Open-air storage with and without composting as post-treatment methods to degrade pharmaceutical residues in anaerobically digested and dewatered sewage sludge.

Over a period of 12 months, the fate of three hormones, 12 antibiotics and 30 pharmaceutically active substances (PhACs) was investigated during open-air storage without and with composting of anaerobically digested and dewatered sewage sludge. The effect of oxidation conditions during storage on degradation of hormones and PhACs in the sludge biomass was also examined. Under summer and winter conditions in Uppsala County, Sweden, two field-scale sludge windrows were constructed: open-air storage of sewage sludge windrow without composting (NO-COM)) and open-air storage windrow with composting (COM). NO-COM achieved effective removal of ∑Hormones (85%) and ∑Antibiotics (95%), but lower removal of ∑PhACs (34%), during the study year. The top layers of the sludge pile had significantly lower concentrations of ∑PhACs (3100-5100 ng/g ash) than deeper layers (8000-11,000 ng/g ash). After one year of composting, the degradation in the COM windrow resulted in concentrations of ∑Hormones (

N-Glycosylation profiling of intact target proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS.

Glycosylation influences the structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. The types and abundance of glycans on glycoproteins can vary due to diseases such as cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role glycans play in modulating protein function, glycosylation analysis could lead to the discovery of novel biomarkers and is of prime importance in controlling the quality of glycoprotein biopharmaceuticals. Here, we present a method for the identification and quantification of glycoforms directly on intact proteins, after immunoaffinity purification from biological fluids. The method was validated and applied to serum transferrin and the biopharmaceutical trastuzumab. The accuracy of the method, expressed as the relative error (RE), ranged from 2.1 (at high concentrations) to 7.9% (at low concentrations), and intra- and inter-day precision, expressed as relative standard deviation (RSD), was 3.2 and 8.2%, respectively. The sensitivity and linearity of the method were suitable for serum analysis and the LOQ was calculated to be 3.1 and 4.4 µg mL-1 for transferrin (TFN) and trastuzumab (TRA), respectively. Its application to transferrin from five healthy human serum samples yielded concentrations between 1.61 and 3.17 mg mL-1, which are in agreement with blood reference levels. In parallel, the structure of the identified glycans was determined by ion mobility spectrometry coupled with tandem mass spectrometry. No chromatographic separation was required and sample preparation was performed in a semi-automatic manner, facilitating the handling of up to 12 samples at a time. This method should be useful for clinical laboratories and for the quality control of large batches of biopharmaceuticals.

Using magnetic core-shell nanoparticles coated with an ionic liquid dispersion assisted by effervescence powder for the micro-solid-phase extraction of four beta blockers from human plasma by ultra high performance liquid chromatography with mass spectrometry detection.

In this work, a novel, efficient, and green sorbent, SiO2 @Fe3 O4 has been created and functionalized with 1-butyl-3-methylimidazolium hexafluorophosphate as an ionic liquid. This sorbent was applied for microextraction of four beta blockers, propranolol, metoprolol, atenolol, and alprenolol with bupivacaine as internal standard from human plasma followed by liquid chromatography with mass spectrometric detection. A mixture of sodium bicarbonate and sodium dihydrogen phosphate was used as an extractant dispersive agent (effervescent power) to enhance the interaction between the magnetic sorbent and analytes. Main affecting parameters on microextraction and elution were optimized. Figures of merit for dispersive solid phase extraction with ionic liquid coated magnetic nanoparticles assisted by effervescent powder were calculated under the optimized conditions. The detection limits for propranolol, metoprolol, atenolol, and alprenolol were found at 0.33, 0.62, 0.03, and 0.44 ng/mL, respectively. For all analytes, good linearity was obtained. Intra- (n = 5) and interday (n = 10) precision were both under 6.3% while the preconcentration factors were obtained in the range between 15-18. The extraction efficiencies for each analyte ranged from 75 to 91%. The method was successfully applied for determination of trace amounts of the beta blockers in human plasma samples.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples.

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin, C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders.

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid.

N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS.

It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 µL of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups.

Benzothiazole, benzotriazole, and their derivates in clothing textiles--a potential source of environmental pollutants and human exposure.

Textiles play an important role in our daily life, and textile production is one of the oldest industries. In the manufacturing chain from natural and/or synthetic fibers to the final clothing products, the use of many different chemicals is ubiquitous. A lot of research has focused on chemicals in textile wastewater, but the knowledge of the actual content of harmful chemicals in clothes sold on the retail market is limited. In this paper, we have focused on eight benzothiazole and benzotriazole derivatives, compounds rated as high production volume chemicals. Twenty-six clothing samples of various textile materials and colors manufactured in 14 different countries were analyzed in textile clothing using liquid chromatography tandem mass spectrometry. Among the investigated textile products, 11 clothes were for babies, toddlers, and children. Eight of the 11 compounds included in the investigation were detected in the textiles. Benzothiazole was present in 23 of 26 investigated garments in concentrations ranging from 0.45 to 51 µg/g textile. The garment with the highest concentration of benzothiazole contained a total amount of 8.3 mg of the chemical. The third highest concentration of benzothiazole (22 µg/g) was detected in a baby body made from "organic cotton" equipped with the "Nordic Ecolabel" ("Svanenmärkt"). It was also found that concentrations of benzothiazoles in general were much higher than those for benzotriazoles. This study implicates that clothing textiles can be a possible route for human exposure to harmful chemicals by skin contact, as well as being a potential source of environmental pollutants via laundering and release to household wastewater.

Community interactions modify the effects of pharmaceutical exposure: a microcosm study on responses to propranolol in Baltic Sea coastal organisms.

This study investigated the uptake and effects of a common human pharmaceutical, propranolol, on the structure and function of a coastal Baltic Sea model community consisting of macroalga (Ceramium tenuicorne), mussels (Mytilus edulis trossulus), amphipods (Gammarus spp.), water and sediment. The most sensitive species, the mussel, was affected to the same extent as in previous single species studies, while the effects on the amphipod and alga were smaller or even positive compared to experiments performed in less complex test systems. The observed cascade of beneficial effects was a result of inter-specific species interactions that buffered for more severe effects. The poor condition of the mussel led to a feeding shift from alga to mussel by the amphipods. The better food quality, due to the dietary shift, counteracted the effects of the exposure. Less amphipod grazing, together with increased levels of nutrients in the water was favourable for the alga, despite the negative effects of propranolol. This microcosm study showed effects on organisms on different organizational levels as well as interactions among the different components resulting in indirect exposure effects of both functional and structural nature. The combination of both direct and indirect effects would not have been detected using simpler single- or even two-species study designs. The observed structural changes would in the natural environment have a long-term influence on ecosystem function, especially in a low-biodiversity ecosystem like the Baltic Sea.

Quinolines in clothing textiles--a source of human exposure and wastewater pollution?

A production process in which the use of various types of chemicals seems to be ubiquitous makes the textile industry a growing problem regarding both public health as well as the environment. Among several substances used at each stage, the present study focuses on the quinolines, a class of compounds involved in the manufacture of dyes, some of which are skin irritants and/or classified as probable human carcinogens. A method was developed for the determination of quinoline derivatives in textile materials comprising ultrasound-assisted solvent extraction, solid phase extraction cleanup, and final analysis by gas chromatography/mass spectrometry. Quinoline and ten quinoline derivatives were determined in 31 textile samples. The clothing samples, diverse in color, material, brand, country of manufacture, and price, and intended for a broad market, were purchased from different shops in Stockholm, Sweden. Quinoline, a possible human carcinogen, was found to be the most abundant compound present in almost all of the samples investigated, reaching a level of 1.9 mg in a single garment, and it was found that quinoline and its derivatives were mainly correlated to polyester material. This study points out the importance of screening textiles with nontarget analysis to investigate the presence of chemicals in an unbiased manner. Focus should be primarily on clothing worn close to the body.

Determination of benzothiazole and benzotriazole derivates in tire and clothing textile samples by high performance liquid chromatography-electrospray ionization tandem mass spectrometry.

A high performance liquid chromatography-tandem mass spectrometry method utilizing electrospray ionization in positive and negative mode has been developed for the separation and detection of benzothiazole and benzotriazole derivates. Ultra-sonication assisted solvent extraction of these compounds has also been developed and the overall method demonstrated on a selected clothing textile and an automobile tire sample. Matrix effects and extraction recoveries, as well as linearity and limits of detection have been evaluated. The calibration curves spanned over more than two orders of magnitude with coefficients of correlation R(2)>0.99 and the limits of detection and the limits of quantification were in the range 1.7-58pg injected and 18-140pg/g, respectively. The extraction recoveries ranged between 69% and 102% and the matrix effects between 75% and 101%. Benzothiazole and benzotriazole derivates were determined in the textile sample and benzothiazole derivatives determined in the tire sample with good analytical performance.

Glycosylation profiling of therapeutic antibodies in serum samples using a microfluidic CD platform and MALDI-MS.

The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.

Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics.

Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process.

High-throughput profiling of N-linked oligosaccharides in therapeutic antibodies using a microfluidic CD platform and MALDI-MS.

Recombinant therapeutic antibodies have shown a great potential in the treatment of several severe medical conditions such as cancer and autoimmune diseases. Glycosylation plays a critical role in biological activity and immunogenic properties of these compounds. The analysis of glycan profiles is therefore necessary in many steps of the development and manufacturing process from early development to quality control of the final product. In this paper, a fast, parallel, and robust sample preparation platform for glycosylation profiling using a microfluidic compact disc (CD) is presented. A sequential process including selective capture of antibody from a crude cell supernatant using protein A beads, enzymatic release of glycans, purification with a graphitized carbon black column, and crystallisation for MALDI-TOF analysis were performed on the CD. Glycosylation profiles of an antibody intended for therapeutic use produced in two different cell lines were compared.

Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system.

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.

Physiological effects of diclofenac, ibuprofen and propranolol on Baltic Sea blue mussels.

Pharmaceuticals are constantly dispersed into the environment and little is known of the effects on non-target organisms. This is an issue of growing concern. In this study, Baltic Sea blue mussels, Mytilus edulis trossulus, were exposed to diclofenac, ibuprofen and propranolol, three pharmaceuticals that are produced and sold in large quantities and have a widespread occurrence in aquatic environments. The mussels were exposed to pharmaceuticals in concentrations ranging from 1 to 10,000 microg l(-1). The pharmaceuticals were added both separately and in combination. Mussels exposed to high concentrations of pharmaceuticals showed a clear response compared to controls. Firstly, they had a significantly lower scope for growth, which indicates that the organisms had a smaller part of their energy available for normal metabolism, and secondly, they had lower byssus strength and lower abundance of byssus threads, resulting in reduced ability to attach to the underlying substrate. Mussels exposed to lower concentrations showed tendencies of the same results. The concentration of diclofenac and propranolol was quantified in the mussels using both liquid chromatography coupled to mass spectrometry (LC-MS). The measurements showed a significantly higher concentration in the organisms as compared to the water the mussels were exposed to; the uptake reached concentrations two orders of magnitudes higher than found in sewage treatment plant effluents. This study showed that common pharmaceuticals are taken up and negatively affect the physiology of a non-target species at levels of two to three orders of magnitudes higher than found in sewage treatment plant effluents.

Screening and quantification of pesticides in water using a dual-function graphitized carbon black disk.

A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS.

SALDI-MS signal enhancement using oxidized graphitized carbon black nanoparticles.

The signal intensity of low-molecular-weight compounds analyzed using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS) was significantly enhanced when oxidized graphitized carbon black (GCB) particles were used as the desorption/ionization surface. The surface of oxidized GCB contains more carboxylic acid groups than non-oxidized GCB. Carboxylic acid groups enhance the efficiency of the ionization process and the desorption of more hydrophobic compounds. A common pharmaceutical compound, propranolol, was successfully extracted from Baltic Sea blue mussels and quantified using oxidized GCB as the SALDI surface, whereas deuterated propranolol was used as the internal standard. The calibration curve showed a wide linear dynamic range of response (0.1-20 microg/mL) and good reproducibility (RSD < 10%). It was not possible to detect propranolol in Baltic Sea blue mussels when non-oxidized GCB was used as the SALDI surface.

Mu-trap for the SALDI-MS screening of organic compounds prior to LC/MS analysis.

A procedure for rapidly screening and quantitatively analyzing organic molecules is presented, in which a miniaturized solid-phase extraction (SPE) cartridge containing 0.6 mg of graphitized carbon black (the GCB-mu-trap) is used for sample pretreatment. Then surface-assisted laser desorption ionization time-of-flight mass spectrometry (SALDI-TOF-MS) screening is followed by liquid chromatography/mass spectrometry (LC/MS) for robust quantitative analysis of samples containing analytes of interest. Liquid samples with volumes up to 100 mL were extracted using the GCB-mu-trap, and SALDI screening was performed by transferring a few particles of the GCB 4 sorbent from the mu-trap onto a stainless steel plate. Analytes were then simply ionized and desorbed by irradiating the GCB 4 particles without any further pretreatment. GCB 4 was found to be an excellent surface for the SALDI analysis of small molecules, providing spectra with very clean backgrounds. The small size of the cartridge (micropipet filter tip) results in enrichment of the analytes on a small surface area, affording low SALDI-TOF-MS detection limits. Furthermore, the removal of just a few particles from the mu-trap does not significantly affect the subsequent quantitative determination. This approach offers considerable reductions in analytical costs by eliminating unnecessary SPE-LC/MS analyses.

Parallel nanoliter microfluidic analysis system.

A parallel nanoliter microfluidic analysis system based on capillary action, centrifugal force, and hydrophobic barriers is described. The precision of 112 parallel volume definition operations is determined to 0.75% CV at 200 nL using the individual sample introduction structure. For 20 nL, the actual measurement error is the dominating factor, with a combined error of 1.9% CV. Individual dispensing as well as dispensing through a common distribution channel is described. The volume definition precision for the common distribution channel is 1.6% CV for 200 nL. Unlike the dominating forces in microliter-sized channel systems, we describe hysteresis effects as exerting a major influence, which needs to be considered in order to control the operation and design of discrete nanoliter fluidics. Hydrophobic patches at the corners of the rectangular channel control corner-enhanced wicking. Excellent flow control of 1 and 2 nL/s is achieved using a predefined spin program.