Supercritical fluid extraction of microbial phospholipid fatty acids from activated sludge

Supercritical carbon dioxide (scCO₂) extraction was applied for the determination of microbial phospholipid fatty acids (PLFA) in activated sludge. Quantification was performed by using gas chromatography–mass spectrometry (GC–MS). The highest extraction yields of PLFA, at a concentration of 7.28 nmol/mg-dry activated sludge, was obtained at a temperature of 80 °C, pressure of 25 MPa and 10% (v/v) methanol for a 15-min extraction time. ScCO₂ extraction results obtained in these conditions were comparable with those obtained by liquid organic solvent extraction (LSE) based on diversity and equalibility indices. The repeatability test showed that the relative standard deviation values were less than 13%. The experimental results show that the scCO₂ extraction saves time and uses much less organic solvent. In addition, scCO₂ extraction is a promising and alternative method for the analysis of microbial community structure in environmental assessment using the PLFA profile.     

Identification of ubiquinones and menaquinones in activated sludge by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A sensitive analytical method has been developed for identification of ubiquinones (UQ-n(Hx)) and menaquinones (MK-n(Hx)) in activated sludge by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry in negative mode (LC-NI-APCI-MS). Extraction and clean-up of samples were carried out on Sep-Pak Plus Silica solid-phase extraction cartridges. Complete separation of quinones was achieved with an ODS analytical column and using isopropyl ether-methanol (17:83, v/v) as the mobile phase. The compositions of ubiquinones and menaquinones were determined directly using combined information on retention time, the molecular ion mass and fragment ion masses. The lowest instrument quantitative detection limits (LODinst) for UQ-6, UQ-10, and Vitamin K1 were estimated to be 0.4, 4 and 0.12 ng (S/N = 10) using LC-NI-APCI-MS in SIM mode, and the lowest method detection limits (LODmeth) achieved by spiking experiment were estimated to be 0.2, 2 and 0.06 microg/g for UQ-6, UQ-10 and Vitamin K1, respectively. On the other hand, the LODinst for UQ-6, UQ-10, and Vitamin K1 were estimated to be 10, 100 and 2 ng (S/N = 10) using LC-NI-APCI-MS in full-scan mode, and the LODmeth were estimated to be 7, 60 and 1.2 microg/g for UQ-6, UQ-10, and Vitamin K1, respectively. Both LC-NI-APCI-MS and LC-UV/DAD were applied in the analysis of an activated sludge extract. UQ-n (n = 6-10), MK-n (n = 6-10), MK-n(H2) (n = 7-10), MK-n(H4) (n = 8-9) and MK-8(H6) were detected by LC-NI-APCI-MS, while UQ-6, UQ-7, MK-7(H), MK-9 and MK-10(H2) were not found by LC-UV/DAD. These results suggest that LC-NI-APCI-MS is more sensitive than LC-UV/DAD for the analysis of quinones in environmental samples such as sediment, activated sludge and bio-film in biological processes and other aquatic environments.     

Pressurized liquid extraction in the analysis of food and biological samples

Originally, the use of the pressurized liquid extraction technique (PLE) was mainly focused on the extraction of environmental pollutants present in soil matrices, sediments, and sewage sludge. However, more recently the distinct advantages of this technique are being exploited in diverse areas, including biology, and the pharmaceutical and food industries. The aim of the present review is to explore recent analytical applications of this extraction technique (PLE) in the extraction of contaminant compounds and matrix components in food and biological samples, placing special emphasis on the strategies followed to obtain a rapid, selective, efficient and reliable extraction process.     

Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge

Supercritical CO2 (scCO2) extraction, with methanol as modifier, was applied to the determination of ubiquinones and menaquinones in activated sludge. Four ubiquinones and 12 menaquinones species were identified based on retention time and UV spectrum in 0.1g dried activated sludge. The optimum extraction conditions were at a pressure of 25 MPa, a temperature of 55 degrees C, and 10% (v/v) methanol for 15 min. At this condition, the concentrations of extracted ubiquinones and menaquinones were found to be 0.181 and 0.326 micromol/g-dry-cell, respectively. The results were comparable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Furthermore, the method was evaluated in term of repeatability, which resulted in an RSD of < or =10%. The experimental results have demonstrated the technique to be simple, fast, and with less consumption of organic solvents. This work shows the potential application of supercritical CO2 extraction to microbial community analysis using quinone profile.     

Simple approach for the determination of brominated flame retardants in environmental solid samples based on solvent extraction and solid-phase microextraction followed by gas chromatography-tandem mass spectrometry

A viable approach for the analysis of polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs) in sewage sludge samples is presented in this paper. The proposed method combines ultrasound-assisted extraction (UAE) of the solid sample and headspace solid-phase microextraction (HS-SPME) of the obtained extract, followed by gas chromatography coupled to tandem mass spectrometry (GC-MS-MS) analysis. Different parameters affecting the extraction process are evaluated and optimized. The addition of a small amount of Florisil to the sample during UAE provides a significant improvement of the chromatographic background and, at the same time, a much more efficient HS-SPME. Extensive method validation is performed using real sewage sludge samples. The proposed method exhibits good performance in terms of linearity and precision, with recoveries exceeding 92% and limits of detection in the sub ng g(-1) level. Practical applicability is demonstrated through the analysis of real contaminated sewage sludge and sediment samples in which some of the target PBDEs are detected and quantified. This proposed combined methodology represents a large time-saving when compared to other classic multi-step solvent extraction methods and it constitutes a suitable approach for the analysis of the target compounds in environmental complex solid samples.     

Pyrolysis-capillary gas chromatography-mass spectrometry for the determination of polyvinyl chloride traces in solid environmental samples

A novel method based on pyrolysis-capillary gas chromatography-mass spectrometry (CGC-MS) was developed for the quantitative analysis of polyvinylchloride (PVC) in solid environmental samples like sludge and dust. The samples are extracted and the extract is fractionated by solid-phase extraction (SPE). Possibly interfering biological and frequently occuring synthetic polymers are removed by this clean-up. The final extract is analyzed by pyrolysis-CGC-MS. Selective detection of PVC is performed by using specific markers in the pyrogram. Quantitation is done on naphthalene. Good linearity was obtained in a range from 0.5 to 100 microg applied to the pyrolyser. The limit of quantitation (LOQ) in sludge and dust samples is 10 mg/kg dry mass. A correlation between PVC and phthalates was made for sewage sludge samples.     

Determination of Cd in sonicate slurries and leachates of biological and environmental materials by FI-CV-AAS

An analytical method for Cd analysis in solid samples which combines the ultrasonic slurry formation with cold vapour generation and atomic absorption spectrometry is described. The samples are suspended in HCl and sonicated until homogeneous and reduced particle size slurry formation. Several aspects were studied: acidity of the medium, sonication time, and slurry formation in different matrices. The procedure described permits the use of direct calibration, with KCN addition as masking agent of interfering ions (Cu, Pb, Ni and Zn) present in the environmental matrices. Supernatant analysis of these last samples experimentally shown that preparation of the suspension with 6 mol l(-1) HCl concentration led to quantitative extraction of Cd. Biological materials analysis needed the use of the standard addition calibration method due to the high matrix effect observed. Supernatant analysis in biological samples does not give a total Cd recovery for all of them. The detection limits observed for Cd were 0.05 and 0.2 mug l(-1) for supernatant and slurry analysis respectively in environmental samples, while in biological samples were 0.2 and 0.6 mug l(-1) for supernatant and slurry analysis, respectively. In all case the better precision was obtained for supernatant analysis (3-6%) than slurry analysis (6-12%). The results obtained by analysing different reference materials (sewage sludge, city waste incineration, Antarctic krill and human hair) showed good agreement with the certified value confirming the validity of such a method for Cd determination instead to wet digestion procedures.     

Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.     

Optimisation of caffeine and antidepressants extraction from sediments and sewage sludge using experimental designs

This study proposes a microwave-assisted method for the simultaneous extraction of highly prescribed antidepressants (citalopram, venlafaxine, fluoxetine, sertraline and amitriptyline) and caffeine from sediments and sewage sludge for subsequent HPLC-PDA analysis. Because the sludge and sediment matrices have high contents of organic material, they strongly interact with the analytes and hinder extraction. Thus, a carefully optimised analytical methodology is required for quantitative extraction. A simplex-centroid design was applied to optimise the solvent composition, and a three-factor central composite rotational design was used to optimise the extraction protocol with regards to pH, amount of solvent and processing time. Samples (in triplicates) were fortified with a standard mixed solution of all the analytes and extracted according to the experimental design in each study. The extraction steps included: 30 s vortexing, 20 s microwave heating at 10 W, cooling to room temperature (25°C) in an ultrasonic bath for 60 s, 2 min centrifugation at 2000 rpm, and filtration. Analysis of variance and lack-of-fit tests were used to assess the significance of data fitting at 95% confidence. The desirability function was the optimisation tool used to obtain the ideal extraction conditions. As a result, a binary mixture of methanol and acetonitrile in 45:55 and 53:47 (v/v) ratios was indicated as the optimum solvent composition for the simultaneous extraction of all the target drugs from the sludge and sediment, respectively. The optimised extraction conditions were: 3 extraction cycles with 4 mL of solvent at pH 3 for sewage sludge extraction and 4 extraction cycles with 3 mL of solvent at pH 11 for sediment extraction. Further, low recoveries were obtained for extractions from sediment as compared to sludge indicating strong interaction of antidepressants and caffeine with the acidic organic components of sediments. It was found that the optimisation of pH of the extraction phase was crucial for the efficient extraction of the analytes from these environmental matrices.     

Statistical tools for the optimization of a highly reproducible method for the analysis of polycyclic aromatic hydrocarbons in sludge samples

The aim of this study is to develop and optimize an analytical method for the determination of 14 priority PAHs in sludge samples based on Accelerated Solvent Extraction (ASE) coupled to RP-HPLC/fluorescence detection. Statistical tools were used to demonstrate the influence of the parameters during the optimization steps. The final parameters were selected to provide analytical errors statistically as low as possible. First, couples of excitation/emission detection wavelengths were tested, and some were finally selected to provide errors lower than 2%. It was then demonstrated that PAH extraction efficiencies are not statistically influenced by the ASE parameters. It was also found that the ASE extraction from sludge samples provides statistically similar results to those obtained with traditional Soxhlet extraction, but with a lower reproducibility error. After optimization, the accuracy of the method was validated with a certified sludge. In conclusion, an optimized analytical procedure has been proposed to monitor PAHs during lab-scale experiments requiring highly repeatable and accurate results from a low sample volume contaminated by PAHs at trace levels.     

State of the art of environmentally friendly sample preparation approaches for determination of PBDEs and metabolites in environmental and biological samples: A critical review

Green chemistry principles for developing methodologies have gained attention in analytical chemistry in recent decades. A growing number of analytical techniques have been proposed for determination of organic persistent pollutants in environmental and biological samples. In this light, the current review aims to present state-of-the-art sample preparation approaches based on green analytical principles proposed for the determination of polybrominated diphenyl ethers (PBDEs) and metabolites (OH-PBDEs and MeO-PBDEs) in environmental and biological samples. Approaches to lower the solvent consumption and accelerate the extraction, such as pressurized liquid extraction, microwave-assisted extraction, and ultrasound-assisted extraction, are discussed in this review. Special attention is paid to miniaturized sample preparation methodologies and strategies proposed to reduce organic solvent consumption. Additionally, extraction techniques based on alternative solvents (surfactants, supercritical fluids, or ionic liquids) are also commented in this work, even though these are scarcely used for determination of PBDEs. In addition to liquid-based extraction techniques, solid-based analytical techniques are also addressed. The development of greener, faster and simpler sample preparation approaches has increased in recent years (2003–2013). Among green extraction techniques, those based on the liquid phase predominate over those based on the solid phase (71% vs. 29%, respectively). For solid samples, solvent assisted extraction techniques are preferred for leaching of PBDEs, and liquid phase microextraction techniques are mostly used for liquid samples. Likewise, green characteristics of the instrumental analysis used after the extraction and clean-up steps are briefly discussed.     

Effects of sludge retention time on membrane fouling and microbial community structure in a membrane bioreactor

In this study, the effect of sludge retention time (SRT) on membrane bio-fouling was investigated in a membrane bioreactor (MBR) equipped with a sequential anoxic/anaerobic reactor. Specific cake resistance (α), trans-membrane pressure (TMP), mixed liquor suspended solids (MLSS), particle sizes, extracellular polymer substances bound in sludge (bound-EPS) and their correlations with membrane bio-fouling were studied at different SRTs. As SRT decreased to 20 days, the bound-EPS per unit of biomass increased, and consequently, the value of α increased, which resulted in the rise of TMP. However, the reduction of the bound-EPS content was relatively small as compared to a significant decrease in the value of α at longer SRTs (above 60 days). These observations suggest that colloidal particles significantly contribute to membrane bio-fouling. In addition, the diversity of the microbial community structure of activated sludge in the MBRs was observed using the respiratory quinone profile. The ubiquinone species containing UQ-8, belonging to the class β-Proteobacteria type were the major constituents of the microbial community structure. The mole fraction of menaquinone MK-6, -7 and -8(H2) increased as SRT increased. Thus, the results of this study indicate that growth of microorganisms belonging to the δ- and ?-subclass of Proteobacteria as well as the members of the Cytophaga–Flavobacterium cluster increased at longer SRTs.     

Extraction and clean-up strategies for the analysis of poly- and perfluoroalkyl substances in environmental and human matrices

The rapidly expanding field of per- and polyfluorinated alkyl substances (PFASs) research has resulted in a wide range of analytical methodologies to determine the human and environmental exposure to PFASs. This paper reviews the currently applied techniques for sample pre-treatment, extraction and clean-up for the analysis of ionic and non-ionic PFASs in human and environmental matrices. Solid phase extraction (SPE) is the method of choice for liquid samples (e.g. water, blood, serum, plasma), and may be automated in an on-line set-up for (large volume) sample enrichment and sample clean-up. Prior to SPE, sample pre-treatment (filtration or centrifugation for water or protein precipitation for blood) may be required. Liquid-liquid extraction can also be used for liquid samples (and does not require above mentioned sample pretreatment). Solid-liquid extraction is the commonly applied method for solid matrices (biota, sludge, soil, sediment), but automation options are limited due to contamination from polytetrafluorethylene tubings and parts applied in extraction equipment. Air is generally preconcentrated on XAD-resins sandwiched between polyurethane foam plugs. Clean-up of crude extracts is essential for destruction and removal of lipids and other co-extractives that may interfere in the instrumental determination. SPE, (fluorous) silica column chromatography, dispersive graphitized carbon and destructive methods such as sulphuric acid or KOH treatment can be applied for clean-up of extracts. Care should be taken to avoid contamination (e.g. from sample bottles, filters, equipment) and losses of PFASs (e.g. adsorption, volatilization) during sampling, extraction and clean-up. Storage at -20 degrees C is generally appropriate for conservation of samples.     

Determination of isoflavones and coumestrol in river water and domestic wastewater sewage treatment plants

Phytoestrogens activate a biological response in vertebrata where they can mime or modulate the action of endogenous estrogens. For this reason they have been subjected to several studies about their physiological effects on humans and many analytical methodologies for their determination in food matrices and physiological fluids have been developed. On the contrary, little information can be found in literature about the presence of isoflavones and coumestrol in the environment, even if it is known that this may have significance. In the present study we investigated the presence of nine selected free and conjugated phytoestrogens in environmental water. A liquid chromatography-mass spectrometry (LC-MS/MS) based analytical methodology was developed and employed for detection of target compounds in surface water and wastewater.The methodology uses solid-phase extraction, followed by high performance liquid chromatography coupled to tandem mass spectrometry using an electrospray (ESI) interface operating in positive ion mode (LC-ESI-MS/MS). The extraction was made with 200 mg, 6 mL OASIS HLB^® cartridges. Recoveries for the selected compounds were in the 67-97% range for all the considered analytes. The method was employed for environmental monitoring. Samples of river water and wastewater collected over a 4-month period were analyzed with the developed procedure. Results showed the presence of isoflavones in most of the samples analyzed. Average concentration of target analytes found in wastewater sewage treatment plant influent ranged from 454 to 12 ng/L. In effluent water and river water the analytes were present at lower concentration.     

Multi‐residue method for the analysis of pharmaceutical compounds in sewage sludge,compost and sediments by sonication‐assisted extraction and LC determination

A method for the simultaneous determination of 16 pharmaceutical compounds in three types of sewage sludge (primary, secondary and anaerobically digested dehydrated sludge), compost and sediment samples is described. Pharmaceutical compounds evaluated were nonsteroidal anti‐inflammatory drugs (acetaminophen, diclofenac, ibuprofen, ketoprofen, naproxen and salicylic acid), antibiotics (sulfamethoxazole and trimethoprim), an anti‐epileptic drug (carbamazepine), a β‐blocker (propranolol), a nervous stimulant (caffeine), estrogens (17α‐ethinylestradiol, 17β‐estradiol, estriol and estrone) and lipid regulators (clofibric acid, metabolite of clofibrate and gemfibrozil). The method is based on the ultrasonic‐assisted extraction, clean‐up by SPE and analytical determination by HPLC with diode array and fluorescence detectors. The best extraction recoveries were achieved in a three‐step extraction procedure with methanol and acetone as extraction solvents. Extraction recoveries of several pharmaceutical compounds as caffeine were highly dependent on the type of sample evaluated. The applicability of the method was tested by analyzing primary, secondary and anaerobically digested dehydrated sludge, compost and sediment samples from Seville (Southern Spain). Ten of the sixteen pharmaceutical compounds were detected in sludge samples and five in compost and sediment samples. The highest concentration levels were recorded for ibuprofen in sewage samples, whereas salicylic acid and 17α‐ethinylestradiol were detected in all of the samples analyzed.     

Determination of nonylphenol and nonylphenol ethoxylates in environmental solid samples by ultrasonic-assisted extraction and high performance liquid chromatography-fluorescence detection

A simple and rapid analytical method for the determination of nonylphenol (NP) and nonylphenol ethoxylates (NPEOx) in solid environmental samples has been developed. This method combines an ultrasonic-assisted extraction procedure in small columns and an enrichment step onto C(18) solid-phase extraction cartridges prior to separation using HPLC with fluorescence detection. Method optimization was carried out using soil samples fortified at different concentration levels (from 0.1 to 100 microg/g). Under optimum conditions, 2g of soil was placed in small glass columns and extraction was performed assisted by sonication (SAESC) at 45 degrees C in two consecutive steps of 15 min using a mixture of H(2)O/MeOH (30/70). The obtained extracts were collected, loaded onto 500 mg C(18) cartridges, and analytes were eluted with 3 x 1 ml of methanol and 1 ml of acetonitrile. Finally, sample extracts were evaporated under a nitrogen stream, redissolved in 500 microl H(2)O/AcN (50/50), and passed though a 0.45 microm nylon filter before final determination by HPLC-FL. The developed procedure allowed to achieve quantitative recoveries for NP and NPEOx, and was properly validated. Finally, the method was applied to the determination of these compounds in soils and other environmental solid samples such as sediments, compost and sludge.     

Determination of glyoxal and methylglyoxal in environmental and biological matrices by stir bar sorptive extraction with in-situ derivatization

Stir bar sorptive extraction with in-situ derivatization using 2,3-diaminonaphthalene (DAN) followed by liquid desorption and high performance liquid chromatography with diode array detection (SBSE(DAN)in-situ-LD-HPLC-DAD) was developed for the determination of glyoxal (Gly) and methylglyoxal (MGly) in environmental and biological matrices. DAN proved very good specificity as in-situ derivatising agent for Gly and MGly in aqueous media, allowing the formation of adducts with remarkable sensitivity, selectivity and the absence of photodegradation. Assays performed on spiked (1.0 microg L(-1)) water samples, under convenient experimental conditions, yielded recoveries of 96.2+/-7.9% for Gly and 96.1+/-6.4% for MGly. The analytical performance showed good accuracy, suitable precision (<12.0%), low detection limits (15 ng L(-1) for Gly and 25 ng L(-1) for MGly adducts) and excellent linear dynamic ranges (r2>0.99) from 0.1 to 120.0 microg L(-1). By using the standard addition method, the application of the present method to tap and swimming-pool water, beer, yeast cells suspension and urine samples allowed very good performance at the trace level. The proposed methodology proved to be a feasible alternative for routine quality control analysis, showing to be easy to implement, reliable, sensitive and with a low sample volume requirement to monitor Gly and MGly in environmental and biological matrices.     

液相萃取-高效液相色谱-串联质谱联用测定污泥中的全氟化合物

全氟化合物是一种新型持久性有机污染物,污水处理厂是其一个主要污染来源。目前还没有建立起一种统一的污泥样品中全氟化合物的分析方法。本文报道了一种基于液相萃取和高效液相色谱-串联质谱联用技术测定污泥中的7种全氟烷基羧酸及其2种不饱和氟调酸前体物、2种全氟烷基磺酸及其5种磺酰胺衍生物前体物的方法。实验对萃取剂(甲醇)的pH值、超声萃取温度与时间、洗脱剂体积进行了优化,确定了中性溶剂、40℃下超声萃取10min,Envicarbon柱净化的前处理方法,并成功地应用于实际污泥样品中全氟化合物的测定。方法的回收率为74%～141%(不饱和氟调酸除外),线性范围为0.1～20μg/L(羧酸系列)及0.25～50μg/L(磺酸系列)内线性关系良好(r20.99),定量限为0.6～30μg/kg(干重)。内标物质的使用可有效消除环境基质引起的仪器离子抑制现象,使定量更加准确。     

Ultra-performance liquid chromatography/tandem mass spectrometry method for the determination of lercanidipine in human plasma

A simple, sensitive and rapid ultra-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Lercanidipine and the internal standard, nicardipine, were extracted from plasma by liquid-liquid extraction using tert-butyl methyl ether as the extraction solvent. UPLC analysis was performed isocratically on an AcQuity UPLC BEH C18 analytical column (2.1 x 50.0 mm i.d., particle size 1.7 microm). The mobile phase consisted of 70% acetonitrile in water containing 0.2% v/v formic acid and pumped at a flow rate of 0.30 mL/min. ESI in positive ion mode, with multiple reaction monitoring (MRM), was chosen for the detection of the analytes. The assay was linear over a concentration range of 0.05-30 ng/mL for lercanidipine with a limit of quantitation of 0.05 ng/mL. Quality control samples (0.05, 0.15, 15 and 25 ng/mL) in five replicates from five of analytical runs demonstrated intra-assay precision (% CV < or =7.3%), inter-assay precision (% CV < or =6.1%) and an overall accuracy (% relative error) of less than 6.2%. A run time of less than 1.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to quantify lercanidipine in human plasma covering a variety of pharmacokinetic or bioequivalence studies.