downloaded by UNIV ILLINOIS URBANA on June 3, 2013 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch021

Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry and Derivatization for the Identification of Polar Carbonyl Disinfection By-Products C. Zwiener, T. Glauner, and F. H . Frimmel Engler-Bunte-Institut, Wasserchemie, Universitat Karlsruhe (TH), Karlsruhe, Germany

Derivatization with 2,4-dinitrophenylhydrazine (DNPH) in combination with LC-ESI/MS/MS was successfully applied to investigate polar disinfection by-products with carbonyl groups. The collision-induced dissociation mass spectra (CID¬ -MS) show distinct fragments of the DNPH moiety which facilitates selective measurements of different carbonyl groups by tandem mass spectrometric experiments like precursor ion scans (e.g. m/z 163 for aldehydes; m/z 152 for ketones, m/z 182 for dicarbonyls and hydroxycarbonyls). Several multifunctional carbonyls could be tentatively identified in treated water samples by putting together all information of HPLC separation, derivatization, precursor ion scans, and CED-MS. However, DNPH derivatization is not a suitable reagent for halogenated carbonyl compounds due to side reactions. The chlorine is substituted by DNPH. O¬ -(carboxymethyl) hydroxylamine (CMHA) is a suitable alternative. CMHA derivatization is specifically aimed at the carbonyl group and does not affect the chlorine. The formed oxime derivatives reveal abundant signal intensity in ESI and meaningful CID-MS with common fragments of the CMHA moiety and diagnosticfragmentsof the carbonyl moiety.

356

© 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.

357


Introduction Disinfection is required for drinking water production to minimize microbial activity and to exclude health effects caused by microorganisms. Chlorine and chlorine containing chemicals are the most common disinfectants used today. In addition, ozonation plays an increasing role in water treatment. Apart from the intended effect the disinfectants can also react with organic and inorganic water constituents to form disinfection by-products (DBP). In the following only organic DBPs will be considered. In the case of chlorine disinfectants trihalomethanes (THM) are in general the most abundant class of DBPs. THMs were first found by Rook (1, 2) and are the only DBPs regulated in the Drinking Water Directive in Europe to a level of 50 μg/L (J). Therefore only THMs serve as indicators for organic DBP formation in drinking water regulations. The U.S. Environmental Protection Agency (EPA) regulates THMs and five haloacetic acids (HAA). The maximum contaminant levels were set to 80 μg/L for THMs and to 60 μg/L for five HAAs (4). Further efforts to identify additional DBPs after application of chlorine, and ozone -based chemicals have been made and led to long lists of compounds (5, 6). Moreover, there are still about 50% of the halogenated DBPs unknown as a mass balance of the identified DBPs and the total organic halogen (TOX) reveals. This applies also to the assimilable organic carbon (AOC) with respect to ozone DBPs (7). A toxicological estimation cannot be performed concerning this missing gap. In particular, the occurrence of the strong mutagen M X ((3chloro-4-dicUoromethyl-5-hydroxy-2(5J^furanone) (8) reveals the importance of identifying even traces of DBPs and of minimizing those in drinking water treatment. Epidemiological studies give indication on effects of DBPs on human reproduction and development, e.g. low birth weight or spontaneous abortion (9) . Further studies concerning DBPs and health effects have been undertaken (10) .

New research is in particular required for highly polar and high-molecular weight compounds. Since the DBP formation primarily occurs by reaction of the disinfectant with natural organic matter (NOM) the properties of the products and intermediates may be expected to range from those of NOM itself (polar, high molecular weight) to those of THMs (volatile, non-polar, low molecular weight). NOM is a polydisperse and heterogeneous mixture of natural aliphatic and aromatic compounds (11). The reaction with disinfectants yields unknown breakdown products and for example the well-known THMs (Figure 1). The breakdown of NOM during oxidation and chlorination could be shown by size exclusion chromatography (SEC) with organic carbon detection (12). The halogenated fraction can be measured as TOX. The focus of our work is on carbonyl compounds as intermediates and end-products of reactions between disinfectants and ΝΟΜ. Reaction pathways of THM and M X formation show the importance of carbonyl compounds as intermediates (13, 14). Furthermore,



Figure 1. DBP formation by the reaction between natural organic matter (NOM) and chlorine or ozone in the presence of bromide ions


Br

ι

α-c-H

α α

ι

α-c-H

α

THMs..

(/ι 00


359 carbonyl compounds occur as end-products (DBPs) of chlorination and ozonation in water treatment. For carbonyl compounds derivatization with 2,4-dinitrophenylhydrazine (DNPH) and HPLC/UV is a well-known method in aerosol science (75). Recently detection with atmospheric pressure chemical ionization (APCI) mass spectrometry was used for DNPH derivatives (16, 17). DNPH was also successfully applied to detect DBPs of ozonated aqueous samples with electrospray ionization (ESI) mass spectrometry (18). The application and optimization of ESI tandem mass spectrometry for the analysis of DNPH derivatives was shown in literature (19, 20). The capabilities and limits of tandem mass spectrometry to identify the derivatives of carbonyl DBPs will be discussed in this work.

Methods

Derivatization DNPH derivatization was carried out by a modified method according to literature (19) by adding 500 μΐ of reagent solution (6.7 mM DNPH) to 50 mL aqueous sample. The reagent solution was prepared by dissolving 20 mg DNPH (Fluka) in 15 mL HCl/water/acetonitrile 2:5:1 (v/v) according to literature (21). The reaction time was at least 12 h at room temperature. CMHA derivatization was carried out by adding 1 mL of a 60 mM aqueous solution of O-(carboxymethyl) hydroxylamine hemihydrochloride (Fluka) to 50 mL aqueous sample. The reaction time was at least 10 h at room temperature. For both reagents standard solutions of pure carbonyl compounds were prepared in water-acetonitrile (Aldrich), 50:50 (v/v).

Extraction and preconcentration of aqueous samples The acidified samples were preconcentrated by SPE on Oasis HLB cartridges (200 mg sorbent, Waters). Elution was done with acetonitrile resulting in preconcentration factors of 25 to 250. More details are described elsewhere (19).


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Analysis Analysis was performed using an Agilent 1100 HPLC system coupled with an electrospray ionization source (Turbolon Spray, Applied Biosystems Sciex) to a triple-quadrupole mass spectrometer (API 3000, Applied Biosystems Sciex). Samples (50 μ!) were injected onto a reversed-phase column (Xterra MS, 150 mm χ 2.1 mm, 5 μπι particles; Waters) at a flow rate of 300 μΏχτάιι. A gradient elution with acetonitrile/water both with 1 mM ammonium acetate was applied from 30 % to 100 % in 25 min. ESI was used with a nebulizer gas flow of 1.3 L/min, a spray voltage of 5000 V and a dry gas temperature of 450 °C. The mass spectrometer was run in different modes at unit resolution. CID-MS were produced by collision with nitrogen gas molecules at a collision gas thickness of 2.19xl0 molecules/cm . The values of the collision energies were chosen to decrease the abundance of the precursor ion at least to one third of its original value. Further details are described elsewhere (19). 17

2

Samples Samples were collected from tap waters (20), swimming pools (19) and Lake Hohloh, a bog lake in the Black Forest near Karlsruhe (11). Ozonation was done at a concentration of 1.2 mg/L for 2 h at room temperature. Subsequent chlorination was carried out with a solution of hypochloric acid (1.8 mg/L chlorine) for 72 h at room temperature in the dark. The residual chlorine was quenched after reaction with an excess of ammonium chloride (Merck).

Results and Discussion Derivatization with DNPH The well-known derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine (DNPH) yields the corresponding hydrazones, which have several advantages for the subsequent analysis compared to the parent carbonyl compounds (Figure 2). The hydrazone derivatives are not charged but easy to ionize by negative electrospray ionization (ESI) yielding the negative deprotonated molecules [M-H]\ Due to a kind of labeling of analytes with carbonyl functional groups with the DNPH moiety they show common fragmentation patterns in tandem mass spectrometry. This can be used to measure selectively DNPH derivatives by tandem mass spectrometric experiments like precursor ion scans. Furthermore the hydrazones are easy to preconcentrate by solid-phase extraction and to separate on reversed-phase HPLC columns. 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.

361


N0

2

Figure 2. Derivatization reaction between 2,4-dinitrophenylhydrazine (DNPH) and a carbonyl compound (R j and R2 represent hydrocarbon moieties)

Tandem mass spectrometric properties of DNPH derivatives The collision-induced dissociation mass spectra (CID-MS) of the DNPH derivatives of an aldehyde, a ketone, a hydroxyketone, and an oxoacid are shown in Figure 3. In general most of the abundant mass fragments below m/z 200 can be assigned to mass fragments of the DNPH moiety only. In this mass range common fragments for different compound classes can be found, like m/z 152 for carbonyl compounds in general, m/z 163 for aldehydes, m/z 182 for ahydroxyaldehydes and dicarbonyls (Figure 4). In particular dicarbonyl compounds show much less fragments than other carbonyl compounds. Aldehydes and ketones show also abundant fragments of the neutral loss of NO (e.g. m/z 263 in Figure 3a) and sometimes of N 0 . Oxoacids show the neutral loss of C 0 (e.g. m/z 281 in Figure 3d). CID mass fragments of further aldehydes and ketones are compiled in Table I. The data reveal that aldehydes in general show abundant ions at m/z 163 and at m/z 152, whereas ketones show only ions at m/z 152. The ions at m/z 205 and m/z 220 represent aldehydes and ketones with 4 or more carbon atoms. The CID mass fragments of the more polar carbonyls are shown in Table II. Hydroxyketones show abundant fragments at m/z 152 and at m/z 182, the dicarbonyls with both carbonyl groups derivatized show almost only onefragmentat m/z 182, the ketoacids ions at m/z 182 and a neutral loss of C 0 . This general scheme gives the possibility to use precursor ion scans for ions at m/z 152, m/z 163 or m/z 182 to selectively measure the DNPH derivatives of different classes of carbonyl compounds in complex samples. 2

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Figure 3. Collision induced dissociation mass spectra (CID-MS) of the DNPH derivatives ofheptanal (a), 3-hydroxy-2-butanone (b), 2-heptanone (c), and 2oxoglutaric acid (d)

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m/z Figure 4. CID-MS of the DNPH derivatives ofglyoxal (a) and methyl glyoxal (b) 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.


Butanal Pentanal Hexanal Heptanal 2-Butanone 2-Pentanone 3-Pentanone 2-Hexanone 2-Heptanone 4-Heptanone

Compound

72.1 86.2 100.1 114.2 72.1 86.1 86.1 100.2 114.2 114.2

Molecular mass Amu [M-HJ- ion of DNPH derivative m/z 251.1 265.1 279.1 293.2 251.1 265.1 265.1 279.1 293.1 293.1

163 (100), 152 (90), 151 (60), 191 (40), 221 (35), 205 163 (100), 152 (94), 151 (51), 191 (40), 205 (52), 235 163 (100), 152 (95), 151 (50), 205 (49), 191 (33), 249 163 (100), 152 (70), 151 (53), 205 (45), 191 (26), 263 178 (31), 221 (44), 151 (20), 181 (20) 152 (80), 205 (56), 235 (42), 178 (23) 152 (35), 205 (100), 235 (16), 178 (15) 152 (75), 249 (42), 220 (25), 178 (20) 152 (60), 263 (28), 220 (15), 178 (13) 152 (95), 263 (32), 206 (30), 179 (14)

(14) (38) (24) (26)

Selected CID-MS fragments of the derivatives m/z (abundance)

Table I. Selected collision-induced dissociation mass spectrometric (CID-MS) fragments of DNPH derivatives of aldehydes and ketones.



Hydroxyacetone 1,3-Dihydroxyacetone 3-Hydroxy-2-butanone 5 -Hydroxy-2-pentanone Glyoxal Methylglyoxal Dimethylglyoxal Glyoxylic acid 2-Oxopropionic acid Ketomalonic acid 2-Oxoglutaric acid

Compound

Molecular mass amu 74.1 90.2 88.1 102.2 58.0 72.1 86.1 74.0 88.0 118.0 146.1

[M-H]- ion of DNPH derivative m/z 253.0 269.0 267.3 281.0 417.1 431.0 445.2 253.0 267.0 297.0 325.0

Selected CID-MS fragments of the derivatives m/z (abundance) 152 (100), 179 (14), 182 (13) 182 (50) 152(100), 182 (60) 152 (100), 252 (25), 179 (11), 220 (10) 182 (100), 234 (10) 182 (100), 248 (5) 182 (100), 265 (15) 182(100), 209(42) 182(100), 223(40) 182 (100), 152 (8) 182 (100), 281 (8), 151 (4), 163 (4)

Table II. Selected collision-induced dissociation mass spectrometric (CID-MS) fragments of DNPH derivatives of hydroxycarbonyls, dicarbonyls, and ketoacids.


Os ON

367


Table ΙΠ. Retention times of the DNPH derivatives of polar carbonyl compounds in the HPLC chromatogram of a standard solution. Compound Glyoxylic acid 2-Oxoglutaric acid 2-Oxopropionic acid Ketomalonic acid Hydroxyacetone 3-Hydroxy-2-butanone 5-Hydroxy-2-pentanone Acetaldehyde Glyoxal Pentanal Methylglyoxal Dimethylglyoxal 4-Heptanone 3-Octanone

Retention timefmin) 2.1 2.1 2.2 2.3 7.8 9.9 10.1 11.9 17.5 18.1 18.9 20.4 20.9 22.4

Selective measurement and identification The chromatograms of precursor ion scans of m/z 182 are shown in Figure 5 for aqueous samples of a bog lake after chlorination and after ozonation plus chlorination. The mass to charge ratio of several abundant peaks could be determined and the peaks at 17.5 min (m/z 417) and at 18.9 min (m/z 431) could be identified as glyoxal and methylglyoxal by comparison of the retention times (Table III) and the CID-MS with a standard. The following shows as an example how DBPs may be identified based on tandem mass spectrometry of DNPH derivatives. Comparison of the retention times for the peak at 7.9 min would lead to the suggestion of hydroxyacetone, but the mass to charge ratio does not fit. Further information was obtained from the CID-MS (product ion scan of m/z 461) of the peak at 7.9 min (Figure 6). The CID-MS shows as expected an abundant ion at m/z 182 indicating a dicarbonyl compound or a hydroxy carbonyl and a neutral loss of 44 indicating a carboxyl group. Putting together a carboxyl group and two carbonyl groups 2,3-dioxopropionic acid and



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Retention time (min) Figure 5. Chromatograms ofprecursor ion scans at m/z 182 of treated brown waterfroma bog lake. - a) Chlorinated; b) Ozonated and chlorinated

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MS and Time of Flight

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