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«Determination of tetracycline residues in soil by pressurized liquid extraction and liquid chromatography tandem mass spectrometry Vicente Andreu · ...»

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Autores (p.o. de firma): V. Andreu, P. Bazquez-Roig, C. Blasco y Y. Picó

Título: “Determination of tetracycline residues in soils by pressurized liquid extraction

and liquid chromatography tandem mass spectrometry”

Revista: Analytical and Bioanalytical Chemistry

Volumen: 394(5) Páginas, inicial: 1329 final: 1339 Fecha: 2009

Determination of tetracycline residues in soil by pressurized

liquid extraction and liquid chromatography tandem mass


Vicente Andreu · Pablo V Roig · Cristina Blasco · Yolanda Picó1 P.V. Roig, C. Blasco · Y. Picó () Laboratori de Bromatologia, Facultat de Farmàcia, Universitat de València, Av, Vicent Andrés s/n, 46100 Burjassot, València, Spain.

E-mail: yolanda.pico@uv.es Tel:+34 96 3543092 Fax:+34 96 3544954 V. Andreu Centro de Investigaciones sobre Desertificacion (CIDE), Camí de la Marjal s/n, 46470 Albal, Valencia, Spain E-mail: Vicente.andreu-perez@uv.es Tel: +34 96 1220540 Fax: +34 96 1270967


An optimized extraction and clean-up method for the analysis of chlortetracycline (CTC), doxycycline (DC), oxytetracycline (OTC), and tetracycline (TC) in soil is presented. Soil extraction in a pressurized liquid extraction (PLE) system, extract clean-up by solid-phase extraction (SPE) and tetracycline determination by liquid chromatography tandem mass spectrometry (LC-MS/MS) provided appropriate efficiency and reproducibility. Different dispersing agents and solvents for soil extraction and several SPE cartridges for clean-up were compared. The best extraction results were obtained using EDTA treated sand, as dispersing agent, and water at 70 º C. The most effective clean-up was obtained using StrataX(TM) sorbent in combination with a strong anion exchange cartridge. Recoveries ranged from 70 to 99 % and precision, as indicated by the relative standard deviations (RSDs), was within the range of 8 -15 %. The limits of quantification (LOQs) by using LC-MS/MS, based on S/N: 10, ranged from 1 μg kg-1 for TC to 5 µg kg-1 for CTC. These results pointed out that this technique is appropriate to determine tetracyclines in soils. Analysis of 100 samples taken in the Valencian Community revealed that in soil up to 5 µg kg-1 CTC, 15 µg kg-1 OTC,18 µg kg-1 TC and 12 µg kg-1 DC could be detected. Detection of the analytes in several samples, which typify the Spanish agricultural soils, should be outlined as most important result of this study.

Antibacterials · Tetracyclines · LC-MS/MS · Pressurized Liquid Extraction · Keywords Environmental analysis· Soil Introduction Pharmaceutical residues in the environment are of increasing concern worldwide because the large number of drugs used in human and veterinary medicine [1-4]. After excretion, these drugs and their metabolites reach the environment by passing sewage treatment plants or by soil amended with sewage sludges or manures [5-7]. New investigations show that more than 45 different drugs can be found in surface waters from the low to the very low µg l-1 concentration range [8-10].

One among the first groups of antibiotics to come into use in human being was the tetracyclines (TCs), these drugs have stood the test of time and are continuing to be useful in treating a broad range of infections [11]. TCs have been found widely disseminated in water and sewage. However, little is know about their occurrence and impact in soils. Some data extracted from the literature show that residues of the commonly used veterinary drugs, tetracycline (TC) and chlortetracycline (CTC) can be detected in soil surface (0-40 cm) fertilized with animal slurry [12-15]. Nevertheless, no leaching of TCs into deeper soil segments or groundwater has beem observed [14,15]. The halflife of oxytetracycline (OTC) in manure was 30 days and the compound was still detectable in this matrix after 5 months. In the manured soils, OTC was detected at concentrations at least 10 times lower than the European Agency for the Evaluation of Medicinal Products threshold (100 µg kg-1) [16].

Detection of TCs in soil, sediments, and water, and the growing concern of their potentially adverse effect on natural ecosystems have resulted in a need to understand their behavior in the soil system.

TCs have multiple ionizable functional groups such that at environmentally relevant pH values, they may exist as a cation, zwitterion, or a net negatively charged ions, which complicates predicting their sorption, availability, and transport. The sorption of OTC, TC, and CTC by several soils varying in pH, clay content and type, cation exchange capacity (CEC), anion exchange capacity (AEC), and soil organic carbon was investigated. Strongest sorption was observed for clays, followed by HS, and then clay-HC [17,18]. Greater sorption by the Ca systems than the K ones and decreased sorption with increasing pH suggests that cation binding and cation exchange contribute to sorption [19-21].

Summarizing, TCs enter in the environment in significant concentrations via repeated amendments with manure or sludges, build up persistent residues, and accumulate in soil. Therefore, TCs may have a potential risk and investigations on their environmental effects are necessary [22].

In a recent review, O’Connor and Aga [7] discuss strategies for sample preparation, extraction, clean-up using solid-phase extraction (SPE) and molecularly-imprinted polymers (MIPs), and analysis of TCs and their transformation products in soils. This review also points out that available information about the environmentally-relevant concentrations of TCs is limited, mostly due to analytical difficulties encountered when trying to analyze trace levels of these compounds in the presence of complex matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become widely used in detecting antibiotics, including TCs. In applying LC-MS/MS for environmental investigations, the analyst is faced with two major challenges: poor detectability of TCs and highly variable matrix interferences, which compromise quantification. Efforts have been directed to attain high-throughput methods able to extract a large number of samples in a short time [15,16,23]. Pressurized liquid extraction (PLE) is a rather new technique that uses solvent at a relatively high pressure and temperature without their critical point being reached. This improves efficiency compared to extractions at room temperature and atmospheric pressure [7]. Recently, O’Connor et al. [24] optimized the extraction of TCs by using rapid and simple PLE procedures with a mixture of acetate buffer (pH 8), and methanol as the extracting solvent in soil. In the same way, Jacobsen et al. [25] reported the use of PLE with mixtures of methanol and citric acid (pH 4.7) to extract TC, macrolide and sulfonamide antibiotics from agricultural soils. Other studies have reported the use PLE to extract both, veterinary drugs from food matrices [26-28] or different contaminants from soils [29,30]. All these studies remarked the technology's benefits in providing rapid and reliable analysis.

The present study focuses on developing a method for the simultaneous determination of four TCs —TC, OTC, CTC and doxycycline (DC)— in soil, using PLE and SPE extraction followed by LC–MS/MS with a triple quadrupole (QqQ) analyzer. Different dispersing agents and solvents for soil extraction and several SPE cartridges for clean-up were compared. The method was applied to soil samples of different locations and after different sludge treatments. To the best of our knowledge, this is the first finding of TC residues in typical Spanish agricultural soils.

Experimental Chemicals and standards TC, OTC, CTC, DC and demeclocycline (DMC) were purchased from Sigma (St. Louis, MO).

DMC was used as internal standard (IS) because it is an obsolete antibiotic. The three epimers, 4epitetracycline (e-TC), 4-epioxytetracycline (e-OTC), and 4-epichlortetracycline (e-CTC), were from Acros (Fisher Scientific, Schwerte, Germany). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). Formic acid, citric acid monohydrate, sodium acetate anhydrous, sodium hydroxide pellets and ethylene diaminetetraacetic disodium salt (EDTA-Na2) were of analytical grade (Aldrich, Madrid, Spain). Deionised water ( 8 MΩ cm resistivity) was obtained from the Milli-Q SP Reagent Water system (Millipore, Bedford, MA, USA). All the solvents and solutions were filtered through a 0.45 μm cellulose filter from Scharlau (Barcelona, Spain) before use. Acidic, neutral and basic alumina (Al2O3) were obtained from Merck, silica gel form scharlau, Florisil® from Aldrich, sea sand from Panreac and anhydrous sodium sulfate (analytical grade) from Scharlau. To block metal impurities, 60 g of solid sorbent was placed in a Buchner funnel and 120 mL of 0.1 M EDTA-Na2 was passed through the sorbent using vacuum. SAX cartridges (strong anion exhange, 500 mg sorbent, 6 ml cartridge) were purchased from Isolute, IST. Oasis® HLB extraction cartridges [poly(divinylbenzene–co-N-pyrrolidone), 6 cc, 200 mg] were from Waters (Milford, MA, USA) and Strata-X® SPE cartridges [surface modified styrene divinylbenzene, 6 cc, 200 mg) from Phenomenex (Torrance, CA, USA).

Sample collection Samples of soil were collected from the Ap horizon (0–20 cm) from 50 fields located in the South surrounding area of the Valencia city (Spain) in early February 2007. The soils are of loamy texture highly carbonated, representing typical Spanish agricultural soils. Some soil characteristics are listed in Table 1. The last fertilization with sewage sludges was in September 2006 according to the data provided by the Conselleria de Medio Ambient. There were no more data on the amount of sludge added to the field or the contamination levels of the sludges by these pharmaceuticals. Two control samples from fields without slurry fertilization since, at least, 5 years were also taken from this region. Samples were taken in plastic bags and immediately transported under cooling to the laboratory. To achieve homogeneous samples, the soils were air dried and sieved through a 2 mm sieve before further handling.

Sample preparation Pressurized liquid extraction (PLE) The extraction of antibacterial agents from soil was performed by PLE, using an ASE 200 system from Dionex (Sunnyvale, CA, USA). The system was operated with pressure resistant steel extraction cells with a volume of 22 ml and lined with glass-fibre filters from Dionex.

Approximately 5 g of soil were added of 10 µl DC (IS) solution of 10 µg ml-1 and blended with 5 g of EDTA-Na2 washed sea sand for 5 minutes in a mortar using a pestle. This mixture was introduced into a stainless steel extraction cell, which was positioned in the PLE system connected to a four-bottle solvent controller. Nitrogen, at a pressure of 10 bar, was supplied to assist the pneumatic system and to purge the extraction cells. The extraction cells were preheated for 2 min, the analytes were extracted with water at 70ºC and 1500 psi for 10 min of static time, in one cycle, at 100 % of flush; then, the extraction cells were purged for 60 s with nitrogen to eliminate any trace of the extraction solvent. The total final volume of extract was approximately 40 ml.

Solid-phase extraction Clean up and pre-concentration was performed using a combination of SAX and Strata-X cartridges. Cartridges were placed in tandem to simultaneously remove negatively charged humic material (SAX) and retain the antibacterial agents (Strata-X). The SAX cartridge was placed on top of the Strata-X cartridge and both columns were conditioned first with 2 ml methanol and then 2 ml water. PLE extracts (40 ml) were passed through both SPE-columns at approximately 5 ml min−1 and after extraction the columns were washed with 2 ml water and dried under vacuum for 15 min.

Then, the SAX cartridge was removed and the antibacterial agents were eluted from the Strata-X sorbent with 2 ml methanol. The eluate was evaporated to dryness using a multi-sample Turbovap LV Evaporator (Zymark, Hoptkinton, MA, USA), and the residue was redissolved in 1 ml methanol-water (10:90).

Liquid chromatography-mass spectrometry A Quattro LC triple quadrupole mass spectrometer from Micromass (Manchester, U.K.), equipped with an LC Alliance 2690 system (Waters) consisted of an autosampler and a quaternary pump, a pneumatically assisted electrospray probe, a Z-spray interface and a Mass Lynx NT software Ver. 4.1 were used. Analysis was performed in positive ion mode. The ESI source values were: capillary voltage, 3.00 kV; extractor, 2 V; RF lens, 0.5 V; source temperature, 120 ºC;

desolvation temperature, 300 ºC, and desolvation and cone gas (nitrogen 99,99% purity) flows, 600 l h-1 and 60 l h-1, respectively. The analyzer settings were resolution, 12.0 (unit resolution) for the first and third quadrupoles; ion energy, 2.0; entrance and exit energies, -1 and 1; multiplier, 650;

collision gas (argon, 99.995 %) pressure 2.79 x 10-3 mbar; interchannel delay, 0.02 s; total scan time, 1.0 s. The mass spectrometer was operated in scan and product ion scan modes to optimize the conditions and select the transitions, and in selected reaction monitoring (SRM) mode to confirm the identity of analytes in the samples by selecting two transitions for each one and to quantify.

Table 2 shows the particular conditions and transitions for each analyte. The analytical column was a Xterra C18 (100 x 2.1 mm I.D., 3.5 µm) from Waters. The mobile phase consisted of methanol and water, both with 10 mM formic acid at 0.2 ml min-1 in gradient that begins with 10 % methanol, increasing linearly in 15 min to 90 % of methanol, maintaining this proportion for 5 min and return to the initial conditions in 10 min. The injected volume was 20 µl.

Method validation Recoveries for the entire procedure were determined using the two control samples taken from fields without fertilization. Soil samples were fortified with CTC, OTC, TC and DC on three concentration levels (approximately 10, 50 and 100 μg kg−1 soil) and the IS at fixed concentration of 100 μg kg−1. Six different extractions were performed at each level. The fortified samples were extracted and analysed using the entire procedure. Recoveries were calculated as the percentage of extracted antibacterial agent compared to the spiked level.

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