Anal Bioanal Chem DOI 10.1007/s00216-014-7921-6
RESEARCH PAPER
Determination of sulfadiazine in phosphate- and DOC-rich agricultural drainage water using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry P. A. Léon Bouyou & Johan J. Weisser & Bjarne W. Strobel
Received: 7 March 2014 / Revised: 12 May 2014 / Accepted: 22 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Trace levels of the veterinary antibiotic compound sulfadiazine (SDZ) can be determined in agricultural drainage water samples with this new method. Optimized sample pretreatment and solid-phase extraction was combined with liquid chromatography coupled to tandem mass spectrometry (SPE LC-MS/MS) using positive electrospray ionization. The linear dynamic range for the LC-MS/MS was assessed from 5 μg/L to 25 mg/L with a 15-point calibration curve displaying a coefficient of correlation r2 =0.9915. Agricultural drainage water spiked at a concentration of 25 ng/L gave recoveries between 63 and 98 % (relative standard deviation 15 %), while at 10 ng/L, it showed a lower recovery of 32 % (relative standard deviation 47 %). The final SPE LC-MS/MS method had a limit of detection (LOD)Method and a limit of quantification (LOQ)Method of 7.5 and 23 ng/L agricultural drainage water, respectively. Determination of SDZ, spiked at a realistic concentration of 50 μg/L, in artificial drainage water (ADW) containing common and high levels of phosphate (0.05, 0.5, and 5 mg/L) gave recoveries between 70 and 92 % (relative standard deviation 7.4–12.9 %). Analysis of the same realistic concentration of SDZ in ADW, spiked with common and high levels of dissolved organic carbon (2, 6, and 15 mg/L) confirmed the possible adaptation of a tandem solid-phase extraction (strong anion exchange (SAX)-hydrophilic-lipophilic balance (HLB)) followed by liquid chromatography-tandem mass spectrometry methodology. P. A. L. Bouyou (*) : B. W. Strobel Section of Environmental Chemistry and Physics, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark e-mail: [email protected] J. J. Weisser Section of Analytical Biosciences, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
Recoveries obtained ranged from 104 to 109 % (relative standard deviation 2.8–5.2 %). The new methods enable determination of the veterinary antibiotic compound SDZ in agricultural drainage water from field experiments and monitoring schemes for phosphate- and dissolved organic carbon (DOC)-rich water samples in intensive farming areas. Keywords SDZ . LC-MS/MS . SPE . Veterinary antibiotic . Sulfonamide
Introduction Intensive farming, designed to maximize production efficiency and thereby resulting in a great concentration of animals in a limited area, implies considerable use of physiologically highly active drug substances for combating parasites, preventing and treating bacterially transmitted diseases, and accelerating meat production. In Denmark, for example, largescale pig productions have become one of the main farming activities, and therefore one of the main environmental concerns. If 60,000 farmers were producing 13 million odd pigs 20 years ago, nowadays some 13,000 farmers produce nearly twice as many. The similar pattern observed all over Europe could then partially explain the dramatic increase in veterinary antibiotic concentrations measured in streams near these areas [1–3]. As most known bioactive substances, sulfonamide antibiotics are not completely eliminated in animal organisms as they are administered in high doses for fast and strong impact and excreted shortly after absorption [4]. Sulfonamides have previously been detected at concentrations up to 500 mg/L in pig slurry [5], 20 mg/kg in pig manure [6], and 0.5 mg/kg in some agricultural soils [7]. Large-scale use of veterinary antibiotics is, however, not only common in Europe [8], but also in the USA [9] and, to the best of our knowledge, in China, Southeast Asia, and Russia.
P.A.L. Bouyou et al.
Sulfadiazine (N1-[2-pyrimidinyl]-sulfanilamide), a fairly hydrophobic sulfonamide antibiotic, was used in the present study as a model compound for the analysis of sulfonamides in agricultural drainage waters. Its physicochemical properties can be found in Table 1. Sulfadiazine (SDZ) was previously found in animal wastes in its active parent form [12, 13] together with the corresponding N4-acetyl-sulfadiazine metabolite and alongside low levels of the 4-hydroxylsulfadiazine metabolite [14]. In storage tanks and in manured soils, however, the metabolites are converted back to the parent SDZ [15, 16], and biodegradation and photodegradation of parent sulfonamides were shown to be of minor importance in such systems [7, 11, 17]. For centuries, animal wastes (manures, slurries, etc.) have been known to be valuable by-products used as a source of organic fertilizers in livestock farming for their high content in organic carbon and nutrients, both essential for plant growth. Manure, which contains high levels of ammonia, increases the soil water pH, which favors the formation of the more mobile anionic SDZ species [18] by means of repulsive forces excluding it from soil pores. The same mobility can be expected in the case of grazing livestock, since the presence of urine and feces increases the soil solution ionic strength resulting in increased competitions for soil sorption sites. As previously described by Halling-Sørensen and coworkers [19], SDZ may then be susceptible to enter the drainage pipe system and ultimately the connected streams through surface runoff and soil infiltration initiated by animal wastes spreading or by grazing livestock. Sulfonamides have been found in groundwater below and surface waters close to intensive agricultural areas in concentrations ranging from 0.01 μg/L to 3.3 mg/L [1–3, 20, 21]. Another study carried out by Thiele-Bruhn and Aust [22] brought evidences of a concomitant increase in dissolved organic matter (DOM) and a decrease in sulfonamides’ sorption to soil. Even though the adsorption of sulfonamides to DOM was shown to be strongly dominated by hydrophobic interactions, Bialk and coworkers [23] described the ability of the anilinic nitrogen of the SDZ structure to covalently cross couple through nucleophilic addition with the OM’s polar regions (phenolic and carboxylic groups, keto-, enol-, and alcoholic functions, lipids, and lignin dimers) [17, 24], which consequently enhances the stability of the sulfonamide-DOM
products. As the recycling of animal wastes increases the soil water pH, the amount of DOM, and the soil water ionic strength, an enhancement of the colloidal transport of SDZ was raised as a reasonable explanation for its mobilization from the top soil to the drainage pipes. Preferential pathways, including macropores, cracks, and fissures also exist in soils [25, 26]. These are characterized by less pronounced adsorption, sedimentation, and sieving processes. Thus, in preferential pathways, SDZ-DOM products may become highly mobile and rapidly transported to field drains and ultimately enter the nearby surface waters. This leaching risk is especially enlarged if the application of animal wastes is followed by heavy precipitation events [2, 27] or if the soil is subject to rapid transport [18]. Solid-phase extraction (SPE) is a common method for extracting analytes from aqueous matrices and has become one of the preferred techniques over the classical liquid-liquid extractions (LLE) [28]. SPE has been successfully applied with SDZ and other sulfonamides using the Oasis® hydrophilic-lipophilic balance (HLB) cartridges, both offline [21, 29, 30] and online [1, 31], with higher recoveries than other SPE sorbents, such as the Oasis® MCX cartridge [32]. Some other studies have introduced the use of a tandem SPE procedure, combining the Agilent strong anion exchange (SAX) and the Oasis® HLB cartridges [20, 25, 30]. These cartridges are placed in tandem to simultaneously remove negatively charged humic material (SAX) and to retain the antibiotic agents (HLB) subsequently eluted in a selective fashion. All in all, these combined steps lower the amount of interfering matrix components. Like most of the sulfonamides, SDZ is highly polar, water soluble and labile, nonvolatile, and with a relatively high molecular weight, and therefore not readily analyzed by GCmass spectrometry (MS). It has been shown to efficiently ionize under positive electrospray conditions (ESI+). Liquid chromatography (LC)-ESI-MS [6, 21] or LC-ESI-tandem mass spectrometry (MS/MS) [20, 29, 32] have therefore been shown to be one of the best choices for separation and analysis of SDZ at trace concentrations in complex matrices. It is well known that matrix interferences are ubiquitous during LC-ESI-MS analysis due to ionization competitions. Nonetheless, matrix effects can be compensated for in various ways: by using appropriate
Table 1 Physicochemical properties of sulfadiazine Compound
Sulfadiazine (SDZ)
Structure
Composition
Mw (g/mol)
pKa
Solubility (mg/L)
Log Kow
Kd (L/kg)
C10H10N4O2S
Average mass, 250.27 mono-isotopic mass, 250.05
pKa1 =1.6 pKa2 =6.4 [10]
77
−0.09 [10]
2.0 [11]
Determination of sulfadiazine in drainage waters
internal standards or matrix matched calibration, alone or combined, or by doing standard addition experiments. Here, LCMS/MS was selected as a platform due to its increased versatility and selectivity, and presumed sensitivity as compared to LC-DAD, LC-FLD, and LC-MS [28]. To the best of our knowledge, no method has previously been published where SDZ was analyzed in agricultural drainage water samples. The aim of this study was to develop a new analytical method using SPE LC-MS/MS for its determination at trace levels in agricultural drainage water. Application of the method for phosphate- and dissolved organic carbon (DOC)-loaded samples proved that the analysis of sulfonamide antibiotics could be carried out in complex samples using the adapted tandem SPE (SAX-HLB) method combined with LC-MS/MS.
Experimental Reagents and standards SDZ (>99 %, Chemical Abstracts Service (CAS) no. 68-35-9) was purchased from Sigma-Aldrich (Schnelldorf, Germany), and the internal standard (I.S.), sulfadiazine-d4 (SDZ-d4, CAS no. 1020719-78-1), was from C/D/N Isotopes Inc. (PointeClaire, QC, Canada). Stock solution of 1,000 mg/L SDZ was prepared in methanol and stored at −20 °C for a maximum of 6 months. Working standard solutions were prepared as 128 mg/L SDZ-d4 solutions in methanol and were also stored at −20 °C. Methanol was of high-performance liquid chromatography (HPLC) grade from Lab-Scan (Dublin, Ireland). Formic acid (∼98 %), sodium hydroxide pellets, and potassium chloride were purchased from Merck KGaA (Darmstadt, Germany), ethanol (96 %) was obtained from Kemetyl A/S (Køge, Denmark), and citric acid monohydrate (99.8 %) was from Sigma-Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate (99.5 %), sodium chloride, calcium chloride dihydrate, and magnesium chloride hexahydrate were from VWR Chemicals (Leuven, Belgium). Ottawa sand standard, of general-purpose grade (20–30 mesh), was from AppliChem GmbH (Darmstadt, Germany); nitric acid (69.0– 70.0 %) was purchased from J. T. Baker (Deventer, The Netherlands), and pure water was produced in-house with a Milli-Q gradient system (Millipore, Bedford, MA, USA). The 615-mg/L DOC stock solution originated from an isolation of a Norwegian spruce forest floor, as previously described by Strobel and coworkers [33]. Drainage water samples The drainage water used for spiking during the method development of the solid-phase extraction was sampled at a tile drain site in Rodstenseje, Odder, Denmark (55° 57′ 40.71″ N
and 10° 9′ 46.92″ E). The water was analyzed, and no background SDZ was detected. Preparation of the artificial agricultural drainage water The agricultural drainage water (ADW) was used for spiking during the method development of the solid-phase extraction with phosphate- and DOC-rich matrices. The composition of the ADW was based on sampling campaigns carried out in Denmark over the last years and was prepared in deionized water with 0.652 mM sodium chloride, 0.0254 mM potassium chloride, 1.84 mM calcium chloride dihydrate, and of 0.255 mM magnesium chloride hexahydrate. Properties of the ADW and of the agricultural drainage water collected at the Rodstenseje site can be found in Table 2. Characterization of water samples The pH of the water samples was determined on the day of collection with a 6.0228.000 pH meter from Methrom (Herisan, Switzerland). Concentrations of Cl − , NO 3 − , H2PO4−, and SO42− were determined by ion chromatography (Methrom 833 IC Handling Unit, 818 IC Pump, 820 IC Separation Centre, 819 IC Detector, Metrosep A Supp 5 column, Herisan, Switzerland) after the samples have been pre-filtered through 0.45-μm regenerated cellulose (RC) filters from La-Pha-Pack® GmbH (Langerwehe, Germany). Total organic carbon (TOC) and DOC were determined by a Shimadzu TIC-500 Total Organic Carbon Analyzer (Kyoto, Japan) after filtration with 0.45-μm nylon (PA) filters from La-Pha-Pack® GmbH (Langerwehe, Germany). Concentrations of cations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent Technologies Inc., Agilent 8800, Palo Alto, CA, USA). Polyatomic interferences were removed with an octopole reaction system (ORS) pressurized with He or H2. External calibration was applied and drift corrected by periodic recalibrations. All water samples were acidified with nitric acid (70 %) to a volumetric amount of 0.57 % prior to ICP-MS analyses. For all samples, repeated analyses showed acceptable uncertainties. Liquid chromatography-tandem mass spectrometry analysis The liquid chromatographic system consisted of a 1100 Series HPLC instrument (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a degasser, a quaternary pump, a cooled autosampler, and a column oven. The mass spectrometer was a PE-Sciex API 2000 triple quadrupole mass spectrometer from Applied Biosystems MDS Sciex Instruments (Foster City, CA, USA) equipped with an ESI interface. The injection volume was 10 μL. Separation was performed on an XTerra RP18 column (100 mm×2.1 mm, 3.5 μm) and a guard column
P.A.L. Bouyou et al. Table 2 Selected properties of the agricultural drainage water samples from the Rodstenseje site in Odder (Denmark) and the artificial drainage water (ADW). All analyses were performed in triplicate Parameter
pH Temperature, °C Total organic carbon, mg/L Dissolved organic carbon, mg/L Phosphate, mg/L Calcium, mg/L Sodium, mg/L Magnesium, mg/L Potassium, mg/L Iron, μg/L Aluminium, μg/L Manganese, μg/L Zinc, μg/L Copper, μg/L Chloride, mmol/L Nitrate, μmol/L Sulfate, μmol/L a
Agricultural drainage water (Odder, Denmark)a
Artificial drainage watera
6.53±0.01 5.5±0.1 2.29±0.19
6.91±0.01 5.2±0.1 0.49±0.02
0.56±0.03
0.41±0.09
0.405±0.005 121±0.8 21.5±0.6 7.69±0.18 6.30±0.22 268±8 146±6 19.8±0.3 13.9±0.3 2.53±0.14 0.752±0.001 478±2 319±0.8
– 105±0.6 21.8±0.8 13.8±0.2 1.59±0.12 143±3 33.9±2.5 1.64±0.16 1.10±0.21 0.37±0.09 1.97±0.03 15±0.2 22.3±0.5
Average value±standard deviation
XTerra RP18 column (20 mm×2.1 mm, 3.5 μm) from Waters (Milford, MA, USA) applying a binary gradient flow rate of 0.2 mL/min at 25 °C. The mobile phase A consisted of 15 % methanol and 0.1 % formic acid in Milli-Q water (v/v), and the mobile phase B, of 95 % methanol and 0.1 % formic acid in Milli-Q water (v/v). The initial mobile-phase blend was 98 % A and 2 % B with 5 min of gradient ending with a composition of 20 % A and 80 % B. This composition returned to the starting conditions within 0.5 min for a period of 9.5 min, giving a total analysis time of 15 min. The retention times for SDZ and SDZ-d4, and the optimized parameters for the LCMS/MS analysis are summarized in Table 3. Electrospray ionization was performed in positive mode (ESI+). The desolvation temperature was set to 450 °C with a nebulizer gas pressure of 32 psi, while the curtain and collision gas
pressures were 14 and 6 psi, respectively. The ion spray voltage was 5,500 V. The multiple reaction monitoring function (MRM) was applied in all analyses. Solid-phase extraction The method development and final extraction from agricultural drainage water samples were performed on Oasis® HLB 6-cm3 200-mg (30 μm) cartridges from Waters (Milford, MA, USA). The evaporator and the IST Vacmaster SPE rack were from Macherey-Nagel GmbH (Düren, Germany). The pump was a VacSafe 15 from Labogene ApS (Lynge, Denmark). Nitrogen (99.8 %) was supplied by Air Liquid (Ballerup, Denmark). A summary of the method is presented in Fig. 1. Most veterinary antibiotics are acidic substances; thus, the acidification to pH 3 in the water samples is required in order to obtain SDZ’s neutral form (cf. pKa values in Table 1). As already shown by Zhou and coworkers [20], working at pH 3 allows for the maximum specific retention of SDZ within the polymeric Oasis® HLB columns, whereas the still negatively charged OM usually present in agricultural drainage water samples can be flushed through or retained, for example, by a SAX cartridge. In the first part of the method development, the Oasis® HLB cartridges were conditioned with 5 mL ethanol followed by 3×1 mL methanol and finally 5 mL citric acid (0.04 M). The acidified (pH 3) water samples were loaded at a rate of 3 mL/min. Initially, the optimal temperature for evaporation of the methanol eluates under a gentle stream of nitrogen was tested between 20 and 60 °C by spiking 2 mL methanol to a realistic concentration of 50 μg/mL SDZ. The washing step of the SPE cartridges, using Milli-Q water, was also investigated for possible loss. For this matter, 100 mL of water pH adjusted to 3 spiked with 50 μg/L SDZ was added to the Oasis® HLB cartridges. After the loading, portions of 1 mL of Milli-Q water were added and analyzed individually. In order to assess the volume of methanol needed to quantitatively elute SDZ from the Oasis® HLB cartridges, 100 mL of spiked Milli-Q water samples containing 50 μg/L SDZ was loaded onto the cartridges and eluted with methanol in small portions (500 μL). Each portion was analyzed separately to obtain elution profiles from
Table 3 Specific settings in the final analytical LC-MS/MS method, applying ESI in positive mode, together with the most intense precursor and product ions for sulfadiazine and its corresponding I.S. Compound
Retention time (min)†
Precursor ion (m/z)
Product ions quantify/qualify (m/z)
SDZ
4.39
251.1
156.1/108.0
SDZ-d4
4.32
255.1
160.1/112.0
Ionization mode
Dwell time (ms)
Declustering potential (V)
Focusing potential (V)
Entrance potential (V)
Collision energy (eV)
Collision cell exit potential (V)
Positive
500
14.0
390.0
10.0
24.0
2.0
Determination of sulfadiazine in drainage waters
Sample preparation 1000 mL water sample is adjusted to pH 3 with formic acid and spiked with 100 µL I.S. (sulfadiazine-d 4) SPE cartridge conditioning 5 mL ethanol 3x1 mL methanol 5 mL citric acid (0.04 M) Low DOC content (< 2 mg/L)
High DOC content (> 2 mg/L)
Sample load Single cartridge SPE setup - Oasis® HLB 6 cm3 200 mg (30 µm)
Sample load Tandem SPE setup Agilent LRC-SAX 1 cm3 100 mg and Oasis® HLB 6 cm3 200 mg (30 µm)
Wash 5 mL Milli-Q water to remove excess acid and wash sorbent Elution 2 mL methanol to elute extracted sulfadiazine Evaporation To dryness under gentle nitrogen stream and re-constitution in 1 mL (1:1, v/v) methanol/Milli-Q water Analysis LC-MS/MS Fig. 1 The final SPE method using either Oasis® HLB 6-cm3 200-mg (30 μm) cartridges or a combination of Agilent Bond Elut LRC-SAX 1cm3 100-mg and Oasis® HLB 6-cm3 200-mg (30 μm) cartridges for 1,000-mL water samples
0.5 to 5 mL. The Oasis® HLB cartridges were also evaluated for SDZ breakthrough at three different sample volumes: 200, 500, and 1,000 mL. Before extraction, the samples were adjusted to pH 3 and spiked with SDZ at a total mass of 50 μg giving concentrations of 250, 100, and 50 μg/L for the 200-, 500-, and 1,000-mL samples, respectively. Two Oasis® HLB cartridges were set up in tandem in order to assess breakthrough. In the second part of the method development, a tandem SPE procedure was developed in ADW containing common and high concentrations of phosphate and DOC. Agilent Bond Elut LRC-SAX 1-cm3 100-mg cartridges were chosen and conditioned in the same way as the Oasis® HLB cartridges and placed on top of the latter for the adapted tandem SPE (SAX-HLB) method (Fig. 1). Both cartridges were eluted with 2 mL methanol, and the eluates were tested for SDZ content.
Filtration and pressurized liquid extraction The filtration of natural drainage water samples was performed on Whatman™ GF/C glass microfiber filters ( 1 . 2 μm ) from GE Healthcare (Little Ch a l f o n t , Buckinghamshire, UK), and the experiments were performed in triplicate. The samples were adjusted to pH 3 using formic acid, and 100 μL working solution of I.S. (SDZ-d4) was added prior to filtration. In order to assess the SDZ loss during the filtration process, the filters were analyzed by pressurized liquid extraction (PLE), following the procedure developed by Jacobsen and coworkers [30]. For this purpose, the filters were frozen at −20 °C for at least 24 h before being freezedried overnight in a Type FD 3 freeze-drier from Heto-Holten A/S (Allerød, Denmark) operating at −48 °C and 10−5 bar. The next step involved SDZ extraction from the freeze-dried filters by PLE, using an ASE 200 system from Dionex (Sunnyvale, CA, USA). The system was operated with 33 mL of pressure-resistant steel extraction cells and lined with cellulose filters from Dionex (Sunnyvale, CA, USA). Initially, 200-mL samples of agricultural drainage water from the Rodstenseje site with pH adjusted to 3 were spiked to a realistic concentration of 50 μg/L SDZ. Conditioning of the filters included soaking in Milli-Q water and thereafter in methanol before resting in Milli-Q water prior to usage. In order to assess the SDZ loss during this step, 100 μL SDZ-d4 (I.S.) was added to the filtrates and the filters, and SPE LCMS/MS and PLE combined with SPE LC-MS/MS procedures were carried out, respectively. Moreover, the filters were weighted to account for their content in sequestered matter. Practically, the used Whatman™ filters were cut in small pieces and mixed with 10-g Ottawa sand and 100 μL of SDZ-d4 at a concentration of 128 mg/L. The extraction buffer consisted of a 1:1 (v/v) mixture of methanol and 0.2 M citric acid buffer with pH adjusted to 4.7 with sodium hydroxide. A first static extraction was operated with approximately 30 mL of extraction buffer at 1,500 psi for 10 min, followed by flushing the extract into a collection vial. Then, a second static extraction was operated with an additional 30 mL of extraction buffer for 3 min and flushed into the same collection vial. The total final volume of the extract was approximately 60 mL, and the extractions were performed at room temperature. In each of the static extraction cycle, 60 % of flush volume and 120-s purge period were applied. Between each run, the extraction cells were cleaned by ultra-sonication for 15 min in a mixture of Milli-Q water/methanol (1:1, v/v), followed by 15 min of ultra-sonication in Milli-Q water. Method validation using standard solutions The linearity, limit of detection (LODInstr) and limit of quantification (LOQInstr) of the LC-MS/MS system were evaluated in an initial step by injecting pure standards into the LC-MS/
P.A.L. Bouyou et al.
MS system. Linearity was investigated using a 15-point calibration curve determined in the range 5 μg/L–25 mg/L. It was set as a criterion that peak area ratios between the qualifier and the quantifier ion transitions should not deviate more than 20 % from a fixed ratio [34]. Intra- (n=27) and inter-day (n=27) instrument precision were determined from relative standard deviations (RSDs) of the peak areas obtained for a triplicate injection during the calibration curve determination. For the LC-MS/MS instrument, the LODInstr and LOQInstr were calculated as recommended by the ICH guideline [35] following the equations LODInstr ¼ 3:3 σS and LOQInstr ¼ 10 σS . Here, σ is the standard deviation of the peak area obtained when analyzing in triplicate the least concentrated SDZ sample during the determination of the calibration curve, and S is the slope of the calibration curve.
Results and discussion Method development Optimization of the LC-MS/MS ionization efficiency of sulfadiazine in an agricultural drainage water matrix The development of an efficient LC separation with an appropriate ionization method coupled to MS/MS was necessary prior to the development of the SPE method. For SDZ, the most intense precursor ion was the [M+ H]+ ion at m/z 251.1. A common fragmentation pattern has already been observed for the sulfonamide family [36], where amino sulfonamides such as SDZ typically produce product ions at m/z 156.1, 108.0, and 92.0 (Fig. 2). The fragmentation of SDZ resulted in a loss of 95 Da corresponding to the cleavage of the sulfonamide bond [36], leading to m/z 156.1. Further loss of SO2 leads to the ion at m/z 92.0, while it is known that m/z 108.0 is formed via a rearrangement [37]. The resulting precursor and product ions are listed in Table 3. The most prominent MRM transition (m/z 156.1) was used for quantification, and the other one (m/z 108.0) was used as a qualifier ion. The corresponding transitions for the SDZ-d4 analog were m/z 255.1→ 160.0→112.0 (Table 3). The overall variation of the quantifier/qualifier ion-ratio stability was investigated in the range 5 μg/L–100 mg/L and showed an overall good consistency with a mean value of 1.35±0.02 (RSD 1.70 % (n = 51)). Nonetheless, the quantifier/qualifier ion-ratios decreased with increasing SDZ concentrations with a logarithmic regression, displaying a coefficient of regression r2 =0.8693 (Fig. 3, Eq. 1). R ¼ −0:0051:log10 ½SDZ þ 1:3497
ð1Þ
Here, [SDZ] represents the concentration of SDZ, and R is the ratio of the quantifier/qualifier peak areas. In other words, Að251:1 156:1Þ represents the ratio of the peak , where A 251:1 R ¼ A 251:1 156:1 ð108:0Þ areas of the precursor ion and of the quantifier product ion, and A 251:1 108:0 is the ratio of the peak areas of the precursor ion and of the qualifier product ion (cf. Table 3). An advantage from using quantifier/qualifier ion ratios would be achieved in the case of impossible quantification using the quantifier ion, due to partly overlapping chromatographic peaks, in the case of matrix interferences. It may then be possible from the peak height ratio of the qualifier/quantifier ions to determine whether or not it corresponds to the correct peak. If this is the case, things can be inverted, and then the qualifier ion can be used for quantification. By using this strategy, the number of samples that cannot be quantified directly can be reduced to close to zero. However, in the rare cases where the quantifier ion peak is totally covered by an interfering peak, it has to be noted that quantification with the qualifier ion implies a loss of specificity, as only one specific ion is involved for identification. At this stage, a complete LC-MS/MS methodology was established and used in all further SPE optimizations. Optimizations were performed by continuously infusing SDZ at a concentration of 20 mg/L in methanol/Milli-Q water (1:1, v/v).
Optimization of the solid-phase extraction Solid-phase extractions using Oasis® HLB 6-cm3 200-mg (30 μm) cartridges have been known to efficiently extract SDZ from aqueous matrices and have previously been successfully applied for wastewater [2, 29], groundwater, and surface water [1, 21] and also for coastal water [32]. Effects of the evaporation, washing, and elution steps on recoveries Before developing the final method for the Oasis® HLB cartridges, an optimization of the three steps preceding the final analysis on LC-MS/MS was required (Fig. 1). The effect of evaporation temperature on the recoveries of SDZ is shown in Fig. 4a. No significant loss was observed up to a temperature of 40 °C, which was the temperature chosen for application in the final method (Fig. 1), and this supports results described by Ye and coworkers [29]. The washing step involved the use of Milli-Q water in order to remove acidic components prior to elution with methanol. No loss of SDZ occurred up to 8 mL (data not shown). The final washing volume was set to 5 mL which corresponds to flushing the sorbent pore volume approximately 19 times (Fig. 1). This result lays within the average volume of MilliQ water previously used for cleaning up SPE cartridges [21, 29, 32]. The elution volume of methanol is a crucial step
Determination of sulfadiazine in drainage waters
Fig. 2 Fragment ions used as quantifiers and qualifiers in tandem mass spectrometry for sulfadiazine according to literature references [36, 37]
to assure a quantitative elution of SDZ trapped into the SPE cartridges. It can be seen that after applying 1.5 mL of solvent, SDZ is quantitatively removed from the cartridge (Fig. 4b). A confirmatory experiment using 2 mL of methanol without a stepwise addition confirmed the quantitative recovery of SDZ from the SPE cartridge. The final elution volume was then set to 2 mL which corresponds to flushing the sorbent pore volume approximately eight times (Fig. 1). The optimization of the methanol elution volume minimized the use of organic solvent compared to
previous methods described by Ye and coworkers [29], Lindsey and coworkers [21], and Na and coworkers [32], who respectively used 3, 5, and 6 mL. Effect of sample size on recoveries In order to be able to reach a low LODMethod assuring a better quality in the assessment of agricultural drainage water contaminations, increasing the sample volume can have an important impact. However, there are some limitations regarding the handling of large volumes; increasing the
1.42 R = -0.0051.log10 [SDZ] + 1.3497 r2 = 0.8693
A(251.1/156.1) / A(251.1/108.0)
1.40
1.38
1.36
1.34
1.32
1.30 0.01
0.1
1
10
100
Sulfadiazine concentration (mg/L)
Fig. 3 Quantifier/qualifier ion-ratio stability assessment during the calibration curve determination for the LC-MS/MS method in the range of 5 μg/L–100 mg/L. The sulfadiazine concentrations are plotted on a logarithmic scale, and the coefficient of regression was assessed by a
logarithmic regression analysis which showed a coefficient r2 =0.8693. The mean value of the quantifier/qualifier ion-ratios was 1.35, and its RSD value was 1.70 % (n=51). Each data point represents a triplicate sample, and error bars represent one standard deviation
P.A.L. Bouyou et al.
b
140
140
120
120
100
100 Recovery (%)
Recovery (%)
a
80
60
80
60
40
40
20
20
0
0 30
40
50
60
70
0
1
2
3
4
5
Methanol (mL)
Temperature (°C)
Fig. 4 a Effect of evaporation temperature on the recovery of sulfadiazine. Methanol was spiked at a concentration of 50 μg/mL and evaporated under a gentle stream of nitrogen prior to reconstitution in 1 mL 1:1 (v/v) mixture of methanol/Milli-Q water. Each experiment was performed in triplicate, and error bars represent one standard deviation.
b Evaluation of the elution volume of methanol required to elute the compounds applied to the Oasis® HLB 6-cm3 200-mg (30 μm) cartridges. The cartridges were loaded with 100-mL spiked Milli-Q water (pH 3) at a concentration level of 50 μg/L of sulfadiazine. Each data point represents a triplicate sample, and error bars represent one standard deviation
sample volume could indeed give rise to breakthrough of SDZ due to a saturation of the SPE sorbent. The data indicated no breakthrough of SDZ for any of the selected volumes (200, 500, and 1,000 mL). Based on these findings, 1,000 mL was applied as a suitable sample volume in the final method (Fig. 1). On the one hand, the use of 1,000 mL samples showed an important improvement compared to previous results using working volumes ranging from 100- to 500-mL samples [21, 29, 32]. In this regard, the ability to handle larger sample volumes provides the reach of a lower absolute LODMethod. However, results assessing the content in particles bearing a size fraction >1.2 μm in the Rodstenseje agricultural drainage water were found to be rather low for this type of water samples (0.45± 0.25 mg). This is probably for such reason that neither breakthrough of SDZ nor clogging of the SPE cartridges were observed up to a sample volume of 1,000 mL. In the first part of the method development, the use of Oasis® HLB cartridges to retain and elute SDZ was optimized. The 1,000-mL water samples were acidified to pH 3, and thereafter loaded at a rate of 3 mL/min onto the Oasis® HLB cartridges. Conditioned with 5 mL ethanol followed by 3×1 mL methanol and finally 5 mL citric acid (0.04 M), the SPE cartridges were washed with 5 mL Milli-Q water before SDZ was eluted with 2 mL methanol. The eluates were evaporated to dryness (evaporation was stopped immediately after the samples became dry) under a gentle stream of nitrogen at 40 °C and redissolved in a final volume of 1 mL methanol/
Milli-Q water (1:1, v/v), prior to LC-MS/MS determination. A summary of the method is presented in Fig. 1. Method validation LC-MS/MS validation The linearity of the 15-point linear calibration curve in the range of 5 μg/L–25 mg/L gave a correlation coefficient r2 of 0.9915, and the obtained LODInstr and LOQInstr values were 0.247 and 0.823 ng/mL, respectively. Therefore, the absolute amount required in the LC-MS/MS system to reach the LOQ of SDZ was 8.23 pg. Monitoring the intra- and inter-day precisions during the LC-MS/MS validation showed very stable results, with values of 1.70 %±1.25 (n= 27) and 2.12 %±1.96 (n=27), respectively. Continuous monitoring of the I.S. during the LC-MS/MS validation showed very stable peak areas. For the I.S., the average area was 5.48·104, and the relative standard deviation was 6 % (n=54). SPE LC-MS/MS method validation When analyzing spiked agricultural drainage water, SDZ-d4 was added before the analysis, and the quantification was based on the ratio (SDZ/SDZ-d4). The I.S. concentration was kept constant at 12.8 mg/L. Validation of the complete SPE LC-MS/MS method was done by spiking agricultural drainage water samples from the Rodstenseje site adjusted to pH 3. Recoveries were determined on six concentrations
Determination of sulfadiazine in drainage waters
based on preliminary LOQs of the entire method for spiked agricultural drainage water. The spiking levels were 1, 5, 10, 25, 50, and 100 ng/L. Calibration was performed as an internal calibration in methanol/Milli-Q water (1:1, v/v). Recoveries from the spiking of agricultural drainage water at concentrations close to the limit of what can be quantified, compensated by SDZ-d4, is given in Table 4. SDZ could be detected down to 10 ng/L, though displaying a significant decrease in recovery when compared to the other concentrations tested. Results also showed that at a concentration of 5 ng/L SDZ could be detected, though not quantified, while at a concentration of 1 ng/L (2,500 times below the threshold), it was not detectable. Results demonstrate a good accuracy with recoveries above 80 % for spiking levels above 25 ng/L. These recovery levels are in accordance with other recovery studies which assessed the successful extraction of SDZ from a wide range of environmental samples, i.e., from surface [20], coastal [32], and groundwater [1], but also from wastewater [29], and from soil [30] and animal manure [6] extracts. In these studies, recoveries ranged from 51 to 96 %. Consequently, the present method demonstrates its ability to extract SDZ to a high extent from agricultural drainage water samples. The data also showed acceptable precision of the method since RSDs are near 20 % or below. Based on these data, the LODMethod and LOQMethod were evaluated. The LODMethod and LOQMethod were evaluated with a signal to noise ratio of 3.3 and 10, respectively, using the peak area of the extract from agricultural drainage water spiked with the lowest concentration of SDZ and injected six times. The concentration was 10 ng/L, and the obtained LODMethod value was 7.48 ng/L. The corresponding LOQ Method value was assessed to be 22.7 ng/L. Therefore, the absolute amount required in the SPE LC-MS/MS method to reach the LOQ of SDZ was 0.23 pg. This is especially important for SDZ because it is known to occur and has previously been detected in groundwater below and surface waters close to intensive agricultural areas [1–3, 20, 21]. These corresponding levels of detection and quantification are in the same order of magnitude (low to medium ng/L range) as other studies focusing on surface, coastal, and
Table 4 Effects of sample concentration on absolute recoveries of sulfadiazine spiked to final concentrations ranging from 1 to 100 ng/L in 1-L agricultural drainage water with pH adjusted to 3. Each experiment was 1 ng/L Recovery (%) SDZ
RESEARCH PAPER
Determination of sulfadiazine in phosphate- and DOC-rich agricultural drainage water using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry P. A. Léon Bouyou & Johan J. Weisser & Bjarne W. Strobel
Received: 7 March 2014 / Revised: 12 May 2014 / Accepted: 22 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Trace levels of the veterinary antibiotic compound sulfadiazine (SDZ) can be determined in agricultural drainage water samples with this new method. Optimized sample pretreatment and solid-phase extraction was combined with liquid chromatography coupled to tandem mass spectrometry (SPE LC-MS/MS) using positive electrospray ionization. The linear dynamic range for the LC-MS/MS was assessed from 5 μg/L to 25 mg/L with a 15-point calibration curve displaying a coefficient of correlation r2 =0.9915. Agricultural drainage water spiked at a concentration of 25 ng/L gave recoveries between 63 and 98 % (relative standard deviation 15 %), while at 10 ng/L, it showed a lower recovery of 32 % (relative standard deviation 47 %). The final SPE LC-MS/MS method had a limit of detection (LOD)Method and a limit of quantification (LOQ)Method of 7.5 and 23 ng/L agricultural drainage water, respectively. Determination of SDZ, spiked at a realistic concentration of 50 μg/L, in artificial drainage water (ADW) containing common and high levels of phosphate (0.05, 0.5, and 5 mg/L) gave recoveries between 70 and 92 % (relative standard deviation 7.4–12.9 %). Analysis of the same realistic concentration of SDZ in ADW, spiked with common and high levels of dissolved organic carbon (2, 6, and 15 mg/L) confirmed the possible adaptation of a tandem solid-phase extraction (strong anion exchange (SAX)-hydrophilic-lipophilic balance (HLB)) followed by liquid chromatography-tandem mass spectrometry methodology. P. A. L. Bouyou (*) : B. W. Strobel Section of Environmental Chemistry and Physics, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark e-mail: [email protected] J. J. Weisser Section of Analytical Biosciences, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
Recoveries obtained ranged from 104 to 109 % (relative standard deviation 2.8–5.2 %). The new methods enable determination of the veterinary antibiotic compound SDZ in agricultural drainage water from field experiments and monitoring schemes for phosphate- and dissolved organic carbon (DOC)-rich water samples in intensive farming areas. Keywords SDZ . LC-MS/MS . SPE . Veterinary antibiotic . Sulfonamide
Introduction Intensive farming, designed to maximize production efficiency and thereby resulting in a great concentration of animals in a limited area, implies considerable use of physiologically highly active drug substances for combating parasites, preventing and treating bacterially transmitted diseases, and accelerating meat production. In Denmark, for example, largescale pig productions have become one of the main farming activities, and therefore one of the main environmental concerns. If 60,000 farmers were producing 13 million odd pigs 20 years ago, nowadays some 13,000 farmers produce nearly twice as many. The similar pattern observed all over Europe could then partially explain the dramatic increase in veterinary antibiotic concentrations measured in streams near these areas [1–3]. As most known bioactive substances, sulfonamide antibiotics are not completely eliminated in animal organisms as they are administered in high doses for fast and strong impact and excreted shortly after absorption [4]. Sulfonamides have previously been detected at concentrations up to 500 mg/L in pig slurry [5], 20 mg/kg in pig manure [6], and 0.5 mg/kg in some agricultural soils [7]. Large-scale use of veterinary antibiotics is, however, not only common in Europe [8], but also in the USA [9] and, to the best of our knowledge, in China, Southeast Asia, and Russia.
P.A.L. Bouyou et al.
Sulfadiazine (N1-[2-pyrimidinyl]-sulfanilamide), a fairly hydrophobic sulfonamide antibiotic, was used in the present study as a model compound for the analysis of sulfonamides in agricultural drainage waters. Its physicochemical properties can be found in Table 1. Sulfadiazine (SDZ) was previously found in animal wastes in its active parent form [12, 13] together with the corresponding N4-acetyl-sulfadiazine metabolite and alongside low levels of the 4-hydroxylsulfadiazine metabolite [14]. In storage tanks and in manured soils, however, the metabolites are converted back to the parent SDZ [15, 16], and biodegradation and photodegradation of parent sulfonamides were shown to be of minor importance in such systems [7, 11, 17]. For centuries, animal wastes (manures, slurries, etc.) have been known to be valuable by-products used as a source of organic fertilizers in livestock farming for their high content in organic carbon and nutrients, both essential for plant growth. Manure, which contains high levels of ammonia, increases the soil water pH, which favors the formation of the more mobile anionic SDZ species [18] by means of repulsive forces excluding it from soil pores. The same mobility can be expected in the case of grazing livestock, since the presence of urine and feces increases the soil solution ionic strength resulting in increased competitions for soil sorption sites. As previously described by Halling-Sørensen and coworkers [19], SDZ may then be susceptible to enter the drainage pipe system and ultimately the connected streams through surface runoff and soil infiltration initiated by animal wastes spreading or by grazing livestock. Sulfonamides have been found in groundwater below and surface waters close to intensive agricultural areas in concentrations ranging from 0.01 μg/L to 3.3 mg/L [1–3, 20, 21]. Another study carried out by Thiele-Bruhn and Aust [22] brought evidences of a concomitant increase in dissolved organic matter (DOM) and a decrease in sulfonamides’ sorption to soil. Even though the adsorption of sulfonamides to DOM was shown to be strongly dominated by hydrophobic interactions, Bialk and coworkers [23] described the ability of the anilinic nitrogen of the SDZ structure to covalently cross couple through nucleophilic addition with the OM’s polar regions (phenolic and carboxylic groups, keto-, enol-, and alcoholic functions, lipids, and lignin dimers) [17, 24], which consequently enhances the stability of the sulfonamide-DOM
products. As the recycling of animal wastes increases the soil water pH, the amount of DOM, and the soil water ionic strength, an enhancement of the colloidal transport of SDZ was raised as a reasonable explanation for its mobilization from the top soil to the drainage pipes. Preferential pathways, including macropores, cracks, and fissures also exist in soils [25, 26]. These are characterized by less pronounced adsorption, sedimentation, and sieving processes. Thus, in preferential pathways, SDZ-DOM products may become highly mobile and rapidly transported to field drains and ultimately enter the nearby surface waters. This leaching risk is especially enlarged if the application of animal wastes is followed by heavy precipitation events [2, 27] or if the soil is subject to rapid transport [18]. Solid-phase extraction (SPE) is a common method for extracting analytes from aqueous matrices and has become one of the preferred techniques over the classical liquid-liquid extractions (LLE) [28]. SPE has been successfully applied with SDZ and other sulfonamides using the Oasis® hydrophilic-lipophilic balance (HLB) cartridges, both offline [21, 29, 30] and online [1, 31], with higher recoveries than other SPE sorbents, such as the Oasis® MCX cartridge [32]. Some other studies have introduced the use of a tandem SPE procedure, combining the Agilent strong anion exchange (SAX) and the Oasis® HLB cartridges [20, 25, 30]. These cartridges are placed in tandem to simultaneously remove negatively charged humic material (SAX) and to retain the antibiotic agents (HLB) subsequently eluted in a selective fashion. All in all, these combined steps lower the amount of interfering matrix components. Like most of the sulfonamides, SDZ is highly polar, water soluble and labile, nonvolatile, and with a relatively high molecular weight, and therefore not readily analyzed by GCmass spectrometry (MS). It has been shown to efficiently ionize under positive electrospray conditions (ESI+). Liquid chromatography (LC)-ESI-MS [6, 21] or LC-ESI-tandem mass spectrometry (MS/MS) [20, 29, 32] have therefore been shown to be one of the best choices for separation and analysis of SDZ at trace concentrations in complex matrices. It is well known that matrix interferences are ubiquitous during LC-ESI-MS analysis due to ionization competitions. Nonetheless, matrix effects can be compensated for in various ways: by using appropriate
Table 1 Physicochemical properties of sulfadiazine Compound
Sulfadiazine (SDZ)
Structure
Composition
Mw (g/mol)
pKa
Solubility (mg/L)
Log Kow
Kd (L/kg)
C10H10N4O2S
Average mass, 250.27 mono-isotopic mass, 250.05
pKa1 =1.6 pKa2 =6.4 [10]
77
−0.09 [10]
2.0 [11]
Determination of sulfadiazine in drainage waters
internal standards or matrix matched calibration, alone or combined, or by doing standard addition experiments. Here, LCMS/MS was selected as a platform due to its increased versatility and selectivity, and presumed sensitivity as compared to LC-DAD, LC-FLD, and LC-MS [28]. To the best of our knowledge, no method has previously been published where SDZ was analyzed in agricultural drainage water samples. The aim of this study was to develop a new analytical method using SPE LC-MS/MS for its determination at trace levels in agricultural drainage water. Application of the method for phosphate- and dissolved organic carbon (DOC)-loaded samples proved that the analysis of sulfonamide antibiotics could be carried out in complex samples using the adapted tandem SPE (SAX-HLB) method combined with LC-MS/MS.
Experimental Reagents and standards SDZ (>99 %, Chemical Abstracts Service (CAS) no. 68-35-9) was purchased from Sigma-Aldrich (Schnelldorf, Germany), and the internal standard (I.S.), sulfadiazine-d4 (SDZ-d4, CAS no. 1020719-78-1), was from C/D/N Isotopes Inc. (PointeClaire, QC, Canada). Stock solution of 1,000 mg/L SDZ was prepared in methanol and stored at −20 °C for a maximum of 6 months. Working standard solutions were prepared as 128 mg/L SDZ-d4 solutions in methanol and were also stored at −20 °C. Methanol was of high-performance liquid chromatography (HPLC) grade from Lab-Scan (Dublin, Ireland). Formic acid (∼98 %), sodium hydroxide pellets, and potassium chloride were purchased from Merck KGaA (Darmstadt, Germany), ethanol (96 %) was obtained from Kemetyl A/S (Køge, Denmark), and citric acid monohydrate (99.8 %) was from Sigma-Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate (99.5 %), sodium chloride, calcium chloride dihydrate, and magnesium chloride hexahydrate were from VWR Chemicals (Leuven, Belgium). Ottawa sand standard, of general-purpose grade (20–30 mesh), was from AppliChem GmbH (Darmstadt, Germany); nitric acid (69.0– 70.0 %) was purchased from J. T. Baker (Deventer, The Netherlands), and pure water was produced in-house with a Milli-Q gradient system (Millipore, Bedford, MA, USA). The 615-mg/L DOC stock solution originated from an isolation of a Norwegian spruce forest floor, as previously described by Strobel and coworkers [33]. Drainage water samples The drainage water used for spiking during the method development of the solid-phase extraction was sampled at a tile drain site in Rodstenseje, Odder, Denmark (55° 57′ 40.71″ N
and 10° 9′ 46.92″ E). The water was analyzed, and no background SDZ was detected. Preparation of the artificial agricultural drainage water The agricultural drainage water (ADW) was used for spiking during the method development of the solid-phase extraction with phosphate- and DOC-rich matrices. The composition of the ADW was based on sampling campaigns carried out in Denmark over the last years and was prepared in deionized water with 0.652 mM sodium chloride, 0.0254 mM potassium chloride, 1.84 mM calcium chloride dihydrate, and of 0.255 mM magnesium chloride hexahydrate. Properties of the ADW and of the agricultural drainage water collected at the Rodstenseje site can be found in Table 2. Characterization of water samples The pH of the water samples was determined on the day of collection with a 6.0228.000 pH meter from Methrom (Herisan, Switzerland). Concentrations of Cl − , NO 3 − , H2PO4−, and SO42− were determined by ion chromatography (Methrom 833 IC Handling Unit, 818 IC Pump, 820 IC Separation Centre, 819 IC Detector, Metrosep A Supp 5 column, Herisan, Switzerland) after the samples have been pre-filtered through 0.45-μm regenerated cellulose (RC) filters from La-Pha-Pack® GmbH (Langerwehe, Germany). Total organic carbon (TOC) and DOC were determined by a Shimadzu TIC-500 Total Organic Carbon Analyzer (Kyoto, Japan) after filtration with 0.45-μm nylon (PA) filters from La-Pha-Pack® GmbH (Langerwehe, Germany). Concentrations of cations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent Technologies Inc., Agilent 8800, Palo Alto, CA, USA). Polyatomic interferences were removed with an octopole reaction system (ORS) pressurized with He or H2. External calibration was applied and drift corrected by periodic recalibrations. All water samples were acidified with nitric acid (70 %) to a volumetric amount of 0.57 % prior to ICP-MS analyses. For all samples, repeated analyses showed acceptable uncertainties. Liquid chromatography-tandem mass spectrometry analysis The liquid chromatographic system consisted of a 1100 Series HPLC instrument (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a degasser, a quaternary pump, a cooled autosampler, and a column oven. The mass spectrometer was a PE-Sciex API 2000 triple quadrupole mass spectrometer from Applied Biosystems MDS Sciex Instruments (Foster City, CA, USA) equipped with an ESI interface. The injection volume was 10 μL. Separation was performed on an XTerra RP18 column (100 mm×2.1 mm, 3.5 μm) and a guard column
P.A.L. Bouyou et al. Table 2 Selected properties of the agricultural drainage water samples from the Rodstenseje site in Odder (Denmark) and the artificial drainage water (ADW). All analyses were performed in triplicate Parameter
pH Temperature, °C Total organic carbon, mg/L Dissolved organic carbon, mg/L Phosphate, mg/L Calcium, mg/L Sodium, mg/L Magnesium, mg/L Potassium, mg/L Iron, μg/L Aluminium, μg/L Manganese, μg/L Zinc, μg/L Copper, μg/L Chloride, mmol/L Nitrate, μmol/L Sulfate, μmol/L a
Agricultural drainage water (Odder, Denmark)a
Artificial drainage watera
6.53±0.01 5.5±0.1 2.29±0.19
6.91±0.01 5.2±0.1 0.49±0.02
0.56±0.03
0.41±0.09
0.405±0.005 121±0.8 21.5±0.6 7.69±0.18 6.30±0.22 268±8 146±6 19.8±0.3 13.9±0.3 2.53±0.14 0.752±0.001 478±2 319±0.8
– 105±0.6 21.8±0.8 13.8±0.2 1.59±0.12 143±3 33.9±2.5 1.64±0.16 1.10±0.21 0.37±0.09 1.97±0.03 15±0.2 22.3±0.5
Average value±standard deviation
XTerra RP18 column (20 mm×2.1 mm, 3.5 μm) from Waters (Milford, MA, USA) applying a binary gradient flow rate of 0.2 mL/min at 25 °C. The mobile phase A consisted of 15 % methanol and 0.1 % formic acid in Milli-Q water (v/v), and the mobile phase B, of 95 % methanol and 0.1 % formic acid in Milli-Q water (v/v). The initial mobile-phase blend was 98 % A and 2 % B with 5 min of gradient ending with a composition of 20 % A and 80 % B. This composition returned to the starting conditions within 0.5 min for a period of 9.5 min, giving a total analysis time of 15 min. The retention times for SDZ and SDZ-d4, and the optimized parameters for the LCMS/MS analysis are summarized in Table 3. Electrospray ionization was performed in positive mode (ESI+). The desolvation temperature was set to 450 °C with a nebulizer gas pressure of 32 psi, while the curtain and collision gas
pressures were 14 and 6 psi, respectively. The ion spray voltage was 5,500 V. The multiple reaction monitoring function (MRM) was applied in all analyses. Solid-phase extraction The method development and final extraction from agricultural drainage water samples were performed on Oasis® HLB 6-cm3 200-mg (30 μm) cartridges from Waters (Milford, MA, USA). The evaporator and the IST Vacmaster SPE rack were from Macherey-Nagel GmbH (Düren, Germany). The pump was a VacSafe 15 from Labogene ApS (Lynge, Denmark). Nitrogen (99.8 %) was supplied by Air Liquid (Ballerup, Denmark). A summary of the method is presented in Fig. 1. Most veterinary antibiotics are acidic substances; thus, the acidification to pH 3 in the water samples is required in order to obtain SDZ’s neutral form (cf. pKa values in Table 1). As already shown by Zhou and coworkers [20], working at pH 3 allows for the maximum specific retention of SDZ within the polymeric Oasis® HLB columns, whereas the still negatively charged OM usually present in agricultural drainage water samples can be flushed through or retained, for example, by a SAX cartridge. In the first part of the method development, the Oasis® HLB cartridges were conditioned with 5 mL ethanol followed by 3×1 mL methanol and finally 5 mL citric acid (0.04 M). The acidified (pH 3) water samples were loaded at a rate of 3 mL/min. Initially, the optimal temperature for evaporation of the methanol eluates under a gentle stream of nitrogen was tested between 20 and 60 °C by spiking 2 mL methanol to a realistic concentration of 50 μg/mL SDZ. The washing step of the SPE cartridges, using Milli-Q water, was also investigated for possible loss. For this matter, 100 mL of water pH adjusted to 3 spiked with 50 μg/L SDZ was added to the Oasis® HLB cartridges. After the loading, portions of 1 mL of Milli-Q water were added and analyzed individually. In order to assess the volume of methanol needed to quantitatively elute SDZ from the Oasis® HLB cartridges, 100 mL of spiked Milli-Q water samples containing 50 μg/L SDZ was loaded onto the cartridges and eluted with methanol in small portions (500 μL). Each portion was analyzed separately to obtain elution profiles from
Table 3 Specific settings in the final analytical LC-MS/MS method, applying ESI in positive mode, together with the most intense precursor and product ions for sulfadiazine and its corresponding I.S. Compound
Retention time (min)†
Precursor ion (m/z)
Product ions quantify/qualify (m/z)
SDZ
4.39
251.1
156.1/108.0
SDZ-d4
4.32
255.1
160.1/112.0
Ionization mode
Dwell time (ms)
Declustering potential (V)
Focusing potential (V)
Entrance potential (V)
Collision energy (eV)
Collision cell exit potential (V)
Positive
500
14.0
390.0
10.0
24.0
2.0
Determination of sulfadiazine in drainage waters
Sample preparation 1000 mL water sample is adjusted to pH 3 with formic acid and spiked with 100 µL I.S. (sulfadiazine-d 4) SPE cartridge conditioning 5 mL ethanol 3x1 mL methanol 5 mL citric acid (0.04 M) Low DOC content (< 2 mg/L)
High DOC content (> 2 mg/L)
Sample load Single cartridge SPE setup - Oasis® HLB 6 cm3 200 mg (30 µm)
Sample load Tandem SPE setup Agilent LRC-SAX 1 cm3 100 mg and Oasis® HLB 6 cm3 200 mg (30 µm)
Wash 5 mL Milli-Q water to remove excess acid and wash sorbent Elution 2 mL methanol to elute extracted sulfadiazine Evaporation To dryness under gentle nitrogen stream and re-constitution in 1 mL (1:1, v/v) methanol/Milli-Q water Analysis LC-MS/MS Fig. 1 The final SPE method using either Oasis® HLB 6-cm3 200-mg (30 μm) cartridges or a combination of Agilent Bond Elut LRC-SAX 1cm3 100-mg and Oasis® HLB 6-cm3 200-mg (30 μm) cartridges for 1,000-mL water samples
0.5 to 5 mL. The Oasis® HLB cartridges were also evaluated for SDZ breakthrough at three different sample volumes: 200, 500, and 1,000 mL. Before extraction, the samples were adjusted to pH 3 and spiked with SDZ at a total mass of 50 μg giving concentrations of 250, 100, and 50 μg/L for the 200-, 500-, and 1,000-mL samples, respectively. Two Oasis® HLB cartridges were set up in tandem in order to assess breakthrough. In the second part of the method development, a tandem SPE procedure was developed in ADW containing common and high concentrations of phosphate and DOC. Agilent Bond Elut LRC-SAX 1-cm3 100-mg cartridges were chosen and conditioned in the same way as the Oasis® HLB cartridges and placed on top of the latter for the adapted tandem SPE (SAX-HLB) method (Fig. 1). Both cartridges were eluted with 2 mL methanol, and the eluates were tested for SDZ content.
Filtration and pressurized liquid extraction The filtration of natural drainage water samples was performed on Whatman™ GF/C glass microfiber filters ( 1 . 2 μm ) from GE Healthcare (Little Ch a l f o n t , Buckinghamshire, UK), and the experiments were performed in triplicate. The samples were adjusted to pH 3 using formic acid, and 100 μL working solution of I.S. (SDZ-d4) was added prior to filtration. In order to assess the SDZ loss during the filtration process, the filters were analyzed by pressurized liquid extraction (PLE), following the procedure developed by Jacobsen and coworkers [30]. For this purpose, the filters were frozen at −20 °C for at least 24 h before being freezedried overnight in a Type FD 3 freeze-drier from Heto-Holten A/S (Allerød, Denmark) operating at −48 °C and 10−5 bar. The next step involved SDZ extraction from the freeze-dried filters by PLE, using an ASE 200 system from Dionex (Sunnyvale, CA, USA). The system was operated with 33 mL of pressure-resistant steel extraction cells and lined with cellulose filters from Dionex (Sunnyvale, CA, USA). Initially, 200-mL samples of agricultural drainage water from the Rodstenseje site with pH adjusted to 3 were spiked to a realistic concentration of 50 μg/L SDZ. Conditioning of the filters included soaking in Milli-Q water and thereafter in methanol before resting in Milli-Q water prior to usage. In order to assess the SDZ loss during this step, 100 μL SDZ-d4 (I.S.) was added to the filtrates and the filters, and SPE LCMS/MS and PLE combined with SPE LC-MS/MS procedures were carried out, respectively. Moreover, the filters were weighted to account for their content in sequestered matter. Practically, the used Whatman™ filters were cut in small pieces and mixed with 10-g Ottawa sand and 100 μL of SDZ-d4 at a concentration of 128 mg/L. The extraction buffer consisted of a 1:1 (v/v) mixture of methanol and 0.2 M citric acid buffer with pH adjusted to 4.7 with sodium hydroxide. A first static extraction was operated with approximately 30 mL of extraction buffer at 1,500 psi for 10 min, followed by flushing the extract into a collection vial. Then, a second static extraction was operated with an additional 30 mL of extraction buffer for 3 min and flushed into the same collection vial. The total final volume of the extract was approximately 60 mL, and the extractions were performed at room temperature. In each of the static extraction cycle, 60 % of flush volume and 120-s purge period were applied. Between each run, the extraction cells were cleaned by ultra-sonication for 15 min in a mixture of Milli-Q water/methanol (1:1, v/v), followed by 15 min of ultra-sonication in Milli-Q water. Method validation using standard solutions The linearity, limit of detection (LODInstr) and limit of quantification (LOQInstr) of the LC-MS/MS system were evaluated in an initial step by injecting pure standards into the LC-MS/
P.A.L. Bouyou et al.
MS system. Linearity was investigated using a 15-point calibration curve determined in the range 5 μg/L–25 mg/L. It was set as a criterion that peak area ratios between the qualifier and the quantifier ion transitions should not deviate more than 20 % from a fixed ratio [34]. Intra- (n=27) and inter-day (n=27) instrument precision were determined from relative standard deviations (RSDs) of the peak areas obtained for a triplicate injection during the calibration curve determination. For the LC-MS/MS instrument, the LODInstr and LOQInstr were calculated as recommended by the ICH guideline [35] following the equations LODInstr ¼ 3:3 σS and LOQInstr ¼ 10 σS . Here, σ is the standard deviation of the peak area obtained when analyzing in triplicate the least concentrated SDZ sample during the determination of the calibration curve, and S is the slope of the calibration curve.
Results and discussion Method development Optimization of the LC-MS/MS ionization efficiency of sulfadiazine in an agricultural drainage water matrix The development of an efficient LC separation with an appropriate ionization method coupled to MS/MS was necessary prior to the development of the SPE method. For SDZ, the most intense precursor ion was the [M+ H]+ ion at m/z 251.1. A common fragmentation pattern has already been observed for the sulfonamide family [36], where amino sulfonamides such as SDZ typically produce product ions at m/z 156.1, 108.0, and 92.0 (Fig. 2). The fragmentation of SDZ resulted in a loss of 95 Da corresponding to the cleavage of the sulfonamide bond [36], leading to m/z 156.1. Further loss of SO2 leads to the ion at m/z 92.0, while it is known that m/z 108.0 is formed via a rearrangement [37]. The resulting precursor and product ions are listed in Table 3. The most prominent MRM transition (m/z 156.1) was used for quantification, and the other one (m/z 108.0) was used as a qualifier ion. The corresponding transitions for the SDZ-d4 analog were m/z 255.1→ 160.0→112.0 (Table 3). The overall variation of the quantifier/qualifier ion-ratio stability was investigated in the range 5 μg/L–100 mg/L and showed an overall good consistency with a mean value of 1.35±0.02 (RSD 1.70 % (n = 51)). Nonetheless, the quantifier/qualifier ion-ratios decreased with increasing SDZ concentrations with a logarithmic regression, displaying a coefficient of regression r2 =0.8693 (Fig. 3, Eq. 1). R ¼ −0:0051:log10 ½SDZ þ 1:3497
ð1Þ
Here, [SDZ] represents the concentration of SDZ, and R is the ratio of the quantifier/qualifier peak areas. In other words, Að251:1 156:1Þ represents the ratio of the peak , where A 251:1 R ¼ A 251:1 156:1 ð108:0Þ areas of the precursor ion and of the quantifier product ion, and A 251:1 108:0 is the ratio of the peak areas of the precursor ion and of the qualifier product ion (cf. Table 3). An advantage from using quantifier/qualifier ion ratios would be achieved in the case of impossible quantification using the quantifier ion, due to partly overlapping chromatographic peaks, in the case of matrix interferences. It may then be possible from the peak height ratio of the qualifier/quantifier ions to determine whether or not it corresponds to the correct peak. If this is the case, things can be inverted, and then the qualifier ion can be used for quantification. By using this strategy, the number of samples that cannot be quantified directly can be reduced to close to zero. However, in the rare cases where the quantifier ion peak is totally covered by an interfering peak, it has to be noted that quantification with the qualifier ion implies a loss of specificity, as only one specific ion is involved for identification. At this stage, a complete LC-MS/MS methodology was established and used in all further SPE optimizations. Optimizations were performed by continuously infusing SDZ at a concentration of 20 mg/L in methanol/Milli-Q water (1:1, v/v).
Optimization of the solid-phase extraction Solid-phase extractions using Oasis® HLB 6-cm3 200-mg (30 μm) cartridges have been known to efficiently extract SDZ from aqueous matrices and have previously been successfully applied for wastewater [2, 29], groundwater, and surface water [1, 21] and also for coastal water [32]. Effects of the evaporation, washing, and elution steps on recoveries Before developing the final method for the Oasis® HLB cartridges, an optimization of the three steps preceding the final analysis on LC-MS/MS was required (Fig. 1). The effect of evaporation temperature on the recoveries of SDZ is shown in Fig. 4a. No significant loss was observed up to a temperature of 40 °C, which was the temperature chosen for application in the final method (Fig. 1), and this supports results described by Ye and coworkers [29]. The washing step involved the use of Milli-Q water in order to remove acidic components prior to elution with methanol. No loss of SDZ occurred up to 8 mL (data not shown). The final washing volume was set to 5 mL which corresponds to flushing the sorbent pore volume approximately 19 times (Fig. 1). This result lays within the average volume of MilliQ water previously used for cleaning up SPE cartridges [21, 29, 32]. The elution volume of methanol is a crucial step
Determination of sulfadiazine in drainage waters
Fig. 2 Fragment ions used as quantifiers and qualifiers in tandem mass spectrometry for sulfadiazine according to literature references [36, 37]
to assure a quantitative elution of SDZ trapped into the SPE cartridges. It can be seen that after applying 1.5 mL of solvent, SDZ is quantitatively removed from the cartridge (Fig. 4b). A confirmatory experiment using 2 mL of methanol without a stepwise addition confirmed the quantitative recovery of SDZ from the SPE cartridge. The final elution volume was then set to 2 mL which corresponds to flushing the sorbent pore volume approximately eight times (Fig. 1). The optimization of the methanol elution volume minimized the use of organic solvent compared to
previous methods described by Ye and coworkers [29], Lindsey and coworkers [21], and Na and coworkers [32], who respectively used 3, 5, and 6 mL. Effect of sample size on recoveries In order to be able to reach a low LODMethod assuring a better quality in the assessment of agricultural drainage water contaminations, increasing the sample volume can have an important impact. However, there are some limitations regarding the handling of large volumes; increasing the
1.42 R = -0.0051.log10 [SDZ] + 1.3497 r2 = 0.8693
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Fig. 3 Quantifier/qualifier ion-ratio stability assessment during the calibration curve determination for the LC-MS/MS method in the range of 5 μg/L–100 mg/L. The sulfadiazine concentrations are plotted on a logarithmic scale, and the coefficient of regression was assessed by a
logarithmic regression analysis which showed a coefficient r2 =0.8693. The mean value of the quantifier/qualifier ion-ratios was 1.35, and its RSD value was 1.70 % (n=51). Each data point represents a triplicate sample, and error bars represent one standard deviation
P.A.L. Bouyou et al.
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Fig. 4 a Effect of evaporation temperature on the recovery of sulfadiazine. Methanol was spiked at a concentration of 50 μg/mL and evaporated under a gentle stream of nitrogen prior to reconstitution in 1 mL 1:1 (v/v) mixture of methanol/Milli-Q water. Each experiment was performed in triplicate, and error bars represent one standard deviation.
b Evaluation of the elution volume of methanol required to elute the compounds applied to the Oasis® HLB 6-cm3 200-mg (30 μm) cartridges. The cartridges were loaded with 100-mL spiked Milli-Q water (pH 3) at a concentration level of 50 μg/L of sulfadiazine. Each data point represents a triplicate sample, and error bars represent one standard deviation
sample volume could indeed give rise to breakthrough of SDZ due to a saturation of the SPE sorbent. The data indicated no breakthrough of SDZ for any of the selected volumes (200, 500, and 1,000 mL). Based on these findings, 1,000 mL was applied as a suitable sample volume in the final method (Fig. 1). On the one hand, the use of 1,000 mL samples showed an important improvement compared to previous results using working volumes ranging from 100- to 500-mL samples [21, 29, 32]. In this regard, the ability to handle larger sample volumes provides the reach of a lower absolute LODMethod. However, results assessing the content in particles bearing a size fraction >1.2 μm in the Rodstenseje agricultural drainage water were found to be rather low for this type of water samples (0.45± 0.25 mg). This is probably for such reason that neither breakthrough of SDZ nor clogging of the SPE cartridges were observed up to a sample volume of 1,000 mL. In the first part of the method development, the use of Oasis® HLB cartridges to retain and elute SDZ was optimized. The 1,000-mL water samples were acidified to pH 3, and thereafter loaded at a rate of 3 mL/min onto the Oasis® HLB cartridges. Conditioned with 5 mL ethanol followed by 3×1 mL methanol and finally 5 mL citric acid (0.04 M), the SPE cartridges were washed with 5 mL Milli-Q water before SDZ was eluted with 2 mL methanol. The eluates were evaporated to dryness (evaporation was stopped immediately after the samples became dry) under a gentle stream of nitrogen at 40 °C and redissolved in a final volume of 1 mL methanol/
Milli-Q water (1:1, v/v), prior to LC-MS/MS determination. A summary of the method is presented in Fig. 1. Method validation LC-MS/MS validation The linearity of the 15-point linear calibration curve in the range of 5 μg/L–25 mg/L gave a correlation coefficient r2 of 0.9915, and the obtained LODInstr and LOQInstr values were 0.247 and 0.823 ng/mL, respectively. Therefore, the absolute amount required in the LC-MS/MS system to reach the LOQ of SDZ was 8.23 pg. Monitoring the intra- and inter-day precisions during the LC-MS/MS validation showed very stable results, with values of 1.70 %±1.25 (n= 27) and 2.12 %±1.96 (n=27), respectively. Continuous monitoring of the I.S. during the LC-MS/MS validation showed very stable peak areas. For the I.S., the average area was 5.48·104, and the relative standard deviation was 6 % (n=54). SPE LC-MS/MS method validation When analyzing spiked agricultural drainage water, SDZ-d4 was added before the analysis, and the quantification was based on the ratio (SDZ/SDZ-d4). The I.S. concentration was kept constant at 12.8 mg/L. Validation of the complete SPE LC-MS/MS method was done by spiking agricultural drainage water samples from the Rodstenseje site adjusted to pH 3. Recoveries were determined on six concentrations
Determination of sulfadiazine in drainage waters
based on preliminary LOQs of the entire method for spiked agricultural drainage water. The spiking levels were 1, 5, 10, 25, 50, and 100 ng/L. Calibration was performed as an internal calibration in methanol/Milli-Q water (1:1, v/v). Recoveries from the spiking of agricultural drainage water at concentrations close to the limit of what can be quantified, compensated by SDZ-d4, is given in Table 4. SDZ could be detected down to 10 ng/L, though displaying a significant decrease in recovery when compared to the other concentrations tested. Results also showed that at a concentration of 5 ng/L SDZ could be detected, though not quantified, while at a concentration of 1 ng/L (2,500 times below the threshold), it was not detectable. Results demonstrate a good accuracy with recoveries above 80 % for spiking levels above 25 ng/L. These recovery levels are in accordance with other recovery studies which assessed the successful extraction of SDZ from a wide range of environmental samples, i.e., from surface [20], coastal [32], and groundwater [1], but also from wastewater [29], and from soil [30] and animal manure [6] extracts. In these studies, recoveries ranged from 51 to 96 %. Consequently, the present method demonstrates its ability to extract SDZ to a high extent from agricultural drainage water samples. The data also showed acceptable precision of the method since RSDs are near 20 % or below. Based on these data, the LODMethod and LOQMethod were evaluated. The LODMethod and LOQMethod were evaluated with a signal to noise ratio of 3.3 and 10, respectively, using the peak area of the extract from agricultural drainage water spiked with the lowest concentration of SDZ and injected six times. The concentration was 10 ng/L, and the obtained LODMethod value was 7.48 ng/L. The corresponding LOQ Method value was assessed to be 22.7 ng/L. Therefore, the absolute amount required in the SPE LC-MS/MS method to reach the LOQ of SDZ was 0.23 pg. This is especially important for SDZ because it is known to occur and has previously been detected in groundwater below and surface waters close to intensive agricultural areas [1–3, 20, 21]. These corresponding levels of detection and quantification are in the same order of magnitude (low to medium ng/L range) as other studies focusing on surface, coastal, and
Table 4 Effects of sample concentration on absolute recoveries of sulfadiazine spiked to final concentrations ranging from 1 to 100 ng/L in 1-L agricultural drainage water with pH adjusted to 3. Each experiment was 1 ng/L Recovery (%) SDZ
