In-Tube Solid-Phase Microextraction and Liquid Chromatography

Anal. Chem. 2009, 81, 5827–5832

In-Tube Solid-Phase Microextraction and Liquid Chromatography Using a Monolithic Column for the Selective Determination of Residual Ethylenediamine in Industrial Cationic Polymers M. C. Prieto-Blanco,† P. Lo´pez-Mahıá,† and P. Campıńs-Falco´*,‡ Departamento Quı´mica Analı´tica, Facultade de Ciencias, Universidade da Corunã, Campus da Zapateira, E-15071 A Corunã, Spain and Departament de Quı´mica Analı´tica, Facultat de Quı´mica, Universitat de Valencia, C/Dr. Moliner 50, E46100 Burjassot, Valencia, Spain The selective determination of the diamine ethylenediamine (EDA) in the presence of a higher amount of residual dimethylamine in cationic polymers has been developed. The strategy uses both a solution derivatization with a selective agent of primary amines such as o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) and an intube solid-phase microextraction (IT-SPME) coupled to liquid chromatography (LC). A 70 cm long, 0.32 mm internal diameter, and 3 µm thick commercially available capillary column coated with 95% polydimethylsiloxane and 5% polydiphenylsiloxane was employed to replace the injection loop of a Rheodyne injection valve. A volume of 1 mL of derivatized sample was passed through the capillary, and 100 µL of water was later used for cleaning and filling the capillary. After, the injection was effected and the desorption of the derivative from the capillary was carried out in the dynamic mode using the mobile phase. Chromatographic separation was performed in less than 2 min using a monolithic silica column Onyx (100 mm × 3.0 mm i.d.) under isocratic conditions. The effect of several parameters affecting derivatization and IT-SPME was investigated. The quantification of EDA was realized over the range of 0.07-2 µg/mL with adequate linearity, accuracy, and reproducibility, and the limit of detection was 20 ng/mL. The method is rapid and low in cost, and sample handling and organic solvent consumption have been minimized. Its application to polymeric cationic surfactants used in water treatment allowed the selective quantification of residual EDA at low microgram per milliliter levels of concentration without off-line preconcentration. Aliphatic amines and polyamines are used like raw materials and intermediates in chemical industries for the production of surfactants, plastics, dyes, and medicines and are used like corrosion inhibitors in power plants. For these reasons, they can be found at different levels in industrial products and also in environmental samples.1 * Corresponding author. E-mail: [email protected]. † Universidade da Corun ã. ‡ Universitat de Valencia. 10.1021/ac900796j CCC: $40.75  2009 American Chemical Society Published on Web 06/12/2009

One industrial process, in which amines are involved, is the synthesis of polymeric cationic surfactants. These products are applied like a flocculant in water treatment, and therefore, their residual products can affect water quality. Cationic polymers synthesized from epichlorohydrin, dimethylamine, and ethylenediamine (EPI-DMA-EDA) are a type of polymers characterized by higher molecular weight and viscosity due to the presence of branched chains. The function of ethylenediamine (EDA) in polymer formation is the branching of the main chain. The effect of the EDA amount on polymer structure and its derived properties have been studied.2-5 Amounts of EDA varying in the range of 0.95-2.13%2 or 0.1-2%3 were sufficient for achieving an effective polymer in wastewater treatment, which is fundamental in decoloring of industrial effluent.4,6 Although the toxicity of the EDA was assessed in some studies,7 it is necessary to develop cost-effective methods for controlling manufactured products. Particularly, this paper contributes to this area for residual EDA studies in cationic polymers. To our knowledge, no such studies have been carried out. A necessary step in the amine analysis by chromatographic techniques for improving both sensibility and chromatographic behavior is the chemical derivatization. o-Phthaldialdehyde combined with compounds containing a thiol group (SH-group) such as N-acetyl-L-cysteine is one of the derivatizing agents most used in the analysis of amino acids and primary amines.8,9 Particularly, EDA was analyzed in workplace air by ion pair high pressure liquid chromatography (HPLC) using dansyl chloride as the (1) Moliner-Martıńez, Y.; Molıńs-Legua, C.; Campıńs- Falco´, P. Talanta 2004, 62, 373. (2) Farinato, R. S.; Calbick, J.; Sorci, G. A.; Florenzano, F. H.; Reed, W. F. Macromolecules. 2005) , 38, 1148. (3) Zagidullin, R. N.; Rasulev, Z. G.; Dmitriev, Y. K.; Muratov, M. M.; Yusupov, A. G.; Vakhitov, Kh. S.; Kul′garin, D. S.; Chernikova, E. A.; Sitdikova, Z. F. Russian Patent 2,245,343, 2005. (4) Gao, B. Y.; Zhang, H.; Yue, Q. Y.; Zou, X. H.; Wang, S. G. Jingxi Huaging 2005, 22, 611. (5) Dragan, S.; Ghimici, L.; Carpov, A.; Chirica, E.; Maftei, M. Mater. Plast. 1990, 27, 72. (6) Temple, R. S.; Stoltz, M. J. U.S. Patent 2,002,130,089, 2002. (7) Yang, R. S. H.; Garman, R. H.; Maronpot, R. R.; McKelvey, J. A.; Weil, C. S.; Woodside, M. D. Fundam. Appl. Toxicol. 1983, 3, 512. (8) Kutlań, D.; Presits, P.; Molna´r-Perl, I. J. Chromatogr., A 2002, 949, 248. (9) Moliner-Martıńez, Y.; Herraéz-Hernańdez, R.; Campıńs- Falco´, P. J. Chromatog., A 2007, 1164, 329.

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derivatizing agent.10 Also, a routine method for analysis of free amine in polymeric amine hardeners containing about 0.3% of EDA with salicylaldehyde-diphenylboron chelate has been proposed.11 In environmental matrices (river water and seawater), C2-C7 aliphatic diamines were analyzed by precolumn derivatization with acetylacetone and were cleaned up by solid-phase extraction.12 Separations with monolithic silica columns are acquiring relevance since this kind of column has a larger permeability to the mobile phase, porosity, and resistance and the need of a lesser sample cleanup than packed columns.13,14 At present, only aromatic amines extracted from water samples using two-step liquid-phase microextraction have been determined using a monolithic silica column.15 Sample preparation research is developing online procedures compatible with chromatographic systems, which require a lower volume of organic solvents and save more time than conventional ones. In-tube solid-phase microextraction (IT-SPME) coupled to liquid chromatography is one of those new procedures. An open coated capillary or a section of a GC capillary column placed in an autosampler unit or as a loop in an injection valve are possible configurations. The advantage of the former is that it allows the automatization of all analytical process easily, but it is possible only with some autosampler models (Agilent 1100 series or Famous).16,17 The preconcentration achieved is limited by the autosampler configuration. The latter one is more versatile and can be coupled to any chromatographic system.18,19 However, its automation requires two HPLC pumps and a switching valve in order to save the amount of sample used. This configuration also permits one to process higher volumes of sample and to work with longer capillaries than those required by the autosampler IT-SPME option. To date, no single strategy or interface device design has proven optimal.20 Although the design of in-tube SPME appears similar to the use of a trapping column in column switching techniques for online sample preparation, equilibrium versus exhaustive extraction remains a fundamental difference between the two methods. The elimination of breakthrough is a vital consideration in the use of a trapping column, whereas equilibrium extraction necessitates that a portion of the analyte remains in the sample after passing through the sorbent. Also, the process can be realized manually. In the present work, a new way to combine chemical derivatization and IT-SPME is proposed. The procedure is useful for the specific determination of residual EDA in the cationic polymer (EPI-DMA-EDA). Different derivatization and extraction schemes could be applied in IT-SPME as their analogues in-fiber SPME or supported solid-phase extraction (SPE) derivatization: (a) adding (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)

Henriks-Eckerman, M. L.; Laijoki, T. J. Chromatogr. 1985, 333, 220. Winkler, E.; Hohaus, E.; Felber, E. J. Chromatogr. 1988, 436, 447. Asan, A.; Isildak, I. Mikrochim. Acta 1999, 132, 13. Guiochon, G. J. Chromatogr., A 2007, 1168, 101. Zou, H.; Huang, X.; Ye, M.; Luo, Q. J. Chromatogr., A 2002, 954, 5. Yazdi, A. S. J. Chromatogr., A 2005, 1082, 136. Gou, Y.; Tragas, C.; Lord, H.; Pawliszyn, J. J. Microcolumn Sep. 2000, 12, 125. Prieto-Blanco, M. C.; Lo´pez-Mahıá, P.; Campıńs-Falco´, P. J. Chromatogr., A 2008, 1188, 118. Cha´fer-Perica´s, C.; Herraéz-Hernańdez, R.; Campıńs- Falco´, P. J. Chromatogr., A 2007, 1141, 10. Campıńs-Falco´, P.; Herraéz-Hernańdez, R.; Verdu´-Andre´s, J.; Cha´fer-Perica´s, C. Anal. Bioanal. Chem. 2009, 394, 557. Lord, H. L. J. Chromatogr., A 2007, 1152, 2.

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the reagent to the samples and subsequent extraction of the derivatives in the coating, (b) extraction of the analytes on the coating and subsequent formation of the derivatives into the coating by flowing the reagent, and (c) loading the coating with the reagent and subsequent extraction/derivatization of the analytes by flowing the sample. Indeed, the most suitable approach depends on the properties of the sample, the analytes, and the reagent, as well as on the characteristics of the reaction to be performed. As mentioned above, the synthesis conditions indicate that dimethylamine (DMA) is the main amine used in the polymerization with epichlorhydrin and that EDA is employed in a smaller amount. For that reason, they could be found in a different concentration range as residual products in the cationic polymer. This fact was verified in a previous paper17 for DMA analysis, and also, the need of developing a specific method for EDA analysis was established. In ref 17, a multiple withdrawal/ expulsion approach was used employing the above-mentioned option c, and therefore, the potential advantage of in-tube SPME for integrating analyte enrichment and derivatization was not fully exploited, as only a few microliters of the sample was processed. In ref 19, an in-valve IT-SPME supported derivatization scheme was proposed for water analysis that permits processing large sample volumes (up to 1.5 mL). In the present paper, a new method is proposed: a selective agent of primary amines o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) for chemical derivatization was selected, and the derivative was concentrated online by IT-SPME and separated using a monolithic column. The paper demonstrates that, bearing in mind the requirements of the analysis needed, option a was optimal. The method combines the following: a solution derivatization with low reagent consumption and wastes, the novel approach of IT-SPME avoiding off-line treatments, and a relatively new packing material of the analytical column for improving analysis time and minimizing markedly organic solvent consumption. We have used the mentioned approach for the following reasons: the characteristics of the matrix (high levels of ionic charge and high density), the level of the EDA in the sample, the presence of the DMA at a concentration higher than for EDA, analysis time, and minimal residues. No in-fiber SPME or SPE procedures have been developed for cationic polymers. The method is proved useful for the specific EDA quantitation as a residual component in industrial manufactured products, and the ratio of DMA/EDA of residual amounts could be established. EXPERIMENTAL SECTION Apparatus. The chromatographic system used consisted of a quaternary pump (Hewlett-Packard 1100 Series) equipped with an injection valve (Rheodyne model 7725) with a 50 µL injection loop. All the components of the system were linked with fused silica tubing (500 µm i.d.). A fluorescence detector (HewlettPackard, 1050 Series) was coupled to a data system (Agilent, HPLC ChemStation) for data acquisition and calculation. The excitation and emission wavelengths used were 330 and 440 nm, respectively. Reagents and Solutions. Ethylendiamine dihydrochloride 98%, poly(dimethylamine-co-epichlorohydrin-co-ethylenediamine) (50% (w/w) solution in water), o-phthaldialdehyde (OPA), N-acetylL-cysteine (NAC), methylamine hydrochloride, ethylamine 70%

Figure 1. Schematic diagram for the online in-tube SPME-LC (liquid chromatography) system. FD: Fluorescence detection. Injection valve in the load position (1) and in the INJECT position (2).

aqueous solution, and propylamine 98% were obtained from SigmaAldrich (Steinheim, Germany). Acetonitrile of HPLC grade (Scharlau, Barcelona, Spain) and water were deionized and filtered through 0.45 µm nylon membranes (Teknokroma, Barcelona, Spain). NAC solution was prepared in water, and OPA solution was prepared in water containing methanol. The stock solution of OPA-NAC was a mixture of OPA and NAC at the same concentration of each compound in the solution (7.4 × 10-2 M), and the final percentage of methanol was 1.4%. The working solution (7.4 × 10-4 M) was prepared by dilution of the stock solution of OPA-NAC. The OPA solution was prepared every 3 days, and the OPA-NAC mixture was prepared daily. All solutions were stored in the dark at 4 °C. Boric acid (1.0 M) containing potassium chloride (1.0 M) was mixed with sodium hydroxide (1.0 M) to obtain a pH ) 10 and a final buffer concentration of 0.5 M.18 A stock solution of EDA was prepared (3000 mg L-1) by dissolving the pure compound in water, and working solutions were made in water by dilution of the stock solution. The EPI-DMA-EDA polymer was prepared in water by weighing the commercial (formulated) polymer (20 mg mL-1). Columns, Mobile Phase, and Chromatographic Conditions. The analytical columns employed were the LiChrospher column (125 × 4.0 mm i.d., 5 µm particle diameter) (Merck, Darmstadt, Germany) and the monolithic C18 column Onyx (100 × 3.0 mm i.d.) (Phenomenex, Torrance, CA). A GC TRB-5 capillary column of 70 cm in length and 0.32 mm in internal diameter coated with 95% polydimethylsiloxane and 5% polydiphenylsiloxane (Teknokroma, Barcelona, Spain) (3 µm of coating thickness) was used for IT-SPME. Chromatographic separation using the monolithic column was carried out in the isocratic elution mode with acetonitrile-water (50/50%) at a flow rate of 1 mL/min. The gradient elution mode

at a flow rate of 1 mL/min was used with the conventional C18 column (LiChrospher). The conditions for the elution program were as follows: the content of acetonitrile was 50% (v/v) at time 0, and then it was linearly increased up to 100% (v/v) at 2 min, it was maintained at 100% (v/v) until 10 min, and it was linearly decreased to 50% at 15 min. The flow was 1 mL/min. Derivatization Procedure. The standard solution of EDA (up to 3 mg/L) or sample (1000 µL), 0.5 M borate buffer (400 µL), and 0.7 mM OPA-NAC (200 µL) were mixed, and the resulting mixture was allowed to react for 10 min. In-Tube Solid-Phase Microextraction. For the interfacing in-tube SPME with the liquid chromatographic system, a piece of commercial GC capillary column (TRB-5) of 70 cm in length was placed in the high-pressure six port valve (Rheodyne model 7725), replacing the injection loop (see Figure 1). Capillary connections were facilitated by the use of a 2.5 cm sleeve of 1/16 in. polyetheretherketone (PEEK) tubing at each end of the capillary. A PEEK tubing internal diameter of 535 µm was suitable to accommodate the capillary used. Normal 1/16 in. stainless steel nuts, ferrules, and connectors were used to complete the connections. In the load position of the valve, 1 mL of derivatized sample solution is manually passed through the capillary column. The inject velocity is fixed at 0.50 mL/min. After, 100 µL of water is processed for cleaning and for replacing the derivatized mixture, and the valve is rotated to the injection position (see Figure 1). The desorption of derivatives is realized by flowing the mobile phase (dynamic mode). RESULTS AND DISCUSSION Optimization of Derivatization and IT-SPME Procedure in Standards and Cationic Polymer. The derivative of EDA was formed in solution and passed through the capillary column for Analytical Chemistry, Vol. 81, No. 14, July 15, 2009

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preconcentration by using the system drawn in Figure 1. Particular parameters of the derivatization including solvent, pH, composition of buffer, and sample/OPA-NAC volume ratio were revised. The influence of IT-SPME on parameters involved in the derivatization such as concentration and excess of derivatizing agent was also studied. Specific parameters of IT-SPME such as processed sample volume, composition, and volume of desorption solvent were optimized. In the literature, the derivatization of primary amines using OPA-NAC is performed in an aqueous medium with borate buffer in the pH range of 9-11 and in the concentration range of 10-2-10-3M for the OPA-NAC mixture.1,9 Molar ratios of OPA-NAC other than 1:1 were not assayed because a significant decrease in the response was obtained for polyamines.21 Saito et al.22 indicated that an excess of OPA in the derivatization step served to catalyze the degradation of the isoindole derivative. This statement could explain the poor stability obtained for EDA in ref 8 because OPA-NAC molar ratios of 1:3 and 1:50 were employed. In initial experiments, it was observed that OPA-NAC concentrations of 10-2-10-3 M degraded the separation and reproducibility of the EDA derivative. We have observed this effect previously for 9-fluorenylmethyl chloroformate (FMOC) reagent,17 and it can be produced for a charge excess in the capillary column. An OPA-NAC concentration of 7 × 10-4 M was selected as optimal. Derivatization was performed using water and mixtures of acetonitrile/water (50/50%) as the solvent. A higher sensibility of EDA derivative was obtained when the derivatization took place entirely in water. Concerning the reaction pH, the peak area of the derivative was duplicated when an increase in pH from 8 to 10 was realized. Also, a decrease in peak tailing was observed when the composition of borate buffer contained potassium chloride. The influence of pH was examined by taking into account the two processes (derivatization and extraction) in conjunction. In a previous work,9 short chain amines were derivatized in 3 min using a greater concentration of OPA-NAC (10-2 M). However, in the optimized conditions stablished here, EDA showed a remarkable increase of sensibility up to 10 min. The longer reaction time did not improve the EDA response markedly. A reaction time of 10 min provided a suitable reproducibility. Several sample/OPA-NAC volume ratios providing molar relations of OPA-NAC/EDA between 6 and 23 were tested for a given EDA concentration and by maintaining constant buffer volume. The results obtained are shown in Figure 2 (line A) for standard solutions. A volume ratio of 2.5 corresponding to the highest molar ratio of OPA-NAC/EDA assayed ()23) provided a lower response, as seen in Figure 1. This response was in accordance with the demonstrated fact that an excess of OPA-NAC can cause a degradation of isoindole and, so, a decrease in response.23 So for standard solutions, sample/OPA-NAC volume ratios between 3 and 10 (providing molar relations of reagent/ analyte between 18 and 6, respectively) gave suitable results. (21) Hanczko´, R.; Ja´mbor, A.; Perl, A.; Molna´r-Perl, I. J. Chromatogr., A 2007, 1163, 25. (22) Campıńs-Falco´, P.; Molins-Legua, C.; Sevilano-Cabeza, A.; Tortajada Genaro, L. A. J. Chromatogr., B 2001, 759, 285. (23) Saito, K.; Horie, M.; Nakazawa, H. Anal. Chem. 1994, 66, 134.

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Figure 2. Effect of sample/OPA-NAC volume ratio in response to the EDA standard (A) and to the spiked cationic polymer with EDA (B). The responses are normalized to a maximum standard response. See Table 1 for the conditions (EDA 0.73 mg/L). Optimal conditions were used except for the volume ratio sample/OPA-NAC. For chromatographic separation, see the Experimental Section. Table 1. Variables Studied and Optimal Values for EDA Determination in Cationic Polymers parameter taken standard or sample EDA concentration OPA-NAC concentration buffer pH sample (or standard)/ OPA-NAC volume ratio standard or sample IT-SPME volume washing and filling IT-SPME volume concentration formulated polymer

range studied

optimum value/ volume

0.02-3 µg/mL

0.06-2 µg/mL/ 1000 µL

10-2-10-4 M

7 × 10-4 M/200 µL

8-10 2.5-10

10/400 µL of buffer 5

100-1500 µL

1000 µL

50-125 µL of water/ 100 µL of water acetronitrile 0.5-20 mg/mL 20 mg/mL

The processed volume of sample and the amount of analyte extracted depends on length, internal diameter, and film thickness of the capillary column.24 We selected a commercial GC capillary column coated with 95% polydimethylsiloxane and 5% polydiphenylsiloxane on the basis of previous papers.17,18 Sample volumes between 0.1 and 1.5 mL were assayed for EDA standards. As expected, an increase of EDA signal was observed when the sample volume passing through the capillary was increased. The improvement achieved with 1.5 mL was similar to that corresponding with 1.0 mL. To achieve the maximun preconcentration factor, a volume of 1.0 mL was selected for processing the sample by IT-SPME. The possibility of using static desorption with a solvent composition different from the mobile phase is a useful procedure when the analytes are strongly adsorbed into the capillary coating.25,26 In this work, dynamic desorption is chosen because it is sufficient to transport the EDA derivative to the analytic column. However, selectivity among the derivatizing agent and the EDA derivative was improved by including a cleaning step with water or an acetonitrile/water mixture through the capillary before the mobile phase was passed. This fact allowed the waste (24) Kataoka, H. Anal. Bioanal. Chem. 2002, 373, 31. (25) Eisert, R.; Pawliszyn, J. Anal. Chem. 1997, 69, 3140. (26) Saito, Y.; Jinno, K. J. Chromatogr., A 2003, 1000, 53.

Figure 3. Chromatograms of the OPA-NAC blank without washing (---), the OPA-NAC blank with water washing (- - -), and the EDA derivatized with water washing (s). See Table 1 for optimal conditions (EDA 0.73 mg/L). For chromatographic separation, see the Experimental Section.

in excess of the derivatizing agent to be partially removed, which improved the life of the analytical column. Water and acetonitrile were tested as washing solvents before the injection step. The organic solvent removed the derivatizing agent and partially removed the EDA derivative. Then, water was selected. Different volumes of water were tested as a function of the derivatized EDA standard volume. Figure 3 shows the chromatograms obtained without and with the cleaning step. The inclusion of this step in the optimized procedure also improved the separation of the EDA derivative. For 1 mL of derivatized standard processed by IT-SPME, 100 µL of water was selected for cleaning and filling the capillary previous to the injection in the analytical column. On the other hand, studies about the miscibility of the EPI-DMA-EDA manufactured product (see Reagents and Solutions section) with water and mobile phases were carried out. The EPI-DMA-EDA manufactured product has an average molecular weight of 75 000 and, therefore, a high viscosity, which makes its manipulation difficult and requires control of the homogeneity of their solutions. A chosen working concentration of polymer must also be miscible in the chromatographic system including IT-SPME. Initial experiments were performed using a polymer concentration of 0.5 mg/mL, but it can be increased to 20 mg/ mL, due to the fact that the derivatization is performed totally in an aqueous medium in which the matrix is soluble. Also, the water volume used in the washing process must remove, at least partially, the polar cationic polymer, improving the life of the analytical column. In preliminary studies, we used the three options for IT-SPME outlined in the introduction section, but only the first one (option a) provided suitable results. This fact confirms that the way to perform derivatization depends on the requirements of the analysis studied. The different behaviors in the EPI-DMA-EDA matrix and in the standard sample were observed in the study of sample/ OPA-NAC volume ratios (Figure 2, line B). The matrix containing EDA was spiked with the same concentration of standard; hence, a response higher than that of the standard might be expected. For sample/OPA-NAC volume ratios higher than 5, the matrix shows a lower response than the standard, possibly due to matrix effects. Cationic polymers have a high density of charge which may produce matrix effects similar to the samples with high amount of salts. Table 1 summarizes the ranges of the studied variables and the optimized conditions.

Figure 4. Chromatograms of EDA derivatized and separated in the C18 packed column (---) and in the monolithic silica column (solid blue line). See Table 1 for conditions (EDA 1.7 mg/L). Optimal conditions were used except the IT-SPME volume was 100 µL and the washing and filling volume was 75 µL. For chromatographic separation, see the Experimental Section. Table 2. Precision Obtained by IT-SPME Procedure precision (% RSD) taken concentration (µg/mL)

intraday (n ) 2)

0.06 0.15 0.30 0.73 2.0

10 6 4 2 2

interday (n ) 5) 10 4

Chromatographic Study and Analytical Parameters. Two analytical columns (monolithic and packed C18) were tested and compared. Using the C18 column, the EDA derivative has a retention time of 2.3 min under gradient elution, but the time necessary to come back to the initial conditions of elution increases the total time of analysis (15 min, see Experimental Section). Using the monolithic column, chosen with an i.d. of 3 mm instead of 4 mm used for the C18 packed column, the derivative of EDA was eluted in 1 min under isocratic conditions maintaining the same flow rate and the initial mixture (50% acetonitrile/50% water) of the gradient elution used with the C18 packed column. The employment of the monolithic column provided a shorter analysis time, less organic solvent consumption, and also, a better peak resolution, as can be seen in Figure 4. A peak was only obtained for EDA-isoindol; no degradation peaks were observed. The calibration equation was y ) b1x + b0, where b1 ± sb1 ) 323 ± 3 and b0 ± sb0 ) 3 ± 3 (R2 ) 0.9991, n ) 10). The linear interval is given in Table 1 (optimal taken standard or sample EDA concentration). This interval was similar to that achieved with the C18 packed column. The interday precision of the slope of the calibration graph was around 10% for three replications made in 3 months. The detection (LOD) and quantification (LOQ) limits were calculated experimentally from solutions containing concentrations providing a signal/noise ratio of 3 and 10, respectively. The obtained values were 20 ng/mL for the LOD and 60 ng/mL for the LOQ and were expressed as the taken standard or the sample EDA concentration. Repeatability and reproducibility were evaluated by calculating intraday and interday coefficients of variations, respectively (Table 2). Similar values were obtained using the C18 packed column. Analytical Chemistry, Vol. 81, No. 14, July 15, 2009

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Figure 5. Chromatograms of the EPI-DMA-EDA cationic polymer (solid blue line) and the polymer fortified with 0.73 mg/L of EDA (---) under the optimal conditions described in Table 1. For chromatographic separation, see the Experimental Section.

The selectivity of the method was tested with other primary amines (methylamine, ethylamine, and propylamine). Their retention times were lower than 1 min; hence, interferences were not found. Application to Real Samples. Although the OPA-NAC solution derivatization is a contrasted procedure for estimating primary amines, direct injection of the EDA derivative formed from the cationic polymer does not permit the analysis of the residual EDA due to a lack of sensitivity. The concentration of the derivative by IT-SPME made it possible to quantify EDA in the EPI-DMA-EDA polymer. The application of the developed method to the EPI-DMAEDA polymer showed the presence of EDA at a concentration of 7 ± 1 µg/g of formulated polymer (Figure 5). The area obtained for the sample was around 3 times the LOQ of the method. The sample of EPI-DMA-EDA was also spiked with 730 ng/mL of EDA and obtained a recovery of 110 ± 10%; consequently, the matrix effect was not present. The two chromatograms obtained are shown in Figure 5. Considering the value obtained for residual DMA in ref 17, the ratio (residual DMA/residual EDA) could be established; the value obtained was 288. This value is compatible with patented polymers containing 0.1% of EDA.3 CONCLUSIONS This paper describes a different way to combine chemical derivatization and IT-SPME for the analysis of amines in cationic

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polymers. IT-SPME allowed the partial removal of the excess of the derivatizing agent and matrix sample and then allowed the life of the analytical column to be improved. This paper demonstrates that the way to perform derivatization depends on parameters such as the sample matrix, amount and behavior of analyte in the sample, interfering agent, derivatization reaction, and analytical instrumentation. The extraction and selective desorption of EDA was performed in a short time (2 min), and the separation using a monolithic column was performed in less than 2 min. Other advantages were the low consumption of reagents and solvents and the generation of little waste. The proposed method allowed the determination of EDA with OPA-NAC in a complex real sample with high levels of ionic charge and in the presence of another residual amine (DMA) at a level of concentration higher than that for EDA by combining IT-SPME and chemical derivatization. The derivatives of EDA formed in solution are injected in the capillary column, and some of the parameters involved in the derivatization such as concentration and the excess of the derivatizing agent are adapted and improved by IT-SPME. The derivatization conditions in which EDA had a similar behavior in both the standard solution and the sample were chosen. EDA was specifically quantified in the EPI-DMA-EDA polymer as a residual component at microgram per milliliter levels, establishing the DMA/EDA ratio of residual amounts (at 288). This paper demonstrates the presence of EDA as a residual compound in the manufactured polymer, although this amine is employed in lower amounts than EPI and DMA in the synthesis of the manufactured product. This contribution can be useful for improving the synthesis or evaluating the risk of using the manufactured product in the environment. ACKNOWLEDGMENT M.C.P.-B. is an Isidro Parga Pondal Fellow, Xunta de Galicia, Spain, and expresses her gratitude to the Xunta de Galicia for the grant of the PGIDIT. The authors are grateful to the Spanish Ministerio de Educacion y Ciencia (Projects CTQ2007-65156/BQU and CTQ2008-01329/BQU).

Received for review April 14, 2009. Accepted June 1, 2009. AC900796J

In-Tube Solid-Phase Microextraction and Liquid Chromatography

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