Anal Bioanal Chem (2015) 407:4219–4226 DOI 10.1007/s00216-015-8581-x
NOTE
Analytical method development for the determination of emerging contaminants in water using supercritical-fluid chromatography coupled with diode-array detection Vilma del Carmen Salvatierra-Stamp & Silvia G. Ceballos-Magaña & Jorge Gonzalez & Valentin Ibarra-Galván & Roberto Muñiz-Valencia
Received: 6 January 2015 / Revised: 11 February 2015 / Accepted: 20 February 2015 / Published online: 24 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract An analytical method using supercritical-fluid chromatography coupled with diode-array detection for the determination of seven emerging contaminants—two pharmaceuticals (carbamazepine and glyburide), three endocrine disruptors (17α-ethinyl estradiol, bisphenol A, and 17β-estradiol), one bactericide (triclosan), and one pesticide (diuron)— was developed and validated. These contaminants were chosen because of their frequency of use and their toxic effects on both humans and the environment. The optimized chromatographic separation on a Viridis BEH 2-EP column achieved baseline resolution for all compounds in less than 10 min. This separation was applied to environmental water samples after sample preparation. The optimized sample treatment involved a preconcentration step by means of solidphase extraction using C18-OH cartridges. The proposed method was validated, finding recoveries higher than 94 % and limits of detection and limits of quantification in the range of 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively. Method validation established the proposed method to be selective, linear, accurate, and precise. Finally, the method was successfully applied to environmental water samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8581-x) contains supplementary material, which is available to authorized users.
: J. Gonzalez : V. Ibarra-Galván : Carmen Salvatierra-Stamp V. del C. Salvatierra-Stamp · J. Gonzalez · V. Ibarra-Galván · R. Muñiz-Valencia (*) Facultad de Ciencias Químicas, Universidad de Colima, Carretera Colima-Coquimatlán km 9, 28400 Coquimatlán, Colima, Mexico e-mail: [email protected] S. G. Ceballos-Magaña Facultad de Ciencias, Universidad de Colima, c/ Bernal Díaz del Castillo 340, 28045 Colima, Mexico
Keywords Emerging contaminants . Supercritical-fluid chromatography . Solid-phase extraction . Environmental water samples
Introduction In recent decades, the occurrence of emerging contaminants (ECs) in the aquatic environment has become a global problem. ECs consist of a vast and expanding array of anthropogenic and natural substances including pharmaceuticals, pesticides, personal-care products, hormones, industrial chemicals, and many other compounds [1]. Although these chemical agents are not traditionally regarded as contaminants, it is now known that they have potential harmful effects on organisms, including carcinogenic, teratogenic, and toxic effects and disruption of the endocrine system [2], which may appear in subsequent generations and result in irreversible effects. Pharmaceuticals and their metabolites have not been viewed as environmental pollutants and there has been little consideration regarding their post-excretion destination. Nevertheless, their presence in the environment depends on many factors including the dosage frequency and amount and the excretion efficiency. Pesticides are frequently used in agriculture, industry, gardening, and domestic activities to limit, inhibit, and prevent the growth of harmful animals, insects, invasive plants, weeds, bacteria, and fungi [3]. Personal-care products (PCPs) are also a large group of ECs, which includes non-prescription and prescription pharmaceuticals for human and veterinary use. Some of the ECs associated with PCPs are widely found in the environment and in humans, for example bisphenol A, triclosan, 17α-ethinyl estradiol, and 17β-estradiol, and are regarded as endocrine-disrupting chemicals [4].
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Environmental water resources are increasingly exposed to EC contamination from many sources. Effluents from wastewater treatment plants could be regarded as one of the most important sources of EC environmental contamination [1, 5]. The occurrence of these pollutants in the environment is usually in a very low concentration range: from ng L−1 to μg L−1 [6]. Maximum contaminant levels (MCL) for ECs are still undefined because of the wide variety of compounds and their different toxicity. Nevertheless, some countries have banned the use and/or reduced the MCL of some compounds on the basis of the water-quality requirements for a particular use (livestock watering, drinking water, waters to maintain aquatic life, waters for use for irrigation, industrial cooling, etc.). Common techniques to clean up and preconcentrate target analytes are stir-bar sorptive extraction [7], liquid–liquid microextraction [8], solid-phase extraction [6], headspace solid-phase microextraction [9], and supercritical-fluid extraction [10]. Among these, solid-phase extraction (SPE) is the most frequently used for the analysis of ECs [4, 6, 11, 12]. Sometimes EC analysis can be a difficult task because of the wide variety of compounds to be analyzed. As can be seen from the large number of publications related to this topic, liquid and gas chromatography are well-established techniques used to analyze these contaminants [13, 14]. Among these, LC is the preferred separation technique. The detection mode most commonly used is mass spectrometry (MS). For example, analytical methods were reported using LC–MS for six carbamate pesticides as ECs in water samples [15], 12 pharmaceutical compounds including their metabolites and degradation products in seawater [16], and eight artificial sweeteners, including aspartame and its metabolites [17]. The use of MS detectors is the ideal technique for the determination and/or confirmation of ECs in environmental samples because of its high selectivity and sensitivity, enabling these compounds to be quantified at a few ng L−1. However, this MS equipment has a high operating cost and is not available in all laboratories for routine analysis. To improve the analysis of ECs in different samples, alternative chromatography techniques, including supercriticalfluid chromatography (SFC), can be used. The use of SFC in this context has attracted increasing interest from researchers. In SFC, carbon dioxide (CO2) is the most commonly used mobile phase because it is non-toxic and non-explosive, and the experimental conditions required to convert CO2 to fluid are easily achievable (the critical temperature and pressure are 31 °C and 1070 psi, respectively) [18]. SFC enables the use of high flow-rates with low pressure falls through the columns, leading to short analysis time and low consumption of organic solvents. All of these characteristics imply sharper peaks, improved resolutions, and shorter equilibration times for columns and thus faster methods. In addition, the increase in organic-solvent cost has made the use of SFC more attractive.
V.C. del Salvatierra-Stamp et al.
According to literature searches, most published methods using SFC are related to the separation of enantiomers in the pharmaceutical industry [19, 20], and there are only a few publications concerning environmental matrices. Among the few reported methods, most are devoted to the analysis of pesticides in food matrices [21]. Furthermore, there is no literature reporting the use of SFC with diode-array detection (DAD) for the simultaneous determination of different EC classes in environmental water samples. The objective of this paper was the development of a SFC analytical method that was affordable, of simple implementation, and sufficiently sensitive to quantify ECs at μg L−1 level in water samples. This method used solid-phase extraction as sample treatment before the analysis with SFC-DAD. The contaminants under study (glyburide, carbamazepine, 17αethinyl estradiol, 17β-estradiol, bisphenol A, diuron, and triclosan) were chosen on the basis of the frequency of their use and their toxic effects on both humans and the environment. The developed method was validated using environmental water samples, with satisfactory results. Finally, the method was successfully applied to environmental water samples.
Materials and methods Chemicals and materials The molecular structures and acronyms of the ECs considered in this study are shown in Fig. 1. All analytical standards (CARB, GLYB, 17EE, 17E, BPA, TRCS, and DIU) and LC–MS grade solvents (methanol and acetonitrile) were purchased from Sigma–Aldrich (Saint Louis, MO, USA). Pressurized liquid CO2 (99.999 %) was purchased from Praxair (Colima, Mexico). The analytical standards were dissolved in methanol (MeOH) to prepare 1000 mg L−1 solutions of individual EC and of a mixture of EC. These stock standard solutions were prepared weekly, stored at 4 °C, and protected from light. The combined stock solution was used for the preparation of working standards solutions at different concentrations and used for different studies. Solid-phase extraction (SPE) cartridges Bond Elut-C18OH (1 g per 6 mL) and HF Bond Elut-C18 (500 mg per 3 mL) from Varian, Agilent (Santa Clara, CA. USA) and Discovery DSC18 (500 mg per 6 mL) SPE cartridges from Sigma– Aldrich (Saint Louis, MO, USA) were used. Ultrapure water with 18.2 MΩ cm resistivity was obtained from a Milli-Q water system from Millipore (Bedford, MA, USA). A Viridis BEH-2-EP column (4.6 mm×100 mm, 5 μm) from Waters (Milford, MA, USA) was also used. Equipment and chromatography conditions A Thermo Scientific Orion 3-Star Plus pH/ATC Triode Refillable Electrode (Guadalajara, Mexico) was used for sample pH
Quantitative SFC-DAD method for emerging contaminants in water
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Fig. 1 Molecular structures, commercial names, and acronyms of the selected compounds
adjustment. For sample preconcentration a Visiprep SPE vacuum manifold from Sigma–Aldrich (Saint Louis, MO, USA) was connected to a vacuum pump model EV-40, from EVAR (Guadalajara, Mexico). The chromatography system was the Acquity Ultra Performance Convergence Chromatography (UPC2) equipped with a binary solvent pump, refrigerated autosampler with a 10 μL loop, back-pressure regulator (BPR), convergence manager, and columns oven coupled with a diode-array detector (DAD) from Waters (Milford, MA, USA). Data collection and analyses were performed using EmpowerTM Pro 3 Software from Waters (Milford, MA, USA). Chromatographic analyses were performed on the Viridis BEH-2-EP column, with 10 μL injection. The temperature of the autosampler was maintained at 15 °C and the flow at 1.4 mL min−1. The BPR was maintained at 2000 psi and
the column temperature at 40 °C. The mobile phase was a mixture of CO2 and acetonitrile (AcN) as organic modifier. The separation was performed in gradient mode as follows: from 0 to 5 min increase from 5 to 30 % AcN; and finally from 5 to 10 min increase from 30 to 40 %. Column reequilibration was performed for 3 min at the initial conditions. UV-absorbance DAD detection for qualitative analysis was performed in the 190–360 nm range, and for quantitative purposes was performed with a fixed wavelength of 215 nm. For the optimization of the mobile phase, a methanolic solution containing a single EC or an appropriate mixture of them (10 μg mL−1) was injected. Peaks were identified and EC peak purities were evaluated by comparing their retention times and UV spectra with those of previously registered compounds. The total run time was less than 10 min.
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Sample collection and preparation
Method validation
Samples were collected from rivers near Colima City, Mexico. All samples were collected from 0.5 to 1 m below the surface in 1 L amber glass bottles, kept in coolers, and transported to the laboratory where they were stored at 4 °C and protected from daylight until analysis. Before extraction, water samples were filtered to remove suspended matter through 0.45 μm highly hydrophilic polyvinylidene fluoride membrane filters from Phenomenex (Torrance, CA, USA). Solid-phase extraction was used for sample treatment. In summary, 150 mL water sample was placed in a 250 mL flask and processed at a flow of 8 mL min−1 and at pH 5.5 through C18-OH cartridges which had previously been conditioned with MeOH (3 mL) and water (6 mL). Afterwards, the elution of ECs was performed using 3 mL MeOH at 1 mL min−1 flow. The eluate was injected into the SFC system. The absolute preconcentration factor was 50. Blank water samples were previously checked for ECs following the above procedure, with negative results.
The validation of the proposed method was performed under ICH and European Commission Decision 2002/657/EC guidelines [22, 23]. According to these guidelines, the properties of an analytical method to be evaluated are selectivity, linearity, precision, accuracy, and limits of detection and quantification. Selectivity was evaluated by qualitative comparison of the retention time of the peaks obtained with those of a standard solution. Simultaneously, the identification of the analytes was confirmed by comparing the corresponding UV spectra of the peaks in the chromatograms of the sample and standard solutions. Linearity was assessed using calibration curves at six concentrations and plotting the peak area versus concentration of each analyte. Precision was expressed as relative standard deviation (RSD) at three different concentrations on three consecutive days. Accuracy, expressed as recovery percentages,
Fig. 2 SFC-DAD chromatogram at 215 nm of a 2 mg L−1 standard mixture solution
Fig. 3 SFC-DAD chromatograms at 215 nm of a 10 mg L−1 spiked EWS (a) and blank EWS (b)
Quantitative SFC-DAD method for emerging contaminants in water
was determined by comparing the concentrations found in spiked environmental water samples with the added concentration. Limits of detection (LOD) and quantification (LOQ) were determined on the basis of the standard deviation of blank-sample responses and the slope of the calibration curve for each analyte.
Results and discussion SFC-DAD optimization Organic modifiers are usually added to CO2 supercritical fluid to increase the solubility of polar compounds, change the eluent strength of the mobile phase, and improve peak shape and sensitivity. In this work, AcN was used as the organic modifier added to the mobile phase. To reduce analysis time and improve resolution, a gradient optimization for the Viridis BEH 2-EP column was performed. After assessing AcN gradients of different steepness, baseline separation in less than 10 min was achieved (Fig. 2). The final SFC gradient conditions are described in the BSample collection and preparation^ section. For optimum separation of ECs, the resolution (Rs) values were higher than 2.3, and peak symmetry values were close to 1, which means that the peak is symmetrical; the exception was GLYB, with a symmetry of 1.53.
Table 1 Correlation coefficients from matrix-matched calibration curves, intra-day precision, accuracy, and limit of detection (LOD) and quantification (LOQ) for ECs using EWS
Compound
Correlation coefficient (r)
TRCS
0.9997
DIU
0.9997
17EE
0.9999
BPA
0.9999
17E
0.9998
CARB
0.9999
GLYB
0.9999
a
Recovery assessed using three spiked EWS at the lowest, medium, and highest concentration of linear ranges
b Limit of detection (LOD) and quantification (LOQ)
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Using the optimum gradient separation, the effect of temperature and back-pressure regulator (BPR) pressure was studied. Changing column temperature and BPR pressure changes the density and viscosity of the mobile phase. These changes could affect the chromatographic separation and sensitivity. In this study, the effect on the separation of the BPR and the column temperature was assessed at 1500 and 2000 psi and at 35 and 40 °C, respectively. On the basis of the observed separations, optimum conditions were identified as 40 °C and 2000 psi. Once the pressure and temperature were established, the effect of flow rates of 1.0 and 1.4 mL min−1 was tested. At 1.0 mL min−1 flow, the resolution between 17EE and BPA decreased significantly. Therefore, the flow was set at 1.4 mL min−1. On the basis of the UV spectra (200–400 nm) of each EC, the detection wavelength was established at 215 nm.
SPE optimization The optimization of solid-phase extraction (SPE) is described in detail in the Electronic Supplementary Material (ESM). In summary, the sample flow through the SPE cartridge was set at 8 mL min−1, the pH was 5.5, no washing step was used, and 3 mL MeOH was used as elution solvent.
Intra-day precision (n=3)
Accuracy
(mg L−1)
RSD (%)
%Recoverya (RSD)
0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00
6.5 5.5 5.3 6.7 5.5 5.4 9.1 7.0 6.0
94.5 (11.7) 101.0 (6.0) 100.6 (5.5) 98.0 (9.3) 94.7 (5.8) 100.6 (5.5) 96.5 (11.1) 103.1 (7.2) 100.0 (6.1)
0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00
7.1 6.1 5.8 8.0 6.7 5.3 8.3 5.5 5.0 7.4 6.0 5.0
100.2 (7.7) 102.6 (6.2) 100.1 (5.8) 99.5 (10.5) 95.6 (7.0) 100.2 (5.3) 98.8 (11.1) 101.9 (5.7) 100.3 (5.1) 96.1 (8.5) 96.0 (6.1) 99.9 (5.1)
LODb (μg L−1)
LOQb (μg L−1)
0.40
1.22
0.43
1.31
1.59
4.83
0.23
0.69
0.71
2.15
0.10
0.31
1.41
4.27
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Method validation
Accuracy
Method validation included the evaluation of selectivity, linearity, precision, accuracy, and limits of detection and quantification.
Accuracy of the method was estimated from recovery experiments of the target analytes at 0.014, 0.2, and 2 mg L−1 concentrations, with three replicates. Recovery (%R) was assessed using the equation:
Selectivity
%R ¼ 100 ðConcentration found=Concentration spikedÞ
Selectivity was assessed by a qualitative comparison of the chromatograms obtained from standards and environmental water samples (EWS), following the procedure described in the BEquipment and chromatography conditions^ and BSample collection and preparation^ sections. Figure 2 shows the chromatogram obtained from a standard mixture of ECs (2 mg L−1), and Fig. 3a,b show EWS spiked with ECs (10 mg L−1) and EWS blank, respectively. As can be seen, possible interferences resulting from the presence of substances in samples were not observed. In addition, a detection and identification process based on retention times and using a diode-array detector was performed. The RSD (n=20) of the retention times for all ECs under the conditions described in the BEquipment and chromatography conditions^ section was lower than 1 %. The UV spectrum of each peak in the chromatogram was stored and subsequently compared with that of the corresponding standard. The chromatograms in Fig. 3a,b did not indicate signals originating from impurities. Therefore, this procedure is adequate for analysis of ECs in EWS.
The recoveries obtained for the target compounds ranged from 94.5 to 103.1 % (Table 1), which are within the acceptance criteria. Limits of detection (LOD) and quantification (LOQ) LOD and LOQ were evaluated by analyzing 20 blank EWS to calculate the standard deviation during the time window in which the analytes are expected. To obtain LOD and LOQ values, these standard deviations were divided by the slope of the calibration curve of each analyte and multiplied by 3.3 and 10, respectively. As shown in Table 1, the LOD and LOQ were in the range of 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively.
Linearity Linearity of the matrix-matched calibration curves was investigated by adding, to EC-free EWS, standards of ECs at 0.014, 0.02, 0.04, 0.10, 0.20, 0.40, 0.60, and 2 mg L−1. After sample preparation, the resulting extracts were analyzed under the conditions described in the BEquipment and chromatography conditions^ section. The results were evaluated by linear regression, plotting each EC peak area (y) versus the concentration (x=mg L−1 for EWS). For each one, a calibration equation y=mx+b was obtained. The linear ranges and regression coefficients (r) are summarized in Table 1. In all cases, r values were higher than 0.999. Precision Precision of the method based on intra-day repeatability was assessed by triplicate analysis of spiked samples at three concentrations of ECs (0.014, 0.2, and 2 mg L−1). The triplicate determinations of each concentration were conducted over three consecutive days. As can be seen from Table 1, the relative standard deviations were lower than 9.2 %.
Fig. 4 Chromatogram of a superficial water sample contaminated with TRCS and BPA
Quantitative SFC-DAD method for emerging contaminants in water
Application to real samples To establish the applicability of the developed method, seven different EWS were analyzed. The quantification was performed using the optimized conditions for SPE and SFCDAD, and the confirmation of the positive findings was obtained by comparing the UV spectra of the peaks present in the samples with those previously obtained from the standards. An example of a chromatogram of superficial water sample with positive analysis is shown in Fig. 4. From the analytical results, TRCS was found in EWS4 and EWS5 at concentrations of 1.3 and 1.2 μg L−1, respectively. TRCS was also found in EWS1, EWS3, EWS6, and EWS7, at concentrations lower than LOQ. DIU, 17E, 17EE, CARB, and GLYB were not detected in the analyzed samples. BPA concentration in EWS4 was 0.70 μg L−1, and in EWS2 was lower than LOQ.
Conclusions A sensitive and fast analytical SFC-DAD method for the simultaneous analysis of seven emerging contaminants (TRCS, DIU, 17EE, 17E, CARB, GLYB, and BPA) in water samples was developed. The separation was performed on a Viridis BEH 2-EP column in less than 10 min. This separation was applied to environmental water samples after sample preparation using solid-phase extraction by means of C18-OH cartridges. The proposed method was validated according to international guidelines. Recovery values were higher than 94 %. Limits of detection and limits of quantification were in the range 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively. Precision values evaluated as RSD were lower than 9.2 %. Method validation therefore established the proposed method to be selective, linear, accurate, and precise. Finally, the method was successfully applied to environmental water samples. Acknowledgments The authors thank Alberto Jauregui and Omar Ramirez from Waters for loaning the Water Acquity UPC2 systems and the column used in this study. Also Salvatierra-Stamp wishes to thank Consejo Nacional de Ciencia y Tecnologia-Mexico for the grant provided. Compliance with ethical standards The authors declare that they have no conflict of interest. The research presented did not involve human participants and/or animals. Also, all authors are informed about this submission.
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4225 2. Eertmans F, Dhooge W, Stuyvaert S, Comhaire F (2003) Endocrine disruptors: effects on male fertility and screening tools for their assessment. Toxicol Vitro 17(5–6):515–524 3. Bottoni P, Grenni P, Lucentini L, Caracciolo AB (2013) Terbuthylazine and other triazines in Italian water resources. Microchem J 107: 136–142 4. Clara M, Strenn B, Saracevic E, Kreuzinger N (2004) Adsorption of bisphenol-A, 17 beta-estradiole and 17 alpha-ethinylestradiole to sewage sludge. Chemosphere 56(9):843–851 5. Santos JL, Aparicio I, Alonso E, Callejón M (2005) Simultaneous determination of pharmaceutically active compounds in wastewater samples by solid phase extraction and high-performance liquid chromatography with diode array and fluorescence detectors. Anal Chim Acta 550(1–2):116–122 6. Jiang JQ, Zhou Z, Sharma VK (2013) Occurrence, transportation, monitoring and treatment of emerging micro-pollutants in waste water — A review from global views. Microchem J 110:292–300 7. Camino-Sánchez FJ, Zafra-Gómez A, Cantarero-Malagón S, Vílchez JL (2012) Validation of a method for the analysis of 77 priority persistent organic pollutants in river water by stir bar sorptive extraction in compliance with the European Water Framework Directive. Talanta 89:322–334 8. Wang C, Wu Q, Wu C, Wang Z (2011) Application of dispersion-solidification liquid-liquid microextraction for the determination of triazole fungicides in environmental water samples by high-performance liquid chromatography. J Hazard Mater 185(1):71–76 9. Tankiewicz M, Morrison C, Biziuk M (2013) Application and optimization of headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–flame-ionization detector (GC–FID) to determine products of the petroleum industry in aqueous samples. Microchem J 108:117–123 10. Ramsey ED, Sun Q, Zhang Z, Guo W, Liu JY, Wu XH (2010) Sustainable oil-in-water analysis using a supercritical fluid carbon dioxide extraction system directly interfaced with infrared spectroscopy. J Environ Sci 22(9):1462–1468 11. Li X, Zheng W, Kelly WR (2013) Occurrence and removal of pharmaceutical and hormone contaminants in rural wastewater treatment lagoons. Sci Total Environ 445–446:22–28 12. Rodriguez-Mozaz S, Lopez de Alda MJ, Barceló D (2007) Advantages and limitations of on-line solid phase extraction coupled to liquid chromatography-mass spectrometry technologies versus biosensors for monitoring of emerging contaminants in water. J Chromatogr A 1152(1–2):97–115 13. Pedrouzo M, Borrull F, Marcé RM, Pocurull E (2011) Analytical methods for personal-care products in environmental waters. Trends Anal Chem 30(5):749–760 14. Fatta D, Achilleos A, Nikolaou A, Meriç S (2007) Analytical methods for tracing pharmaceutical residues in water and wastewater. Trends Anal Chem 26(6):515–533 15. Shi Z, Hu J, Li Q, Zhang S, Liang Y, Zhang H (2014) Graphene based solid phase extraction combined with ultra high performance liquid chromatography-tandem mass spectrometry for carbamate pesticides analysis in environmental water samples. J Chromatogr A 1355: 219–227 16. Paíga P, Lolić A, Hellebuyck F, Santos LH, Correia M, Delerue-Matos C (2014) Development of a SPE-UHPLC-MS/MS methodology for the determination of non-steroidal anti-inflammatory and analgesic pharmaceuticals in seawater. J Pharm Biomed Anal. doi:10.1016/j. jpba.2014.06.017 17. Berset JD, Ochsenbein N (2012) Stability considerations of aspartame in the direct analysis of artificial sweeteners in water samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Chemosphere 88(5):563–569
4226 18. Taylor LT (2009) Supercritical fluid chromatography for the 21st century. J Supercrit Fluids 47(3):566–573 19. De Klerck K, Mangelings D, Vander Heyden Y (2012) Supercritical fluid chromatography for the enantioseparation of pharmaceuticals. J Pharm Biomed Anal 69:77–92 20. Kalíková K, Slechtová T, Vozka J, Tesařová E (2014) Supercritical fluid chromatography as a tool for enantioselective separation; a review. Anal Chim Acta 821:1–33
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Analytical method development for the determination of emerging contaminants in water using supercritical-fluid chromatography coupled with diode-array detection Vilma del Carmen Salvatierra-Stamp & Silvia G. Ceballos-Magaña & Jorge Gonzalez & Valentin Ibarra-Galván & Roberto Muñiz-Valencia
Received: 6 January 2015 / Revised: 11 February 2015 / Accepted: 20 February 2015 / Published online: 24 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract An analytical method using supercritical-fluid chromatography coupled with diode-array detection for the determination of seven emerging contaminants—two pharmaceuticals (carbamazepine and glyburide), three endocrine disruptors (17α-ethinyl estradiol, bisphenol A, and 17β-estradiol), one bactericide (triclosan), and one pesticide (diuron)— was developed and validated. These contaminants were chosen because of their frequency of use and their toxic effects on both humans and the environment. The optimized chromatographic separation on a Viridis BEH 2-EP column achieved baseline resolution for all compounds in less than 10 min. This separation was applied to environmental water samples after sample preparation. The optimized sample treatment involved a preconcentration step by means of solidphase extraction using C18-OH cartridges. The proposed method was validated, finding recoveries higher than 94 % and limits of detection and limits of quantification in the range of 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively. Method validation established the proposed method to be selective, linear, accurate, and precise. Finally, the method was successfully applied to environmental water samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8581-x) contains supplementary material, which is available to authorized users.
: J. Gonzalez : V. Ibarra-Galván : Carmen Salvatierra-Stamp V. del C. Salvatierra-Stamp · J. Gonzalez · V. Ibarra-Galván · R. Muñiz-Valencia (*) Facultad de Ciencias Químicas, Universidad de Colima, Carretera Colima-Coquimatlán km 9, 28400 Coquimatlán, Colima, Mexico e-mail: [email protected] S. G. Ceballos-Magaña Facultad de Ciencias, Universidad de Colima, c/ Bernal Díaz del Castillo 340, 28045 Colima, Mexico
Keywords Emerging contaminants . Supercritical-fluid chromatography . Solid-phase extraction . Environmental water samples
Introduction In recent decades, the occurrence of emerging contaminants (ECs) in the aquatic environment has become a global problem. ECs consist of a vast and expanding array of anthropogenic and natural substances including pharmaceuticals, pesticides, personal-care products, hormones, industrial chemicals, and many other compounds [1]. Although these chemical agents are not traditionally regarded as contaminants, it is now known that they have potential harmful effects on organisms, including carcinogenic, teratogenic, and toxic effects and disruption of the endocrine system [2], which may appear in subsequent generations and result in irreversible effects. Pharmaceuticals and their metabolites have not been viewed as environmental pollutants and there has been little consideration regarding their post-excretion destination. Nevertheless, their presence in the environment depends on many factors including the dosage frequency and amount and the excretion efficiency. Pesticides are frequently used in agriculture, industry, gardening, and domestic activities to limit, inhibit, and prevent the growth of harmful animals, insects, invasive plants, weeds, bacteria, and fungi [3]. Personal-care products (PCPs) are also a large group of ECs, which includes non-prescription and prescription pharmaceuticals for human and veterinary use. Some of the ECs associated with PCPs are widely found in the environment and in humans, for example bisphenol A, triclosan, 17α-ethinyl estradiol, and 17β-estradiol, and are regarded as endocrine-disrupting chemicals [4].
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Environmental water resources are increasingly exposed to EC contamination from many sources. Effluents from wastewater treatment plants could be regarded as one of the most important sources of EC environmental contamination [1, 5]. The occurrence of these pollutants in the environment is usually in a very low concentration range: from ng L−1 to μg L−1 [6]. Maximum contaminant levels (MCL) for ECs are still undefined because of the wide variety of compounds and their different toxicity. Nevertheless, some countries have banned the use and/or reduced the MCL of some compounds on the basis of the water-quality requirements for a particular use (livestock watering, drinking water, waters to maintain aquatic life, waters for use for irrigation, industrial cooling, etc.). Common techniques to clean up and preconcentrate target analytes are stir-bar sorptive extraction [7], liquid–liquid microextraction [8], solid-phase extraction [6], headspace solid-phase microextraction [9], and supercritical-fluid extraction [10]. Among these, solid-phase extraction (SPE) is the most frequently used for the analysis of ECs [4, 6, 11, 12]. Sometimes EC analysis can be a difficult task because of the wide variety of compounds to be analyzed. As can be seen from the large number of publications related to this topic, liquid and gas chromatography are well-established techniques used to analyze these contaminants [13, 14]. Among these, LC is the preferred separation technique. The detection mode most commonly used is mass spectrometry (MS). For example, analytical methods were reported using LC–MS for six carbamate pesticides as ECs in water samples [15], 12 pharmaceutical compounds including their metabolites and degradation products in seawater [16], and eight artificial sweeteners, including aspartame and its metabolites [17]. The use of MS detectors is the ideal technique for the determination and/or confirmation of ECs in environmental samples because of its high selectivity and sensitivity, enabling these compounds to be quantified at a few ng L−1. However, this MS equipment has a high operating cost and is not available in all laboratories for routine analysis. To improve the analysis of ECs in different samples, alternative chromatography techniques, including supercriticalfluid chromatography (SFC), can be used. The use of SFC in this context has attracted increasing interest from researchers. In SFC, carbon dioxide (CO2) is the most commonly used mobile phase because it is non-toxic and non-explosive, and the experimental conditions required to convert CO2 to fluid are easily achievable (the critical temperature and pressure are 31 °C and 1070 psi, respectively) [18]. SFC enables the use of high flow-rates with low pressure falls through the columns, leading to short analysis time and low consumption of organic solvents. All of these characteristics imply sharper peaks, improved resolutions, and shorter equilibration times for columns and thus faster methods. In addition, the increase in organic-solvent cost has made the use of SFC more attractive.
V.C. del Salvatierra-Stamp et al.
According to literature searches, most published methods using SFC are related to the separation of enantiomers in the pharmaceutical industry [19, 20], and there are only a few publications concerning environmental matrices. Among the few reported methods, most are devoted to the analysis of pesticides in food matrices [21]. Furthermore, there is no literature reporting the use of SFC with diode-array detection (DAD) for the simultaneous determination of different EC classes in environmental water samples. The objective of this paper was the development of a SFC analytical method that was affordable, of simple implementation, and sufficiently sensitive to quantify ECs at μg L−1 level in water samples. This method used solid-phase extraction as sample treatment before the analysis with SFC-DAD. The contaminants under study (glyburide, carbamazepine, 17αethinyl estradiol, 17β-estradiol, bisphenol A, diuron, and triclosan) were chosen on the basis of the frequency of their use and their toxic effects on both humans and the environment. The developed method was validated using environmental water samples, with satisfactory results. Finally, the method was successfully applied to environmental water samples.
Materials and methods Chemicals and materials The molecular structures and acronyms of the ECs considered in this study are shown in Fig. 1. All analytical standards (CARB, GLYB, 17EE, 17E, BPA, TRCS, and DIU) and LC–MS grade solvents (methanol and acetonitrile) were purchased from Sigma–Aldrich (Saint Louis, MO, USA). Pressurized liquid CO2 (99.999 %) was purchased from Praxair (Colima, Mexico). The analytical standards were dissolved in methanol (MeOH) to prepare 1000 mg L−1 solutions of individual EC and of a mixture of EC. These stock standard solutions were prepared weekly, stored at 4 °C, and protected from light. The combined stock solution was used for the preparation of working standards solutions at different concentrations and used for different studies. Solid-phase extraction (SPE) cartridges Bond Elut-C18OH (1 g per 6 mL) and HF Bond Elut-C18 (500 mg per 3 mL) from Varian, Agilent (Santa Clara, CA. USA) and Discovery DSC18 (500 mg per 6 mL) SPE cartridges from Sigma– Aldrich (Saint Louis, MO, USA) were used. Ultrapure water with 18.2 MΩ cm resistivity was obtained from a Milli-Q water system from Millipore (Bedford, MA, USA). A Viridis BEH-2-EP column (4.6 mm×100 mm, 5 μm) from Waters (Milford, MA, USA) was also used. Equipment and chromatography conditions A Thermo Scientific Orion 3-Star Plus pH/ATC Triode Refillable Electrode (Guadalajara, Mexico) was used for sample pH
Quantitative SFC-DAD method for emerging contaminants in water
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Fig. 1 Molecular structures, commercial names, and acronyms of the selected compounds
adjustment. For sample preconcentration a Visiprep SPE vacuum manifold from Sigma–Aldrich (Saint Louis, MO, USA) was connected to a vacuum pump model EV-40, from EVAR (Guadalajara, Mexico). The chromatography system was the Acquity Ultra Performance Convergence Chromatography (UPC2) equipped with a binary solvent pump, refrigerated autosampler with a 10 μL loop, back-pressure regulator (BPR), convergence manager, and columns oven coupled with a diode-array detector (DAD) from Waters (Milford, MA, USA). Data collection and analyses were performed using EmpowerTM Pro 3 Software from Waters (Milford, MA, USA). Chromatographic analyses were performed on the Viridis BEH-2-EP column, with 10 μL injection. The temperature of the autosampler was maintained at 15 °C and the flow at 1.4 mL min−1. The BPR was maintained at 2000 psi and
the column temperature at 40 °C. The mobile phase was a mixture of CO2 and acetonitrile (AcN) as organic modifier. The separation was performed in gradient mode as follows: from 0 to 5 min increase from 5 to 30 % AcN; and finally from 5 to 10 min increase from 30 to 40 %. Column reequilibration was performed for 3 min at the initial conditions. UV-absorbance DAD detection for qualitative analysis was performed in the 190–360 nm range, and for quantitative purposes was performed with a fixed wavelength of 215 nm. For the optimization of the mobile phase, a methanolic solution containing a single EC or an appropriate mixture of them (10 μg mL−1) was injected. Peaks were identified and EC peak purities were evaluated by comparing their retention times and UV spectra with those of previously registered compounds. The total run time was less than 10 min.
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Sample collection and preparation
Method validation
Samples were collected from rivers near Colima City, Mexico. All samples were collected from 0.5 to 1 m below the surface in 1 L amber glass bottles, kept in coolers, and transported to the laboratory where they were stored at 4 °C and protected from daylight until analysis. Before extraction, water samples were filtered to remove suspended matter through 0.45 μm highly hydrophilic polyvinylidene fluoride membrane filters from Phenomenex (Torrance, CA, USA). Solid-phase extraction was used for sample treatment. In summary, 150 mL water sample was placed in a 250 mL flask and processed at a flow of 8 mL min−1 and at pH 5.5 through C18-OH cartridges which had previously been conditioned with MeOH (3 mL) and water (6 mL). Afterwards, the elution of ECs was performed using 3 mL MeOH at 1 mL min−1 flow. The eluate was injected into the SFC system. The absolute preconcentration factor was 50. Blank water samples were previously checked for ECs following the above procedure, with negative results.
The validation of the proposed method was performed under ICH and European Commission Decision 2002/657/EC guidelines [22, 23]. According to these guidelines, the properties of an analytical method to be evaluated are selectivity, linearity, precision, accuracy, and limits of detection and quantification. Selectivity was evaluated by qualitative comparison of the retention time of the peaks obtained with those of a standard solution. Simultaneously, the identification of the analytes was confirmed by comparing the corresponding UV spectra of the peaks in the chromatograms of the sample and standard solutions. Linearity was assessed using calibration curves at six concentrations and plotting the peak area versus concentration of each analyte. Precision was expressed as relative standard deviation (RSD) at three different concentrations on three consecutive days. Accuracy, expressed as recovery percentages,
Fig. 2 SFC-DAD chromatogram at 215 nm of a 2 mg L−1 standard mixture solution
Fig. 3 SFC-DAD chromatograms at 215 nm of a 10 mg L−1 spiked EWS (a) and blank EWS (b)
Quantitative SFC-DAD method for emerging contaminants in water
was determined by comparing the concentrations found in spiked environmental water samples with the added concentration. Limits of detection (LOD) and quantification (LOQ) were determined on the basis of the standard deviation of blank-sample responses and the slope of the calibration curve for each analyte.
Results and discussion SFC-DAD optimization Organic modifiers are usually added to CO2 supercritical fluid to increase the solubility of polar compounds, change the eluent strength of the mobile phase, and improve peak shape and sensitivity. In this work, AcN was used as the organic modifier added to the mobile phase. To reduce analysis time and improve resolution, a gradient optimization for the Viridis BEH 2-EP column was performed. After assessing AcN gradients of different steepness, baseline separation in less than 10 min was achieved (Fig. 2). The final SFC gradient conditions are described in the BSample collection and preparation^ section. For optimum separation of ECs, the resolution (Rs) values were higher than 2.3, and peak symmetry values were close to 1, which means that the peak is symmetrical; the exception was GLYB, with a symmetry of 1.53.
Table 1 Correlation coefficients from matrix-matched calibration curves, intra-day precision, accuracy, and limit of detection (LOD) and quantification (LOQ) for ECs using EWS
Compound
Correlation coefficient (r)
TRCS
0.9997
DIU
0.9997
17EE
0.9999
BPA
0.9999
17E
0.9998
CARB
0.9999
GLYB
0.9999
a
Recovery assessed using three spiked EWS at the lowest, medium, and highest concentration of linear ranges
b Limit of detection (LOD) and quantification (LOQ)
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Using the optimum gradient separation, the effect of temperature and back-pressure regulator (BPR) pressure was studied. Changing column temperature and BPR pressure changes the density and viscosity of the mobile phase. These changes could affect the chromatographic separation and sensitivity. In this study, the effect on the separation of the BPR and the column temperature was assessed at 1500 and 2000 psi and at 35 and 40 °C, respectively. On the basis of the observed separations, optimum conditions were identified as 40 °C and 2000 psi. Once the pressure and temperature were established, the effect of flow rates of 1.0 and 1.4 mL min−1 was tested. At 1.0 mL min−1 flow, the resolution between 17EE and BPA decreased significantly. Therefore, the flow was set at 1.4 mL min−1. On the basis of the UV spectra (200–400 nm) of each EC, the detection wavelength was established at 215 nm.
SPE optimization The optimization of solid-phase extraction (SPE) is described in detail in the Electronic Supplementary Material (ESM). In summary, the sample flow through the SPE cartridge was set at 8 mL min−1, the pH was 5.5, no washing step was used, and 3 mL MeOH was used as elution solvent.
Intra-day precision (n=3)
Accuracy
(mg L−1)
RSD (%)
%Recoverya (RSD)
0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00
6.5 5.5 5.3 6.7 5.5 5.4 9.1 7.0 6.0
94.5 (11.7) 101.0 (6.0) 100.6 (5.5) 98.0 (9.3) 94.7 (5.8) 100.6 (5.5) 96.5 (11.1) 103.1 (7.2) 100.0 (6.1)
0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00 0.014 0.20 2.00
7.1 6.1 5.8 8.0 6.7 5.3 8.3 5.5 5.0 7.4 6.0 5.0
100.2 (7.7) 102.6 (6.2) 100.1 (5.8) 99.5 (10.5) 95.6 (7.0) 100.2 (5.3) 98.8 (11.1) 101.9 (5.7) 100.3 (5.1) 96.1 (8.5) 96.0 (6.1) 99.9 (5.1)
LODb (μg L−1)
LOQb (μg L−1)
0.40
1.22
0.43
1.31
1.59
4.83
0.23
0.69
0.71
2.15
0.10
0.31
1.41
4.27
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Method validation
Accuracy
Method validation included the evaluation of selectivity, linearity, precision, accuracy, and limits of detection and quantification.
Accuracy of the method was estimated from recovery experiments of the target analytes at 0.014, 0.2, and 2 mg L−1 concentrations, with three replicates. Recovery (%R) was assessed using the equation:
Selectivity
%R ¼ 100 ðConcentration found=Concentration spikedÞ
Selectivity was assessed by a qualitative comparison of the chromatograms obtained from standards and environmental water samples (EWS), following the procedure described in the BEquipment and chromatography conditions^ and BSample collection and preparation^ sections. Figure 2 shows the chromatogram obtained from a standard mixture of ECs (2 mg L−1), and Fig. 3a,b show EWS spiked with ECs (10 mg L−1) and EWS blank, respectively. As can be seen, possible interferences resulting from the presence of substances in samples were not observed. In addition, a detection and identification process based on retention times and using a diode-array detector was performed. The RSD (n=20) of the retention times for all ECs under the conditions described in the BEquipment and chromatography conditions^ section was lower than 1 %. The UV spectrum of each peak in the chromatogram was stored and subsequently compared with that of the corresponding standard. The chromatograms in Fig. 3a,b did not indicate signals originating from impurities. Therefore, this procedure is adequate for analysis of ECs in EWS.
The recoveries obtained for the target compounds ranged from 94.5 to 103.1 % (Table 1), which are within the acceptance criteria. Limits of detection (LOD) and quantification (LOQ) LOD and LOQ were evaluated by analyzing 20 blank EWS to calculate the standard deviation during the time window in which the analytes are expected. To obtain LOD and LOQ values, these standard deviations were divided by the slope of the calibration curve of each analyte and multiplied by 3.3 and 10, respectively. As shown in Table 1, the LOD and LOQ were in the range of 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively.
Linearity Linearity of the matrix-matched calibration curves was investigated by adding, to EC-free EWS, standards of ECs at 0.014, 0.02, 0.04, 0.10, 0.20, 0.40, 0.60, and 2 mg L−1. After sample preparation, the resulting extracts were analyzed under the conditions described in the BEquipment and chromatography conditions^ section. The results were evaluated by linear regression, plotting each EC peak area (y) versus the concentration (x=mg L−1 for EWS). For each one, a calibration equation y=mx+b was obtained. The linear ranges and regression coefficients (r) are summarized in Table 1. In all cases, r values were higher than 0.999. Precision Precision of the method based on intra-day repeatability was assessed by triplicate analysis of spiked samples at three concentrations of ECs (0.014, 0.2, and 2 mg L−1). The triplicate determinations of each concentration were conducted over three consecutive days. As can be seen from Table 1, the relative standard deviations were lower than 9.2 %.
Fig. 4 Chromatogram of a superficial water sample contaminated with TRCS and BPA
Quantitative SFC-DAD method for emerging contaminants in water
Application to real samples To establish the applicability of the developed method, seven different EWS were analyzed. The quantification was performed using the optimized conditions for SPE and SFCDAD, and the confirmation of the positive findings was obtained by comparing the UV spectra of the peaks present in the samples with those previously obtained from the standards. An example of a chromatogram of superficial water sample with positive analysis is shown in Fig. 4. From the analytical results, TRCS was found in EWS4 and EWS5 at concentrations of 1.3 and 1.2 μg L−1, respectively. TRCS was also found in EWS1, EWS3, EWS6, and EWS7, at concentrations lower than LOQ. DIU, 17E, 17EE, CARB, and GLYB were not detected in the analyzed samples. BPA concentration in EWS4 was 0.70 μg L−1, and in EWS2 was lower than LOQ.
Conclusions A sensitive and fast analytical SFC-DAD method for the simultaneous analysis of seven emerging contaminants (TRCS, DIU, 17EE, 17E, CARB, GLYB, and BPA) in water samples was developed. The separation was performed on a Viridis BEH 2-EP column in less than 10 min. This separation was applied to environmental water samples after sample preparation using solid-phase extraction by means of C18-OH cartridges. The proposed method was validated according to international guidelines. Recovery values were higher than 94 %. Limits of detection and limits of quantification were in the range 0.10–1.59 μg L−1 and 0.31–4.83 μg L−1, respectively. Precision values evaluated as RSD were lower than 9.2 %. Method validation therefore established the proposed method to be selective, linear, accurate, and precise. Finally, the method was successfully applied to environmental water samples. Acknowledgments The authors thank Alberto Jauregui and Omar Ramirez from Waters for loaning the Water Acquity UPC2 systems and the column used in this study. Also Salvatierra-Stamp wishes to thank Consejo Nacional de Ciencia y Tecnologia-Mexico for the grant provided. Compliance with ethical standards The authors declare that they have no conflict of interest. The research presented did not involve human participants and/or animals. Also, all authors are informed about this submission.
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