MS and Time of Flight

Chapter 11

Identification of Labile Polar Organic Contaminants by Atmospheric-Pressure Ionization Tandem Mass Spectrometry 1

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Edward T. Furlong , Imma Ferrer , Paul M. Gates , Jeffery D. Cahill , and E. Michael Thurman Downloaded by COLUMBIA UNIV on September 2, 2012 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch011

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National Water Quality Laboratory, U.S. Geological Survey, P.O. Box 25046, Denver, CO 80225 Current address: [email protected] Current address: U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, KS 66049 2

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Mass spectrometry, in single or multiple stages, can be coupled 40 high-performance liquid chromatography by an atmospheric-pressure ionization interface (HPLC/MS or HPLC/MS/MS), thus providing a sensitive and selective tool for the identification and quantitation of labile polar organic contaminants in aquatic and sedimentary environments. An example is the identification of unknown compounds in a water sample showing substantial toxicity to plankton. This sample was analyzed by full-scan HPLC/MS with atmospheric-pressure chemical ionization in the positive mode. Iterative, data-dependent HPLC/MS/MS analyses identified characteristic fragment ions for these compounds and confirmed the presence of an extended series of nonylphenol ethoxylate surfactant homologues at microgram¬ -per-liter concentrations in these agriculturally influenced samples. An additional example is provided by the use of ion¬ -trap HPLC/MS/MS with electrospray ionization to identify pharmaceuticals and cationic surfactants in sediment extracts at concentrations ranging from 2 to 200 ng/g. The MS/MS capabilities of this instrument permitted positive identification at subnanogram-per-gram concentrations in the presence of substantial coextracted interferences.

U.S. government work. Published 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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Downloaded by COLUMBIA UNIV on September 2, 2012 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch011

Introduction For several years, the combination of high-performance liquid chromatography with mass spectrometry (HPLC/MS) by using an atmosphericpressure ionization interface has been proposed as the ideal tool for sensitive and selective identification and quantitation of labile polar organic contaminants, such as pesticides, in aquatic and sedimentary environments (1). Although numerous applications for identifying and quantifying pesticides, drinking-water disinfection byproducts, surfactants, and other contaminant classes at less than l ^ g / L concentrations have been reported (2-5), there has not been a parallel increase in use of HPLC/MS for routine environmental monitoring. Although at least one large-scale study has used routine monitoring by HPLC/MS (6), two problems seem to impede wider implementation. The first problem is limited fragmentation in the atmospheric-pressure ionization (API) source, which can preclude unambiguous identification. The second problem is coeluting sample matrix components that also can interfere with identification, or can affect analyte ionization efficiency, which result in signal suppression or enhancement, thus introducing a positive or negative bias into quantitation. Limited fragmentation and matrix effects are not limited to environmental) analysis (7-14). Approaches often used in gas chromatography/mass spectrometry, such as the incorporation of isotopically labeled internal standards into the instrumental analysis, can reduce but not eliminate the impact of limitedfragmentationand matrix effects on HPLC/MS analysis. As a result, these matrix-associated problems have hindered the wider acceptance of HPLC/MS for routine monitoring of labile polar organic contaminants. Tandem mass spectrometry (MS/MS), using either quadrupole or ion-trap (IT) mass spectrometers, when coupled with high-performance liquid chromatography (HPLC/MS/MS) addresses some of these shortcomings. In MS/MS, a precursor ion of a single m/z is selected from all the ions produced and transmitted to the mass analyzer. In HPLC/MS/MS under positive electrospray ionization conditions, this precursor ion typically is a protonated molecule or adduct ion of the compound of interest. This precursor ion is fragmented, and one or more of the characteristic product fragment ions are analyzed and used for specific identification. This selective, multistage analysis provides a higher order of mass spectral information that can be used to identify and quantify unknown compounds in the presence of complex sample matrixes, a shortcoming for HPLC coupled to a single level of MS analysis. In some cases, chromatographic separation is not required, which provides the additional advantage of rapid analysis. In this chapter, two examples of the unique capabilities of quadrupole and ion-trap tandem mass spectrometers for environmental HPLC/MS/MS analysis are described. Quadrupole HPLC/MS/MS is applied to a complex agricultural


177 runoff sample where MS/MS was used to determine the identity of high molecular weight nonionic nonylphenol ethoxylate surfactants (NPEOs). Iontrap HPLC/MS/MS is applied to the identification and quantitation of pharmaceuticals and antimicrobial surfactants in sediment extracts containing a complex mixture of natural and anthropogenic organic compounds.


Methods Trace organic constituents in the agricultural water samples were isolated using C solid-phase extraction cartridges eluted with 95:5 methanol:water, and the extracts concentrated. Commercial mixtures of NPEOs used to confirm identification in water samples were diluted and used as is. The sample extracts and standards were analyzed by injection of 5-μί aliquots into a stream of 30percent acetonitrile in water, at a flow rate of 0.6 mL/min. The flow-injection stream was interfaced to a triple-quadrupole mass spectrometer by an atmospheric-pressure chemical ionization (APCI) interface. The APCI vaporizer was held at 475°C, the capillary at 225°C, and the sheath gas pressure was 551.6 kPa. The MS was operated in the positive ion mode. Pharmaceuticals and antimicrobials in sediment were extracted by pressurized-fluid extraction of wet sediment with a 60-percent acetomtrile:40percent water mixture, using the procedure of Ferrer and Furlong (15). The sediment extracts were analyzed without further isolation or purification, although they were diluted to the composition of the starting eluent of the HPLC gradient (15-percent acetonitrile in water). A 150 mm by 2.0 mm i.d. octadecylsilane reversed-phase (5-μπι particle diameter) HPLC column was used. A linear gradient from 15- to 100-percent acetonitrile in an aqueous ammonium formate/formic acid buffer (10 mM, pH 3.7) was used to separate the pharmaceuticals. Each extract was analyzed twice. The first analysis used a quadrupole HPLC/MS system with electrospray ionization operated in the positive ion mode and with selected-ion monitoring to enhance sensitivity and decrease chemical noise. The second analysis made use of ion-trap mass spectrometry (ITMS), also with electrospray ionization operated in the positive ion mode. For the ITMS, two analyses were required, the first by full-scan MS analysis, followed by an MS/MS analysis based on full-scan MS results. The full-scan MS analysis was used to identify the protonated molecule or adduct ion for each pharmaceutical. This step was followed by an MS/MS analysis of each precursor ion to confirm the identification of each pharmaceutical. Antimicrobial surfactants were determined using only ion-trap tandem mass spectrometry, described by Ferrer and Furlong (15). 8


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Results


Agricultural Samples An initial full-scan MS analysis (Figure 1) of the surface-water sample, suspected to contain agricultural runoff, suggested that the sample potentially contained three homologous series of compounds. Each series was separated by a diagnostic 44 mass-to-charge units (m/z), which in turn suggested three series of polyethoxylated polymers (16). The first series, ranging from 353 to 573 m/z, was suspected to be a series of nonylphenol ethoxylate (NPEO) surfactant homologues containing 3 to 8 ethoxy units, based on the expected protonated molecules for NPEO surfactants. The mass spectrum of the protonated molecules of a mixture of NPEO surfactants was determined by full-scan MS analysis of a commercial NPEO mixture, which indicated that the suspected NPEO protonated molecules in the water samples also were present in the standard (Figure 2). However, the distributions of NPEO homologues in this and other commercial mixtures were substantially biased towards higher NPEO homologues relative to the homologues present in the environmental samples. Tandem mass spectrometry then was used to confirm identification of NPEOs in this sample by comparison of MS and MS/MS spectra from the environmental sample to MS and MS/MS spectra of a commercial mixture of NPEOs (Figures 3 and 4). In both standard and environmental samples, the same series of putative protonated molecules were observed, each separated by 44 m/z. In the MS/MS spectra of the environmental sample and commercial mixture, the same product fragment ions are observed, although there are differences between the relative abundances of the same mass ion in each spectrum. These differences likely are a function of the absolute abundance of the protonated molecular ion used for the MS/MS analysis (m/z 485) of the environmental sample in relation to the pure standard. The pattern of NPEO homologues in the environmental water samples did not closely match any of the standards and might reflect biotic or abiotic alteration of the NPEO signature under environmental conditions. A less abundant second series differing by m/z 44, from 370 to 529 m/z, tentatively was identified as a homologous series of polyglycol ethers (PGEs), described as minor components of commercial NPEO mixtures (17). The number of ethoxy units in the homologous series ranges from 8 to 12. A third homologous series, tentatively identified as carboxylated degradation products of NPEOs (CNPEOs), also was observed. The CNPEO homologues contain 6 to 8 ethoxy units. The CNPEOs previously have been reported as primary degradation products of the NPEOs in wastewater, surface water, and agricultural settings (18-20), although homologues with lower degrees of


179 485N-6

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Ν-χ: N P E O homologue with χ ethoxy units Ρ-χ: P G E homologue with χ ethoxy units CN-x: carboxylated μ. 80 N P E O homologue with Ζ xcarboxy units LU ϋ

N-5 441

ISM 397

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P-11 502

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CN-6 542

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MS and Time of Flight

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