Monitoring leachables from single-use bioreactor bags for mammalian cell culture by dispersive liquid-liquid microextraction followed by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry.

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production.

Determination of selected parabens, benzophenones, triclosan and triclocarban in agricultural soils after and before treatment with compost from sewage sludge: A lixiviation study.

An accurate and sensitive method for the determination of selected EDCs in soil and compost from wastewater treatment plants is developed and validated. Five parabens, six benzophenone-UV filters and the antibacterials triclosan and triclocarban were selected as target analytes. The parameters for ultrasound-assisted extraction were thoroughly optimized. After extraction, the analytes were detected and quantified using ultra-high performance liquid chromatography tandem mass spectrometry. Ethylparaben (ring-(13)C6 labelled) and deuterated benzophenone (BP-d10) were used as internal standards. The method was validated using matrix-matched calibration and recovery assays with spiked samples. The limits of detection ranged from 0.03 to 0.40 ng g(-1) and the limits of quantification from 0.1 to 1.0 ng g(-1), while precision in terms of relative standard deviation was between 9% and 21%. Recovery rates ranged from 83% to 107%. The validated method was applied for the study of the behavior of the selected compounds in agricultural soils treated and un-treated with compost from WWTP. A lixiviation study was developed in both agricultural soil and treated soil and first order kinetic models of their disappearance at different depths are proposed. The application of organic composts in the soil leads to an increase of the disappearance rate of the studied compounds. The lixiviation study also shows the risk of pollution of groundwater aquifers after disposal or waste of these EDCs in agricultural soils is not high.

New method for the determination of parabens and bisphenol A in human milk samples using ultrasound-assisted extraction and clean-up with dispersive sorbents prior to UHPLC-MS/MS analysis.

A sensitive and accurate analytical method for the determination of methyl-, ethyl-, propyl- and butylparaben and bisphenol A in human milk samples has been developed and validated. The combination of ultrasound-assisted extraction (UAE) and a simplified and rapid clean-up technique that uses sorbent materials has been successfully applied for the preparation of samples prior to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The analytes were extracted from freeze-dried human milk samples using acetonitrile and ultrasonic radiation (three 15-min cycles at 70% amplitude), and further cleaned-up with C18 sorbents. The most influential parameters affecting the UAE method and the clean-up steps were optimized using design of experiments. Negative electrospray ionization (ESI) in the selected reaction monitoring (SRM) mode was used for MS detection. The use of two reactions for each compound allowed simultaneous quantification and identification in one run. The analytes were separated in less than 10min. Deuterium-labeled ethylparaben-d5 (EPB-d5) and deuterium-labeled bisphenol A-d16 (BPA-d16) were used as surrogates. The limits of quantification ranged from 0.4 to 0.7ngmL(-1), while inter- and intra-day variability was under 11.1% in all cases. In the absence of certified reference materials, recovery assays with spiked samples using matrix-matched calibration were used to validate the method. Recovery rates ranged from 93.8% to 112.2%. The proposed method was satisfactorily applied for the determination of four selected parabens and bisphenol A in human milk samples obtained from nursing mothers living in the province of Granada (Spain).

Improved sample treatment for the determination of 17 strong sorbed quinolone antibiotics from compost by ultra high performance liquid chromatography tandem mass spectrometry.

The use of compost from sewage sludge for agricultural application is nowadays increasing, since composting is recognized as one of the most important recycling options for this material, being a source of nutrients for plants but also of contamination by persistent pollutants. In the present work, a multi-residue analytical method for the determination of 17 quinolone antibiotic residues in compost using multivariate optimization strategies and ultra high performance liquid chromatography-tandem mass spectrometry has been developed. It is based on the use of microwave-assisted extraction at drastic conditions with ACN:m-phosphoric acid (1% w/v) for 5 min at 120°C, in order to achieve a quantitative extraction of the compounds (>76% of extraction recovery). Extracts were cleaned-up by salt-assisted liquid-liquid extraction (SALLE) with NaCl at pH 1.5 (with HClO4) and then using a dispersive sorbent (PSA). After LC separation, the MS conditions, in positive electrospray ionization mode (ESI), were individually optimized for each analyte to obtain maximum sensitivity in the selected reaction monitoring mode (SRM). The analytes were separated in less than 7 min. Cincophen was used as surrogate standard. The limits of detection ranged from 0.2 to 0.5 ng g(-1), and the limits of the quantification from 0.5 to 1.5 ng g(-1), while intra- and inter-day variability (% RSD) was under 7% in all cases. A recovery assay was performed with spiked samples. Recoveries ranging from 95.3% to 106.2% were obtained. Cleanup procedure reduced significantly matrix effects, which constitutes an important achievement, considering the important drawbacks of matrix components in quality and validation parameters. This method was applied to several commercial compost samples. Only 6 of the studied antibiotics were not detected in any of the samples. The antibiotics with the highest concentrations were ciprofloxacin (836 ng g(-1)), ofloxacin (719 ng g(-1)), and enrofloxacin (674 ng g(-1)), which were also the only ones found in all the analyzed samples. The results showed that this method could also be potentially adapted for the analysis of other strong sorbed basic pharmaceuticals in solid environmental matrices.

Determination of benzophenone-UV filters in human milk samples using ultrasound-assisted extraction and clean-up with dispersive sorbents followed by UHPLC-MS/MS analysis.

A new sample preparation method for the determination of five benzophenone UV-filters in human breast milk has been developed. The procedure involves the lyophilization of the sample, and its subsequent extraction by ultrasound sonication using acetonitrile. In order to reduce matrix effects produced by milk components that are coextracted, mainly proteins, sugars and lipids, a further clean-up step with a mixture of dispersive-SPE sorbents, C18 and PSA, was applied. Extraction parameters were optimized using experimental design, and the compounds were detected and quantified by ultrahigh performance liquid-chromatography tandem mass spectrometry (UHPLC-MS/MS) in positive ESI mode. Analytes were separated in 10 min. BP-d10 was used as internal standard. The limits of detection (LODs) were between 0.1 and 0.2 ng mL(-1), and the limits of quantification (LOQs) were between 0.3 and 0.6 ng mL(-1) for the target analytes. The inter- and intra-day variability was <12%. The method was validated using matrix-matched calibration and recovery assays with spiked samples. Recovery rates were between 90.9 and 109.5%. The method was successfully applied for the determination of these compounds in human milk samples collected from volunteers lactating mothers with no known occupational exposure to these compounds who live in the province of Granada (Spain). The analytical method developed here may be useful for the development of more in-depth studies on the prenatal exposure and biomonitoring of these commonly used UV-filters.

Analysis of quinolone antibiotic derivatives in sewage sludge samples by liquid chromatography-tandem mass spectrometry: comparison of the efficiency of three extraction techniques.

This work presents a comparison of three extraction techniques -ultrasound-assisted extraction (USE), microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE) - and evaluates their efficiency in the determination of quinolone antibiotics in sewage sludge samples. Extraction parameters for each technique were optimized using design of experiments, and the compounds were detected and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), operating in positive electrospray ionization (ESI) mode. The use of two selected reaction monitoring transitions for each compound allowed simultaneous quantification and identification in one run. Analytes were separated in less than 10 min. Marbofloxacin and cincophen were used as surrogates for amphoteric and acid quinolones, respectively. The limits of detection (LODs) were between 2 and 5 ng g(-1), and the limits of quantification (LOQs) were between 4 and 18 ng g(-1) for the various analytes. The inter- and intra-day variability was <7%. Due to the absence of certified reference materials (CRMs), the method was validated using matrix-matched calibration and a recovery assay with spiked samples. Recovery rates were between 97.9% and 104.8%. Statistical comparison demonstrated no significant differences between the three extraction techniques. The methods were successfully applied for the determination of quinolones in sewage sludge samples collected from different wastewater treatments plants (WWTPs) located in the province of Granada (Spain). The analytical methods developed here may be useful for the development of more in-depth studies on the occurrence and fate of these commonly used pharmaceuticals in WWTPs and in the environment.

Removal and degradation characteristics of quinolone antibiotics in laboratory-scale activated sludge reactors under aerobic, nitrifying and anoxic conditions.

This work describes the removal of 6 quinolone antibiotics from wastewaters under different redox conditions (aerobic, nitrifying and anoxic) through batch experiments in laboratory scale activated sludge reactors using mixed liquor from a membrane bioreactor pilot plant (MBR). The main removal pathways for antibiotics from wastewaters involved in each treatment are described. Mass balances indicated that sorption on sludge played a dominating role in the elimination of antibiotics. Sorption potential depended on the redox conditions, being lower in nitrifying (Kd, 414-876 L kg(-1)) and anoxic (Kd, 471-930 L kg(-1)) sludge in comparison with aerobic sludge (Kd, 534-1137 L kg(-1)). Kd was higher for piperazinylic quinolones. Redox conditions also influenced biodegradation, a secondary pathway, which followed first-order kinetics with degradation rates constants ranging from 1.8·10(-3) to 8.2·10(-3) h(-1). Biodegradation rates under anoxic conditions were negligible. The experimental results have also demonstrated much higher removal efficiency by biodegradation (36.2-60.0%) under nitrifying conditions in comparison with aerobic conditions (14.9-43.8%). The addition of allylthiourea, an ammonia monooxygenase inhibitor, inhibited nitrification completely and reduced significantly the biodegradation of target antibiotics (16.5-29.3%). The residual biodegradation in the presence of allylthiourea may be due to the activity of heterotrophs in the enriched nitrifier culture. The removal of the selected antibiotics under the studied redox conditions depended significantly on the bacteria composition of the sludge. These results suggest that despite the known persistence of this group of antibiotics it is possible to enhance their degradation using nitrifying conditions, which at adequate working conditions as high SRT, typical in MBR, become a promising alternative for improving quinolones removal from environment.

Removal of quinolone antibiotics from wastewaters by sorption and biological degradation in laboratory-scale membrane bioreactors.

Laboratory-scale batch experiments were developed to investigate the main removal routes for 6 commonly found quinolones (ciprofloxacin, moxifloxacin, norfloxacin, ofloxacin, pipemidic acid, and piromidic acid), in wastewaters from a wastewater treatment plant, at µg L(-1) levels in an aerobic sludge system from a membrane bioreactor (MBR) pilot plant. It was demonstrated that sorption and biotransformation were the main removal routes for the target antibiotics over other possible pathways, as volatilization or hydrolysis, under the experimental conditions. Mass balances indicated that sorption on sludge played a dominant role in the elimination of antibiotics from waters. The sorption coefficient K(d) depended strongly on temperature and on the quinolone type and were higher at lower temperatures and for piperazinylic quinolones. K(d) values were between 516 and 3746 L kg(-1) in the temperature range of 9-38°C. Higher mixed liquor suspended solids (MLSS) increased quinolone removal efficiency mainly by sorption. Quinolone biodegradation constituted a secondary pathway, and could be described by first-order kinetics with degradation-rate constants ranging from 8.0 × 10(-4)h(-1) to 1.4 × 10(-2)h(-1) within the same temperature range and MLSS from 7000 to 15,000 mg L(-1). Biodegradation depended on the MLSS and temperature, but also on the initial chemical oxygen demand (COD). Higher biodegradation rates were observed at higher MLSS and temperature, as well as at low initial COD. Ciprofloxacin and moxifloxacin registered the highest biodegradation percentages (52.8% and 47.2%, respectively, at 38°C and 15,000 mg L(-1) MLSS), which is evidence that, despite the known persistence of this group of antibiotics and removal from waters mainly by sorption, it was possible to improve their removal by biodegradation, with an appropriate selection of conditions and control of process variables, as a preliminary step towards the elimination of these antibiotics from the environment. Further research is needed on the possibilities of removing sorbed antibiotics from sludge.

Improved sample treatment for the determination of bisphenol A and its chlorinated derivatives in sewage sludge samples by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry.

A selective, sensitive, robust and accurate method for the determination of bisphenol A (BPA) and its chlorinated derivatives in sewage sludge samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is presented. Prior to instrumental analysis, an extraction procedure using pressurized liquid extraction (PLE) was carried out in order to obtain the highest recoveries and improve sensitivity. After LC separation, the MS conditions, in negative atmospheric pressurized chemical ionization (APCI) mode, were individually optimized for each analyte to obtain maximum sensitivity in the selected reaction monitoring (SRM) mode. The use of two reactions for each compound allowed simultaneous quantification and identification in one run. The analytes were separated in less than 6 min. BPA-d(16) was used as internal standard. The limits of detection of the method ranged from 4 to 8 ng g(-1) and the limits of quantification from 14 to 26 ng g(-1), while inter- and intra-day variability was under 6% in all cases. Due to the absence of certified materials, the method was validated using matrix-matched calibration and a recovery assay with spiked samples. Recovery rates ranged from 97.7% to 100.6%. The method was satisfactorily applied for the determination BPA and its chlorinated derivatives in sewage sludge samples collected from wastewater treatment plants (WWTPs) located in the province of Granada (Spain). The sludge samples came from a conventional activated sludge (AS) plant and from a membrane bioreactor (MBR) pilot plant.

Analysis of bisphenol A and its chlorinated derivatives in sewage sludge samples. Comparison of the efficiency of three extraction techniques.

This paper presents a comparison of three extraction techniques--ultrasound-assisted extraction, microwave-assisted extraction and pressurized liquid extraction--in order to evaluate their efficiency in the determination of bisphenol A and its chlorinated derivatives in sewage sludge samples. Extraction parameters for each technique were accurately optimized to achieve the highest recoveries. The selected compounds were detected and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), operating in negative atmospheric pressure chemical ionization (APCI) and in multiple reaction monitoring (MRM) mode. The analytes were separated in less than 6 min. BPA-d16 was used as internal standard. Three selective, sensitive, robust and accurate analytical methods were developed. The limits of detection (LODs) of the methods ranged from 2 to 9 ng g⁻¹ and the limits of quantification (LOQs) from 8 to 26 ng g⁻¹, while inter- and intra-day variability was under 6% in all cases. Given the absence of certified reference materials (CRMs), the methods were validated separately by using matrix-matched calibration and recovery assays with spiked samples. Recovery rates ranged from 97.7% to 103.1%. The sewage sludge samples used for experiments were collected from two different wastewater treatments plants (WWTPs) located in the province of Granada (South-East Spain). The statistical comparison of the methods demonstrated no statistically significant differences between the extraction techniques for the determination of BPA and chlorinated derivatives in sludge samples.