Determination of five beta-blockers in wastewaters by coupled-column liquid chromatography and fluorescence detection

Analytica Chimica Acta 666 (2010) 38–44

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Determination of five beta-blockers in wastewaters by coupled-column liquid chromatography and fluorescence detection P. Parrilla Vázquez ∗ , M. Martínez Galera, A. Serrano Guirado, M.M. Parrilla Vázquez Department of Analytical Chemistry, University of Almería, La Ca˜ nada de San Urbano, 04120 Almería, Spain

a r t i c l e

i n f o

Article history: Received 21 December 2009 Received in revised form 23 February 2010 Accepted 24 March 2010 Available online 30 March 2010 Keywords: Liquid chromatography Coupled columns Large volume injection Beta-blockers Wastewater

a b s t r a c t A simple multidimensional system for direct injection of large volumes has been developed for the determination of five beta-blockers (atenolol, nadolol, metoprolol, bisoprolol and betaxolol) in wastewater using fluorescence detection. A C18 50 mm × 4.6 mm i.d. column coupled to a RP Amide C16 150 mm × 4.6 mm i.d. column for analyte clean-up and determination were used, respectively. The capability of a first column for eliminating large, interfering molecules, combined with an optimised, coupled-column liquid chromatography separation procedure (LC–LC), large volume injection (LVI) and fluorescence detection (FD), gave excellent sensitivity and selectivity for the target analytes. The LVILC–LC-FD method combines analyte isolation, preconcentration and determination into a single step. Detection limits obtained in wastewater were lower than, or equal to, 0.0020 ␮g L−1 . Limits of quantification (LOQs) obtained in the matrix according to IUPAC, ranged between 0.0052 and 0.0089 ␮g L−1 , whereas LOQs calculated according to EURACHEM Guidance, varied between 0.4 and 0.6 ␮g L−1 . Accuracy values ranged from 82 to 107% (n = 3) and relative standard deviation (RSD) values ranged from 0.8 to 9%. The LVI-LC–LC-FD method was applied for determining the target analytes in wastewater samples obtained in Almería (Spain). © 2010 Elsevier B.V. All rights reserved.

1. Introduction Beta-blockers are prescription drugs used in the treatment of hypertension, angina pectoris and arrhythmia [1]. They work by blocking the effects of adrenaline on the body’s beta-receptors, thereby slowing the nerve impulses to the heart and reducing its workload. Most of these compounds are discharged into the environment on a continual basis via domestic or industrial sewage systems and wet-weather runoff. After human consumption occurs, beta-blockers are first subjected to metabolism and, thereafter, the excreted metabolites, as well as unaltered parent compounds, can then be subjected to further transformations in sewage treatment plants. However, many of these compounds survive biodegradation and are discharged into receiving waters. Furthermore, metabolic conjugates can even be converted back to their free parent forms. Once the substances enter the environmental waters, they may have dramatic effects on aquatic life, e.g. high toxicity and high potency, affecting key biological functions such as reproduction [2,3]. Metoprolol is reported as being the main beta-blocker found in surface waters at high concentrations of 2200 ng L−1 [3–6]. Hirsch

∗ Corresponding author. Tel.: +34 50 015613; fax: +34 50 015483. E-mail address: [email protected] (P.P. Vázquez). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.03.049

et al. [7] and Ternes [4] identified metoprolol, betaxolol, bisoprolol and nadolol in sewage treatment plant effluents. Atenolol has been found at a level of 3.4 ␮g L−1 in effluent wastewaters from a hospital situated in the same geographical area as the proposed work (Almería, Spain) [8]. There is a need for fast, sensitive and low cost analytical methods for the determination of beta-blockers. These compounds are thermolabile and non-volatile, and they have previously been analyzed by gas chromatography–mass spectrometry after derivatization, which makes the sample preparation laborious and time consuming [9,10]. Liquid chromatography–tandem mass spectrometry combined with the necessary sample concentration/clean-up is indicated as the technique of choice for analyzing polar and thermo labile compounds such as beta-blockers [10–12] because of its specificity and selectivity. Extraction of beta-blockers from water samples has usually been performed by off-line solid-phase extraction (SPE) using Oasis HLB [13,14] and C18 [15] cartridges. The need to reduce the overall sample preparation time, as well as the quantities of organic solvents needed for the extraction of organic pollutants from environmental samples has led to the development of several new extraction approaches. The adverse environmental impact of analytical methodologies has been reduced in three different ways: (1) reduction of the amount of solvents required in sample pretreatment;

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(2) reduction in the amount and the toxicity of solvents and reagents employed in the measurement step, especially by automation and miniaturization; and (3) development of alternative direct analytical methodologies not requiring solvents or reagents. Coupled chromatographic techniques are an advantageous alternative available for the trace-level determination of pollutants in environmental samples. In particular, coupled-column reversed-phase liquid chromatography combined with large volume injections (LVI), is a powerful and appropriate technique for the rapid, sensitive and selective determination of polar pollutants in environmental samples [16], which allows automated sample processing using the separation power of a first column (C-1) and analysis in an analytical column C-2. Besides the opportunity of enlarging the sample injection volume to improve sensitivity, it offers the possibility of removing a large excess of early-eluting polar interferences encountered in environmental analysis. This latter capability is the key to success for enhancing selectivity in the analysis of polar compounds. Fluorometric detection (FD) is generally more sensitive than the classical UV absorption and less expensive than MS detection. In addition, fluorescence detectors are very selective, overcoming matrix interference, when compared with other spectrophotometric techniques based only on absorption measurements. This is because both excitation and emission wavelength maxima are available to characterize a specific compound. Fluorometric methods are also selective because only a limited number of organic compounds fluoresce in comparison to the large number of molecules absorbing light [17]. The aim of this work was to develop a coupled-column liquid chromatography combined with large volume injection and fluorescence detection (LVI-LC–LC-FD) method to analyze five betablockers, including atenolol, nadolol, metoprolol, bisoprolol and betaxolol in wastewater samples. These drugs are currently in use in Spain and are thus potential contaminants of our aquatic environment. The methodology used avoids the use of large amounts of organic solvents in preconcentration and extraction steps and, additionally, offers advantages such as: - incorporating sample extraction, concentration and sample introduction into a single procedure; - reducing the amounts of solvents used; - increasing sensitivity and selectivity; - decreasing the time; - facilitating automation; - lowering environmental toxicity; - lowering cost; and - improving safety.

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2.2. Standard solutions Stock solutions of the analytes were prepared by weighing the appropriate amount of each beta-blocker and dissolving it in a volume of MeOH to give a final concentration of 400 mg L−1 . From the stock solutions, mixtures of 10 mg L−1 of each beta-blocker were prepared in MeOH. These solutions were kept at 4 ◦ C and used to prepare standard working solutions in MeOH:water (5:95, v/v). For recovery determinations, wastewater samples were spiked at concentration levels of 3, 6 and 9 ␮g L−1 , simulating real waters containing the target analytes. Then, 0.0, 2.0, 4.0 and 6.0 ␮g L−1 of each analyte were added to four aliquots of each previously spiked sample for calibration by the standard addition method. Finally, 2 mL of each sample were injected into the chromatograph. Spiked wastewater samples were centrifugated at 5000 × g and then, were filtered through 0.45 ␮m cellulose acetate filters before analysis. 2.3. Equipment The system consisted of a Rheodyne (Cotati, CA, USA) Model 7725 sample injector equipped with a 2 mL loop, used to perform large volume injections, a Rheodyne Type 7000 high-pressure column-switching valve (HP), a Model 510 isocratic LC pump (P-1), a Model 600 gradient LC pump (P-2) from Waters (Milford, MA, USA) and a programmable fluorescence detector of variable wavelength, Model 474 from Waters. Data treatment of chromatographic peaks was performed using Millennium32 software (Waters). A schematic diagram of the separation procedure involved in coupled-column RPLC is shown in Fig. 1. For coupled-column LC analysis, a 50 mm × 4.6 mm i.d. column packed with 5 ␮m Aquasil C18 from Thermo Electron (Bellefonte, PA, USA) as the first column (C-1) and a 150 mm × 4.6 mm i.d. column packed with 5 ␮m Discovery® RP Amide C16 from Supelco (Bellefonte, PA, USA) as the analytical column (C-2) were used. A 50 mm × 4.6 mm i.d. column packed with 5 ␮m Hypersil Elite C18 from Thermo Electron and a 50 mm × 4.6 mm i.d. column packed with 5 ␮m GFF-II (ISRP, Pinkerton) from Regis (Morton Grove, IL, USA) were also used in the optimization step. 2.4. Sampling Effluent wastewater samples were collected from four municipal wastewater treatment plants in Almería, a city of 190,000 inhabitants. The samples were collected in 2.5 L amber glass bottles and transported to the laboratory in ice. Wastewater samples were centrifugated at 5000 × g and were filtered through 0.45 ␮m cellulose acetate filters. Subsequently, wastewater samples were stored at 4 ◦ C until analysis which was performed within 24 h in order to avoid any degradation.

2. Experimental 2.5. LVI-LC–LC-FD analysis conditions 2.1. Chemicals and solvents All beta-blocker standards in the study were of analytical grade (>98%) and purchased from Sigma (St. Louis, MO, USA). The HPLCgrade methanol (MeOH) was obtained from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q water purification system from Millipore (Bedford, MA, USA). The phosphate buffer (pH 3) was prepared by dissolving 3.4 g KH2 PO4 from Fluka (Buchs, Switzerland) and diluting to a final volume of 1 L. The pH was adjusted with 0.1 M orthophosphoric acid from Panreac (Barcelona, Spain). The mobile phase was filtered through 0.45 ␮m cellulose acetate (buffer) or PTFE (MeOH) from Millipore and degassed with helium prior to and during use.

Wastewater samples (2 mL) were directly injected into a C1 column with a phosphate buffer (25 mM, pH 3):MeOH (90:10, v/v) as mobile phase (M-1) delivered by pump 1 (Fig. 1A). After sample loading with 4 mL M-1 (injection volume included), the system was switched to the state shown in Fig. 1B, coupling C-1 on-line with C-2 for 7 min and transferring the fraction containing the five analytes from C-1 to C-2 with mobile phase M-2 [a phosphate buffer–methanol gradient (Table 1)]. After transfer, the switching valve was returned to its initial position, allowing C1 to be rinsed and re-equilibrated with the mobile phase M-1. Simultaneously, the analytes were separated on C-2 using M-2 (Fig. 1C). Total chromatographic analysis time was 20 min. The mobile phases were adjusted to a flow of 1 and 1.5 mL min−1 for

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Fig. 1. Schematic LC–LC system representing three steps: (A) sample injection, (B) transfer step, and (C) reconditioning and analytical separation step. IS, sample injector; C-1 and C-2, first and second separation column, respectively; M-1 and M-2, mobile phases; S1 and S2, high-pressure valve position; I1 and I2, interferences; A, target analytes; HV, high-pressure valve; D, detector.

M-1 and M-2, respectively. A 2 mL loop was used. The fluorometric detection was performed at an excitation wavelength (ex ) of 230 nm and at an emission wavelength (em ) of 302 nm for all analytes. Table 1 Mobile phase gradient (M-2) used for the determination of the studied compounds. Time (min)

Phosphate buffer (%)

Methanol (%)

0 4 10 16 20

70 70 50 50 70

30 30 50 50 30

3. Results and discussion 3.1. Fluorescence detection The maxima excitation and emission wavelengths of the target beta-blockers were located between 227–232 and 300–302 nm, respectively. As all beta-blockers under study showed closely located excitation maxima (and emission maxima), we selected a ex and em of 230 and 302 nm, respectively, as the best compromise wavelengths. Batch studies showed that the fluorescence signal for the target beta-blockers was the same in binary aqueous mixtures with MeOH as in 100% water. Therefore, samples were injected

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without MeOH addition to avoid dilution in the wastewater samples. 3.2. Selection of coupled columns One important advantage of using multidimensional LC techniques is to perform an efficient clean-up in the first column (C-1) and a good separation in the second column (C-2). In addition, this methodology allows automated sample processing, once it is injected into the chromatographic system. In this method, several types of columns such as 5 ␮m Hypersil C18 column (50 mm × 4.6 mm id), 5 ␮m Pinkerton GFF II column (50 mm × 4.6 mm id) and 5 ␮m Aquasil C18 column (50 mm × 4.6 mm id) were tested for use as C-1. The Aquasil column was selected because it provides significant removal of earlyeluting matrix interferences. In addition, a (150 mm × 4.6 mm id) 5 ␮m Discovery® RP Amide C16 column, used as C-2, allowed the necessary separation for the five analytes. 3.3. Optimization of M-1 The composition and the flow rate of the mobile phase M1 through C-1 were adjusted to provide a good separation between analytes and interferents. In this way, different phosphate buffer:methanol mixtures with methanol content ranging from 0 to 50% (v/v) were evaluated. A decrease in the percentage of MeOH in the mobile phase increased the retention of analytes and interferents on C-1. This effect of MeOH percentage on retention times was higher for the less polar analytes. However, owing to the small retention shown by atenolol and nadolol, the organic solvent was decreased in mobile phase M-1, in such a way that a mobile phase of phosphate buffer:methanol (90:10, v/v) at a flow rate of 1 mL min−1 was a good compromise between clean-up and preventing elution of the first-eluting analyte. 3.4. Selection of injection volume The injection volume was studied as it is related to the sensitivity of the method. For this purpose, C-1 was connected to the fluorescence detector and mobile phase M-1 was passed through it. Different injection volumes (50–5000 ␮L) were tested to introduce decreasing concentrations (400–4 ␮g L−1 ) of standards, in such a way that the amount of analyte injected was kept constant at 0.02 ␮g. From the results obtained, it was stated that an increase in the retention time of the analytes was achieved when the injection volume increased for all of the analytes (Fig. 2). This study showed the possibility of performing LVI with good retention times and peak shape (width and asymmetry) even with a 5000 ␮L loop. However, a sample volume of 2000 ␮L was selected as a compromise between the required sensitivity and speed of analysis. 3.5. Separation of beta-blockers in C-2 Beta-blockers are basic in nature with pKa values in the range 7.1–9.7, and, at a neutral pH, they exist largely in their ionized form. The log Kow values of the target compounds ranged between 0.16 for atenolol and 3.3 for betaxolol, and their water solubility ranged between 451 mg L−1 for betaxolol and 13,300 mg L−1 for atenolol. Atenolol and nadolol are highly soluble compounds and betaxolol is the least-soluble compound of the target beta-blockers. The chromatography of basic beta-blockers has traditionally been performed using bonded C18-silica phases [8,18,19] and buffer salts are needed to provide an adequate retention of analytes in the stationary phase. The pH of the mobile phase can also be a determining factor. At a pH below 3, since the pKa of normal silanols is in the 5–7

Fig. 2. Chromatograms corresponding to 0.02 ␮g of atenolol, injected with different loops.

range, the majority of silanol sites should be in neutral form, so the interactions with the protonated basic compounds should be minimized. The result is that low pHs may cause early elution of basic beta-blockers in the chromatogram. Therefore, for the simultaneous determination of target beta-blockers, and to use the same pH in all of the experiments, a pH 3 buffer (phosphoric acid–potassium phosphate) was used as aqueous solvent in M-1 and M-2. With the aim of comparing their performance, two different columns, a 5 ␮m Gemini C18 (150 mm × 4.6 mm i.d.) from Waters and a 5 ␮m Discovery® RP C16 (150 mm × 4.6 mm i.d.) from Supelco, were tested as C-2. Different isocratic and gradient conditions were assayed with phosphate buffer:methanol ratios ranging from 0:100 to 100:0 (v/v). Experiments indicated that the last mentioned column provided the best separation with the highest fluorescence signals for the analytes. It must be noted that the flow rate and eluotropic strength of M-2 is usually higher than that of M-1 in order to enhance sensitivity by means of peak compression and to reduce the analysis time by reducing the transfer step. In addition, its composition must be adequate enough to provide satisfactory separation of analytes on C-2. However, there was a limited choice of M-2 because of the expected poor retention of atenolol and nadolol. The gradient for M-2 shown in Table 1 was selected as optimum because it provides enough peak compression and adequate peak resolution, as well as a sensitivity enhancement. 3.6. Optimization of clean-up and transferring time Once the selected parameters were fixed, the final columnswitching conditions were found by connecting C-1 directly to the detector, the high-pressure valve being set at position S1 (Fig. 1A) and injecting 2 mL of a 10 ␮g L−1 mixture of the analytes. Taking into account that the clean-up time is defined as the interval elapsed until the more polar analyte starts to elute from C-1, using the mobile phase M-1, 4 min were obtained for this parameter. It is necessary to point out that only 400 ␮L of MeOH were used for the clean-up step versus several millilitres used for preconditioning and eluting the cartridges when SPE is applied. Fig. 3A shows that the FD chromatogram of a wastewater blank does not present any fluorescence peak. However, to check the presence of matrix interferences, which would not show fluorescence but coelute with the analytes, 2000 ␮L of wastewater were injected using a photodiode array detector. Fig. 3B shows that most of the matrix compounds were eluted before 4 min. Thus, a clean-up time of 4 min offers the possibility of removing the

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Fig. 3. (A) FD chromatogram of a wastewater blank (ex = 230 nm and em = 302 nm). Injection volume = 2000 ␮L. (B) UV chromatogram of the same wastewater blank ( = 205 nm). Injection volume = 2000 ␮L.

large excess of early-eluting polar interferences, which contributes to decreasing the possible influence of the matrix background on the fluorescence signal. In addition, the time of life of C-2 is enlarged. The transferring time is the time needed for the delivery of the last, and most retained beta-blocker from C-1 into C-2. To calculate this parameter, the LC–LC system was connected with the highpressure valve set to position S2 (Fig. 1B) using the mobile phase M-2 circulating from C-1 to C-2. Transferring with a mobile phase M-2 which guaranteed peak compression is crucial in avoiding dispersion and peak broadening in C-2. However, a mobile phase M-2 adequate to get optimal peak compression involves the use of high percentages of organic solvent. On the other hand, beta-blockers are polar analytes that are not sufficiently retained on reversed phases (C-2) when the mobile phases used contain high percentages of organic solvent. Therefore, M-2 must be a compromise between both situations. Using the gradient program described in Section 2.5, the transferring time was found to be 7 min. Fig. 4 shows a chromatogram obtained by direct injection of 2000 ␮L of a standard solution containing 2 ␮g L−1 of each betablocker and a chromatogram obtained by injecting 2000 ␮L of a standard solution also containing 2 ␮g L−1 of each beta-blocker and which has undergone column switching –both of them being obtained with the same mobile phase. It can be seen that the column switching approach does not make the separation on C-2 more difficult.

Fig. 4. Chromatograms of a standard solution containing 2 ␮g L−1 of each compound. Peak assignment: 1, atenolol; 2, nadolol; 3, metoprolol; 4, bisoprolol; 5, betaxolol. Chromatogram (A) LVI-LC-FD without column switching (separation on the C-2 column) and (b) LVI-LC–LC-FD (column switching).

3.7. Validation of the analytical method The proposed analytical method was validated using solventbased and matrix-matched standards. To obtain them, Milli-Q and wastewater samples not containing the target analytes were spiked with known amounts of each compound prior to analysis. The limits of detection (LODs), limits of quantification (LOQs), precision (RSDs), linearity, and recovery, were all studied. LODs and LOQs were calculated statistically according to IUPAC as 3.84- and 10-fold the blank standard deviation divided by the slope of the calibration graph [20], respectively. In addition, the LOQs were calculated according to the EURACHEM Guidance, as the analyte concentration whose RSD (obtained with three successive injections of the same solution) was equal to 10% [21]. The results obtained for these parameters using Milli-Q and wastewater are shown in Tables 2 and 3. LOQs obtained by using EURACHEM Guidance were higher than those obtained by the IUPAC criterion in agreement with the results found by other authors [22]. On the other hand, LOQs obtained using the criterion proposed by the EURACHEM Guidance estimated more realistic values, as they are based on the measurement of replicated standards or spiked real samples instead of on a blank measurement, as is the case of the IUPAC criterion. The LODs were in the same order as those reported in the literature for the determination of beta-blockers in wastewa-

Table 2 Analytical figures of merit obtained using Milli-Q water. Compound

Atenolol Nadolol Metoprolol Bisoprolol Betaxolol a b

Linear range (␮g L−1 )

0.2–20 0.4–20 0.4–20 0.6–20 0.3–20

IUPAC criterion. EURACHEM criterion (RSD 10%).

R2

0.9997 0.9924 0.9991 0.9995 0.9932

Regression equation

Y = 153,975X − 9836 Y = 129,654X − 15321 Y = 143,883X + 23034 Y = 165,441X + 14603 Y = 204,472X + 9973

LODa (␮g L−1 )

0.0010 0.0035 0.0039 0.0015 0.0011

LOQa (␮g L−1 )

0.0026 0.0091 0.0102 0.0039 0.0029

LOQb (␮g L−1 )

0.2 0.4 0.4 0.6 0.3

RSD (%) 3 (␮g L−1 )

7 (␮g L−1 )

6.2 6.1 3.2 3.9 3.4

6.9 6.6 3.9 2.3 5.6

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Table 3 Analytical figures of merit obtained using wastewater. Linear range (␮g L−1 )

Compound

Atenolol Nadolol Metoprolol Bisoprolol Betaxolol a b

0.4–20 0.4–20 0.6–20 0.5–20 0.4–20

R2

LODa (␮g L−1 )

Regression equation

0.9933 0.9997 0.9939 0.9945 0.9910

Y = 135,021X + 3200 Y = 84,741X + 9532 Y = 32,440X + 5831 Y = 173,403X + 9411 Y = 78,397X + 12530

0.0033 0.0034 0.0027 0.0026 0.0020

LOQa (␮g L−1 )

0.0086 0.0089 0.0070 0.0068 0.0052

LOQb (␮g L−1 )

0.4 0.4 0.6 0.5 0.4

RSD (%) 3 (␮g L−1 )

7 (␮g L−1 )

3.1 9.2 9.2 4.1 3.4

4.9 3.2 3.9 4.3 5.2

IUPAC criterion. EURACHEM criterion (RSD 10%).

Table 4 Mean recovery percentages and RSD (%) for the determination of five beta-blockers in wastewater at three concentration levels (n = 3). Sample

Sample 1 Sample 2 Sample 3 a

Atenolol

Nadolol

Metoprolol

Bisoprolol

Betaxolol

␮g L−1

Recovery (%)a

␮g L−1

Recovery (%)a

␮g L−1

Recovery (%)a

␮g L−1

Recovery (%)a

␮g L−1

Recovery (%)a

3 6 9

81.5 (3.1) 107.3 (0.9) 93.3 (1.3)

3 6 9

86.7 (9.1) 102.8 (2.6) 98.4 (1.3)

3 6 9

89.2 (9.1) 84.3 (0.9) 106.4 (2.2)

3 6 9

86.2 (4.0) 104.1 (3.3) 102.8 (1.4)

3 6 9

98.9 (3.4) 98.5 (4.8) 92.1 (0.8)

RSD (%) in parentheses.

ter samples by other commonly used techniques, such as liquid chromatography–tandem mass spectrometry [3,23,24]. However, in the above procedures, wastewater samples were extracted by off-line SPE. The linear range was established for each beta-blocker, the lower limit being the LOQ calculated according to the last criterion and the upper limit being the concentration for which the signal deviates from linearity by 3–5% [25]. Calibration curves showed good linear relationship (r2 > 0.9910) between 0.4 and 20 ␮g L−1 (six standards covering the whole range were used and each point of the calibration graph was obtained in triplicate). The method was also checked for the condition of uniform variance (homoscedasticity) over the linear range [25]. For calibration experiments, this means that the precision of the measurements is independent of the concentration. Each calibration graph was obtained in triplicate and the comparison of standard deviations showed that the dispersion of the measurements was independent of the analyte concentration. The presence of constant systematic errors due to additive signals from matrices was checked by comparing the intercepts of calibration graphs built using solvent-based and matrix-matched standards [25], with no evidence of significant differences. Relative systematic errors due to matrix effect or matrix interferences may affect the slope of calibration graphs [25]. With the aim of checking changes in the analytical signal due to matrix effect, calibration graph slopes obtained by using matrix-matched and solvent-based standards were compared for each beta-blocker by means of a t-test [25]. Significant differences were observed between the slopes of

Fig. 5. LVI-LC–LC-FD chromatograms of (a) a wastewater blank and (b) the same wastewater sample spiked at 1 ␮g L−1 . Peak assignment: 1, atenolol; 2, nadolol; 3, metoprolol; 4, bisoprolol; 5, betaxolol.

both equations for all compounds. For this reason, matrix-matched standards were used throughout for quantification. In order to establish the accuracy and precision of the overall method, three replicates of wastewater samples were spiked and analyzed using the standard addition method as described in Section 2.2. Mean recovery and RSDs percentages for the three concentration levels are summarized in Table 4. In the results, good precision was obtained for all analytes (RSD% < 10%) and accuracy values were acceptable for all beta-blockers (between 81.5 and 107.3%), which are within the range expected for beta-blocker analysis [3]. 3.8. Analysis of wastewater samples Beta-blockers have frequently been found in monitoring programmes of surface and wastewaters at ng L−1 and ␮g L−1 levels, respectively [4–8,11]. A total of four different wastewater samples from Almería (Spain) were analyzed by the proposed method, but beta-blocker residues were not detected. Fig. 5 shows two chromatograms corresponding to a real wastewater sample and a real wastewater sample spiked with 1 ␮g L−1 . It can be seen that no peaks appear at the retention times of the analytes. 4. Conclusions A rapid, sensitive and selective analytical method has been developed, using LC–LC with fluorescence detection, for the determination of atenolol, nadolol, metoprolol, bisoprolol and betaxolol. This method allows the use of large volume injection (LVI) applied to beta-blocker analysis in wastewater samples. Low enough LOQ values were obtained to permit analysis of the beta-blockers at ng L−1 levels. The method compares favourably with others previously developed in terms of sensitivity, materials and time required. The reduced sample handling and the short run time made possible the analysis of at least 30 samples per day. With regard to the robustness of the method, the two columns maintained their performance during all the experiments and readjustments of column-switching conditions (clean-up and transfer time) were unnecessary, even after 6 months. This methodology does not require manipulation of the wastewater samples before injection. The automation of the system is subject to a minimum of human error and contamination. Significant reductions in costs for sample pre-treatment (solvent and

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