simultaneous detection of sulfamethoxazole, diclofenac, carbamazepine, and bezafibrate by solid...

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Journal of Applied Spectroscopy, Vol. 81, No. 2, May, 2014 (Russian Original Vol. 81, No. 2, March–April, 2014) SIMULTANEOUS DETECTION OF SULFAMETHOXAZOLE, DICLOFENAC, CARBAMAZEPINE, AND BEZAFIBRATE BY SOLID PHASE EXTRACTION AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION Z. Zhou and J.-Q. Jiang * UDC 543.544:615.45 A method of solid phase extraction (SPE) coupled with high performance liquid chromatography and diode array detection (HPLC-DAD) was studied for the simultaneous determination of sulfamethoxazole (SMX), diclofenac (DCF), carbamazepine (CBZ), and bezabrate (BZF) in test solutions. The target compounds were extracted by SPE from samples, and the resulting elutes were analyzed using a HPLC-DAD system at wavelengths of 270, 280, 290, and 230 nm for SMX, DCF, CBZ, and BZF, respectively. This method shows good recoveries for SMX, DCF, CBZ, and BZF with mean recoveries of 89.7 ± 9.3%, 86.1 ± 7.6%, 95.0 ± 6.5%, and 94.0 ± 5.4%, respectively. Keywords: sulfamethoxazole, bezabrate, carbamazepine, diclofenac, high performance liquid chromatography and diode array detection, pharmaceuticals, solid phase extraction. Introduction. Since the 1990s, pharmaceutical residues in various therapeutic classes have been widely detected in the aquatic environment at trace levels (usually in ng/l–μg/l) [1–3]. Such emerging micro-pollutants have caused global concerns regarding their potential harm to human beings and the eco-system [4–7]. Therefore, preventing pharmaceutical products from entering the environment is of public interest. However, conventional wastewater treatment processes, particularly the activated sludge, are less efcient in removing most pharmaceutical products [8–10]. Thus, a number of studies have focused on the research and development of advanced water treatment technologies to tackle the issue of the ubiquitous occurrence of pharmaceuticals, with main focus on ozonation [11–13] and hydroxyl radical-based advanced oxidation processes (AOPs) [14–16]. On the other hand, development of reliable and robust analytical methods is crucial to monitor pharmaceuticals in the environment and to understand the performance of treatment technologies. Currently, solid phase extraction (SPE) and liquid chromatography (LC) coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are commonly used for sample preparation and analysis [17, 18]. Indeed, LC-MS and LC-MS/ MS are effective in detecting various pharmaceuticals in complex water matrices, but it is time-consuming and complex in instrumentation. In previous studies, a simple SPE coupled with UV/Vis spectrophotometric method was developed to analyze ciprooxacin and investigate the performance of potassium ferrate (VI) in treating test solutions containing trace levels of ciprooxacin [19, 20]. Potassium ferrate (VI) (K 2 FeO 4 ) is a promising dual-functional chemical which has been applied in many water and wastewater treatment units [21–25]. However, one drawback of SPE-UV/Vis spectrophotometry is that it cannot be used to analyze mixed compounds simultaneously. Therefore, the objective of this study was to develop a SPE method and a high performance liquid chromatography with diode array detection (HPLC-DAD) method to simultaneously detect mixed pharmaceuticals from test solutions, offering a validated analytical method for further studying the performance of ferrate (VI) in the treatment of mixed pharmaceuticals in test solutions. The target pharmaceuticals selected were sulfamethoxazole (SMX), diclofenac (DCF), carbamazepine (CBZ), and bezabrate (BZF) (Table 1): School of Engineering and Built Environment, Glasgow Caledonian University, Glasgow G4 0BA, Scotland, United Kingdom; e-mail: [email protected]. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 81, No. 2, pp. 278–282, March–April, 2014. Original article submitted July 22, 2013. 0021-9037/14/8102-0273 ©2014 Springer Science+Business Media New York 273 _____________________ * To whom correspondence should be addressed.

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Journal of Applied Spectroscopy, Vol. 81, No. 2, May, 2014 (Russian Original Vol. 81, No. 2, March–April, 2014)

SIMULTANEOUS DETECTION OF SULFAMETHOXAZOLE, DICLOFENAC,CARBAMAZEPINE, AND BEZAFIBRATE BY SOLID PHASE EXTRACTIONAND HIGH PERFORMANCE LIQUID CHROMATOGRAPHYWITH DIODE ARRAY DETECTION

Z. Zhou and J.-Q. Jiang* UDC 543.544:615.45

A method of solid phase extraction (SPE) coupled with high performance liquid chromatography and diode array detection (HPLC-DAD) was studied for the simultaneous determination of sulfamethoxazole (SMX), diclofenac (DCF), carbamazepine (CBZ), and bezafi brate (BZF) in test solutions. The target compounds were extracted by SPE from samples, and the resulting elutes were analyzed using a HPLC-DAD system at wavelengths of 270, 280, 290, and 230 nm for SMX, DCF, CBZ, and BZF, respectively. This method shows good recoveries for SMX, DCF, CBZ, and BZF with mean recoveries of 89.7 ± 9.3%, 86.1 ± 7.6%, 95.0 ± 6.5%, and 94.0 ± 5.4%, respectively.

Keywords: sulfamethoxazole, bezafi brate, carbamazepine, diclofenac, high performance liquid chromatography and diode array detection, pharmaceuticals, solid phase extraction.

Introduction. Since the 1990s, pharmaceutical residues in various therapeutic classes have been widely detected in the aquatic environment at trace levels (usually in ng/l–μg/l) [1–3]. Such emerging micro-pollutants have caused global concerns regarding their potential harm to human beings and the eco-system [4–7]. Therefore, preventing pharmaceutical products from entering the environment is of public interest. However, conventional wastewater treatment processes, particularly the activated sludge, are less effi cient in removing most pharmaceutical products [8–10]. Thus, a number of studies have focused on the research and development of advanced water treatment technologies to tackle the issue of the ubiquitous occurrence of pharmaceuticals, with main focus on ozonation [11–13] and hydroxyl radical-based advanced oxidation processes (AOPs) [14–16]. On the other hand, development of reliable and robust analytical methods is crucial to monitor pharmaceuticals in the environment and to understand the performance of treatment technologies. Currently, solid phase extraction (SPE) and liquid chromatography (LC) coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are commonly used for sample preparation and analysis [17, 18]. Indeed, LC-MS and LC-MS/MS are effective in detecting various pharmaceuticals in complex water matrices, but it is time-consuming and complex in instrumentation.

In previous studies, a simple SPE coupled with UV/Vis spectrophotometric method was developed to analyze ciprofl oxacin and investigate the performance of potassium ferrate (VI) in treating test solutions containing trace levels of ciprofl oxacin [19, 20]. Potassium ferrate (VI) (K2FeO4) is a promising dual-functional chemical which has been applied in many water and wastewater treatment units [21–25]. However, one drawback of SPE-UV/Vis spectrophotometry is that it cannot be used to analyze mixed compounds simultaneously. Therefore, the objective of this study was to develop a SPE method and a high performance liquid chromatography with diode array detection (HPLC-DAD) method to simultaneously detect mixed pharmaceuticals from test solutions, offering a validated analytical method for further studying the performance of ferrate (VI) in the treatment of mixed pharmaceuticals in test solutions. The target pharmaceuticals selected were sulfamethoxazole (SMX), diclofenac (DCF), carbamazepine (CBZ), and bezafi brate (BZF) (Table 1):

School of Engineering and Built Environment, Glasgow Caledonian University, Glasgow G4 0BA, Scotland, United Kingdom; e-mail: [email protected]. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 81, No. 2, pp. 278–282, March–April, 2014. Original article submitted July 22, 2013.

0021-9037/14/8102-0273 ©2014 Springer Science+Business Media New York 273

_____________________

*To whom correspondence should be addressed.

274

Materials and Methods. Analytical grade SMX, DCF, CBZ, and BZF were purchased from Sigma-Aldrich (UK). Other chemicals and reagents were obtained from Fisher Scientifi c (UK). All chemicals and reagents were used without further purifi cation. Experimental water was generated by ELGA PureLab Option-R 7/15 pure water system (Veolia Water, France). Stock solutions of the target compounds were prepared separately in methanol at 100 mg/l and stored in the freezer at −18°C. To assess the accuracy and precision of the method, test solutions of the target compounds were prepared and subjected to all the analytical processes. The concentrations of each target compound were set at two levels: 100 and 10 μg/l. Five parallel samples were prepared for each concentration.

Test solutions were fi ltered using 0.45 μm membrane fi lters (Milipore, USA), acidifi ed to pH 2.5 by 2 M H2SO4, and then subject to SPE extraction. The SPE cartridges employed were reversed phase Strata-X 1 g/12 ml Giga Tubes (Phenomenex, UK). For 100 and 10 μg/l samples, 100 and 500 ml solution were used for the extraction, respectively. Generally, the extraction protocol was: (1) conditioning: 6 ml methanol; (2) equilibrating: 6 ml water; (3) loading samples: desired amount of water samples under vacuum at a fl ow rate of 5–10 ml/min; (4) washing: 2 × 6 ml water; (5) drying: 15 min under gentle nitrogen fl ow; and (6) eluting: 2 × 6 ml 2:49:49 (v/v/v) formic acid/methanol/acetonitrile. The elutes were evaporated to dryness at 50°C using a DB-2A Dri-Block (Techne, UK), and then re-constituted to 1 ml by 50:50 (v/v) methanol/water. The fi nal enriched samples were fi ltered using 0.45 μm Puradisc syringe fi lters (Whatman, USA) and then subject to HPLC-UV analysis.

An Agilent 1100 system (Agilent Technologies, USA) consisting of a degasser, a binary pump, a thermo stated column oven and a UV-Vis diode array detector (DAD) was employed for the measurement of the target compounds. Fifty microliters of the samples was manually injected into a 2.6 μm, 100 × 2.10 mm reversed phase Kinetex XB-C18 column (Phenomenex, UK). The column was kept at 25°C and eluted with 0.1% formic acid in deionized water (solvent A) and acetonitrile (solvent B) at a fl ow rate of 0.2 ml/min. The percentage of solvent B was initially 20% and was then increased gradually to 45% over the next 3 min. The percentage of solvent B was then raised to 45% in 3 min, held at this percentage for 15 min and fi nally lowered to 20% in 1 min. Before the next injection, the system was allowed to equilibrate for 10 min. The UV wavelengths for the assay of BZF, SMX, DCF, and CBZ were predetermined in a Jenway 6500 spectrophotometer (Jenway, UK) and set at 230, 270, 280, and 290 nm, respectively.

Calibration curves were performed by injecting standard solutions with four compounds prepared in 50:50 (v/v) methanol/water, following the protocol stated above. The target compounds at concentrations of 100 and 10 μg/l were extracted by SPE and concentrated by 100 and 500 times, respectively. The resulting solution volume was 1 ml with a concentration of 10 and 5 mg/l, respectively. Therefore, the concentrations of each compound for the calibration were set at 0, 1, 2, 4, 6, 8, and 10 mg/l. Calibration curves were performed in triplication.

Results and Discussion. Four compounds were separated in the HPLC using the C18 column. The gradient elution of the column presented a clear separation of all chemicals in 20 min. Figure 1 gives the chromatographs of the four compounds at their specifi c wavelengths. The detection wavelengths of the target compounds were similar to many other studies [27–30].

TABLE 1. Information about SMX, DCF, CBZ, and BZF

Name Therapeutic class CAS NO pKa log PSulfamethoxazole Antibiotics 723-46-6 5.7 [26] 0.89*

Diclofenac Anti-infl ammatory 15307-86-5 4.15* 4.51*

Carbamazepine Antiepileptics 298-46-4 — 2.45*

Bezafi brate Lipid regulator 41859-67-0 3.6 [26] 4.25*

*From SRC PhysProp Database.

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TABLE 2. Recoveries of Target Compounds with Different Concentrations

Test solutions BZF SMX DCF CBZ

100 μg/l 93.0 ± 5.1% 94.8 ± 3.9% 86.5 ± 10.2% 97.0 ± 5.7%

10 μg/l 95.0 ± 6.0% 84.6 ± 10.7% 85.7 ± 5.2% 93.0 ± 7.2%

Overall recovery 94.0 ± 5.4% 89.7 ± 9.3% 86.1 ± 7.6% 95.0 ± 6.5%

Fig. 1. LC chromatographs of test solutions with BZF, SMX, DCF, and CBZ at 10 mg/l.

Fig. 2. Calibration curves of target analytes by HPLC-UV: BZF (a), SMX (b), CBZ (c), and DCF (d).

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SMX was eluted fi rstly from the column with a retention time of 5.3 min. There was a shoulder near the main peak of SMX. CBZ was eluted secondly at 9.4 min. BZF came out after CBZ at 12.4 min, and DCF was fi nally eluted at 17.0 min.

Calibrations of all four compounds were performed simultaneously. Calibration curves covering 1–10 mg/l are generated by linear regression analysis and presented in Fig. 2. All four calibration curves exhibited good coeffi cients of correlation (r2 > 0.99).

The concentrations of target analytes present in the test solutions were quantifi ed based on their specifi c linear regression equations in Fig. 2. Recoveries of each sample were calculated based on detected and spiked quantities of target analytes in the samples. The recoveries of each compound are presented in Table 2. The results showed good recoveries for BZF and CBZ, with overall recoveries 94.0 ± 5.4% and 95.0 ± 6.5%, respectively. Moreover, the recoveries of BZF and CBZ at two concentration levels were also higher than 90%. For SMX and DCF, the recoveries were slightly lower than BZF and CBZ, with values 89.7 ± 9.3% and 86.1 ± 7.6%, respectively. For SMX, the recovery for an initial concentration of 100 μg/l was 94.8 ± 3.9%, while the recovery dropped by about 10% to 84.6 ± 10.7% for samples with an initial concentration of 10 μg/l. For DCF, the recoveries of two concentration levels were close, with the value for 100 μg/l slightly higher than that for 10 μg/l, with values of 86.5 ± 10.2% and 85.7 ± 5.2%, respectively.

TABLE 3. Comparison of Recoveries with Other Analytical Methods

Method Water matrix Recovery, % ReferenceSMX

SPE + HPLC-MS/MS Sewage, surface water 120 [31]SPE + LC-MS/MS Sewage 105 ± 10 [32]SPE + LC-MS/MS Surface water 63 [33]HPLC-DAD Pure water 88.4–97.7 [27]SPE + LC-MS/MS Sewage, surface water 65 ± 2.3 [34]HPLC-DAD Sewage 90–95 [35]SPE + HPLC-DAD Pure water 89.7 ± 9.3 This study

DCFLiquid liquid extraction + HPLC-UV Human plasma 95.1 [36]Micro-extraction + HPLC-DAD Sewage, tap water 71.7–117.3 [28]SPE + HPLC-MS/MS Sewage, surface water 62 [31]SPE + LC-MS/MS Surface water 80 [33]HPLC-DAD Pure water 76.9–83.4 [27]SPE + HPLC-DAD Pure water 86.1 ± 7.6 This study

CBZSPE + HPLC-DAD Human plasma (80±2.8)–(91.9±4.9) [37]Liquid liquid extraction + HPLC-UV Urine 101 [38]SPE + LC-MS/MS Surface water 89 [33]HPLC-DAD Pure water 73.5–85.8 [27]SPE + LC-MS/MS Sewage, surface water 98 ± 7.2 [34]SPE + HPLC-DAD Pure water 95.0 ± 6.5 This study

BZFLiquid liquid extraction + HPLC-UV Human plasma 90–107 [39]SPE + LC-MS/MS Surface water 80 [33]SPE + LC-MS/MS Sewage, surface water 76 ± 2.6 [34]SPE + HPLC-DAD Pure water 94.0 ± 5.4 This study

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In comparison with other studies using HPLC-UV or LC-MS/MS (Table 3), the established method shows good recoveries of the target compounds. Though HPLC-UV has limitations in analyzing real wastewater samples of complex compositions, the good recovery and the time-economic features indicate that the method is reliable for measuring residual concentrations of SMX, DCF, CBZ, and BZF in test solutions.

Conclusions. A SPE coupled with HPLC-UV method was established in this study, which offers simultaneous determination of SMX, DCF, CBZ and BZF in test solutions with good recoveries. The mean recoveries of this analytical method for SMX, DCF, CBZ, and BZF were 89.7 ± 9.3%, 86.1 ± 7.6%, 95.0 ± 6.5%, and 94.0 ± 5.4%. It is an alternative technique to LC-MS or LC-MS/MS used for measuring pharmaceutical compounds.

Acknowledgments. The authors thank the Glasgow Caledonian University Research Committee for the PhD studentship to Zhengwei Zhou.

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