Determination of Chlorophenols in water by LC-MS/MS. Case study: 2-amino-4-chlorophenol

Transcription

1 Deteration of Chlorophenols in water by LC-MS/MS. Case study: 2-ao-4-chlorophenol Marta Sofia Tavares da Fonseca Lopes Mourato Instituto Superior Técnico, Lisbon, Portugal November 214 Abstract Chlorophenols (CP s) are described as having high toxicity, carcinogenic and mutagenic properties, consisting their monitoring in a key piece to reduce contaation of the environment and human health problems. The main objective of this work is to implement a new analytical methodology for the deteration of CP s in water, focusing on a particular compound, 2-ao-4-chlorophenol. The remaining compounds studied are: 2- chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol and 4-chloro-3-methylphenol. The methodology developed involves solid phase extraction followed by analysis by high performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (SPE-ESI-LS-MS/MS). The choice of this method is due to the properties of the target compounds, specifically to its high polarity. The optimization process of the method starts with obtaining the optimum conditions for mass spectrometry that allow the formation of the parent ion and the product ions, followed by selection of the conditions for chromatographic separation, especially the choice of chromatographic column and mobile phase. The sample extraction technique using SPE with 6 ml and 2 mg SDB (styrene divinylbenzene copolymer) cartridges was designed only for the compound 2-ao-4-chlorophenol. The proposed methodology allows only the quantification of the compound 2-ao-4-chlorophenol, with recoveries of 73 to 1 ml of Milli-Q water fortified with standard of 1 mg/l. Key words: Chlorophenols, SPE, HPLC, MS/MS. 1. Introduction In recent decades discharge into the environment of large amounts of chemical compounds of synthetic origin from the industrial activities, agricultural, medical and domestic has been detrimental to living organisms. Chlorophenols (CP s) fit in the above scenario, some of which are considered priority pollutants in waters by the U.S. Environmental Protection Agency (EPA) and also by the European Community (EC). Although with varying toxicity are all described as having high toxicity, mutagenic and carcinogenic properties. Thus the presence of CP s in wastewater and water for drinking purposes is subjected to a tight legislation. CP s are organochlorine compounds having in its constitution the phenol with chlorine atoms that can go up to five (mono-1-chlorophenol, di-2- chlorophenol, tri-3-chlorophenol, tetra-4-chlorophenol and penta-5-chlorophenol). In total there are nineteen different CP s distinguished from each other by the number of chlorine atoms and also by the 1

2 position that they occupy relative to the hydroxyl group. [1] The main sources of CP s are the process of chlorination of phenol, hydrolysis of chlorobenzenes, the bleaching process of wood pulp and degradation of pesticides. [2] Among the different CP s, pentachlorophenol (PCP) have been widely used as a wood preservative. [3] The case study 2-ao-4- chlorophenol (2-A-4-CP) is used as an intermediate for the synthesis of dyes, pigments and pharmaceuticals, for example consisting of a Chlorzoxazone muscle relaxant. [4] Current analytical methods of US-EPA (64, 65 and 841) for the deteration of phenolic compounds are based on liquid-liquid extraction (LLE) followed by gas chromatography (GC) using different detectors such as electron capture detector (ECD), flame ionization detector (FID) or mass spectrometry detector (MS). [5] There is, however, a growing tendency to replace these techniques by solid phase extraction (SPE) and liquid chromatography (LC) in order to avoid handling large amounts of toxic organic solvents and eliate the need for the derivatization before analysis. The development of interfaces at atmospheric pressure (API), has overcome some of the obstacles associated with the LC-MS coupling leading to an increased use of such systems for the analysis of phenolic compounds. [5] Other emerging approaches are solid phase microextraction (SPME) and the combination of SPE with supercritical fluid chromatography (SFE). [5] In alternative to the classical methods of chromatography, capillary electrophoresis (CE) has been used, in various forms, for the deteration of phenolic compounds. [5] Biological techniques such as immunoassays and biosensors arise from the need to develop rapid, portable and cost-effective technologies that can be applied e.g. as screening methods of the analytes prior to chromatographic analysis. [5] In the present study a system of high performance liquid chromatography (HPLC) coupled to a tandem mass spectrometer (MS/MS) equipped with an electrospray ionization (ESI) probe was used. The type of MS/MS setup used consists of two quadrupole analyzers separated by a collision cell (hexapole). The implementation process of the proposed analytical methodology involved the optimization of mass spectrometry conditions, the chromatographic conditions and the sample extraction technique Electrospray Ionization tandem mass spectrometry The basic principle of ESI is the generation of gas phase ions from the conversion of analytes in solution. First, the HPLC eluent containing the analytes is sprayed through a heated nebulizer gas (typically N 2) through a small metal capillary which is maintained at a high potential and atmospheric pressure. Capillary outlet formed small highly charged droplets (aerosol) that are desolvated using a continuous stream of heated inert gas (desolvation gas), usually N 2. The droplet size is reduced as there desolvation occurs until the repulsive force between like charges exceeds the cohesive forces of the liquid phase (surface tension). Thereafter follows a series of bursts to obtain solvent free analyte ions. These ions are then sent by a set of focusing devices (lenses) for the mass analyzer. [6],[7] Figure 1 illustrates the functioning of the ionization source ESI. Figure 1. Schematic representation of the ionization source ESI. [8] This technique is particularly suited for thermally labile organic polar compounds that cannot be analyzed by GC-MS. [7] One of the drawbacks of working with this type of interface is the possibility of ion suppression or enhancement. These effects are 2

3 caused by the existing competition between ions ejected during the desolvation. [8] Depending on the characteristics of the analyte, ionization may occur in positive or negative mode. In the case of positive ESI can be added volatile acids (typically formic or acetic acid) to promote the protonation of the analyte. In the ESI negative mode may be used with the ammonium hydroxide to promote deprotonation, if necessary. Alternatively, use may be volatile buffers (usually acetate or ammonium formate) to control the ph. [9] Figure 2 presents a representative schematic of tandem mass spectrometry. In the first quadrupole (MS1) between the gas-phase ions from the ionization source is selected parent ion according to its ratio m/z. This ion is forwarded to the collision cell (Q) where it undergoes fragmentation typically induced by collision with an inert gas called collision gas (usually Ar). The product ions generated are directed to the second quadrupole (MS2) where the mass scan for obtaining mass spectra occurs. [1] The Milli-Q water was obtained from Millipore system. The HPLC-grade methanol 99,9 was purchased from Carlo Erba Reagents and HPLCgrade acetonitrile 98, formic acid 98, phosphoric acid 85, ammonium acetate 98 and sodium chloride 99,5 from Sigma. The HPLC-grade methanol was used for preparation of the all standard stock solutions with a concentration of about 1 g/l. From these solutions were prepared individual standard solutions for infusion into the mass spectrometer with a concentration of about,5 mg/l. The calibration solutions with concentrations between 2 mg/l and 1 mg/l for 2-A-4-CP and between,25 mg/l and 1 mg/l for the other CP s were obtained by serial dilutions from standard stock solutions and prepared in the first line of the gradient used in the chromatographic system Instrumentation SPE experiments were performed using an extractor system from Variant. The HPLC system Model 2695 Separations Module and the Micromass Quattro Micro API mass spectrometer were both obtained from Waters. The system was controlled by MassLynx 4. software Solid-phase extraction Figure 2. Schematic representation of tandem mass spectrometry (MS / MS). [11] 2. Experimental 2.1. Reagents and standards Pure standards were purchased: 2-ao-4- chlorophenol (2-A-4-CP) 97 from Acros, 2- chlorophenol (2-CP) 99,3, 3-chlorophenol (3-CP) 98,1, 4-chlorophenol (4-CP) 99,3, 2,4- dichlorophenol (2,4-DCP) 99,4, 2,4,5- trichlorophenol (2,4,5-TCP) 99,6, 2,4,6- trichlorophenol (2,4,6-TCP) 99,6, pentachlorophenol (PCP) 99 and 4-chloro-3- methylphenol (4-C-3-MP) 99,3 from Fluka. The cartridges Telos SDB (6 ml/2 mg) were washed and conditioned sequentially with 2 ml of Milli-Q water, two aliquots of 2 ml of methanol, 2 ml of Milli-Q water and 2 ml of Milli-Q water acidified to ph 2 with a aqueous solution of phosphoric acid (1:1). The sample acidified (5 ml or 1 ml) to ph 2 with the solution mentioned above and was passed through the cartridge and then washed with three aliquots of 2 ml of Milli-Q water. Acidification of the samples is necessary to prevent the partial deprotonation of CP s. [12] Elution was carried out with five aliquots of 2 ml of methanol, which were then evaporated to 1 µl by a gentle stream of nitrogen. The final volume was made up to 1 ml with the buffer solution of ammonium acetate (2,5 mm, ph 6,8). 3

4 2.4. Liquid Chromatography conditions An XBridge TM Phenyl column (15x2.1 mm I.D., 3,5 µm particle size) maintained at 4 C was used for the chromatographic separation at a flow rate of,2 ml/, injecting 5 µl onto the column. The mobile phase of (A) buffer solution of ammonium acetate (2,5 mm, ph 6,8) and (B) acetonitrile, at,2 ml/, was used. The elution programme started at an initial composition of 9:1 (v/v) and the acetonitrile content was increased to 2:8 (v/v) in 2 and held at this composition for 2. Finally, the mobile phase was returned to the initial composition in 5, and the column was equilibrated with the initial mobile phase composition for Mass Spectrometry conditions The detection was conducted using electrospray probe (ESI) and the optimal parameter settings for the nine chlorophenols studied were as follows: capillary voltage, 3,5 kv; extractor voltage, 2 V; RF Lens,,5 V; Source temperature, 12 C; desolvation temperature, 27 C; desolvation gas (N 2) flow, 45 L/h; Cone gas flow, 6 L/h. Argon (Ar) was used as collision gas. The other optimized parameters necessary to define the method in Masslynx 4. software are presented in Table 1. Table 1. Data for defining the method in Masslynx 4. software for analyzing CP s under study by LC-ESI-MS / MS. Compound ESI RT window () Parent ion (m/z) Cone voltage (V) 2-A-4-CP Positive CP Negative CP Negative CP Negative PCP Negative C-3-MP Negative ,4-DCP Negative ,4,5-TCP Negative ,4,6-TCP Negative Collision energy (ev) Product ion (m/z) Results and discussion 3.1. Optimization of the conditions for LC- ESI-MS / MS For the optimization process conditions of mass spectrometry, more particularly to obtaining the parent ion, the first parameter to optimize is the ionization mode. For all compounds, except 2-A-4- CP, the analysis was carried out in negative ionization mode ([M - H] - ) according to information present in the literature. [13] In the case of 2-A-4-CP was found that the intensity corresponding to the parent ion signal is increased when made positive ionization, i.e. protonation of the ao group using formic acid (1). After defining the ionization type, different cone voltages were tested with the aim to establish the optimal conditions for CP s analyzed, i.e. the operative parameters for obtaining the 4

5 highest signal for the parent ion with less noise baseline. To obtain the product ions, different collision energy were applied in order to detere the value which gives the product ions with good signal strength and at the same time ensuring that at least 1 of the parent ion intensity is maintained. Sometimes, a slight increase in collision energy resulted not only in the fragmentation of the parent ion, but also the fragmentation of product ions which contributed to an increase in the noise level. The opposite scenario was also observed, i.e. the application of collision energy unable to induce fragmentation of the parent ion. For most CP s, the parent ions obtained suggest the loss in the structure of the parent ion of one or more HCl. For the case study (2-A-4-CP), the product ion of m/z equal to 126 suggests the loss of protonated ae. The analysis of most of CP s studied was conducted by selecting two product ions: one for quantification (more intense) and the other for qualification (the second most intense), as shown in Table 2. From the point of view of mass spectrometry objectives were met, i.e. the characteristic ions (parent ion and product ions) of analyzed CP s were obtained with good signal strength. Then the HPLC conditions were optimized. These stand out as key parts for good chromatographic performance, the choice of column chromatography to be used and its operating temperature, the type of mobile phase and elution. After testing different columns available, XBridge TM Phenyl (15x2.1 mm I.D., 3,5 µm particle size) column was chosen because it allowed to obtain intense and defined peaks with good resolution and a reasonable run time. After choosing the chromatographic column, the optimization of its operating temperature, testing temperatures of 3, 4 and 45 C was done. It was opted to use the lowest temperature that led to better outcomes, in this case 4 C, with the aim of increasing the lifetime of the column. The use of the pair (A) buffer solution of 1 mm ammonium acetate: (B) acetonitrile as a mobile phase and gradient elution led to good results for CP s studied. However, tests were carried out with buffer solutions of ammonium acetate with different concentrations, namely 5, 2,5 and 1 mm, in order to reduce the probability of clogging the capillary by precipitation of ammonium acetate and also ion suppression, this is a characteristic effect of the type of interface used in this work (ESI). With the decrease in buffer concentration it was found that peaks showed more tail. The dissolution of analytes may cause changes in the ph of the mobile phase, which is imized by increasing the concentration of buffer solution. In medium without ph control, the degree of ionization of CP s studied varies along the chromatographic band which usually results in peaks with tails. [14] Despite the small effect of tail noted, the buffer solution of 2,5 mm (ph 6,8) ammonium acetate was chosen because it allowed to obtain a good sign for analyzed CP's, in particular for 2-A-4-CP and at the same time contributes for decreasing the risk of degradation of key constituents for the proper functioning of equipment and imize the effect of ion suppression. It was concluded from the point of view of the chromatographic separation that conditions used allowed obtaining defined and intense peaks and showed a good separation of CP s analyzed, with the exception of isomers (Table 2). Table 2. Retention times, ions for quantification and qualification for the CP s studied. Compound Retention time () Ion for quantification (m/z) Ion for qualification (m/z) 2-A-4-CP 1, CP 12, CP 12, CP 12, PCP 13, C-3-MP 14, ,4-DCP 15, ,4,5-TCP 16, ,4,6-TCP 17,

6 3.2. Quantification The calibration curves (CC) were obtained from an external standard method of representing the response of the analytical system (area) depending on the analyte concentration. Are presented in Table 3 the parameters for the CC obtained by linear regression for the 2-A-4-CP, in a total of 12 working sessions. Table 3. Data obtained for the linear regression in the working range 2-1 mg/l for 2-A-4-CP using the methodology LC-ESI-MS / MS Coefficient of deteration R 2 Coefficient rate Y Intercept,9986 6,1(7),4(2) ,9996 5,2(6),1(9) ,9996 4,3(4),1(7) ,9734 3,(2),9(2) ,9868 4,3(6),9(3) ,9969 2,4(8),2(6) ,9993 5,2(7),2(5) ,988 6,7(5) 1,3(6) ,9945 6,7(4),9(2) ,9942 3,1(6),4(4) ,9988 4,6(5),3() ,999 9,5(3),5(4) By observing the data on linear regressions and analyzing the results for the Mandel test [15] it can be seen that the CC shows good linearity for the 2-A-4- CP in the concentration range 2-1 mg / L. The daily calibration is justified by the lack of reproducibility of a work session to another, reflected by differences in the values of slope. It was found that the analytical threshold corresponds to the limit of quantification (L.Q.), and that it is equivalent to the first point of the CC, ie L.Q. equal to 2 mg/ L. The first point of the CC was based on the criterion that the signal/noise ratio should be greater than 1 for the purpose of quantification Ion Quantification/Ion Qualification ratio The ion quantification/ion Qualification ratio is used to qualify the analyte. Its variation was evaluated along the linear working range for compound 2-A-4-CP, yielding an average value of 6, and a relative standard deviation of 1,9. According to the obtained results it can be concluded that there is good repeatability Solid-phase extraction Different extraction techniques such as liquidliquid extraction (LLE) and solid-phase extraction (SPE) using disks and cartridges were tested. However, only the method SPE with cartridges was successful with regard to the case study (2-A-4-CP). The best results in terms of recovery of 2-A-4-CP were obtained for the cartridges of the type SDB (6 ml/2 mg). Recovery experiments with these cartridges using different volumes of Milli-Q water (5 ml and 1 ml) were performed. In Table 4 are shown for each volume fortified with 1 mg/l of 2-A-4-CP the average recovery percentages obtained in a total of four experiments (n=4) and the relative standard deviation (RSD). Table 4. Results for the recovery experiments with SDB cartridges (6 ml/2 mg) for different volumes of Milli-Q water fortified with 1 mg/l of compound 2-A- 4-CP. Sample volume (ml) Recovery () (n=4) RSD According to the results obtained it was decided to use a sample volume of 1 ml, in order to lower 6

7 the L.Q. for 2-A-4-CP (assug a recovery of 73 was obtained as L.Q. of the method the value of 27 µg/l). No further testing was possible for higher volumes of Milli-Q water due to the limited amount of available cartridges. In short, the extraction method using SPE with SDB cartridges (6 ml/2 mg) is more robust, i.e. allows to obtain reliable and reproducible results. The recovery experiments contributed to assess the accuracy of the method Sample Results Among the different types of water, surface waters was chosen to be analyzed, taking into account the limit of quantification (L.Q.) obtained for 2-A-4-CP and the low concentration factor. The collection of a sample of water from the Tagus River near the Expo Park area was done in low ride to imize sample dilution by junction with seawater. The sample was filtered in order to remove suspended matter. The extraction for a sample volume of 1 ml was performed following the procedure of SPE mentioned above. Recovery assays were performed in duplicate for each fortification level, 4 mg/l and 1 mg/l of 2-A-4-CP. Additionally an analysis was carried out by ion chromatography to detere the amount of chlorides present in the sample, yielding a very high value, about 26 g/l. By observation of the chromatograms obtained (Figure 3) was verified the absence of any sign referring to the compound 2-A-4-CP. This result could possibly be due to the presence of interfering compounds in the analyzed matrix. It is known that the mechanism of retention of the stationary phase used (styrene divinylbenzene copolymer) is by adsorption. As such, it is thought that binding sites of the adsorbent may have interacted with both the molecule of the analyte and/or interfering substances. Thus, the high amount of chlorides in the matrix and the possibility of substances (e.g., humic substances quite common in estuary waters) that interact with the analyte may have contributed to the failure of extraction of the analyte. [16] (a) (b) (c) (d) _8 Smooth(Mn,1x2) Teste41_AmostraFiltrada_1mL_4mgL _8 Smooth(Mn,1x2) Teste41_AmostraFiltrada_1mL_4mgL _9 Smooth(Mn,1x2) Teste42_AmostraFiltrada_1mL_4mgL _9 Smooth(Mn,1x2) > e > Teste42_AmostraFiltrada_1mL_4mgL _11 Smooth(Mn,1x2) > e+3 Another sample analyzed was a sample of water from the Tagus River but cog from the Chamusca zone. The amount of chloride was much less than for the other sample, about 22 mg/l. Was carried out the same extraction procedure, but due to the limited amount of SPE cartridges of 6 ml with 2 mg of adsorbent were used cartridges with the same type of adsorbent (SDB) but with half the capacity (3 ml with 1 mg of adsorbent). For new cartridges were tested two sample volumes (5 ml and 1 ml) by perforg four recovery assays to 5 ml and e > 126 Teste44_AmostraFiltrada_1mL_1mgL e > e _11 Smooth(Mn,1x2) Teste44_AmostraFiltrada_1mL_1mgL _1 Smooth(Mn,1x2) Teste43_AmostraFiltrada_1mL_1mgL _1 Smooth(Mn,1x2) Teste43_AmostraFiltrada_1mL_1mgL > e > e e Figure 3. Chromatograms obtained for duplicate analysis.57 of 2. a sample of water from 1.8 the Tagus River mg / L and 2. (c) and 4. (d) mg 8. / L of 1. compound A > 126 near the Expo Park area fortified with (a) and (b) - 4 CP. 7

8 ml fortified with 4 mg/l and 5 ml and 1 ml fortified with 1 mg/l of compound 2-A-4-CP. In Figure 4 it can be seen the presence of the analyte signal referring to the two levels of fortification used. This result seems to be related to the fact that the matrix presented in its constitution a chloride concentration much lower than the sample previously analyzed. the amount of adsorbent, i.e. with lower retaining capacity may contribute to lower recoveries of the analyte as compared to the values in Table 4. The results may also be affected by interference from chlorides that although at a concentration much lower than that found in the Tagus estuary can still compete with the analyte for the adsorbent. Table 5. (a) _8 Smooth(Mn,1x2) Teste45_AmostraRTChamusca_cartucho1mg_5mL_4mgL _ao_4Clorofenol > e+4 Results for the recovery experiments with SDB cartridges (3 ml/1 mg) for different volumes of sample of water from the Tagus River near the Chamusca zone fortified with 4 mg/l and 1 mg/l of compound 2-A-4-CP. (b) (c) (d) _8 Smooth(Mn,1x2) Teste45_AmostraRTChamusca_cartucho1mg_5mL_4mgL 144 > _1 Smooth(Mn,1x2) 2_ao_4Clorofenol 2.331e+3 Teste47_AmostraRTChamusca_cartucho1mg_1mL_4mgL 144 > _ao_4Clorofenol;1.52;2856.7;3982; e In Table 5 are shown the results for the recovery experiments for sample of water from the Tagus River near Chamusca zone. Since it was not possible to repeat these experiments because no more cartridges it were available is difficult to draw conclusions from the values shown in Table 5. Moreover, the use of cartridges with half volume and _1 Smooth(Mn,1x2) Teste47_AmostraRTChamusca_cartucho1mg_1mL_4mgL _9 Smooth(Mn,1x2) Teste46_AmostraRTChamusca_cartucho1mg_5mL_1mgL 2_ao_4Clorofenol _ao_4Clorofenol _9 Smooth(Mn,1x2) Teste46_AmostraRTChamusca_cartucho1mg_5mL_1mgL _11 Smooth(Mn,1x2) 144 > e > Teste48_AmostraRTChamusca_cartucho1mg_1mL_1mgL 2_ao_4Clorofenol 1 2_ao_4Clorofenol _11 Smooth(Mn,1x2) Teste48_AmostraRTChamusca_cartucho1mg_1mL_1mgL 5.646e > e > e (a) and (b) - 4 mg / L and (c) and (d) - 1 mg / L of 144 > 126 2_ao_4Clorofenol 6.576e Figure 4. Chromatograms obtained for the analysis of a sample of water (5 ml and 1 ml) from the Tagus River near the Chamusca zone fortified with compound 2 -A 4-CP Sample volume (ml) Recovery () for fortification level of 4 mg/l Recovery () for fortification level of 1 mg/l Conclusions and Future work This study shows that it is possible to detere the 2-A-4-CP by the methodology SPE-LC-ESI- MS/MS implemented. The chromatographic method LC-ESI-MS/MS developed shows good linearity, expressed by good correlation coefficients and in accordance with the results obtained by Mandel test. The linear working range obtained for compound 2-A-4-CP was between 2 and 1 mg/l and L.Q. equal to 2 mg/l. From the study of variation of the ratio quantification ion/qualification ion it was concluded that it is stable (relative standard deviation of 1,9) all along the linear working range for the case study. As to regards the technique of solid-phase extraction according to the results obtained it is concluded that the type cartridges SDB (copolymer of styrene and divinylbenzene) with 6 ml and 2 mg of adsorbent are those that enable to recover the analyte of interest (2-A-4-CP). The extraction procedure is very fast and simple and makes use of small amounts of solvent which is an advantage from an environmental point of view. The recovery 8

9 obtained under repeatability conditions, to the maximum limit of the calibration curve in testing with Milli-Q water (1 ml) was 73. Concerning to the water samples from the Tagus River analyzed was not possible to draw conclusions, given the small number of trials. However, the presence of high amounts of chloride and possibly other interfering substances in the matrix may have contributed to the failure of testing for water from the Tagus River from the Expo Park area. The agencies responsible for protecting the environment and human health present worldwide impose different limits for CP s in water. Thus, according to this fact and given that there is no legislation implemented for the 2-A-4-CP, it cannot be inferred if the limit of quantification obtained for this compound is high or not. In the case of having a reasonable amount of SDB type SPE cartridges with 6 ml and 2 mg of adsorbent, tests for various sample volumes should be performed in order to detere the maximum capacity of the cartridges. Thus we would try to reach a compromise between the volume of sample that did not compromise the recovery of the analyte of interest and at the same time contributes to reduce the limit of quantification, thereby extending the range of waters to be analyzed. Another option could pass through the acquisition of extraction disks, suitable for the analytes studied, to enable the use of a greater volume of the sample used with the cartridges, for example SDB-RPS (copolymer of polystyrene and divinylbenzene modified with sulphonic acid groups), which according to the literature can give good results in the extraction of phenolic compounds. [5] For all the other CP s study, the linear range obtained was,25 1 mg/l. It was found that the method is not selective for the isomers 2- chlorophenol, 3-chlorophenol, 4-chlorophenol and 2,4,5-trichlorophenol, 2,4,6-trichlorophenol. It is found that the thresholds obtained are higher than those obtained by GC-MS methodology existing in LAIST. Moreover, GC-MS only does not separate the isomers 3-chlorophenol and 4-chlorophenol. Thus, it is concluded that the chromatographic method implemented is not suitable for the deteration of CP s studied with the exception of 2-A-4-CP. Acknowledgements I would like to thank to Prof. Margarida Romão and Eng. Georgina Sarmento for all the support, guidance and encouragement. My sincere thanks to my colleagues at LAIST, in particular to technical Hélder Gageiro and Ana Fernandes for all the friendship and the knowledge transmitted. To my parents, my sister and my grandparents and extended family appreciate all the support, encouragement and all your patience during this important stage of my life. A final word of thanks goes to my colleagues and friends Ana e Fátima. References [1] Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service, Toxicological profile for chlorophenols, ATSDR, [2] Czaplicka, M., Sources and transformations of chlorophenols in the natural environment, Science T. Environ., vol. 322, pp , 24 [3] Jáuregui, O.; Galceran, M.T., Chapter 6 Phenols, Handbook of Analytical Separations, vol. 3, pp , 21 [4] echem/aminochlorophenol.htm, 18/1/214 [5] Puig D.; Barceló, D., Deteration of phenolic compounds in water and waste water, Trends Anal. Chem., vol. 15, pp , 1996 [6] 13/1/214 [7] s-spectrometry/sample-introduction-andionization/esi-apci, 13/1/214 9

10 [8] Drug Development Services, Mass Spectrometry in Bioanalysis, Particle Sciences, vol. 4, 29 [9] Iglesias, A.H; Introduction to the coupling liquid chromatography mass spectrometry, Waters Technologies, Brasil [1] Chiaradia, M.C.; Collins, C.H.; Jardim, I.F., The state of the art of chromatography associated with the tandem mass spectrometry for toxic compound analysis in food, Quim. Nova, vol. 31, pp , 28 [11] 13/1/214 [12] Bladek, J.; Sliwakowski, M.; III/Phenols: Solid- Phase Extraction, Academic Press, pp , 2 [13] Loos, R.; Hanke, G.; Eisenreich, S. J., Multicomponent analysis of polar water pollutants using sequential solid-phase extraction followed by LC- ESI-MS, J. Environ. Monit., vol. 5, pp , 23 [14] Neto, A. J. Troubleshooting: Problems with the shape of the peaks in liquid chromatography Part 4, Scientia Chromatographica, vol. 2, nº 3, pp , 21 [15] Validation of internal methods of chemical analysis test, RELACRE Guide No. 13, 1st ed., 2 [16] Cunha, Ana C.; Thesis to obtain the degree of Doctor of Chemistry: "Development of Analytical Procedure for Deteration of Drugs and Pesticides in Environmental Aqueous Sample", Federal University of Rio Grande do Sul, 25 1