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CHAPTER 11 ANALYSIS OF SELECTED EMERGING POLLUTANTS
(STEROID SEX HORMONES, DRUGS AND ALKYLPHENOLIC SURFACTANTS) IN THE AQUATIC ENVIRONMENT BY LC-MS
AND LC-MS-MS
María J. Lopez de Alda1, Silvia Díaz-Cruz2, Mira Petrovic1, Damià Barceló1
1Department of Environmental Chemistry, IIQAB-CSIC, c/ Jordi Girona 18-26, 08034 Barcelona, Spain,
2Department of Analytical Chemistry, Faculty of Chemistry, University of Barcelona, c/Diagonal 647, 08028,Barcelona, Spain
ABSTRACT Alkylphenolic surfactants, steroid sex hormones, and pharmaceuticals constitute three classes of emerging pollutants of particular interest in the field of environmental monitoring due to the volume used of these substances and their activity as either endocrine disruptors or as causative agents of bacterial resistance, as is the case of antibiotics. Today, the technique of choice for analysis of these groups of substances is liquid-chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS-MS). This technique provides both good sensitivity and selectivity in the analysis of polar trace-level environmental pollutants and presents certain advantages with respect to other commonly employed analytical techniques such as GC-MS. In this article, the LC-MS-MS methods published so far for the determination of alkylphenolic surfactants, steroid sex hormones and drugs in the aquatic environment, are reviewed. Emphasis is put on practical considerations regarding the analysis of these groups of substances by using different mass spectrometers (single quadrupole, ion trap and triple quadrupole instruments, etc.), interfaces and ionization and monitoring modes. Sample preparation aspects are also briefly discussed. Future trends on the application of LC-MS based novel technologies are finally outlined.
Chapter 11
1 INTRODUCTION In general, legislation is the main driving force in environmental analytical chemistry. Environmentalists have centred on the study of chemicals whose presence in the environment has been regulated through the various lists of criteria or priority pollutants included in the different legislations.
The development of new and more sensitive methods for both detecting chemicals and determining their biological effects has, however, shifted the attention of the scientific community towards new, unregulated contaminants that were previously undetected or had not been considered as a risk. This is the case of the so-called emerging contaminants. Emerging contaminants are defined as newly identified or previously unrecognised pollutants [1], and include products used in everyday life, such as surfactants, pharmaceuticals and personal care products, gasoline additives, plasticizers, etc. (see Table 1).
TABLE 1. Emerging compound classes
Compound class Examples
Pharmaceuticals Veterinary and human antibiotics Analgesics and antiinflamatory drugs Psychiatric drugs Lipid regulators β-blockers X-ray contrasts
Trimethoprim, erytromycine, lincomycin, sulfamethaxozole Codein, ibuprofene, acetaminophen, acetylsalicilyc acid, diclofenac, fenoprofen Diazepam Bezafibrate, clofibric acid, fenofibric acid Metoprolol, propanolol, timolol Iopromide, iopamidol, diatrizoate
Steroids and hormones (contraceptives) Estradiol, estrone, estriol, diethylstilbestrol Personal care products Fragrances Sun-screen agents Insect repellants
Nitro, polycyclic and macrocyclic musks, Benzophenone, methylbenzylidene camphor N,N-diethyltoluamide
Antiseptics Triclorsan, Chlorophene Surfactants and surfactant metabolites Alkylphenol ethoxylates, alkylphenols (nonylphnol and
octylphenol), alkylphenol carboxylates Flame retardants Polybrominated diphenyl ethers (PBDEs),
Tris(2-chloroethyl)phosphate Industrial additives and agents Chelating agents (EDTA), aromatic sulfonates
Gasoline additives Dialkyl ethers, Methyl-t-butyl ether (MTBE) Disinfection by-products Iodo-trihalomethanes, bromoacids, bromoacetonitriles,
bromoaldehydes, cyanoformaldehyde, bromate
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Of all the emerging contaminants, antibiotics, because of the potential genetic selection of resistant bacteria, and endocrine disrupting compounds (EDCs), because of the potential induction of estrogenic effects, such as feminization and hermaphroditism, in aquatic organisms, and ultimately in humans, are probably the biggest worry. Among EDCs, steroids and alkylphenol ethoxylate surfactants (APEOs) are the compound classes most frequently identified as the substances responsible for the estrogenic effects observed in wildlife [2-5].
Due to the physical and chemical characteristics of the above mentioned compound classes (high to moderate polarity and hydrophilicity), the technique of choice for their analysis is LC-MS and LC-MS-MS [6]. Before the advent of LC-MS, many of these polar compounds were difficult and sometimes impossible to measure.
In the last decades, LC-MS and LC-MS-MS have experienced impressive progress, both in terms of technology development and application. Today, the interfaces most widely used for the LC-MS analysis of steroids, drugs, and surfactants, and organic pollutants in general, are electrospray (ESI) and atmospheric pressure chemical ionisation (APCI). ESI is particularly well suited for analysis of polar compounds whereas APCI is very effective in the analysis of medium- and low-polarity substances.
As mass analysers, both single and triple quadrupole mass spectrometers, and to a much lesser extent orthogonal-acceleration time-of-flight (oaTOF), ion trap, and sector-field MS instruments, have been used. Both LC-MS and LC-MS-MS, operated in the selected ion monitoring (SIM) mode and in the selected reaction monitoring mode (SRM), respectively, offer good sensitivity and selectivity in the analysis of environmental pollutants. However, in the case of very complex matrices, such as wastewater and sludge, even when using SRM detection, both false negative results, due to matrix ionisation suppression effects, and false positive results, due to insufficient selectivity, can be obtained [7].
An approach for increasing the selectivity, and avoiding false positive findings is the use of LC-TOF-MS or LC-Q-TOF-MS (combination of a quadrupole front-end and an oa-TOF back-end) [7,8,9]. However, neither of these two advance techniques (LC-oa-TOF-MS and LC-Q-TOF-MS) have been routinely employed yet for the qualitative or quantitative determination of steroids, drugs, and alkylphenolic surfactants in environmental samples, probably due to their low quantitation range. However, the new generation of LC-TOF-MS systems looks promising in this respect, since sensitivity and linear range are similar to those of LC-MS-MS.
Compound identification is also possible in the full scan mode and after collision induced dissociation with single quadrupole and ion trap instruments, and in the precursor-ion, product-ion, and neutral loss scan modes with triple quadrupole instruments, but these techniques often lack sufficient sensitivity for the trace analysis of environmental pollutants [7].
In this article, the LC-MS and LC-MS-MS-based methods so far applied to the environmental monitoring of steroid sex hormones, drugs and alkylphenolic surfactants, are reviewed.
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2 SAMPLE PREPARATION The analysis of micropollutants in the environment constitutes a difficult task, first, because of the complexity of the matrices, and second, because of the normally very low concentrations of the target compounds. In essentially all cases of interest, substantial analyte enrichment is necessary to isolate the target compounds from the matrix and to achieve the LODs required. A typical analytical procedure includes, therefore, various sample preparation steps, such as filtration, extraction, purification, and evaporation. In the following section, the main sample pretreatment procedures applied in the analytical determination by LC-MS or LC-MS-MS of steroid sex hormones and related synthetic compounds, drugs, and alkylphenolic surfactants, in aquatic environmental samples, are briefly discussed. 2.1 Steroid sex hormones and related synthetic compounds. 2.1.1 Aqueous samples The analytical methods published in the literature for the analysis of estrogens and progestogens in wastewater have been recently reviewed by López de Alda and Barceló [10]. According to this review, neither the filtration of the water samples nor the evaporation of the extracts leads to significant losses of the analytes. Thus, of the various steps involved in the sample preparation procedure, the extraction/purification is the most critical.
Extraction of both estrogens and progestogens from water has been usually carried out by off-line solid-phase extraction (SPE) with either disks or most frequently cartridges (see Table 2). The advantages and disadvantages of some commercially available sorbents, cartridges and devices for the off-line and on-line SPE of estrogens and progestogens from environmental matrices have discussed recently [11].
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TABLE 2. Survey of LC-MS and LC-MS-MS methods for quantitative determination of steroid sex hormones and related synthetic compounds in aquatic environmental samples.
Compounds Matrix Extraction Detection method
LOD (ng/l, ng/g)
Ref.
E2, E3, E1, EE, DES, PROG, LEV, NOR
Water on-line SPE (HySphere-Resin-GP col.)
ESI-MS <1 [11]
E2, E3, E1 River water SPE (SDB-SC disk) ESI(NI)-MS 1-50 [12] E2, E1 STP effluent SPE (LiChrolut EN+C18
col.) + Immunoaffinity
extraction
ESI(NI)-MS 0.07-0.18 [13]
E2, EE Aquaria water SPE (Sep-Pak C18) APCI(PI)-MS 0.6-1 [14] E2, E3, E1, EE,
DES River water SPE (C18 col.) IS(NI)-MS 3.2-10.6 [15]
E2, E3, E1, EE River water,
STP influent and effluent
SPE (Carbograph-4 col.)
ESI(NI)-MS-MS 0.08-0.6* [16]
E2, E3, E1, EE, E3-3G, E2-3G, E1-3G, E3-16G, E2-17G, E3-3S, E2-3S, E1-3S.
Sewage and river water
SPE (Carbograph-4) ESI(NI)-MS-MS 0.003-15 [17]
E2, E3, E1, EE, DES
Water (tank, river, STP effl.)
SPE (C18 col.) ESI(NI)-MS-MS 5 [18]
E2, E3, E1, EE STP influent
and effluent SPE
(ENVI-CARB col.) APCI(PI)-MS-
MS 0.5-1* [19]
E2, E3, E1, EE, MES, equilin, testosterone, dihydrotestoste-rone, cyproterone
River water SPE (C18 col.) APCI(PI)-MS-MS
1-10 [20]
E2, E3, E1, EE, DES, PROG, LEV, NOR
River sediment PLE (acetone:metha-nol 1:1),
+ SPE (LiChrospher ADS C4)
ESI-MS (SIM) 0.5-5 [21]
E2, E3, E1, EE, DES, PROG, LEV, NOR
River sediment Ultrasonication (acetone:methanol 1:1)
+ SPE (C18 col.)
ESI-MS (SIM) 0.04-1 []
Abbreviations (not included in the text): Ref., reference; SFE, supercritical fluid extraction; E2, estradiol; E3, estriol; E1, estrone; EE, ethynyl estradiol; DES, diethylstilbestrol; PROG, progesterone; LEV, levonorgestrel; NOR, norethindrone; STP, sewage treatment plant; col., column or cartridge; ACN, acetonitrile; IS, ionspray or pneumatically assisted electrospray E3-3G, estriol 3-(β-D-glucuronide); E2-3G, 17β-estradiol 3-(β-D-glucuronide); E1-3G, estrone 3-(β-D-glucuronide); E3-16G, estriol 16α-(β-D-glucuronide); E2-17G, β-estradiol 17-(β-D-glucuronide); E3-3S, estriol 3-sulfate; E2-3S, estradiol 3-sulfate; E1-3S, estrone 3-sulfate; MES, mestranol
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Octadecyl (C18)-bonded silica has been the adsorbent most widely employed, although polymeric sorbents and graphitized carbon black (GCB) have also been used. Promising results in the field of immunoaffinity extraction have also been obtained by Ferguson et al. [13]and by Tozzi et al. [23].
Attempts to develop fully automated methodologies for the on-line SPE and analysis of estrogens in water have been made in a few occasions [11,24-26], but in only two of them, LC-MS has been used for final determination [11,24]. In the most recent of these works [11], 10×2 mm I.D. HySphere-Resin-GP cartridges (Spark Holland, The Netherlands), were selected out of three other sorbents −C18 BAKER (J.T. Baker, Deventer, The Netherlands), PLRP-S (Polymer Laboratories, Church Stretton), and Oasis HLB (Waters, Milford, MA, USA)- for the on-line SPE of the most environmentally relevant estrogens from water using the automated sample preparation system Prospekt (Spark Holland). With this methodology, estrogens could be detected at concentrations as low as 1 ng/l, in up to 16 samples in a fully automated, unattended way, using an ESI interface in the negative mode of ionisation. 2.1.2 Solid samples The analytical methods described so far in the literature for the determination of estrogens and progestogens in fresh water sediments [27] and in solid environmental samples in general [28] have been reviewed in two recently published articles. These reviews also cover the determination of other classes of EDCs (alkylphenols, polychlorinated compounds (dioxins, furans, and biphenyls), polybrominated diphenyl ethers, and phthalates) [27] and drugs [28], but they do not specifically discuss the use of LC-MS methods. Estrogens and progestogens have been seldom analyzed in sludge, soils and sediments, and in only a few occasions final determination has been carried out by LC-MS [21-29].
Extraction of both natural and synthetic estrogens from river sediments has always been performed with acetone:methanol 1:1, using either sonication [22] or pressurized liquid extraction (PLE) [21], and clean -up of the extracts has been carried out by SPE with C18 columns (see Table 2). The use of a novel methodology based on column switching LC-MS using restricted access materials (RAM) for further, integrated clean-up and analysis has been described by Petrovic et al. [21]. In this work, different LiChrospher ADS RAM precolumns (Merck, Germany), with C4, C8 and C18 modification of inner pore surface were tested, and the ADS C4 precolumn was found to be the most convenient in terms of recovery, selectivity and sensitivity. With this methodology, steroid sex hormones and other pollutants, such as alkylphenolic compounds and bisphenol A, could be detected in sediments at levels of 0.5¯5 ng/g due to the efficient removal of co-extracted matrix components and the consequent reduction of ion suppression effects.
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2.2 Drugs 2.2.1 Aqueous samples Previous to extraction water samples are filtered and pH adjusted to values ranging from acid to alkaline pH (2, 3, 5, 7 and 9) depending on the acid (tetracyclines) or alkaline (phenazone-type) nature of the drugs under study. To avoid photodegradation, which affects particularly some compounds, such as fluoroquinolones and tetracyclines (TCs), samples are stored at low temperatures (c.a. 4ºC) in amber glass bottles until extraction. In most instances, extraction and preconcentration has been performed by SPE, with some exceptions which use lyophilisation [30,31] (see Table 3). For SPE, several adsorbent materials have been employed being the reverse phase. A precaution leading to a notable improvement of extraction efficiencies is the silanisation, for instance with dimethyldichlorosilane [32], of all glassware coming into contact with either the water sample or the extract, or the use of other container materials, such as PTFE. These preventive measures are essential in the analysis of polar compounds, such as betablockers, and of compounds with probed chelating capabilities, such as TCs [33]. Additional approaches to prevent chelation of metals are washing off the cartridge using a diluted HCL solution and adding a strong quelator to the sample, for instance Na2EDTA [34]. In addition, cartridge materials used for analysis of TCs must not contain silanol groups, since they have been found to bind irreversibly to this class of antibiotics. For extraction of TCs from water, solid phase micro-extraction (SPME) ‘on-line’ with LC-MS has also been tested [35].
2.2.2 Solid samples The presence of pharmaceutical products in soil, sediment and sludge has scarcely being investigated as compared to aquatic media. To date, only one review devoted to the analysis of solid environmental matrices is available [28].
Extraction of drugs from solid matrices has normally been performed by sonication or by simple blending or stirring of the sample with polar organic solvents or mixtures of them, or with aqueous solutions. The use of more advanced extraction techniques, such as PLE, has been reported by Reddersen et at. [36] for the analysis of phenazone in sludge.
Cleanup of the extracts, when performed, has been carried out by liquid-liquid extraction (LLE), gel permeation chromatography (GPC), semi-preparative LC, and, in most instances, by SPE. With the exception of Brambilla et al. [37] who used strong cation exchange (SCX) cartridges, SPE cleanup of the extracts has always been performed with reversed phase adsorbents.
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TABLE 3: Survey of LC-MS methods used for quantitative analysis of human and veterinary drugs. Compounds Matrix Extraction Detection
method LOD (ng/l, ng/g)
Ref.
TCs, SAs, trimethoprim, tylosin, ibuprofen, ciprofloxacin, enrofloxacine, fluoxetine, gemfibrozil, among others
Stream waters Tandem SPE Oasis HLB/MCX cartridges SPE Oasis HLB
ESI(PI)-MS ESI(PI)-MS-MS
Not reported
[38]
Analgesics (phenazone), β-blockers (propanolol), broncholitics (clenbuterol), antineoplastics (ifosfamide) lipid lowering agents (simvastatin), X-ray contrast media (iopamidol) Antibiotics: SAs (sulfadiazine) Macrolides (erythromycin) PENs (amoxicillin)
Tap and surface waters
SPE PPL Bond-Elut SPE LiChrolut EN SPE Isolut ENV+
ESI(PI)-MS-MS ESI(PI)-MS-MS ESI(PI)-MS-MS
8-44 8-16 3.7-11 6.4-15 15-21
[39]
Caffeine, aminoantipyrine, propyphenazone, diazepam, nifedipine, glibenclamide, omeprazole, oxyphenbutazone, phenylbutazone, dihydrocarbamazepine
Groundwater, wastewaters
SPE Isolute C18
ESI(PI)-MS-MS 10-50 [33]
TCs, PENs, SAs, macrolid antibiotics, trimethoprim chloramphenicol
Surface waters SPE (except TCs) LiChrolut EN LiChrolut C18 Lyophiliz.
ESI(PI)-MS-MS Except for Chloramphenicol (ESI(NI))
20-50 [31]
Clofibric acid, penicillin V, naproxen, benzafibrate, carbamazepine, diclofenac sodium, ibuprofen, mefenamic acid.
River waters SPE Bondesil ODS 40 µm.
ESI-MS APCI-MS
0.04-1.1 [40]
Ibuprofen, ketoprofen, naproxen, diclofenac, salicylic acid, gemfibrozil
River waters, wastewaters
SPE LiChrolut EN Oasis HLB
ESI(NI)-MS Not reported
[41]
TCs Tylosin
Fertilized soil, Dried liquid manure
Soil water, Ground water
LLE SPE
Baker SDB1
ESI(PI)-MS-MS 5
50 100 100
[42]
TCs Groundwater, confined animal wastewater
SPE HLB ESI(PI)-MS-MS 200-380 [43]
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2.3 Alkylphenolic compounds 2.3.1 Aqueous samples For preconcentration of alkylphenolic surfactants from aqueous samples, SPE is considered the most appropriate technique. Octadecyl (C18) bonded silica has been the SPE material most widely employed for extraction of both neutral and acidic alkylphenolic compounds [13,44-48] (extraction efficiencies higher than 80%), although strong anionic exchange (SAX) [49] and GCB have also been employed [50] (see Table 4). SPE procedures based on the use of two cartridges of different SPE material (C18 and polymeric) coupled in series [51,52] and on sequential selective elution for the analysis of alkylphenolic compounds and their acidic and neutral degradation products have also been reported [53,54].
2.3.2 Solid samples A review on the sample preparation methods for the determination of APEOs and their degradation products in solid environmental matrices has been recently published by Petrovic and Barceló [55]. The common approach for extracting alkyphenolic compounds from solid samples includes either traditional exhaustive extraction techniques, such as Soxhlet extraction or sonication, or advanced extraction techniques based on elevated temperatures and pressures, such as PLE or microwave-assisted extraction (MAE) (Table 4). Typical solvents for the extraction of APEOs and alkylphenols (APs) have been methanol, dichloromethane, dichloromethane/hexane, hexane/acetone or hexane-i-propanol mixtures [55].
A modification of PLE, extraction with pressurized (supercritical) hot water, as well as extraction with hot water under subcritical conditions, have also been used for the analysis of polar alkylphenolic compounds [56]. For extract clean-up, the common approach is based on SPE using different types of commercially packed cartridges (C18, CN or NH2). Recently, a column-switching system using precolumns packed with RAM was successfully employed for the simultaneous analysis of alkyphenolic compounds and steroid sex hormones in sediments [21].
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TABLE 4. Survey of LC-MS and LC-MS-MS methods for quantitative determination of alkylphenol ethoxylates and their metabolites in aquatic environmental samples. Compound Matrix Extraction Clean-up Separation and
detection method LOD (ng/l, ng/g)
Ref.
NPEC, NP, halogenated derivatives
River water, drinking water, sludge
SPE-C18 PLE (MeOH-
acetone, 1:1)
SPE-C18 RP-LC-ESI-MS-MS 1-2 ng/l 0.5-1.5 ng/g
[57]
OP Fish tissue, water
MAE (DCM-MeOH, 2:1)
SPE (Sep-Pak C18)
SPE-NH2 RP-APCI-ESI-MS 100 ng/l 10 ng/g
(muscle) 50 ng/g
(liver)
[58]
NPEO STP samples
SPE-C18 Sequential elution (4 fractions)
FIA-APCI-MS not reported [53,54]
NPEO, NPEC, CAPEC
Wastewater SPE-GCB - RP-LC-ESI-MS Not reported
[59]
NPEO, NPEC, NP
waste water, river water, sludge
SPE-C18 Sonication (DCM-MeOH
(3:7)
SPE-C18 RP-LC-APCI-MS 80-200 ng/l [5152]
APEO, APEC, AP, halogenated derivatives
River water sediment, sludge,
Sonication (DCM-MeOH
(3:7)
SPE-C18 RP-LC-ESI-MS 20-50 ng/l 5-25 ng/g
(sludge) 2-10 ng/g
(sediment)
[60]
NPEO, OPEO
(nEO=6-15)
Wastewater, sludge
LLE (DCM) - RP-LC-ESI-MS 100 ng/g [61]
1. SPE-NH2 2. RP-LC
fractionation
RP-LC-ESI-MS 0.04-0.92 ng/l
[13,62]
APEO (nEO=1-3), APs, XNPs
Estuarine sediment
High-temperature continuous flow sonication (MeOH)
RP-LC fractionation (2 columns in series)
Mixed mode LC-ESI-MS
21.5 NP 0.78-37.3
NPEO
[63]
C-18 1-5 ng/g [64] APEOs, APECs, APs, halogenated derivatives
River sediment, sludge
PLE
RAM (ADS C4)
RP-LC-ESI-MS
0.5-5 ng/g [21]
NPEO (nEO=1-19)
Marine sediment
Soxhlet (hexane-IPA
70:30)
SPE-CN NP-LC-ESI-MS 2-10 [65]
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3 ANALYSIS 3.1 Steroid sex hormones and related synthetic compounds The use of LC-MS or LC-MS-MS for the environmental analysis of steroids has gained in popularity in the last years. The main advantage of this technique is that the enzymatic hydrolysis, required for the immunoassay analysis of both conjugated (glucuronides, sulfates, etc.) and unconjugated estrogens and progestogens, and the derivatization that normally precedes a subsequent GC-MS analysis, can be avoided.
Prior to MS detection, the LC separation of both conjugated and unconjugated estrogens and progestogens, has always been performed on octadecyl silica stationary phases (see Table 2).
As mobile phases, mixtures of water-methanol and, more frequently, water-acetonitrile, have normally been used. The use of mobile phases additives, such as acetic acid 0.1% [20], formic acid 0.2 % [14], ammonium acetate 10 mM [18], or methanolic ammonia (added post-column) [16,17] (see Table 2), for improving LC separation and/or MS sensitivity, has been reported in some works. However, according various studies carried out to investigate in detail the influence of different mobile phase compositions on the ionisation efficiency, a mixture of water and acetonitrile, without addition of bases or buffer systems is the best choice for optimal determination of estrogens [15, 66].
For LC-MS analysis of steroid sex hormones and related synthetic compounds, both APCI, and to a greater extent, ESI have been used. As indicated in Table 2, the LC-MS analysis of estrogens has been carried out in most instances with an ESI interface operating in the negative ion mode of ionisation (NI). With this technique the sensitivity achieved in the analysis of estrogens is considerably better than that attained with ESI in the positive ion mode of ionisation (PI) and with APCI in the NI mode [16,66]. Figure 1 illustrates representative SRM chromatograms obtained from the analysis of environmentally relevant pollutants by LC-ESI-MS-MS. The base peak selected for quantitation of estrogens in the SIM mode, or as precursor for collisionally induced dissociation in the SRM mode, corresponds to the deprotonated molecular ion [M-H]-.
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10101010
10101010
1010101010
1010101010
1010101010
1010101010
1111111111
1010101010
1010101010
1010101010
1010101010
3333333333
1010101010
1010101010
2828282828
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00
Time
0
%
4
0
%
0
%
3
0
%
2
0
%
2
0
%
0
%
7
0
%
6
0
%
287 > 17110,22
271 > 14513,67
295 > 14514,64
269 > 14515,23
267 > 22216,06
18,82
313 > 25321,59
299 > 17114,12
313 > 24516,28
315 > 29720,49
0
0
%
0
%
447 > 1138,29
2,34
349 > 2699,45
2,32
Estradiol-
17-glucuronide
Estrone-3-sulfate
Estriol
Estradiol
Ethynyl Estradiol
Estrone
Diethylstilbestrol
Estradiol-17-acetate
Norethindrone
Levonorgestrel
Progesterone
a)
b)
c)
d)
g)
f)
e)
j)
i)
h)
k)
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00
Time
0
%
4
0
%
0
%
3
0
%
2
0
%
2
0
%
0
%
7
0
%
6
0
%
287 > 17110,22
271 > 14513,67
295 > 14514,64
269 > 14515,23
267 > 22216,06
18,82
313 > 25321,59
299 > 17114,12
313 > 24516,28
315 > 29720,49
0
0
%
0
%
447 > 1138,29
2,34
349 > 2699,45
2,32
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00
Time
0
%
4
0
%
0
%
3
0
%
2
0
%
2
0
%
0
%
7
0
%
6
0
%
287 > 17110,22
271 > 14513,67
295 > 14514,64
269 > 14515,23
267 > 22216,06
18,82
313 > 25321,59
299 > 17114,12
313 > 24516,28
315 > 29720,49
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00
Time
0
%
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00
Time
0
%
4
0
%
4
0
%
0
%
3
0
%
0
%
3
0
%
2
0
%
2
0
%
2
0
%
0
%
2
0
%
0
%
7
0
%
7
0
%
6
0
%
287 > 171
6
0
%
287 > 17110,22
271 > 145
10,22
271 > 14513,67
295 > 145
13,67
295 > 14514,64
269 > 145
14,64
269 > 14515,23
267 > 222
15,23
267 > 22216,06
18,82
313 > 253
16,06
18,82
313 > 25321,59
299 > 171
21,59
299 > 17114,12
313 > 245
14,12
313 > 24516,28
315 > 297
16,28
315 > 29720,49
0
0
%
0
%
447 > 1138,29
2,34
349 > 2699,45
2,320
0
%
0
0
%
0
%
447 > 1138,29
2,34
349 > 2699,45
2,32
Estradiol-
17-glucuronide
Estrone-3-sulfate
Estriol
Estradiol
Ethynyl Estradiol
Estrone
Diethylstilbestrol
Estradiol-17-acetate
Norethindrone
Levonorgestrel
Progesterone
a)
b)
c)
d)
g)
f)
e)
j)
i)
h)
k)
Figure 1. Reconstructed ion chromatograms corresponding to the LC-ESI-MS-MS
analysis of a 100 ng/ml mixture of estrogens and progetsogens in methanol. Column: Purospher STAR RP-18 (125 × 2 mm, 5 µm). Mobile phase: gradient acetonitrile/water. Flow-rate: 0.2 ml/min.
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As compared to estrogens, progestogens have been much less studied [22,29,66], and their analysis by LC-MS-MS has been attempted only once [67]. According to the work published by López de Alda and Barceló [66], progestogens can be detected in the positive ion mode of operation with both APCI and ESI. The base peak used for quantitation of progestogens corresponds to the protonated analyte molecule when APCI is used as interface and to adducts of the analyte molecule with one sodium atom when the interface employed is ESI. However, the sensitivity reported for the analysis of progestogens with the ESI interface is about 10-fold better than that of the APCI interface.
Table 5 lists the SRM transitions most frequently used in the analysis of estrogens (both free and conjugated) and progestogens by LC-ESI-MS-MS. Two transitions, one for quantitation and another one for confirmation, are usually recorded for compound (Table 5). TABLE 5. SRM transitions used in the LC-ESI-MS-MS analysis of the most environmentally relevant estrogens and progestogens in the aquatic environment. Compound Ion
mode 1st SRM transition
(m/z) 2nd SRM transition
(m/z) Ref.
Free estrogens E2 NI 271→183 271→145 [16,17,18,67] E3 NI 287→171 287→145 [16,17,18,67] E1 NI 269→145 269→143 [16,17,18,67] EE NI 295→159 295→145 [16,17,18,67] DES NI 267→222 267→237 [67] Conjugated estrogens
E3-3G NI 463→287 463→113 [17] E3-3S NI 367→287 367→80 [17] E3-16G NI 463→287 463→85 [17] E2-3G NI 447→271 447→113 [17] E2-3S NI 351→271 351→80 [17] E2-17G NI 447→271 447→85 [17, 67] E1-3G NI 445→269 445→113 [17] E1-3S NI 349→269 349→145 [17, 67] Progestogens Norethindrone PI 299→171 299→145 [67] Levonorgestrel PI 313→245 313→184 [67] Progesterone PI 315→297 315→279 [67]
In a work recently carried out by Díaz-Cruz et al. the instrumental sensitivity of
various mass spectrometric techniques optimized for analysis of estrogens and progestogens has been intercompared and the limits of detection obtained were found to improve in the order LC-APCI-MS-MS (5->1000 ng/ml) < GC-MS (1-20 ng/ml) < LC-ESI(NI)-MS(SIM) (0.1-20 ng/ml) < LC-ESI(NI)-MS-MS(SRM) (0.1-10 ng/ml) [67].
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3.2 Drugs For LC-MS and LC-MS-MS analysis of pharmaceuticals, ESI has been the ionisation technique of choice. Ahrer et al. [40] compared the performance of APCI and ESI in the analysis of diverse drugs (paracetamol, clofibric acid, penicillin V, carbamazepine, etc.) and found ESI to provide the best LODs (0.05 – 1.1 µg/l). The same interfaces, in both the positive and the negative ionization modes, were evaluated by Lindsey et al. [34] for the analysis of 11 sulfonamide and TCs antimicrobials. Under optimised conditions both ESI and APCI in the positive ion mode worked well. However, ESI was finally selected, because it provided the best sensitivity towards chlortetracycline.
Both LC-ESI(PI)-MS [38] and LC-ESI(PI)-MS-MS [39] have been used for the analysis of a large number of pharmaceuticals in different extensive monitoring programs, such as that carried out by Kolpin et al. from the U.S. Geological Survey in streams across U.S. during 1999 and 2000 [38], and that performed by Sacher et al. in ground waters across the city of Baden-Württemberg (Germany) in year 2000 [39].
LC separation has been carried out with different columns, mostly C18. As mobile phases, mixtures of water-methanol and water-acetonitrile at different pH, and sometimes modified with acetate [39,40,42,68], formate [38,39,42], or formic acid [43] for improved sensitivity, have been used.
MS and MS-MS determinations have normally been carried out in the SIM mode and the SRM mode, respectively, and in most instances the proton adduct of the analyte molecule [M+H]+ has been selected as precursor ion (see Table 6).
In the analysis of drugs residues in environmental solid matrices MS detection has not been as widespread used as UV or fluorescence, and, when applied, ESI has been the interface chosen. The latest and most advanced work on drug analysis in soils, has been conducted by Hamscher et al. [42], which reports the development of a new method to determine persistent TCs residues in soil fertilised with manure by LC- MS-MS and confirmation by MS-MS-MS.
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TABLE 6: m/z and base peaks of precursor and product ions used in the LC-ESI(PI)-MS-MS analysis of several betablockers, β2-simpathomimetics, antibiotics and other neutral pharmaceuticals.
Compound Precursor ion Product ion 1 Product ion 2
Salbutamola 240 [M+H]+ 222 [M-H2O+H]+ 148 [166-H2O]+ Terbutalina 226 [M+H]+ 152 [M-H2O-tert.butil++2H]+ 125 Fenoterola 304 [M+H]+ 135 [propylphenol]+ 107 [methylenphenol]+ Timolola 317 [M+H]+ 261 [M-tert.butyl++2H]+ 244 [M-tert.butylamine+H]+
Clenbuterola 277 [M-H]+ 203 [M-H2O-tert.butyl++2H]+ 259 [M-H2O+H]+ Celiprolola 380 [M+H]+ 251 (cleavage of side chain) 307 [M-tert.butylamine+H]+ Clarithromycinb 750 [M+H]+ 116 [cladinose-OCH3+H]+ 592 [M-desosamine+H]+
Roxitromycinb 838 [M+H]+ 158 [desosamine+H]+ 680 [M-desosamine+H]+ Sulfamethazineb 279 [M+H]+ 124 [aminodimethylpyridine+H]+ 186 [aminophenyl]+
Trimethoprimb 293 [M+H]+ 123 [M-trimetoxyphenyl]+ 231 [M-2CH3O+H]+
Chloramphenicolb 323 [M-H]+ 152 [nitrobenzylalcohol carbanion]-
176 [194-H2O]-
Chlortetracyclineb 479 [M+H]+ 444 [M-H2O- NH3+H]+ 462 [M-NH3+H]+ Doxycyclineb 445 [M+H]+ 428 [M-NH3+H]+ 410 [M-H2O-NH3+H]+ Oxytetracyclineb 461 [M+H]+ 426 [M-H2O-NH3+H]+ 443 [M-H2O+H]+ Tetracyclineb 445 [M+H]+ 410 [M-H2O-NH3+H]+ 427 [M-H2O+H]+ Cloxacillinb 453 [M+NH4]+ 160 [cleavage in β-lactam+H]+ 277 [cleavage in β-
lactam+H]+ Dicloxacillinb 487 [M+NH4]+ 160 [cleavage in β-lactam+H]+ 311 [cleavage in β-
lactam+H]+ Methicillinb 381 [M+H]+ 165 [dimethoxybenzaldehyd]+ 222 [cleavage in β-
lactam+H]+ Nafcillinb 432 [M+NH4]+ 171 [ethoxynaphthyl]+ 199
[ethoxynaphthylcarbonyl]+ Oxacillinb 419 [M+NH4]+ 144 [phenylisoxazolyl+H]+ 243 [M-
methylphenylisoxazolyl]+
Caffeinec 195 [M+H]+ 138 [M-CH3-N-CO+H]+ 110 [M-CO-N(CH3)-CO+H]+ Omeprazolec 346 [M+H]+ 136 [M-COH3-(C7N2H4)-SO-
CH2]+ 198 [M-COH3-C7N2H4]+
Erythromycinb 716[M-H2O+H]+
522 [M-desosamine-2H2O+H]+ 558 [M-desosamine- H2O+H]+
a Data from Ref. [69]. b Data from Ref. [31]. c Data from Ref. [33]. 3.3 Alkylphenolic compounds The LC separation of APEOs and their metabolites may be carried out either by normal phase (NP)-LC or by reversed phase (RP)-LC (see Table 4). In normal-phase systems, the APEOs are separated according to the increasing number of ethylene oxide units, while corresponding homologues with the same number of ethoxy units but different alkyl substituents co-elute (i.e. octylphenol ethoxylates (OPEO) and nonlyphenol ethoxylates (NPEO)). RP-LC that allows separation according to the character of the hydrophobic moiety is particularly well suited to separate alkyl-homologues, while the various oligomers containing the same hydrophobic moiety elute in one peak. Eluting all the oligomers into one peak has the advantages of increasing the peak intensity and therefore, increasing the sensitivity of determination. However, in RP-LC-ESI-MS, the interference of isobaric doubly charged ions [M+2Na]2+ of highly ethoxylated APEOs, that interfere with singly charged ions of less ethoxylated APEOs (e.g. odd numbered pairs NPE15O/NPE5O), is reported to cause an error up to 40% in the quantification of
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less ethoxylated NPEOs [65]. Intermediate resolution can be obtained by using C1-RP columns which also provide separation according to the number of ethoxy groups. Recently, Ferguson et al. [63] reported on the application of a mixed-mode LC separation, which operates with both size-exclusion and reversed-phase mechanisms, for the comprehensive analysis of NPEOs and NP in sediment and sewage samples.
With regards to detection, ESI and APCI are the ionisation methods of choice for determination of non-ionic surfactants APEOs. The trace analysis of APEOs and their metabolites by LC-MS or LC-MS-MS using API has been recently reviewed by Petrovic et al. [70]. ESI-MS methods, combined with NP-LC [65,71,72] or RP-LC [13,21,50,60 ,73] have been frequently used for the quantitative analysis of ethoxylates in environmental and wastewater samples. Several authors have also reported the identification and determination of APEOs in industrial blends, wastewaters and environmental samples by APCI-MS using FIA [53,54] or preceded by LC, using both RP [51,52,74,75] and NP [76,77] separation.
Generally, ESI interface is more often used for the analysis of alkylphenolic compounds due to the higher sensitivity, especially for alkylphenols [78]. Using an ESI interface APEOs show a great affinity for alkali metal ions, yielding almost exclusively evenly spaced sodium adducts [M+Na]+. However, formation of distinct adducts with ions originating from the buffer, the sample and/or the introduction system (e.g. H+, Na+, K+), water clusters (especially when using APCI interface), dimeric complexes and doubly charged ions, such as disodium adducts [13,50,70] are also reported. The use of mobile phase additives (e.g. ammonium acetate or formate, formic, acetic or trifluoroacetic acid, ammonium hydroxide) assures the reproducible adduct formation.
Halogenated APEOs, formed during chlorination at wastewater and drinking water treatment plants, have also been analysed by LC-ESI-MS [60]. Like their non-halogenated precursors they show a great affinity for alkali metal ions yielding exclusively evenly-spaced (∆44 daltons) sodium adduct peaks [M+Na]+ with no further structurally significant fragmentation. For this group of compounds the characteristic doublet signal of brominated and chlorinated compounds, respectively due to different contribution of their isotopes (79Br:81Br=100:98 and 35Cl:37Cl=100:33, respectively) allows identification of isobaric oligomers (e.g. ClAPnEO and BrAPn-1EO).
LC-MS-MS has been seldom used in the analysis of APEOs. The precursor ion scanning of m/z 121 and 133 and SRM applying FIA-APCI(PI)-MS-MS were used for a rapid screening of NPEOs in wastewaters and industrial blends [53,54]. The precursor scan of m/z 121 is characteristic for ethoxylates with 1 to 4 chain units, while m/z 133 is characteristic for NPE5O - NPE16O [79,80]. APCI-MS-MS showed ethoxy chain fragments at m/z 89, 133, 177 and the diagnostic fragment of OPEOs at m/z 277 and at m/z 291 for NPEOs [81,82].
Persistent acidic metabolic products of APEOs, alkylphenoxy carboxylates (APnEC) and dicarboxylates (CAPEC) were detected, in both, the NI mode [59,60,70,83, and the PI mode [84]. In the NI mode, using ESI, APECs give two types of ions, one corresponding to the deprotonated molecule [M-H]− and the other to [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. Neutral losses of the carboxylated ethoxy chain and carboxylated alkyl chain, respectively, and methanol loss followed by formation of acylium ions, were found to be typical fragmentation patterns for methylated CAPECs detected by ESI in the PI mode [84].
MS-MS spectra of APECs [83,57,85] shows intense signal at m/z 219 (for NPEC) and m/z 205 (for OPEC) that is produced after the loss of the carboxylated
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(ethoxy) chain, and sequential fragmentation of the alkyl chain resulting in ions m/z 133 and 147.
Using an ESI in the negative ion mode, alkylphenols (OP and NP) give exclusively deprotonated molecules (m/z 205 for OP and m/z 219 for NP), whereas 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 [58]. Alkylphenols give, in addition to the [M–H]− ion, fragment m/z 133, resulting from the loss of a C5H12 (OP) and C6H14 (NP) group. However, the sensitivity of detection, using an APCI source was reported to be approximately 40-50 times lower than that obtained with an ESI source [78]. An example of LC-MS-MS analysis of alkylphenolic compounds in sewage sludge is shown in Figure 2.
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time0
100
%
0
100
%
0
100
%
0
100
%
219 > 1334.82e4
20.10
263 > 2052.35e3
6.80
321 > 2192.00e4
9.12
277 > 219
2.03e4
8.44
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time0
100
%
0
100
%
0
100
%
0
100
%
219 > 1334.82e4
20.10
263 > 2052.35e3
6.80
321 > 2192.00e4
9.12
277 > 219
2.03e4
8.44
a)
d)
c)
b)
Figure 2. LC-MS-MS chromatogram of a sewage sludge sample. Traces: a) SRM channel m/z 219 → 133 - NP (tR=20.10); b) SRM channel m/z 263 → 205 - OPE1C (tR=6.80 min); c) SRM channel m/z 321 → 219 NPE2C (tR=9.12); d) SRM channel m/z 277 → 219 – NPE1C (tR= 8.44 min).
Product ion scan of deprotonated molecules of brominated NPECs and NP [57]
yields intense signals at m/z 79 and m/z 81 corresponding to the [Br]− (ratio of isotopes 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. For chlorinated NPs and NPECs the predominant fragmentation 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. Fragment corresponding to [Cl]− was produced only when sufficient collision energy was applied.
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4 CONCLUSIONS AND FUTURE PERSPECTIVES The impressive progress experienced by the LC-MS technology has made possible the routine environmental analysis of a great number of polar compounds, which were previously difficult or even impossible to analyze, such as many pharmaceuticals and alkylphenolic surfactants, at the ng/l and even pg/l level.
However, despite the high sensitivity and selectivity of LC-MS-based methodologies, and in particular of LC-MS-MS, both false positive and negative findings can still occur due to matrix interferences and ionization suppression effects, respectively. In this respect, the introduction of various incipient technologies both in the field of sample treatment, such as the selective supports based on the use of immunosorbents or molecular imprinted polymers, and in the analytical area, such as the oa-TOF-MS and Q-TOF-MS instruments, is expected to significantly improve the reliability of results.
On the other hand, up to the present, the most important value and application of current LC-MS techniques is the determination of known, target compounds. Since the capacity of these techniques for screening and identification of unknowns is relatively low, most efforts have focused on the detection of parent compounds, while the analysis of metabolites and transformation products has been limited to some few groups of compounds, such APEOs. To this end, a technology that holds promise for identification of new contaminants, in addition to oa-TOF-MS and Q-TOF-MS, is on-line LC-nuclear magnetic resonance (NMR)-MS. However, before its application to trace–level environmental analysis can be seriously considered, the low sensitivity of NMR as compared to MS, has to be addressed [86,87,88].
ACKNOWLEDGEMENTS This work has been supported by the Energy, Environmental and Sustainable Development (EESD) Program (Project AWACSS EVK1-CT-2000-00045), and by CICYT (Project PPPQ2000-3006-CE). María J. López de Alda and Mira Petrovic acknowledge Ramon y Cajal contracts from the Spanish Ministry of Science and Technology. REFERENCES
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