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Development and validation of an SPE methodology combined with LCMS/MS for the determination of four ionophores in aqueous environmental matrices a
a
a
a
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Søren Alex Bak , Martin Hansen , Kristine A. Krogh , Asbjørn b
Brandt , Bent Halling-Sørensen & Erland Björklund a
Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark b
Department of Veterinary Medicine, Danish Health and Medicines Authority, Axel Heides Gade 1, 2300, Copenhagen, Denmark Published online: 25 Feb 2013.
To cite this article: International Journal of Environmental Analytical Chemistry (2013): Development and validation of an SPE methodology combined with LC-MS/MS for the determination of four ionophores in aqueous environmental matrices, International Journal of Environmental Analytical Chemistry, DOI: 10.1080/03067319.2013.763250 To link to this article: http://dx.doi.org/10.1080/03067319.2013.763250
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Intern. J. Environ. Anal. Chem., 2013 http://dx.doi.org/10.1080/03067319.2013.763250
Development and validation of an SPE methodology combined with LC-MS/MS for the determination of four ionophores in aqueous environmental matrices
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Søren Alex Baka*, Martin Hansena, Kristine A. Krogha, Asbjørn Brandtb, Bent Halling-Sørensena and Erland Björklunda a
Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark; bDepartment of Veterinary Medicine, Danish Health and Medicines Authority, Axel Heides Gade 1, 2300 Copenhagen, Denmark (Received 25 September 2012; final version received 10 December 2012) A multi-residue analytical methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in aqueous environmental matrices, using nigericin as internal standard. The samples were filtrated prior to solid phase extraction. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry applying electro spray ionisation. The absolute recoveries ranged from 92 to 110% (relative standard deviation: 2–14%) for spiked river water. The final method allowed for detection of ionophores down to a few ng/L in natural water bodies with LOQs for the entire methodology being 40, 49, 67, and 14 ng/L for lasalocid, monensin, salinomycin, and narasin, respectively. Keywords: ionophores; pharmaceuticals; anticoccidials; antibiotics; SPE; LC-MS/MS
1. Introduction Anticoccidial agents or coccidiostatics are used as antibiotic and antiparasitic growth promoters in livestock production [1]. These agents have been used heavily since the market introduction of the ionophores subclass in the 1970s [2]. Since 2006 anticoccidials are the only antibacterial substances still authorised as feed additives within the European Union [3]. The primary use of anticoccidials is for prevention and treatment of the disease coccidiosis caused by unicellular intestinal parasites. In modern livestock production, the conditions are favourable for growth of these parasites. Adverse clinical effects of enteritis such as loss of weight, diarrhoea and high mortality, can eliminate an entire animal production [4]. After the ban of other antibiotic growth promoters, ionophores are used extensively worldwide as prophylactic chemotherapeutics and growth promoters in livestock production. In 2011, the total consumption of ionophores (active compounds) in Denmark exceeded 19.8 tons with salinomycin (SAL) being the most heavily applied, corresponding to 12.4 tons, followed by 7.8 tons of monensin (MON), 6.1 tons of narasin (NAR) and 0.5 ton lasalocid (LAS) [5]. In Norway, 9 tons of NAR were applied in 2010 [6], while in the United Kingdom 169 tons of ionophores were used in 2009 [7] and estimates from the Republic of Korea suggests that more than 800 tons ionophores are administered yearly [8]. In the long term, this may cause problems with resistance development in the treatment of coccidiosis [9]. Physicochemical characteristics of the ionophores are presented in Table 1. *Corresponding author. Email:
[email protected] © 2013 Taylor & Francis
670.9
751.0
765.0
725.0
Monensin (MON) C36H62O11
Salinomycin (SAL) C42H70O11
Narasin (NAR) C43H72O11
Nigericin (NIG) C40H68O11
Mw [g/mol] 590.8
Chemical structure
Lasalocid (LAS) C34H54O8
Compound
4.0
4.4
4.4
4.2
2.6
pKa pH 7
No data
102
294
63
10
Swater,
[mg/L]
No data
4.8 (8)
3.3 (5) 2.9 (7) 2.6 (9)
4.2 (5) 2.8 (7) 3.9 (9)
2.8 (7)
Log Kow (pH)
Table 1. Physicochemical properties of the investigated ionophores: Name, abbreviation, chemical composition and structure, molecular weight (Mw), acid dissociation constant (pKa) – Calculated using MarvinSketch™, water solubility (Swater) and octanol-water distribution coefficient (Kow) of the analysed ionophores.
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Apart from causing resistance in animal production there may also be an environmental concern [10] as it has been reported that as much as 94% of the radioactivity in an ingested dose of 14C-monensin was excreted in the faeces as monensin or biological active transformation products [11]. In 2003, SAL was determined in manure from storage tanks from treated pigs [12] and in soil samples from the field fertilised with the liquid manure from the tanks [13]. Recently, it was concluded that there is a lack of knowledge on the environmental occurrence of these compounds despite their intense use [14,15]. A few reports have suggested ionophores to be emerging environmental contaminants in agricultural run-off waters, surface waters and ground waters due to their increased application as feed additives in modern livestock production [16]. More recently, it was demonstrated that the ionophore NAR was transported from the agricultural soil and into carrots [17]. Some analytical methods have been developed for the detection of MON in a single residue method in groundwater [14,15,18], or included in multi-residue methods for several pharmaceuticals in run-off water [19–23] and groundwater [24]. MON and LAS were also part of a multiresidue solid phase extraction (SPE) LC-MS/MS method developed by Hao et al. [25]. Additionally a methodology for the determination of MON and SAL, and 26 other antibiotics was presented for surface and waste water samples in an Australian study [26]. In total two strategies have been published for multi-residue analysis of three ionophores in aqueous matrices. The first was published by Cha et al. in [27] and is a SPE LC-MS/MS method for determination of MON, SAL and LAS in surface water samples [27]. A year later, the same group reported improved recoveries and LOQ with the same set-up [28]. In 2010, Ramsey et al. used supercritical fluid extraction combined with LC-MS/MS for extraction of LAS, MON and NAR without any application [29]. Recently, Thompson et al. also presented a multi-residue methodology using an on-line large-volume injection approach with 5 mL water samples, for determination of the four ionophores LAS, MON, SAL, NAR and two avermectins in environmental aqueous samples [30]. None of the above published multi-residues methodologies using SPE includes an internal standard with ionophoric attributes and in most of the previous methods only one or two ionophores have been analysed alternatively the methods have a sparse description of method development parameters or validation. The development and validation of a robust multi-residue analytical method to determine all used ionophores in aqueous matrices down to the lower ng per litre range is therefore needed to provide better data to assess the environmental occurrence and fate of ionophores. Consequently, the aim of this paper is to develop such a method for the ionophores LAS, MON, SAL, and NAR including the use of an internal standard. The developed method consists of a solid phase extraction combined with high-performance liquid chromatography tandem in space mass spectrometry.
2. Experimental 2.1 Chemicals and materials Lasalocid A sodium (purity 91%), monensin sodium (90%) and nigericin sodium (98%) were purchased from Sigma-Aldrich (Germany). Salinomycin sodium salt 2.5 hydrate (93%) was from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and narasin (97%) was kindly donated by Eli Lilly (Indianapolis, IN, USA). Stable isotopic standards of ionophores are unavailable; this is why nigericin (NIG) was used as internal standard (IS) in the present study as NIG is not used as a feed additive. Stock solutions of the ionophores were prepared in ethanol at a concentration level of 1 mg/mL and were stored at −18°C for a maximum of 12 months. Acetonitrile, acetone, ethanol, pentane and methanol (MeOH) were HPLC grade and purchased from Lab-Scan (Dublin, Ireland). Sodium chloride (98%) and formic acid (98%)
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were purchased from Merck (Darmstadt, Germany). Nitrogen with a purity of 99.8% was supplied by Air Liquide (Ballerup, Denmark). Pure water was produced in house using a Milli-Q gradient system (Millipore, Bedford, MA, USA). The pH of water samples and mobile phases was measured with a 780 pH Meter from Methrom (Herisan, Switzerland).
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2.2 Sample collection (and site description) 2.2.1 Urban and suburban water samples for method development Urban surface water used for spiking during method development of the SPE procedure was sampled at a lake in Copenhagen, Site A (+55° 42′ 8.00″, +12° 22′ 55.00″). The lake consists of surface water from Copenhagen and is without any connection to agricultural water. Additionally water from a suburban river site in Copenhagen (Site B; +55° 43′ 18.90″, +12° 20′ 8.75″) was spiked and used in the method development. All water samples were collected in 5 L glass containers. Prior to spiking the waters were pre-analysed and no ionophores were detected in these samples. 2.2.2 Agriculture water samples for occurrence studies Water samples from eight different Danish agriculture sites were collected in 5 L glass containers and analysed (Figure 1). Site C (+55° 16′ 39.17″, +11° 35′ 56.10″) was a brackish lake while sampling sites D-F were located in a small sweet water creek (Lindes Å) running through an agricultural field close to a chicken farm. Site D is the influx, site E is the middle and efflux of the field. The last four samples were collected from rivers up streams (Site G; +55° 35′ 39.75″, +11° 18′ 28.93″) and down streams (H–J) from a larger lake system (Tissø). Site H (+55° 33′ 51.10″, +11° 11′ 52.97″) was the exit river (Halleby Aa) from the lake. Site I (+55° 35′ 6.20″, +11° 13′ 10.37″) is a small creek origin next to a chicken farm and runs into the Halleby Aa. Site J (+55° 33′ 51.10″, +11° 11′ 52.97″) is the efflux of the agricultural fields.
Figure 1.
Sample sites at Zealand in Denmark.
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2.3 Solid phase extraction (SPE) 2.3.1 Method development For the SPE experiments, the samples were first pH adjusted to 7.0 ± 0.1 with 0.5 M HCl, then split into subsamples of 1 L. The subsamples were spiked at three concentration levels 50, 200 or 500 ng of each of the ionophores and 200 ng of IS. Hereafter the 1 L samples were vacuumfiltrated through a CF/D 2.7 µm filter from WhatmanTM (Little Chalfont, UK) and 10 mL MeOH was applied to the sample bottle for a short wash. The MeOH was then transferred to the water samples through the same filter to wash of any potential residues of ionophores bond to the filter. Three SPE cartridge materials were evaluated; Oasis® HLB (200 mg, 6 cm3, 30 μm) from Waters (Milford, Massachusetts, USA), and C8 and C18 (500 mg, 3 mL) from Varian (Harbour City, USA). The HLB cartridges were conditioned with 3 mL acetone, 3 mL methanol and two times 3 mL of tap water (adjusted to pH 7), whereas the C8 and C18 cartridges were conditioned with 3 mL heptane, 3 mL acetone, 3 mL methanol and two times 3 mL of tap water (pH 7). A VacMaster manifold from IST (Glamorgan, United Kingdom) with a Comfort Heto Master Jet pump, Type SUE 330Q from Heto-Holten A/S (Allerød, Denmark) connected was used for SPE. The reported absolute recoveries for evaluation of ionophores were obtained by the method described by Hansen et al. [16]. Hereby, ion suppression and matrix effect are circumvented and the performance of the chosen SPE cartridges can be directly evaluated.
2.3.2 Final method In the final method used for extraction of real samples, one litre samples were pH-adjusted to 7.0 with 0.5 M HCl and 200 ng of internal standard (IS) was added prior to filtering and SPE. After filtration using a CF/D 2.7 µm filter from WhatmanTM, all filters were rinsed with MeOH which was added to the water samples resulting in a content of maximum 1% methanol. HLB (200 mg, 6 cm3) SPE cartridges were conditioned with 3 mL acetone, 3 mL methanol and two times 3 mL of tap water (adjusted to pH 7). Thereafter, the samples were loaded on the SPE cartridges using Teflon tubings and a manifold vacuum system at a rate of 5–10 mL/min. Then, the SPE cartridges were air-dried for 30 min and stored at –18°C if needed. The compounds were eluted with 6 mL of methanol. This eluate was evaporated to dryness under a gentle stream of nitrogen at 60°C and reconstituted in 1000 µL of a 3:7 ratio of mobile phase A:B from the HPLC method, before injection into the LC-MS/MS.
2.4 Liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS) An 1100 Series HPLC instrument (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a binary pump, a degasser, a cooled auto sampler at 4°C and a column oven was applied. The HPLC system was coupled to a PE-Sciex API 3000 triple quadrupole mass spectrometer from Applied Biosystems MDS Sciex Instruments (Ontario, Canada) equipped with a Turbo IonSpray interface. Using a 10 μL injection volume the separation was performed on an XTerra RP C18 column (2.1 × 100 mm, 3.5 µm) with C18 column guard (2.1 × 20 mm, 3.5 µm) both from Waters (Milford, MA, USA) applying a binary gradient at a flow rate of 300 µL/min. The column oven was optimized to 70°C to lower the pressure to below 120 bars. Mobile phase A contained 95:5 Milli-Q water:acetonitrile and 10 mM formic acid and 50 µM NaCl. Mobile phase B consisted of 5:95 Milli-Q water:acetonitrile and 10 mM formic acid and 50 µM NaCl. The initial mobile phase composition was 30% A and 70% B with a 0.5 min equilibration period at the start conditions, followed by an 11.5 min gradient ending with a composition of 1% A and 99% B. This composition was held for 1 min thereafter returning to the starting conditions giving
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Table 2. Compound specific mass spectrometry parameters for the optimized method. The focusing potential, Entrance potential and Collision cell exit potential were set at 225 V, 10 V, and 20 V, respectively, for all compounds. Compound
Dwell time (ms)
Declustering potential (V)
Collision energy (eV)
Precursor ion [H+Na]+ (m/z)
Product ions quantify/ qualify (m/z)
Lasalocid Monensin Salinomycin Narasin Nigericin
1000 1000 400 400 400
80 65 100 100 100
45/50 57/78 65/70 65/70 75/80
613.40 693.40 773.5 787.5 747.5
577.4/377.4 675.4/461.5 531.4/431.4 531.4/431.4 703.4/501.4
a total analysis time of 17 min. Electrospray ionisation was performed in positive ionisation mode (ESI +) and the multiple reaction monitoring (MRM) function was applied in all analyses. IonSpray voltage was 5 kV. Nitrogen was used as collision, curtain and nebulizer gas with a gas flow of 8 L/min. Source temperature and spray voltage was 400°C and 4.5 kV. A Valco valve was used as a diverter between the HPLC and the MS, and was set to infuse into the MS from 2.4 to 12.0 min. The analysis is divide into three segments for monitoring of LAS (0–7.0 min), MON (7.0–8.5 min) and SAL; NAR and NIG in the last segment (8.5–17.0 min). The optimised parameters for the LC–MS/MS analysis are summarized as compound specific settings in Table 2. 2.5 Method validation The instrumental limit of detection (LODInstr) for the LC–MS/MS system was estimated from calibration curves by injecting standards dissolved in a 3:7 ratio of mobile phase A:B and then verified by injecting corresponding concentration. All standards and samples were analysed three times and the concentrations were determined from a five-point calibration curve in the range of 20–400 pg/µL for the five ionophores. The linearity of the corresponding calibration curves gave correlation coefficients (r) above 0.9931. The LODInstr and instrumental limit of quantification (LOQInstr) of the LC–MS/MS system were calculated from the linear regression by 3 and 10 times, respectively, of the concentration of the lowest calibration standard multiplied by the standard deviation divided by the average response value of the six samples analyses of the lowest calibration standards. Validation of the whole method (SPE and LC-MS/MS) was done by spiking the urban surface water (Site A) and suburban river water (Site B) samples with all analytes. Recoveries were determined at three water concentration levels, 50, 200, and 500 ng/L for LAS, MON, SAL, and NAR, respectively. 200 ng IS was added per sample prior to filtration and SPE. All extractions were done in triplicate. Day-to-day variation was evaluated extracting the same concentration levels 3 days later. For the entire method the limit of detection (LODMethod) and limit of quantification (LOQMethod) were determined as described for the instrumental limits of detection and quantifications, respectively.
3. Results and discussion 3.1 Method development 3.1.1 Optimisation of liquid chromatography–tandem mass spectrometry The first step in developing an analytical methodology for determination of ionophores was to being able to detect the analytes using MS. Full scan in both positive and negative ionisation
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modes was tested using direct infusion (10 µL/min) of a 1 ng/µL standard of the analytes in order to optimise the MS/MS conditions. The ionophores ability to bind sodium results in formation of sodium adducts even with the use of LC–MS graded solvents. Therefore in positive MS/MS mode detection the sodium ion was best suited as the parent ions. To assure the formation of the sodium adducts a small amount of sodium (50 µM) is added to the mobile phase. The sodium adducts [M + Na]+, at 613.4, 693.4, 773.5, 787.5 and 747.5 (m/z) for LAS, MON, SAL, NAR and NIG, respectively, were identified by multiple reaction monitoring (MRM) quantification with two daughter transitions per compound (Table 2). Fragmentation was produced by collision-induced dissociation (CID) and the product ions yielding the highest signal from the Q3 scanning were used for quantification (Table 2). The optimized chromatographic separation of the compounds is shown for a standard solution in Figure 2. Moreover an increased MS-sensitivity was obtainable by dividing the MRM scanning into three segments; 0–7.0 min monitoring for LAS and 7.0–8.5 min for MON and 8.5–17.0 min monitoring for the remaining analytes. These setting were incorporated into the optimized methodology. 3.1.2 Effects of the evaporation step and sorption to glass on recoveries The optimal temperature for evaporation of the samples after SPE was investigated at five different temperatures: 30, 40, 50, 60, 70°C. Five mL of methanol (the optimized SPE elution solvent) was spiked with 200 ng of each of the five ionophores and was evaporated under a gentle stream of nitrogen to dryness at the tested temperature and immediately reconstituted in
100
% LAS
SAL
NAR
MON 75
50
NIG
25
0
0
5
10
16
Time [Min]
Figure 2. Chromatogram of 500 pg of the ionophores lasalocid (LAS), monensin (MON), salinomycin (SAL), narasin (NAR) and nigericin (NIG, IS), respectively, using the optimized LC–MS/MS conditions and three segments for detection of LAS (0–7.0 min), MON (7.0–8.5 min) and SAL. NAR and NIG were identified in the last segment (8.5–17.0 min).
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1 mL mixture of mobile phase A:B (3:7). No major losses were observed up to a temperature of 60°C. At 70°C, the recoveries decreased by around 20–35% for LAS, SAL and NIG, while the recoveries for MON and NAR remained above 92% (data not shown). A temperature of 60°C was applied in the final method allowing a fast evaporation but assuring minimal losses. Experiments also demonstrated that sorption of ionophores to the glass bottles was minor, maximum 5% for the different ionophores at pH 7.
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3.1.3 Optimisation of solid-phase extraction 3.1.3.1 Investigation of SPE materials. Initially the two SPE sorbent C8 and C18 were investigated using pentane, acetone, methanol and acetonitrile as elution solvents. The C8 sorbent showed a low ability to retain the ionophores and C8 was therefore not further evaluated (data not shown). Analysing the three succeeding 10 mL-aliquots of each elution solvent yields a cumulative recovery profile (Figure 3). Elution of the ionophores from the C18 sorbent demonstrated that pentane was not able to elute any of the compounds (Figure 3.). Applying acetone, MON could be partly eluted when 30 mL was used. Thereafter, when eluting with MeOH the absolute recoveries of the ionophores were 23%, 24% and 53% for SAL, NAR and LAS, respectively, and around 75% for MON and NIG. The recoveries were not improved by using up to 30 mL acetonitrile as eluate instead of MeOH. As the experiments presented in Figure 3 were not fully satisfying, further attempts were made to optimize the SPE methodology. This included both testing of another stationary phase as well as applying different pH values. In the above experiments pH were set to 7.0. However, it should be stressed that the pH of the sample is an important parameter to optimise as all the ionophores have a carboxylic functional group. The reported pKa values from the producers is determined in a mixture of water and organic solvent (>60%) and were in the range from 4.4 to 7.9 [16]. The
Figure 3. Effect of elution solvents on the recoveries of the five ionophores spiked at a concentration of 200 ng on C18 SPE Cartridges (500 mg, adjusted to pH 7.0), n = 2. The recoveries are cumulative for elution with 10 mL in each step.
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aqueous-based pKa values (Table 1) are expected to be one to two log units lower [31]. Using MarvinSketch [32], Sassman et al. calculated the pKa values of LAS and MON to be 2.6 and 4.2, which they concluded as best available software for modelling the pKa values in aqueous solutions [33]. Using MarvinSketch we calculated the pKa values of 4.2 and 4.1 for SAL and NAR, respectively. The pKa of SAL is 2 log units lower then the determined pKa of 6.4 in organic solvent [34]. NAR (4-Methylsalinomycin) is expected to have similar pka value in water do to the close structurally composition, even through the reported pKa is 7.9 for NAR in organic solvent [34]. Lasalocid with a pKa value of 2.6 is the most acidic compound and at pH 3 it is partly neutral (30%), while all others are on their acidic uncharged form. Concerning SPE materials, apart from C18, also HLB was chosen to test the effects on recovery at different pH values. Tap water samples were adjusted to pH 3.0 and 7.0 and spiked to 40 ng of each ionophore and added to the C18 and HLB SPE cartridges. The elution volume was 10 mL of MeOH. Base on the above findings methanol was chosen as elution solvent in these experiments. In Figure 4, it can be seen that the recoveries of the ionophores are affected by decreasing the pH. Overall for the HLB cartridge at pH 7.0, the recoveries are all above 80% for the ionophores and the variations were between 10 and 24%. Using C18 sorbent, the ionophores also showed better recoveries at neutral conditions than acidic conditions, but generally the recoveries were lower and the variation was higher than for the HLB sorbent (data not shown). Based on these experiments, HLB cartridges were applied in the final method using MeOH as elution solvent. Similar observation regarding the effect of pH on the SPE recoveries is described by Hao et al. [20]. They found for spiked surface waters recoveries of 4%, 75% and 51% for MON at pH 2.5, 7.2 and 9.0, respectively. Two other papers have used neutral pH, Cha et al. [27] adjusted water samples to pH 7.5 [27] and Thompson et al. used 7.0 [30]. In contrast, Watkinson et al. adjusted to pH 3 prior to SPE with recoveries of 63 and 64% for MON and SAL, respectively [26]. In the studies without pH-adjustment, the reported pH values of the analysed water samples were in the range of 7.0 to 8.7 so for these applications the evaluated recoveries are
Figure 4. Evaluation of the effect of pH on the recoveries of HLB sorbent. The five ionophores spiked with 40 ng and adjusted to 3.0 and 7.0, respectively. n = 3.
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only for the unprotonated ionophore. Based on the above studies and our data, pH in the order of 7–8 seems best suited to achieve good recoveries from HLB. 3.1.3.2 Effect of elution volume. The elution volume of methanol is a crucial step to assure a quantitative removal of the compounds. To assess the sufficient volume needed, HLB (200 mg) cartridges were loaded with spiked tap water (pH = 7.0) containing 500 ng of each analyte and eluted by adding 1 mL methanol in 8 steps. Thereafter, the 1 mL portions were analysed separately to obtain elution profiles with 1 mL steps from 1 to 8 mL. The elution profile showed that after applying 3 mL of MeOH the compounds were quantitatively removed from the cartridge except for LAS, which was first removed after adding a total of 6 mL methanol (data not shown). Therefore, the final elution volume was set to 6 mL, which corresponds to flushing the sorbent pore volume approximately 22 times. Previously, it was found that 30 mL of MeOH is required to obtain the maximum recovery of ionophores from the C18 SPE cartridge while only 6 mL is required to quantitatively elute them on the HLB SPE cartridge. Also NAR appears to be most strongly retained on the C18 cartridge while LAS requires the largest volume of methanol for elution from the HLB cartridge. 3.1.3.3 Effects of filtration. Prior to SPE of environmental samples a filtration step is often needed. Therefore, the effect of the filtration on the overall absolute recoveries of the ionophores was examined. This was achieved by spiking tap water (adjusted to pH 7) at a concentration of 200 ng/L. The absolute recoveries for the filtration were obtained by comparing the ratio of the concentration between samples spiked before and after the filtration. The samples spiked after the filtration were compared to post SPE spiked samples to calculate the SPE recoveries. Finally, the absolute recoveries for the sample preparation and extraction were determined using the concentration from the pre filtration and post SPE samples. The results are presented in Figure 5.
Figure 5. Absolute recoveries study of the filtration and SPE. 1 L tap water samples adjusted to pH 7 were spiked with 200 ng. The Method recoveries were determined form the ratio of concentration of the pre filtration and post SPE samples. Error bars represent the relative standard deviation (n = 3).
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The filtration results in a loss below 20%. The overall recoveries of the filtration and SPE were 73–92% with RDS in the range from 6 to 13%. Washing of the filter with MeOH after the filtration was found to be important for all ionophores. In the final method, a 10 mL MeOH filter-washing step is applied, corresponding to a total concentration of less than 1% MeOH in the 1 L-samples before SPE, which does not affect the SPE efficiency. None of the methods from the literature mentioned above have investigated the loss caused by filtration, even though it is necessary for most environmental samples prior to SPE.
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3.2 Method validation 3.2.1 LC–MS/MS validation The linearity was assessed using a five-point calibration curve in the concentration range of 20– 400 pg/µL for the five ionophores injecting 10 µL on the LC–MS/MS system. The obtained correlation coefficients (r) for LAS, MON, SAL, NAR and NIG were 0.9932, 0.9959, 0.9965, 0.9973, and 0.9974, respectively. Evaluation of the LODInstr and LOQInstr were performed as described in Section 2.5. The obtained LODInstr values were 5.6, 12.5, 4.9, 8.4, and 13.8 pg/µL for LAS, MON, SAL, NAR and NIG, respectively and LOQInstr values were 18.7, 41.8, 16.4, 28.0, and 46.0 pg/µL in the same order of appearance. This corresponds to absolute amounts of 187, 418, 164, 280, and 460 pg required for injection in the LC–MS/MS system to reach the LOQ. Day-to-day variation of the LC–MS/MS system was obtained by continuous monitoring of the IS during the LC–MS/MS validation and showed minimal variation over 20 days (less than 11% RSD, n = 12, data not shown).
3.2.2 SPE LC–MS/MS validation Validation of the entire SPE LC–MS/MS method was performed by spiking 1 L urban surface water samples from site A (adjusted to pH 7) with the four ionophores at three concentration levels (50, 200, and 500 ng). Furthermore, 1 L urban river water samples were spiked with 200 ng level. When analysing water samples NIG was used as IS and was added as 200 ng NIG before the filtration and the quantification was based on an internal standard calibration. In these experiments, the IS was used to compensate for losses occurring during the sample procedure but also compensated for any variation caused by the LC–MS/MS system. Absolute recoveries for the optimised method are listed in Table 3. Table 3 gives the absolute recoveries from the three spiking levels and the method demonstrates a good accuracy with absolute recoveries above 90% for all compounds and when using the IS, the relative recoveries were even better, particularly for LAS and NAR. The precision of the methodology is below 20%, which is acceptable. The LODMethod were calculated as described above to be 12, 15, 20 and 4 ng/L for LAS, MON, SAL, and NAR, respectively. Corresponding LOQMethod values were 40, 49, 67, and 14 ng/L in the same order of appearance.
3.3 Application to real samples The presented method was applied to agriculture water samples from site C–J (see Figure 1.). All samples were analysed in triplicate. Analysis showed that the concentrations of all ionophores were below the LODMethod meaning that their environmental concentrations do not exceed low ppt-levels (ng/L). These low concentrations could be due to ionophores only being present in very low amounts because of heavy rain the weeks before sampling, causing
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Table 3. Absolute recoveries and relative standard deviations for spiked urban surface water (site A) and urban river water (site B), pH 7, filter wash, n = 3 for all concentration levels, with the exception of NIG 200 ng at site A (n = 9). Lasalocid
Monensin
Salinomycin
Narasin
Nigericin
Abs rec ±RSD Abs rec ±RSD Abs rec ±RSD Abs rec ±RSD Abs rec ±RSD [%] [%] [%] [%] [%] [%] [%] [%] [%] [%]
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50 ng (site A) 200 ng (site A) 500 ng (site A) 200 ng (site B) 200 ng (site B)a
108 99 107 85 92
9 14 6 9 2
129 100 132 95 103
11 2 6 1 7
113 97 123 101 110
15 19 7 10 13
95 92 97 90 99
3 19 6 6 14
95
6
93
4
Notes: aThe absolute recoveries evaluated using values corrected by the IS.
dilution. Furthermore, the samples were not collected immediately after the chicken litter was spread at the agricultural fields, meaning that the ionophores may have been washed out. The ionophores might also have been degraded into degradation products that are not currently included in the presented methodology. Another issue could be that the sampling was not representative for the actual distribution in the surface waters. More samples will be taken to reveal the possible occurrence of ionophores in Danish environmental water bodies. It should be stressed that the LOQs of the presented methods are below the surface water PEC (0.5–2.7 µg/L) and effect concentrations in the literature i.e. this method enables quantification of the ionophores at levels expected to be found in environmental matrices as well as relevant effect levels [35]. 4. Conclusions The described SPE LC-MS/MS method, using internal standard calibration, allows for the determination of four ionophores in aqueous matrices down to the lower ng per litre range. The method has shown to be reliable, robust and accurate for the determination of the ionophores and NIG was demonstrated to be a suitable IS for the four ionophores. The recoveries of the ionophore in river surface water were 92 ± 2%, 103 ± 7%, 110 ± 13%, 99 ± 14% and 93 ± 4%, for LAS, MON, SAL, NAR and NIG (IS) respectively. No matrix effect was seen with spiked surface waters. The measured ionophores concentrations in Danish environmental water bodies are below known effect concentration, but more studies are underway to reveal possible contamination at levels exceeding effect concentrations. Acknowledgments This project was supported by The Danish Medicines Agency and The Drug Research Academy (University of Copenhagen).
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