The Determination of Anaerobic Biodegradation Products of Aromatic

Downloaded by PENNSYLVANIA STATE UNIV on September 2, 2012 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch019

Chapter 19

The Determination of Anaerobic Biodegradation Products of Aromatic Hydrocarbons in Groundwater Using LC/MS/MS 1

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Michael S. Young , ClaudeR.Mallet , David Mauro , Sam Fogel , Ashok Jain , and William Hoynak 4

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Waters Corporation, 34 Maple Street, Milford, MA 01757 META Environmental Inc., 49 Clarendon Street, Watertown, MA 02472 Bioremediation Consulting Inc., 39 Clarendon Street, Watertown, MA 02472 Electric Power Research Institute, 3412 Hillview Avenue, Palo Alto, CA 94304 Northeast utilities, P.O. Box 270, Hartford, CT 06141 2

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This paper describes chromatographic and spectroscopic analysis in support of an investigation of the natural biodegradation of monocyclic and polycyclic aromatic hydrocarbons (ΜΑΗ, PAH) in groundwater. Most of the parent aromatic compounds may be readily determined in complex matrices using either GC or LC. Although GC/MS is a preferred technique for organic environmental analysis, it is not amenable to the analysis of highly polar, labile or non -volatilecompounds, without cumbersome derivatization. The degradation products of MAHs and PAHs are often acidic or otherwise highly polar in nature and are therefore more easily determined using LC. In this presentation we will discuss the utility of LC/MS and LC/MS/MS for the straightforward analysis of the biodegradation products of MAHs and PAHs.

© 2003 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.

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Introduction The reduction in mass and/or concentration of a compound in the environment over time or distance as a result of naturally occurring physical, chemical, or biological processes is known as natural attenuation. Utilization of natural attenuation has gained technical and regulatory acceptance as an appropriate remedial approach at some sites. Many natural processes can occur simultaneously to affect the fate of contaminants in soil and groundwater. Among these processes are sorption, precipitation, chemical stabilization, chemical degradation, biological stabilization, and biological degradation. Biological degradation is the most frequent process governing the environmental fate of organic compounds. Recently, anaerobic biodegradative processes of monoaromatic and polycyclic aromatic hydrocarbons have been shown to generate biochemical intermediates. The presence of selected alkylbenzene and PAH metabolites in groundwater and soil provides evidence that biological degradation is reducing the mass of the parent MAHs and PAHs at that site. The objective of this paper is to demonstrate the utility of LC/MS/MS techniques for the straightforward determination of these metabolites.

Manufactured Gas Plant (MGP) Waste Contaminants The manufactured gas industry produced gas from coal or oil for lighting and heating as well as by-products that served as fuel or feed stocks for the production of chemicals. Over the years, some MGP by-products were released to the environment and exist in soil and sediment or relic plant structures to this day. These by-products primarily included tars, oils, or lampblack, however tars and tar sludge were released in the greatest volumes. Tar is complex mixture of chemicals formed at high temperature under low oxygen conditions. Those chemicals mostly include aromatic hydrocarbons with one to several rings. However, tars also contain aliphatic hydrocarbons, organic acids, nitrogen-, sulfur-, and oxygen-containing heterocyclic compounds, water, particulate carbon, and other substances. In the environment, tars will disperse as a separate organic phase and the chemicals in tar will partition into the vapor



327

and aqueous phases based on their vapor pressures and aqueous solubilities, respectively. Once in the vapor phase or dissolved in groundwater or surface water, tar chemicals will further disperse and will be degraded by chemical and biological mechanisms. Groundwater was collected from several locations at a former MGP site for use in this study. The groundwater samples contained various amounts of dissolved tar chemicals. Of the many aromatic hydrocarbons found at MGP site groundwater, naphthalene, methylnaphthalenes, and certain monoaromatic compounds are often among the most abundant. Because they are more water soluble than the higher molecular weight polycyclic aromatic hydrocarbons (PAH), the mono and diaromatic hydrocarbons are usually the predominant contaminants in groundwater at such sites. Consider the GC analysis presented in Figure 1. This chromatogram was obtained from one of the groundwater samples analyzed in this study; naphthalene, methylnaphthalenes, and indan are the most abundant hydrocarbons.

naphthalene

CQindan 2-methylnaphthalene 1 -methylnaphthalene

_!

ALA

10 Minutes

15

20

25

30

Figure 1. Gas chromatogram of a groundwater samplefroman MGP site showing typical aromatic hydrocarbons found in such samples

Anaerobic degradation of naphthalene and other P A H has been demonstrated in microcosm studies (1). The following compounds have been identified as free acid intermediates of the anaerobic degradation of 2-



328 methylnaphthalene ; naphthyl-2-methylsuccinic acid, naphthyl-2methylenesuccinic acid, 2-naphthoic acid, and 5,6,7,8-tetrahydro-2-naphthoic acid (2). 2-Naphthoic acid and reduced naphthoic acids have also been identified as metabolites of anaerobic naphthalene degradation (3,4). Benzylsuccinic acid, a metabolite of the degradation of toluene, may also be present at PAH degradation sites. Structures for some of these compounds are given in Figure 2. Although the anaerobic biodégradation of indan is not so well documented, this study did identify an unknown compound likely to be a degradate of indan. Note that naphthylmethylsuccinic and benzylsuccinic acids are conjugates to which additional carbon has been added to the metabolite structure according to the biochemical pathways given in reference 2.

Η

οστ°°

COOH

00

οστ " COOH

naphthyl-2-methylsuccinic acid naphthyl-2-methyenesuccinic acid

m^COOH COOH benzylsuccinic acid COOH 2-naphthoic acid

^j^^ 5,6,7,8-tetrahydro-2-naphthoic acid

Figure 2. Structures of Some Possible Anaerobic Dégradâtes at PAH Sites

Because the metabolites are often acidic and much more polar in nature than the PAH compounds from which they were derived, the analytical methods utilized for determination of PAH in environmental samples are usually not appropriate for determination of the metabolites without modification. For example, derivatization is necessary for the GC/MS analysis of most of these compounds. However, electrospray mass-spectrometry interfaced to liquid ehromatograph (LC/MS) is a modern technique that is well suited for the


329 analysis of ionizable compounds such as naphthoic acid. This paper reports die results of the use of LC/MS and LC/MS/MS to demonstrate that dégradâtes of naphthalene or methylnaphthalenes are present in groundwater samples taken at a site contaminated with aromatic hydrocarbons.


Benefits of LC/MS/MS Analysis There are two main benefits to the use of triple quadrupole MS/MS in multiple reaction monitoring (MRM) mode compared with a single quadrupole MS in single ion recording (SIR) mode. In M R M mode, a specific transition between a precursor and product ion is monitored. The first benefit of this technique is a lower background signal compared with selected ion recording (SIR) using a single quadrupole mass spectrometer; the resulting gain in signal to noise ratio can result in more than a tenfold increase in useful sensitivity. The other main benefit of the M R M analysis is the sure knowledge that the monitored product ion is present only as a result of the fragmentation of the precursor. For confirmation of an analyte identity, multiple M R M transitions are monitored if possible. For benzylsuccinic acid, the transition from m/z 207 (M-l, precursor) to m/z 163 is the primary transition resulting from the loss of the carboxylate functionality. However, using a higher collision energy, the transition from m/z 207 to m/z 91 (C H ) may be monitored to help confirm the identity of the compound. Another approach utilized in this study was to monitor a product-ion scan spectrum resulting from a particular precursor ion. Compared with M R M mode, a more complete mass-spectrum is obtained that may be used to obtain qualitative structural information for unknown compounds. However, because the second quadrupole is operated in a full-scan mode, the sensitivity of the product ion mode is less than for M R M mode. For example, a conservative quantitation limit for 2-naphthoic acid is below 0.05 μg/L using M R M mode and approximately 0.5 μg/L using the product-ion scan mode. +

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Experimental

Solid-Phase Extraction (SPE) of Aqueous Samples The groundwater sample (400 mL) was adjusted to below pH 2 by addition of 1 mL of concentrated HC1. A reversed-phase SPE cartridge (Oasis HLB, 3cc,



330 60 mg., Waters Corp.) was conditioned with 1 mL of methanol followed by 1 mL of water. The acidified sample was loaded onto the cartridge at a rate of 5 mL/min. The cartridge was washed with 1 mL of 25:75 methanol/0.1 M HC1. The cartridge was then air-dried by vacuum and was eluted with 3 mL of 90:10 methyl-i-butyl ether (MTBE)/methanol. A 100 μΐ aliquot was removed for hydrocarbon analysis by GC/FID. The remaining eluent was evaporated and the residue was reconstituted in 400 μΙ, of 10:90 acetonitrile/water. The recoveries of 2-naphthoic acid and benzylsuccinic acid were better than 80 % for a typical groundwater sample spiked at a level of 5 μg/L.

LC/MS and LC/MS/MS Analysis The LC/MS system was comprised of a Waters 2690 Separations Module (Waters Corporation, Milford, MA) interfaced to a Micromass Quattro Ultima (Micromass Ltd., Manchester, UK) triple quadrupole spectrometer operated in the negative electrospray mode (ESI-). Analyses were performed using fullscan, M R M and product-scan modes. The analytical column was an XTerra MSC , 100 χ 2.1 mm, 3.5 μΜ particle size (Waters Corp.). Mobile phase A was 20 mM ammonium formate (pH 4.5) and mobile phase Β was acetonitrile. The gradient was 85 % A to 10 % A in 10 minutes. A 20 μΐ, aliquot of the reconstituted eluent was injected for LC/MS/MS analysis; 40μΙ. was injected for full-scan LC/MS analysis. 18

Full-Scan Mode Full-scan LC/MS analysis was accomplished using cone voltages of 13 and 22 V.

MRM Mode Table I summarizes the cone voltages and collision energies utilized for the M R M analysis. These parameters were optimized using actual standard materials, if available. For suspected unknowns, the parameters were estimated from the behavior of similar compounds.


331 Product-Scan Mode


The cone voltage was maintained at 15 V. Product-ion scans were obtained using collision energies of 13 and 22 eV. The precursor ions chosen for this mode were consistent with those chosen for the M R M analyses (see Table I).

Table I. LC/MS/MS conditions for the MRM analysis of groundwater samples from MGP sites Compound

Precursor ion (m/z)

Product ion (m/z)

Naphthoic acid

170.9

126.9

15

20

Tetrahydronaphthoic acid

174.9

130.9

15

20

Benzylsuccinic acid

206.9

162.9

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15

Methylnaphthoicacid

144.9

140.9

15

20

Naphthylmethylsuccinic acid

256.9

212.9

15

15

Collision Energy (eV)

Cone Voltage (V)

GC/FID Analysis GC analysis was accomplished using an HP 5890 Series II gas chromatograph (Agilent Technologies, Wilmington, DE) equipped with a flame-ionization detector (FID). The analytical column was an Rtx-5, 30 m, 0.32 mm, 0.5 μιη df (Restek Corp., Bellafonte, PA). The carrier gas was helium at a flow rate of 1.5 mL/min. The oven temperature was held at 35° C for 2 minutes, programmed at 107minute to 310°, and then held at 310° for 15 minutes. A 1 μΐ, volume was injected in the split/splitless mode with the purge valve on time set at 0.5 minutes.

Calibration Standards 2-naphthoic acid and 1-naphthoic acid were obtained from Sigma-Aldrich (Aldrich, Milwaukee, WI). Benzylsuccinic acid was obtained from SigmaAldrich (Sigma, St. Louis, MO). Aromatic hydrocarbon standards were


332 obtained from Restek (Bellefonte, PA). Standard materials for other suspeeted metabolites were not commercially available.


Results The suspected metabolites discussed below were detected in every groundwater sample with measurable aromatic hydrocarbon contamination. The metabolites chosen for quantitation in this study were the naphthoic acids and benzylsuccinic acid. These compounds are known metabolites of die anaerobic degradation of naphthalene, methylnaphthalenes and toluene and standard materials for these metabolites were readily available. Some other suspected metabolites of naphthalene or methylnaphthalene degradation are 5,6,7,8tetrahydronaphthalene, naphthylmethylsuccinic acid, and methylnaphthoic acid. Although no standard materials were available for these suspected metabolites, efforts were made to identify these compounds or similar compounds using the combination of full-scan LC/MS and product-ion scan LC/MS/MS. Results are summarized in Table II.

Table Π. Results obtained for six groundwater samples taken at an MGP site (pgfL) Sample 1-Naphthoic 2-Naphthoic acid acid

Unknown MW205

Total Naphathalenesl

Indanl

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0.43

0.37

1.5

2700

1400

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3.5

6.5

2.1

3200

550

03

0.19

0.59

0.2

650

200

04

n.d.

n.d.

n.d.

n.d.

n.d.

05

1.3

2.5

0.22

3500

1100

06

1.6

0.73

0.40

6500

1100

Spike

0.32 (64%)

0.35 (70%)

—

—

—

Blank

n.d.

n.d.

n.d.

n.d.

n.d.

1

Result determined using GC/FED

n.d. = not detected


333 Naphthoic Acids


Both 1-naphthoic acid and 2-naphthoic acids were detected and quantified against pure standard materials. The chromatograms shown in Figure 3 are obtained using LC/MS/MS in the M R M mode. These are the only metabolites found in the groundwater samples that were confirmed against pure standards.

standard

-τ sample 4

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minutes Figure 3. LC/MS/MS (MRM) chromatogram of a groundwater sample from an MGP site showing the presence of naphthoic acids

Indan Metabolite (Similar to Benzylsuccinic Acid) Benzylsuccinic acid (BSA) was not identified in these samples. However, a compound was identified that is apparently very similar in structure to BSA. This suspected metabolite is present at concentrations similar to that of the naphthoic acids. Figure 4 shows the product spectrum obtained at 13 eV for the unknown compared with the spectrum obtained for BSA under the same conditions.


334 M-H-C0

2

163.0 100

benzylsuccinic acid "9

M-H-2CO, M-H-C0 -H 0 2

119.0

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4

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2

M-l+H 0

0

2


188.9

_

M

H

207.0

Figure 4. LC/MS/MS product-ion mass-spectrum obtained for possible indan metabolite showing the similarity with spectrum obtainedfor benzylsuccinic acid

5,6,7,8-Tetrahydronaphthalene 5,6,7,8-Tetrahydronaphthalene was not available at the time of this study. One substance was identified in the extracts with a mass-spectrum possibly consistent with this compound.

Naphthlymethylsuccinic acid A number of compounds were identified at very trace levels with massspectra that may be consistent with this compound. The target ions are m/z 213 (M-H -C0 ), m/z 169 (M-H -2C0 ) and m/z 141 ( C H ) . The apparent concentrations of these compounds are well below 100 μg/L. +

+

2

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2

n

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335 Methylnaphthoic acid (or Naphthylacetic acid)


A compound was identified with a product-ion mass spectrum consistent with methylnaphthoic acid or naphthylacetic acid. The mass-spectrum presented in Figure 5 was obtained with collision cell energy of 13 eV.

141.0

100k

1.85.0 l|llll| ||| l^l IIH IIU lll#|| || | |.f.lll,ll|l |... l., l

The Determination of Anaerobic Biodegradation Products of Aromatic

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