Extractions with superheated water

As the temperature of liquid water is raised under pressure, between 100 and 374 degrees C, the polarity decreases markedly and it can be used as an extraction solvent for a wide range of analytes. Most interest has been in its application for the determination of PAHs, PCBs, and pesticides from environmental samples, where it gives comparable results to Soxhlet extraction but more rapidly and without the use of significant volumes of organic solvents. Unlike SPE, n-alkanes are not extracted unless the pressure is reduced and steam is used. Other applications have included the extraction of essential oils from plant material where it preferentially extracts the economically more important oxygenated components compared to steam distillation. The aqueous extract has been concentrated in a number of different methods (solvent extraction, SPE, SPME, extraction disc) or the extraction can be linked on-line to LC or GC. In many cases the superheated water extraction is cleaner, faster and cheaper than the conventional extraction methods.     

Phenolic-compound-extraction systems for fruit and vegetable samples

This paper reviews the phenolic-compound-extraction systems used to analyse fruit and vegetable samples over the last 10 years. Phenolic compounds are naturally occurring antioxidants, usually found in fruits and vegetables. Sample preparation for analytical studies is necessary to determine the polyphenolic composition in these matrices. The most widely used extraction system is liquid-liquid extraction (LLE), which is an inexpensive method since it involves the use of organic solvents, but it requires long extraction times, giving rise to possible extract degradation. Likewise, solid-phase extraction (SPE) can be used in liquid samples. Modern techniques, which have been replacing conventional ones, include: supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). These alternative techniques reduce considerably the use of solvents and accelerate the extraction process.     

Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.     

Immunoassay methods for paralytic shellfish poisoning toxins

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.     

A Green Deep Eutectic Solvent-Based Ultrasound-Assisted Method to Extract Astaxanthin from Shrimp Byproducts

The extraction of valuable compounds from waste products using green and inexpensive solvents is a significant strategy for sample preparation. Accordingly, in the present study, the use of deep eutectic solvents, an emerging green approach, was used to extract astaxanthin, a well-known and widely-used antioxidant, from shrimp byproducts. After evaluating different combinations of extraction conditions and deep eutectic solvents, an ultrasonication method was established and optimized by a systematic investigation of the influencing factors. A comparison of the amount of astaxanthin (102 μg g^{? 1} ) extracted using a traditional organic solvent, ethanol, showed that more astaxanthin (146 μg g^{? 1} ) was obtained using the reported eco-friendly method. The excellent properties of deep eutectic solvents highlight their advantages as promising inexpensive green solvents for the extraction and determination of a range of bioactive compounds from natural products.     

Drug level monitoring: antidepressants.

The determination of antidepressant drugs which act by blocking neuronal uptake of biogenic amines, because of their widespread use and high toxicity, remains one of the most commonly requested drug assays in clinical laboratories. Easy to use immunoassay reagents for the estimation of these drugs are commercially available. However, immunoassays have not been universally accepted because of high probability of these reagents producing false negative and false positive results. At present, column liquid chromatography with absorbance detection and coupled with solid-phase extraction is the most viable technique for a general procedure for the identification and determination of these drugs. The technique of liquid chromatography is economical, environmental friendly since water-miscible and biodegradable solvents can be used for extraction of drugs and their chromatographic separation, and amenable to full automation. New techniques of separation, such as supercritical fluid chromatography and capillary zone electrophoresis, have not yet been applied for the determination of therapeutic concentrations of antidepressants.     

Determination of pesticides by enzyme immunoassay

Immunochemical methods of analysis, which are based on the binding of an antigen (pesticide) molecule to specific antibodies, are finding increasing use for determining pesticides in various samples (water, soil, food products, and biological fluids). Among these, enzyme-linked immunosorbent assay (ELISA), which combines the unique specificity of immunoassay with the high sensitivity of the detection of an enzymatic marker, is the most widely used method. Moreover, in ELISA, the components of an immunochemical reaction are separated; as a consequence, the effect of interfering components in the sample (a so-called matrix effect) is reduced. In this review, the principles of enzyme immunoassay for pesticides are considered, and the determination of pesticides in environmental samples and food products is exemplified. The main directions of the further development of immunoassay techniques for determining pesticides are also discussed.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 3, 2005, pp. 230–246.Original Russian Text Copyright © 2005 by Morozova, Levashova, Eremin.     

On-line coupled superheated water extraction (SWE) and superheated water chromatography (SWC)

Superheated water extraction has been linked directly to a superheated water chromatographic separation so that the process of sample extraction and separation can be achieved without the need for organic solvents at any stage. A model matrix spiked with pharmaceuticals and antioxidants was extracted and the extracted components were collected on a cold polystyrene-divinylbenzene trap. The analytes were then sequentially released by raising the temperature in stages. Each fraction was passed on-line to a polystyrene divinylbenzene analytical column and was eluted with superheated water using a thermal gradient.     

Rapid extraction and quantitative detection of the herbicide diuron in surface water by a hapten-functionalized carbon nanotubes based electrochemical analyzer

A solid phase extraction micro-cartridge containing a non-polar polystyrene absorbent matrix was coupled with an electrochemical immunoassay analyzer (EIA) and used for the ultra-sensitive detection of the phenyl urea herbicide diuron in real samples. The EIA was fabricated by using carboxylated carbon nanotubes (CNTs) functionalized with a hapten molecule (an amine functionalized diuron derivative). Screen printed electrodes (SPE) were modified with these haptenized CNTs and specific in-house generated anti diuron antibodies were used for bio-interface development. The immunodetection was realized in a competitive electrochemical immunoassay format using alkaline phosphatase labeled secondary anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of an electrochemically active product, 1-naphthol, which was monitored by using differential pulse voltammetry (DPV). The assay exhibited excellent sensitivity and specificity having a dynamic response range of 0.01 pg mL(-1) to 10 μg mL(-1) for diuron with a limit of detection of around 0.1 pg mL(-1) (n = 3) in standard water samples. The micro-cartridge coupled hapten-CNTs modified SPE provided an effective and efficient electrochemical immunoassay for the real-time monitoring of pesticides samples with a very high degree of sensitivity.     

Immunobiosensor--an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs.

A rapid immunoassay using an optical biosensor (BIAcore) for determining the presence of sulfamethazine (SMT) residues in pig bile was developed. The assay was used in a routine screening laboratory alongside a previously described biosensor method for sulfadiazine (SDZ). Sulfonamide bile concentrations, determined by enzyme immunoassay (EIA), have already been shown suitable for use in predicting the extent of sulfonamide accumulation in kidney. The ability of immunobiosensor based bile screening to predict violative tissue residues (greater than the maximum residue limit; MRL) was compared with results achieved using two conventional EIAs for two of these drug residues (SMT and SDZ). Analysis of 2081 samples for both sulphonamide residues, over an 8 month period, showed the false positive prediction rate of biosensor analysis to be 0.14% and 0.34% for SMT and SDZ, respectively, compared with false positive rates of 1.54% and 1.44% by EIA. Biosensor analysis showed no false negative predictions for either SMT or SDZ while EIA showed a false negative prediction rate of 0.14% for SMT and 0.24% for SDZ. The present study has clearly demonstrated that immunobiosensor assays can be developed for veterinary drug residue screening programmes. These methods have the potential for generating faster and more reliable results than conventional immunoassay methods.     

Development and Box–Behnken design optimization of a green extraction method natural deep eutectic solvent-based for phenolic compounds from barley malt rootlets

In recent years, attention has been turned finding new sources of phenolic compounds, antioxidant molecules, main by-products from the agri-food chain like barley malt rootlets (BMRs). Traditionally, phenolic compounds are extracted from food matrices using different procedures, for example, solid–liquid, liquid–liquid, or solid-phase extraction techniques employing organic solvents. With the advent of green chemistry, attention has been paid to the search for green, nontoxic, inexpensive, and nonflammable solvents and the natural deep eutectic solvents (NADESs) respect these characteristics. The aim of this project was to develop and optimize an environmentally friendly, inexpensive, and rapid extraction method for phenolic compounds from BMRs using natural DESs as extractive solvents. Several natural DESs were tested as extractive solvents and, among them, the best results in terms of total phenolic content were obtained using a choline chloride-malic acid (1:2 molar ratio)-based mixture. Box–Behnken experimental design guaranteed the extraction of 9.51 ± 0.83 gallic acid equivalent/g of BMRs, under the following optimal extraction conditions: 1:21 solid-to-liquid ratio, 80°C as extraction temperature, 43 min as the time of extraction, and 29% as a percentage of added water in the NADESs. Phenolic acids and flavonoids were detected in the BMRs extract through HPLC-PDA/MS analysis.     

Detection of analyte binding to microarrays using gold nanoparticle labels and a desktop scanner

Microarray hybridization or antibody binding can be detected by many techniques, however, only a few are suitable for widespread use since many of these detection techniques rely on bulky and expensive instruments. Here, we describe the usefulness of a simple and inexpensive detection method based on gold nanoparticle labeled antibodies visualized by a commercial, office desktop flatbed scanner. Scanning electron microscopy studies showed that the signal from the flatbed scanner was proportional to the surface density of the bound antibody-gold conjugates, and that the flatbed scanner could detect six attomoles of antibody-gold conjugates. This detection system was used in a competitive immunoassay to measure the concentration of the pesticide metabolite 2,6-dichlorobenzamide (BAM) in water samples. The results showed that the gold labeled antibodies functioned comparably with a fluorescent based immunoassay for detecting BAM in water. A qualitative immunoassay based on gold-labeled antibodies could determine if a water sample contained BAM above and below 60-70 ng L(-1), which is below the maximum allowed BAM concentration for drinking water (100 ng L(-1)) according to European Union legislation.     

Characterization of a New Monoclonal Antibody Against Mercury(II)

Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunised with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunised mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 μg/L HgCl2 (= 2.1 μg/L Hg²⁺) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).     

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Electrochemical immunoassays

Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests. Moreover, for opaque or optically dense matrices electrochemical methods are superior. Potentiometric, capacitive and amperometric transducers have been applied for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range and low detection limit, amperometric transducers are preferred. Competitive and noncompetitive amperometric immunoassays have been developed with redox compounds or enzymes as labels. This review will give an overview of the most frequently applied principles in electrochemical immunoassays. The potential of an indirect competitive amperometric immunoassay for the determination of creatinine within nanomolar range and the circumvention of the most serious problem in electrochemical immunoassays, namely regeneration, will be discussed.     

Determination of steroid hormones in biological and environmental samples using green microextraction techniques: an overview

Residues of steroid hormones have become a cause for concern because they can affect the biological activity of non-target organisms. Steroid hormones are a potential risk for wildlife and humans through the consumption of contaminated food or water. Their determination requires extraction and clean-up steps, prior to detection, to reach low concentration levels. In recent years, a great effort has been made to develop new analytical methodologies, such as microextraction techniques, that reduce environmental pollution. Researchers have modified old methods to incorporate procedures that use less-hazardous chemicals or that use smaller amounts of them. They are able to do direct analysis using miniaturised equipment and reduced amounts of solvents and wastes. These accomplishments are the main objectives of green analytical chemistry. In this overview, we focus on microextraction techniques for the determination of steroid hormones in biological (e.g., human urine, human serum, fish, shrimp and prawn tissue and milk) and environmental (e.g., wastewaters, surface waters, tap waters, river waters, sewage sludges, marine sediments and river sediments) samples. We comment on the most recent applications in sorptive-microextraction modes, such as solid phase microextraction (SPME) with molecularly imprinted polymers (MIPs), in-tube solid-phase microextraction (IT-SPME), stir-bar sorptive extraction (SBSE) and microextraction in packed sorbent (MEPS). We also describe liquid-phase microextraction (LPME) approaches reported in the literature that are applied to the determination of steroid hormones.     

Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry

Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 microg/mL, respectively. The linear ranges of the calibration curves for EIA and HPLC-DAD were 0.02-0.5 and 0.1-500 microg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and HPLC were 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay were <9.31%, and recovery range for all concentrations was between 71 and 105%. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The r2 values for UPLC-MS/MS with HPLC-DAD and with EIA were 0.9566 and 0.9489, respectively.     

Using natural deep eutectic solvents for the extraction of metabolites in Byrsonima intermedia leaves

Natural deep eutectic solvents have been used as an alternative to organic solvents for the extraction of plants metabolites, allowing for the extraction of compounds of different polarities, while being inexpensive, non‐toxic, and easy to prepare. This work presents the comparison of the chromatographic profiles by high‐performance liquid chromatography with diode‐array detection obtained from Byrsonima intermedia (Malpighiaceae) using five choline chloride‐based natural deep eutectic solvents, in addition to the most used traditional extraction solvents, methanol/water 7:3 and ethanol/water 7:3 v/v. A reference extract was used to tentatively identify compounds by high‐performance liquid chromatography with tandem mass spectrometry. The water content appeared to be important for the extraction efficiency and the mixture choline chloride/glycerol was shown to be the best candidate for efficiently extracting this matrix when compared with the traditional extraction media in addition to being far greener as shown by the environmental analysis tool. Seven phenolic compounds (digalloyl quinic acid, proanthocyanidin dimer, galloylproanthocyanidin dimer, quercetin‐O‐hexoside, galloyl quercetin hexoside, quercetin‐O‐pentoside, and galloyl quercetin pentoside) were tentatively identified in all extracts. Moreover, the influence of these solvents on the antioxidant activity of the extracts was studied and the results for choline chloride/glycerol extracts were very similar to that of the traditional extraction solvents.     

Comparison of direct thermal desorption with water distillation and superheated water extraction for the analysis of volatile components of Rosa damascena Mill. using GCxGC-TOF/MS

The composition of the volatile components from Rosa damascena Mill. was investigated using comprehensive two-dimensional gas chromatography with time of flight mass spectrometry (TOF/MS). The samples were collected from Turkey and were extracted by water distillation (WD), superheated water extraction (SWE) and direct thermal desorption (DTD). It was found that superheated water extraction gave a slightly higher oil yield than water distillation. The major compounds found in volatiles of R. damascena Mill. were linalool, phenylethylalcohol, citronellol, nerol and geraniol. Phenylethylalcohol content was significantly higher using the DTD (36.52%) and SWE (38.14%) techniques compared to the WD (1.92%) technique. A lower content of monoterpene alcohols was found in volatiles extracted using the DTD method (73.69%) compared to the SWE (86.51%) and WD (86.56%) techniques reflecting the main finding that DTD extracts showed a greater total number of different components than either of the other two methods. The number of volatile components identified with a percentage higher than 0.05% were 54, 37, and 34 for the DTD, SWE and WD techniques, respectively. This highlighted DTD as a promising method for qualitative analysis of rose oil which can yield comprehensive results without the traditional obligation for costly and time consuming extraction techniques.     

Superheated water extraction of cholesterol from solid food

A method based on superheated water extraction has been developed for the removal of cholesterol from solid food thus providing a clean approach by avoiding the use of organic solvents. A preconcentration step was also studied with the aim of solving the problem derived from low-cholesterol-content samples and dilution effect. The research also involved the optimisation of the parameters affecting the extraction process by a central composite experimental design as well as a univariate study of the optimisation of the preconcentration step. The time required for total removal of the target compound was 60 min. The method was validated using a certified reference material (NIST-CRM 1845) and was used to analyse food samples within a wide range of cholesterol concentrations. The efficiency for the CRM was 105%. The precision of the method yielded values less than 6.5% (expressed as relative standard deviation) in all instances.