A core–shell titanium dioxide polyaniline nanocomposite for the needle‐trap extraction of volatile organic compounds in urine samples

We synthesized a titanium dioxide–polyaniline core–shell nanocomposite and implemented it as an efficient sorbent for the needle‐trap extraction of some volatile organic compounds from urine samples. Polyaniline was synthesized, in the form of the emeraldine base, dissolved in dimethyl acetamide followed by diluting with water at pH 2.8, using the interfacial polymerization method. The TiO₂ nanoparticles were encapsulated inside the conducting polymer shell, by adapting the in situ dispersing approach. The surface characteristics of the nanocomposite were investigated by Fourier transform infrared spectrometry, scanning electron microscopy, and transmission electron microscopy. After obtaining acceptable preliminary results, some selected volatile compounds, including chloroform, benzene, toluene, ethylbenzene, xylene, and chlorobenzenes were used as model analytes to validate the enrichment properties of the prepared sorbent in conjunction with gas chromatography mass spectrometric detection. Important parameters influencing the extraction process such as extraction temperature, ionic strength, sampling flow rate, extraction time, desorption temperature, and time were optimized. The limits of detection and limits of quantification values were in the range of 0.5–3  and 2–5 ng/L, respectively, using time‐scheduled selected ion monitoring mode. The relative standard deviation percent with three replicates was in the range of 5–10%. The applicability of the developed needle‐trap method was examined by analyzing urine samples and the relative recovery percentages for the spiked samples were in the range of 81–105%.     

Solid‐phase extraction using chloropropyl functionalized sol–gel hybrid sorbent for simultaneous determination of organophosphorus pesticides in selected fruit samples

A new sol–gel hybrid methyltrimethoxysilane‐chloropropyltriethoxysilane was prepared as sorbent for solid‐phase extraction. The extraction efficiency of the prepared sol–gel hybrid methyltrimethoxysilane‐chloropropyltriethoxysilane was assessed by using three selected organophosphorus pesticides, namely, chlorpyrifos, profenofos, and malathion. Gas chromatography–mass spectrometry was used for detection of organophosphorus pesticides. Several vital parameters were optimized to identify the best extraction conditions. Under the optimum extraction conditions, solid‐phase extraction‐methyltrimethoxysilane‐chloropropyltriethoxysilane method showed good linearity range (0.05‐1 μg/mL) with coefficient of determination more than 0.995. The limits of detection obtained were in the range of 0.01–0.07 μg/mL and limits of quantification ranging from 0.03 to 0.21 μg/mL. The limits of detection obtained for the developed method were 2.3–6.5× lower than the limits of detection of commercial octadecyl silica sorbent. Real samples analysis was carried out by applying the developed method on red apple and purple grape samples. The developed method exhibited good recoveries (88.33–120.7%) with low relative standard deviations ranging from 1.6 to 3.3% compared to commercial octadecyl silica sorbent, which showed acceptable recoveries (70.3–100.2%) and relative standard deviations (6.3–8.8%). The solid‐phase extraction‐methyltrimethoxysilane‐chloropropyltriethoxysilane method is presented as an alternative extraction method for determination of organophosphorus pesticides.     

Isolation of free phenolic compounds from arboreal leaves by use of the Florisil/C<Subscript>18</Subscript> system

In studies of the phenolic compounds present in leaves and needles, GC and GC–MS have so far been applied only sporadically. This is probably because of the greater difficulties encountered in preparing the samples for this method than those used for liquid chromatography. When preparing a sample for gas chromatography the analyst is faced with two difficult stages—separation of the compound from the matrix without losses (stage 1) so that the final sample can be derivatized to make it suitable for analysis on a non-polar capillary column of the gas chromatograph (stage 2). This paper presents a procedure for extraction of phenolic compounds from the matrix by means of a Florisil/C₁₈ sorbent system and their analysis by GC. After passage through the adsorbents the recovery ranges from 32% for ferulic acid to 88% for gentisic acid. It was found that this extraction method and the GC analysis are very precise (particularly for samples of a mass <1 g) and can be used for quantification. The high-precision quantification of 15 phenolic acids, shikimic acid, and six other compounds present in pine needles has been achieved. The conditions used for GC analysis and construction of calibration curves for quantitative determination are given.     

Multi-walled carbon nanotube-based dispersive solid phase extraction with following back-extraction for HPLC/UV determination of Rosmarinic acid in lemon balm and Rosemary plant samples

In this paper, dispersive solid phase extraction with following back-extraction and HPLC/UV determination of Rosmarinic acid have been investigated. Multi-walled carbon nanotubes were used as sorbent in the suggested method. The effectiveness of some parameters such as extraction time, sorbent dosage, pH and ionic strength of the aqueous solution on Rosmarinic acid adsorption were studied. The extraction time of 5.0 min, adsorbent weight of 5.0 mg, and pH of 2.5 were obtained as the optimum experimental parameters. Adsorption of Rosmarinic acid molecules was almost independent from ionic strength. For back-extraction purpose a basic solution containing methanol was brought into contact with sorbent to desorb Rosmarinic acid molecules from sorbent containing adsorbed Rosmarinic acid molecules. Linear range of HPLC method for Rosmarinic acid quantification was 1.80–21.62 mg/L (R² = 0.998). The relative standard deviation, limit of detection, and limit of quantification for the suggested method were 0.81%, 0.68 mg/L, and 2.86 mg/L, respectively. The proposed method was applied for extraction and HPLC/UV determination of Rosmarinic acid in Lemon balm and Rosemary plant samples. Comparison between obtained chromatograms before adsorption and after desorption processes showed that suggested extraction/back-extraction process has good selectivity and efficiency for separation of Rosmarinic acid molecules from interfering molecules existing in the plant extracts. The proposed method is efficient, low cost, fast, and simple for separation, extraction, and determination of Rosmarinic acid.     

Optimization of a sensitive method for the determination of nitro musk fragrances in waters by solid-phase microextraction and gas chromatography with micro electron capture detection using factorial experimental design

A solid-phase microextraction method (SPME) followed by gas chromatography with micro electron capture detection for determining trace levels of nitro musk fragrances in residual waters was optimized. Four nitro musks, musk xylene, musk moskene, musk tibetene and musk ketone, were selected for the optimization of the method. Factors affecting the extraction process were studied using a multivariate approach. Two extraction modes (direct SPME and headspace SPME) were tried at different extraction temperatures using two fiber coatings [Carboxen–polydimethylsiloxane (CAR/PDMS) and polydimethylsiloxane–divinylbenzene (PDMS/DVB)] selected among five commercial tested fibers. Sample agitation and the salting-out effect were also factors studied. The main effects and interactions between the factors were studied for all the target compounds. An extraction temperature of 100 °C and sampling the headspace over the sample, using either CAR/PDMS or PDMS/DVB as fiber coatings, were found to be the experimental conditions that led to a more effective extraction. High sensitivity, with detection limits in the low nanogram per liter range, and good linearity and repeatability were achieved for all nitro musks. Since the method proposed performed well for real samples, it was applied to different water samples, including wastewater and sewage, in which some of the target compounds (musk xylene and musk ketone) were detected and quantified. Figure Stardardized Pareto charts for the main effects and interactions     

Application of micro‐solid‐phase extraction for the on‐site extraction of heterocyclic aromatic amines in seawater

An efficient on‐site extraction technique to determine carcinogenic heterocyclic aromatic amines in seawater has been reported. A micro‐solid‐phase extraction device placed inside a portable battery‐operated pump was used for the on‐site extraction of seawater samples. Before on‐site applications, parameters that influence the extraction efficiency (extraction time, type of sorbent materials, suitable desorption solvent, desorption time, and sample volume) were investigated and optimized in the laboratory. The developed method was then used for the on‐site sampling of heterocyclic aromatic amines determination in seawater samples close to distillation plant. Once the on‐site extraction completed, the small extraction device with the analytes was brought back to the laboratory for analysis using high‐performance liquid chromatography with fluorescence detection. Based on the optimized conditions, the calibration curves were linear over the concentration range of 0.05–20 μg/L with correlation coefficients up to 0.996. The limits of detection were 0.004–0.026 μg/L, and the reproducibility values were between 1.3 and 7.5%. To evaluate the extraction efficiency, a comparison was made with conventional solid‐phase extraction and it was applied to various fortified real seawater samples. The average relative recoveries obtained from the spiked seawater samples varied in the range 79.9–95.2%.     

Nitrogen doped nano porous graphene as a sorbent for separation and preconcentration trace amounts of Pb,Cd and Cr by Ultrasonic assisted in‐syringe dispersive micro solid phase extraction

Nitrogen doped nano porous graphene was used as an efficient sorbent in solid‐phase extraction process for simultaneous separation and pre‐concentration of metal ions lead (II), cadmium(II), and chromium(III)) in biological samples. Ultrasonic assisted in‐syringe dispersive micro solid phase extraction coupled with micro sampling atomic absorption spectrometry was utilized for the determination of metal ions. Nitrogen doped nano porous graphene was synthesized as a nano sorbent by chemical vapour deposition method. Methane and aniline were used as carbon and nitrogen sources. The characterization of sorbent was performed by field emission scanning electron microscope, transmission electron microscopy, atomic force microscope, fourier transform infrared, chemical element analysis and raman analysis. Effective parameters on the extraction efficiency such as pH, sorbent dosage, eluent volume and eluent concentration were optimized by central composite design and desirability function. Experimental results indicate that the optimal conditions for this extraction were pH = 6.4, 1.42 mg of sorbent, 100 μL of eluent, and 0.84 mol L^‐1 of eluent concentration. The detection limits are as low as 1.5, 0.3 and 0.9 μg L^‐1 for lead, cadmium, and chromium, respectively. The intra‐day precisions were 3.6, 4.38 and 2.94 and Inter‐day precision were 4.83, 5.26 and 4.52 for lead, cadmium, and chromium, respectively. Method performance was investigated by determination of mentioned heavy metals in complicated biological matrixes such as human plasma, urine and saliva samples with good recoveries.     

Effectiveness of in-needle extraction device for liquid samples

In this work in-needle technique as the method for liquid samples preparation which faces with the problem of high flow resistance produced by a sorbent layer is considered. The influence of parameters (needle size, sorbent particle size and length of sorbent layer) of in-needle device on the flow rate was examined. Acceptable sampling conditions were selected on the basis of experiments and their chemometric evaluation. Darcy law was used to describe flow velocity through sorbent in the needle. Kozeny–Carman model and Brinkman model led to results significantly different from those obtained experimentally. Baaren model gave the most realistic results. Diameter of grain, porosity and a constant termed C were taken into account in this model. Incorporation of an additional parameter – “constant C” provides the completion of the model and allows predicting effectively the permeability of the extraction system. The so-called “constant C” is not constant but characteristic for a given system.     

Solubility parameter used to predict the effectiveness of monolithic in-needle extraction (MINE) device for the direct analysis of liquid samples

The sorbent/eluent systems combined from three macroporous poly(styrene–divinylbenzene) (PS-DVB) monoliths and four solvents as eluents were used for the extraction of phenol, 4-chlorophenol and p-benzochinon from water samples. Monolithic in-needle extraction (MINE) devices were used in the preparation of a series of test water samples for chromatographic analysis. The extraction of phenolic compounds from water samples was carried out by pumping liquid samples through the MINE device. Solubility parameter concept was applied for estimation of effectiveness of MINE. Solubility parameters for (PS-DVB) monoliths were determined according to Small, considering different molar fraction of the monomers used for synthesis. Effectiveness of these systems was estimated according to difference of solubility parameter value in analyte/sorbent, sorbent/eluent, analyte/eluent pairs. The procedure enabling easy prediction of, e.g. the strength of the interactions between the analyte and sorbent, eluent efficiency or the extraction efficiency in MINE system was proposed.     

Magnetic solid-phase extraction of hydrophobic analytes in environmental samples by a surface hydrophilic carbon-ferromagnetic nanocomposite

A new sorbent of carbon-ferromagnetic nanocomposite was proposed for the extraction of polycyclic aromatic hydrocarbons (PAHs) in environmental samples. The sorbent was specially designed with a hydrophobic sublayer and a hydrophilic surface, which endows the sorbent some unique features. The former shows high extraction capability for the PAHs and the latter provides benign compatibility with the sample matrix. The sorbent can be easily dispersed in aqueous solutions for extraction and no additional stirring or shaking was necessary to facilitate the dispersion, which may bring operational convenience especially for on-site sampling and extraction. Parameters affecting the extraction efficiency were investigated in detail. The optimal conditions were as follows: 10mg of nanoparticles, 40mmol/L of sodium chloride, 30min of extraction time without shaking, hexane as the desorption solvent and 15min as the desorption-sonication time. The results demonstrate that enrichment factors ranging from 35- to 133-fold were obtained for the analytes. The limits of detection and the limits of quantification are in the range of 0.015-0.335ng/mL and 0.05-1.14ng/mL, respectively. Finally, the new sorbent was successfully used for the extraction of PAHs in lake water samples.     

Hypercrosslinked strong anion‐exchange resin for extraction of acidic pharmaceuticals from environmental water

Two novel high‐specific surface area polymeric sorbents (HXLPP‐SAXa and HXLPP‐SAXb) were synthesised and evaluated as solid‐phase extraction sorbents. The novel sorbents under study are based on hypercrosslinked polymer microspheres and designed specifically to offer ion‐exchange properties; the specific polymers of interest in the current work have been chemically modified in such a way as to impart a tuneable level of strong anion‐exchange character onto the sorbents. The novel sorbents were applied as strong anion‐exchange sorbents in solid‐phase extraction studies, with the goal being to selectively extract a group of acidic compounds from complex environmental samples in an efficient manner. Out of two HXLPP‐SAX resins evaluated in this study, it was found that the sorbent with the lower ion‐exchange capacity (HXLPP‐SAXa) gave rise to the best overall performance characteristics and, indeed, was found to compare favourably to the solid‐phase extraction performance of commercial strong anion‐exchange sorbents. When the HXLPP‐SAXa sorbent was applied to the solid‐phase extraction of environmental water samples, the result showed quantitative and selective extraction of low levels of acidic pharmaceuticals from 500 mL of river water and 100 mL of effluent wastewater.     

Extraction of trace nitrophenols in environmental water samples using boronate affinity sorbent

In this research, the applicability of a new sorbent based on boronate affinity material is demonstrated. For this purpose, six strong polar nitrophenols were selected as models which are difficult to be extracted in neutral form (only based on hydrophobic interactions). The extracted nitrophenols were separated and determined by high-performance liquid chromatography with diode array detection. The sorbent was synthesized by in situ copolymerization of 3-acrylamidophenylboronic acid and divinylbenzene using dimethyl sulfoxide and azobisisobutyronitrile as porogen solvent and initiator, respectively. The effect of the preparation parameters in the polymerization mixture on extraction performance was investigated in detail. The size and morphology of the sorbent have been characterized via different techniques such as infrared spectroscopy, elemental analysis, scanning electron microscopy and mercury intrusion porosimetry. The important parameters influencing the extraction efficiency were studied and optimized thoroughly. Under the optimum extraction conditions, the limits of detection (S/N = 3) and limits of quantification (S/N = 10) for the target nitrophenols were 0.097–0.28 and 0.32–0.92 μg/L, respectively. The precision of the proposed method was evaluated in terms of intra- and inter-assay variability calculated as RSD, and it was found that the RSDs were all below 9%. Finally, the developed method was successfully applied for environmental water samples such as wastewater, tap, lake and river water. The recoveries varied within the range of 71.2–115% with RSD below 11% in all cases. The results well demonstrate that the new boronate affinity sorbent can extract nitrophenols effectively through multi-interactions including boron–nitrogen coordination, hydrogen-bond and hydrophobic interactions between sorbent and analytes.     

Application of microcrystalline cellulose as an efficient and cheap sorbent for the extraction of metoprolol from plasma and wastewater before HPLC–MS/MS determination

A dispersive solid-phase extraction method based on a new sorbent has been performed on plasma and wastewater samples to determine metoprolol by high-performance liquid chromatography–tandem mass spectrometry. In this study, the analyte was adsorbed from the samples onto microcrystalline cellulose as a green and efficient sorbent and then eluted for use in the determination step. In the mass spectrometer, the analyte was detected in the positive mode and selectivity of the analysis was increased by sequential mass analysis through multiple reaction monitoring. All of the effective parameters in the extraction of metoprolol from plasma and wastewater were optimized. Under optimal conditions the method was linear in the ranges of 1–1,000 and 0.1–1,000 ng/ml in plasma and wastewater samples, respectively. The detection limits of the method were 0.30 and 0.03 ng/ml in plasma and wastewater samples, respectively. The data showed that the method provides low detection limit, wide linear range, good precision and high extraction recovery. Finally several plasma and wastewater samples were successfully analyzed using the method. The use of a small amount of a green and inexpensive sorbent and a low volume of plasma without the need for further pretreatment steps are the main advantages of the method.     

Aptamer-targeted magnetic nanospheres as a solid-phase extraction sorbent for determination of ochratoxin A in food samples

A sorbent based on the aptamer for ochratoxin A was immobilized onto magnetic nanospheres (MNS) and used to develop a magnetic solid-phase extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography separation and fluorescence detection. Specific retention of ochratoxin A by the sorbent was demonstrated, and the capacity of the MNS-aptamer sorbent was determined. The efficacy of this new approach was successfully evaluated through comparison with solid-phase extraction on commercial C18 cartridge. Several different food samples fortified in the range of with 2.5-50 μg/kg yielded mean recoveries from 67% to 90%, respectively. Finally, this oligosorbent was applied to the selective extraction of ochratoxin A from unfortified food samples.     

Development of natural sorbent based micro-solid-phase extraction for determination of phthalate esters in milk samples

In the present study, a natural sorbent based micro-solid phase extraction (μ-SPE) was developed for determination of phthalate esters in milk samples. For the first time, an efficient and cost effective natural material (seed powder of Moringa oleifera) was employed as sorbent in μ-SPE. The sorbent was found to be naturally enriched with variety of functional groups and having a network of interconnected fibers. This method of extraction integrates different steps such as removal of proteins and fatty stuff, extraction and pre-concentration of target analytes into a single step. Thirteen phthalate esters were selected as target compounds for the development and evaluation of method. Some key parameters affecting the extraction efficiency were optimized, including selection of membrane, selection and amount of sorbent, extraction time, desorption solvent, volume of desorption solvent, desorption time and effect of salt addition. Under the optimum conditions, very good linearity was achieved for all the analytes with coefficient of determinations (R²) ranging between 0.9768 and 0.9977. The limits of detection ranged from 0.01 to 1.2 μg L⁻¹. Proposed method showed satisfactory reproducibility with relative standard deviations ranging from 3.6% to 10.2% (n = 7). Finally, the developed method was applied to tetra pack and bottled milk samples for the determination of phthalate esters. The performance of natural sorbent based μ-SPE was better or comparable to the methods reported in the literature.     

Experimental study on solvent-less sample preparation methods: Membrane extraction with a sorbent interface, thermal membrane desorption application and purge-and-trap

Different solvent-free sample preparation techniques for the enrichment of volatile and semivolatile organic compounds from aqueous samples for subsequent gas chromatographic separation and detection are compared. The methods under study are purge-and-trap, membrane extraction with a sorbent interface in two different configurations, and thermal membrane desorption application. The study has been performed with polar as well as with non-polar compounds in respect to sampling yield, enrichment, repeatability and analysis cycle rate. All experiments have been performed with a mobile GC–MS system.     

Dispersive solid phase extraction of metronidazole and clarithromycin from human plasma using a β-cyclodextrin grafted polyethylene polymer composite

In this work, for the first time, a polymeric composite based on β-cyclodextrin grafted with polyethylene has been prepared through ball milling and used as an efficient sorbent for dispersive solid phase extraction of metronidazole and clarithromycin from plasma samples. The prepared sorbent was characterized using Fourier transform infrared spectrophotometry, X-ray diffraction, and scanning electron microscopy. In the extraction process, after precipitating the proteins, the sorbent was added into the sample solution, and the mixture was vortexed to facilitate and speed up the sorption of the analytes onto the sorbent surface. After centrifuging, the sorbent particles were contacted with methanol to elute the analytes under the vortexing process. After this step, an aliquot of the eluate was taken and injected into high-performance liquid chromatography–diode array detector for quantitative analysis. Under the optimum extraction conditions, the extraction recoveries for metronidazole and clarithromycin were 76 and 83%, respectively. The limits of detection were 2.6 and 2.2 ng/ml for metronidazole and clarithromycin, respectively. The repeatability of the offered approach, expressed as relative standard deviation, was equal to or less than 4.7%. Finally, the method was successfully applied to plasma samples of the patients treated with metronidazole and clarithromycin.     

Sorbent-based sampling methods for volatile and semi-volatile organic compounds in air: Part 1: Sorbent-based air monitoring options

Sorbent tubes/traps are widely used in combination with gas chromatographic (GC) analytical methods to monitor the vapour-phase fraction of organic compounds in air. Target compounds range in volatility from acetylene and freons to phthalates and PCBs and include apolar, polar and reactive species. Airborne vapour concentrations will vary depending on the nature of the location, nearby pollution sources, weather conditions, etc. Levels can range from low percent concentrations in stack and vent emissions to low part per trillion (ppt) levels in ultra-clean outdoor locations. Hundreds, even thousands of different compounds may be present in any given atmosphere. GC is commonly used in combination with mass spectrometry (MS) detection especially for environmental monitoring or for screening uncharacterised workplace atmospheres. Given the complexity and variability of organic vapours in air, no one sampling approach suits every monitoring scenario. A variety of different sampling strategies and sorbent media have been developed to address specific applications. Key sorbent-based examples include: active (pumped) sampling onto tubes packed with one or more sorbents held at ambient temperature; diffusive (passive) sampling onto sorbent tubes/cartridges; on-line sampling of air/gas streams into cooled sorbent traps; and transfer of air samples from containers (canisters, Tedlar^® bags, etc.) into cooled sorbent focusing traps. Whichever sampling approach is selected, subsequent analysis almost always involves either solvent extraction or thermal desorption (TD) prior to GC(/MS) analysis. The overall performance of the air monitoring method will depend heavily on appropriate selection of key sampling and analytical parameters. This comprehensive review of air monitoring using sorbent tubes/traps is divided into 2 parts. (1) Sorbent-based air sampling option. (2) Sorbent selection and other aspects of optimizing sorbent-based air monitoring methods. The paper presents current state-of-the-art and recent developments in relevant areas such as sorbent research, sampler design, enhanced approaches to analytical quality assurance and on-tube derivatisation.     

The evaluation of different sorbents for the preconcentration of phenoxyacetic acid herbicides and their metabolites from soils

A procedure using alkaline extraction, solid-phase extraction (SPE) and HPLC is developed to analyze the polar herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) together with their main metabolites in soils. An ion-pairing HPLC method is used for the determination as it permits the baseline separation of these highly polar herbicides and their main metabolites. The use of a highly cross-linked polystyrene-divinylbenzene sorbent (PS-DVB) gives the best results for the analysis of these compounds. This sorbent allows the direct preconcentration of the analytes at the high pH values obtained after quantitative alkaline extraction of the herbicides from soil samples. Different parameters are evaluated for the SPE preconcentration step. The high polarity of the main analytes of interest (2,4-D and MCPA) makes it necessary to work at low flow rates (< or =0.5 mL min(-1)) in order for these compounds to be retained by the PS-DVB sorbent. A two stage desorption from the SPE sorbent is required to obtain the analytes in solvents that are appropriate for HPLC determination. A first desorption with a 50:50 methanol:water mixture elutes the most polar analytes (2,4-D, MCPA and 2CP). The second elution step with methanol permits the analysis of the other phenol derivatives. The humic and fulvic substances present in the soil are not efficiently retained by PS-DVB sorbents at alkaline pH's and so do not interfere in the analysis. This method has been successfully applied in the analysis of soil samples from a golf course treated with a commercial product containing esters of 2,4-D and MCPA as the active components.     

Comparison of Hydrophilic Polymeric Sorbents for On-Line Solid-Phase Extraction of Polar Compounds from Aqueous Samples

Two 4-vinylimidazole-divinylbenzene (4VIm-DVB) polymers were synthesized and applied as sorbents for on-line solid-phase extraction (SPE) followed by liquid chromatography for analyzing polar compounds in aqueous samples. The new sorbents (4VIm-DVB) were compared to another sorbent that had been previously synthesized by our group (N-vinylimidazole-divinylbenzene (NVIm-DVB)) and to the commercial OASIS^® HLB and Strata^TM X. All the sorbents enabled 100 mL of sample to be on-line concentrated with good recoveries for the studied polar compounds. Real water samples were analyzed using NVIm-DVB and OASIS^® HLB as SPE sorbent, for which the best results were obtained.