Determination of Estrone and 17β-Estradiol in Water Samples Using Dispersive Liquid–Liquid Microextraction Followed by LC

A new method, termed dispersive liquid–liquid microextraction (DLLME), was developed for the extraction and pre-concentration of estrone (E1) and 17β-estradiol (E2) in water samples. The samples were extracted by 0.50 mL methanol (disperser solvent) containing 25.0 μL tetrachloroethane (extraction solvent). Important factors such as the volume and type of extraction and disperser solvent, extraction time and salt effect were studied. Under optimum conditions, the enrichment factors and the limits of detection were 347 and 0.2 ng mL^?1 for E1, and 203 and 0.1 ng mL^?1 for E2, respectively. The linear range was 0.5–5,000 ng mL^?1. Compared to other methods, DLLME–LC–VWD has advantages for E1 and E2 analysis in water: high enrichment factor, low cost, simplicity, quick and easy operation.     

Utilization of water-contained surfactant-based ultrasound-assisted microextraction followed by liquid chromatography for determination of polycyclic aromatic hydrocarbons and benzene in commercial oil sample

In this work, a microextraction method, water-contained surfactant-based ultrasound-assisted, followed by high-performance liquid chromatography (HPLC) was developed for determination of five polycyclic aromatic hydrocarbons (PAHs) and benzene in commercial oil samples. During the microextraction method, a micellar solution as the only extraction solvent was injected into the oil sample in a conical bottom glass tube and formed a cloudy solution. The dispersion process was accelerated by applying ultrasound irradiation. Phase separation was done by centrifugation and then the lower sediment phase was directly analyzed by HPLC. A chemometrics approach was applied for the optimization of the extraction condition. Under the optimum conditions, the proposed method showed good linearity within the different ranges for different analytes (e.g., 0.10–200 ng mL^?1 for phenanthrene), the square of the correlation coefficient was higher than 0.999 and the appropriate limit of detection was in the range of 0.04–0.41 ng mL^?1. The recoveries in all cases were above 95 %.     

Determination of Copper in Water by Ionic Liquid Based Microextraction and Chemiluminescence Detection

A versatile, sensitive, and green method based ultrasound-assisted, temperature-controlled, dispersive liquid–liquid microextraction with an ionic liquid and chemiluminescence detection was used for the determination of copper(II) at the ultra-trace level. After complexation by dithizone, copper(II) was extracted into the ionic liquid. Using high temperature and ultrasonic agitation, the copper complex easily migrated into the ionic liquid phase because of the larger contact area. After back extraction, the determination was performed by chemiluminescence based on the catalyzing effect of copper(II) on the decomposition of hydrogen peroxide with rhodamine B. Important parameters that affected the extraction efficiency and chemiluminescence intensity were optimized. Under the optimum conditions, a limit of detection for copper of 0.8 ng L^?1 was obtained with a linear calibration relationship. The method was applied to analyze environmental water samples for copper(II) with satisfactory results.     

Dispersive Liquid–Liquid Microextraction Coupled with High-Performance Liquid Chromatography for Determination of Coumarin Compounds in Radix Angelicae Dahuricae

In this paper, two methods, organic solvent dispersive liquid–liquid microextraction (OS-DLLME) and ionic liquid dispersive liquid–liquid microextraction (IL-DLLME), coupled with high-performance liquid chromatography have been critically compared and introduced for the analysis of the eight coumarin compounds (psoralen, isopsoralen, bergapten, isobergapten, oxypeucedanin, imperatorin, osthole, and isoimperatorin) in Radix Angelicae Dahuricae samples. Experimental conditions have been investigated for both OS-DLLME and IL-DLLME. Under optimal conditions, the detection limits of the eight coumarin compounds obtained by OS-DLLME and IL-DLLME ranged between 0.002–0.026 ng mL⁻¹ and 0.013–0.66 ng mL⁻¹, respectively. The relative standard deviations (RSDs, n = 9) were lower than 8.7 and 8.4% with enrichment factors in the range of 145–380 and 130–230 folds for OS-DLLME and IL-DLLME, respectively. The results showed that there were no significant deviations between the two DLLME methods for the determination of the eight coumarin compounds. Both methods were simple, fast, efficient, and inexpensive. However, compared with IL-DLLME, the OS-DLLME technique exhibited a higher extraction capacity for the eight target analytes.

     

Optimization of temperature-controlled ionic liquid homogenous liquid phase microextraction followed by high performance liquid chromatography for analysis of diclofenac and mefenamic acid in urine sample

In this work, a temperature-controlled ionic liquid homogeneous liquid phase microextraction (TCIL-HLPME) technique followed by HPLC–UV was applied for preconcentration and determination of diclofenac (DIC) and mefenamic acid (MEF) in urine samples. 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄mim][PF₆]) was used as the optimum extraction solvent. Experimental design and response surface methodology was used for the optimization process. Firstly, a screening step, using Plackett-Burman design, was carried out to find the significant factors on the extraction efficiency and subsequently, a central composite design (CCD) was employed to find the optimum values of these parameters. The optimal conditions were obtained as extraction solvent volume of 105 µL; sample pH of 2.0, extraction time of 6 min, centrifugation time of 5 min; heating time of 2 min; heating temperature of 50 °C and 20 % of NaCl. Under optimized conditions, the preconcentration factors of 82 and 60 were obtained for DIC and MEF, respectively. The detections limits of 20 and 30 ng mL^?1 were achieved for DIC and MEF by the proposed method, respectively. The calibration curves were linear in the range of 40–1000 and 60–1000 ng mL^?1 for DIC and MEF, respectively. The intra- and inter-assay precisions (RSD %, n = 3) were in the range of 3.5–4.4 % and 7.3–8.0 % at the concentration level of 100 ng mL^?1, respectively. The validated method was successfully applied for the analysis of target analytes in some urine samples.     

UHPLC–MS/MS method for the determination of bisphenol A and its chlorinated derivatives,bisphenol S,parabens, and benzophenones in human urine samples

In the present work, a new method based on a sample treatment by dispersive liquid–liquid microextraction (DLLME) for the extraction of six bisphenols (bisphenol A, bisphenol S, and monochloro-, dichloro-, trichloro-, and tetrachlorobisphenol A), four parabens (methyl-, ethyl-, propyl-, and butylparaben), and six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8, and 4-hydroxybenzophenone) in human urine samples, followed by ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis, is validated. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-¹³C₆, benzophenone-d₁₀, and bisphenol A-d₁₆ were used as surrogates. Limits of quantification ranging from 0.1 to 0.6 ng mL^?1 and interday variabilities (evaluated as relative standard deviations) from 2.0 to 13.8 % were obtained. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94 to 106 %. A good linearity, for concentrations up to 300 ng mL^?1 for parabens and 40 ng mL^?1 for benzophenones and bisphenols, was also obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.     

Multivariate optimization of the hollow fiber-based liquid phase microextraction of lead in human blood and urine samples using graphite furnace atomic absorption spectrometry

The present study developed a liquid-phase microextraction based on hollow fiber coupled with graphite furnace atomic absorption spectrometry for the effective extraction and quantitation of lead from urine and blood samples. A multivariate design was used for the optimization of the experimental conditions to ensure high extraction efficiency. Six factors (solvent type, chelating agent, time extraction, temperature, donor phase pH, and acceptor phase pH) were obtained by screening eleven factors of the Plackett–Burman design; these were optimized using the central composite design of response surface methodology. The optimum conditions of donor phase pH, acceptor phase pH, temperature, and extraction time were 5, 9.5, 40 °C, and 120 min, respectively. In addition, oleic acid containing dicyclohexyl-18-krone-6 was used for the membrane phase. Under optimal conditions, the enrichment factor, limit of detection, and limit of quantification were obtained in the ranges of 21.3–18.7, 0.001–0.002 ng mL^?1, and 0.008–0.01 ng mL^?1, respectively, in urine and blood samples. The linearity of the calibration curve was established for the concentration of Pb in the range of 1–50 ng mL^?1 (r²?=?0.9983). Finally, the performance of the developed method was evaluated for the determination of lead in urine and blood samples, and satisfactory results were obtained (RSDs <?10% with recovery >?95).     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Analysis of dextromethorphan and pseudoephedrine in human plasma and urine samples using hollow fiber-based liquid–liquid–liquid microextraction and corona discharge ion mobility spectrometry

We report on the use of hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) followed by corona discharge ion mobility spectrometry for the determination of dextromethorphan and pseudoephedrine in urine and plasma samples. The effects of pH of the donor phase, stirring rate, ionic strength and extraction time on HF-LLLME were optimized. Under the optimized conditions, the linear range of the calibration curves for dextromethorphan in plasma and urine, respectively, are from 1.5 to 150 and from 1 to 100 ng mL^?1. The ranges for pseudoephedrine, in turn, are from 30 to 300 and from 20 to 200 ng mL^?1. Correlation coefficients are better than 0.9903. The limits of detection are 0.6 and 0.3 ng mL^?1 for dextromethorphan, and 8.6 and 4.2 ng mL^?1 for pseudoephedrine in plasma and urine samples, respectively. The relative standard deviations range from 6 to 8%.

Determination of Alkylphenols in Water by Dispersive Liquid–Liquid Microextraction Based on Solid Formation without a Disperser

Dispersive liquid–liquid microextraction based on solid formation without a disperser combined with high-performance liquid chromatography has been developed for the determination of 4-tert-butylphenol, 4-n-nonylphenol, and 4-tert-octylphenol. This method is rapid, easy, and uses only 10 µL of a low toxicity organic solvent (1-hexadecanethiol) for the extraction solvent and no disperser solvent. The extraction time and centrifugation time require less than 10 min. The linear range was 1–500 ng mL^?1 for 4-tert-butylphenol, 2–1000 ng mL^?1 for 4-tert-octylphenol, and 5–500 ng mL^?1 for 4-n-nonylphenol with r² ≥ 0.9986. The detection limits were between 0.2 and 1.5 ng mL^?1. The recoveries of lake and river water samples were in the range of 79% to 108%, and the relative standard deviations were 5% to 10%.     

Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

A Chemiluminescence Enzyme‐Linked Immuno‐Sorbent Assay (CL‐ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti‐thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC₅₀ and the limit of detection (LOD) values were 60 ng mL^?1 and 9 ng mL^?1, respectively, similar to those of colorimetric ELISA with a calibration range of 9–15,000 ng mL^?1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid‐liquid extraction or the graphitized carbon‐based solid phase extraction.

The calibration curves in honeybee extracts from liquid‐liquid procedure gave an IC₅₀ of 141 ng mL^?1 and a LOD of 17 ng mL^?1. In case of extracts obtained by SPE these values were 139 ng mL^?1 and 15 ng mL^?1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL^?1 of thiram was 72% for SPE, higher than for liquid‐liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid‐liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.     

Determination of Protoberberine Alkaloids in Coptis chinensis by Microextraction and High Performance Liquid Chromatography

Sample preparation technique based on an organic filter membrane (pH-resolved filter membrane microextraction) (pH-RFMME) was developed, coupled with high-performance liquid chromatography, and used to determine protoberberine alkaloids (jatrorrhizine, epiberberine, coptisine, palmatine, and berberine) in Coptis chinensis at different pH values through a one-step procedure. This green procedure provides a desirable sample pretreatment technology. The main variables affecting the extraction such as filter membrane area (or volumes of extraction solvents), sample pH, eluent pH, ionic strength, extraction stirring rate, extraction time, and sample volume were optimized. Under the optimized conditions, the enrichment factors of the analytes were 40.4–52.0, the linear ranges were 3.2–6250 ng · mL^?1 for jatrorrhizine and epiberberine, 6.0–12000 ng · mL^?1 for coptisine, 1.8–3600 ng · mL^?1 for palmatine, and 18.8–18800 ng · mL^?1 for berberine, with r ² ≥ 0.9945. The limits of detection were less than 0.3 ng · mL^?1. Satisfactory recoveries (84.8%–115.5%) and precision (1.8%–10.0%) were also achieved. These results confirmed that pH-RFMME is a simple, rapid, practical, and environmentally friendly method to isolate analytes that exhibit significant differences in acidity or alkalinity from complex samples.     

Application of dispersive liquid–liquid microextraction for the determination of donepezil in urine by HPLC using a core–shell column

A rapid and novel method combining dispersive liquid–liquid microextraction and high-performance liquid chromatography coupled with fluorescence detection was developed for the determination of donepezil in human urine. Parameters affecting extraction efficiency and chromatographic determination, such as the type and volume of the extraction and disperser solvent, pH of sample for dispersive liquid–liquid microextraction, mobile-phase composition, pH, column oven temperature, and flow rate for chromatographic determination, were evaluated and optimized. Using a C₁₈ core–shell column (7.5 × 4.6?mm, 2.7?μm), the determination of donepezil was accomplished within 5?min. Under optimum conditions, developed method was linear in the range of 0.5–25?ng?mL^?1 with the correlation coefficient >0.99. Limit of detection was 0.15?ng?mL^?1. The relative standard deviation at three concentration levels (2, 12.5, and 20?ng?mL^?1) was less than 11% with accuracy in the range of 96.9–102.8%. The results of this study demonstrate that the use of dispersive liquid–liquid microextraction and core–shell column can be considered as a powerful tool for the analysis of donepezil in human urine.     

Development of molecularly imprinted-solid phase extraction combined with dispersive liquid–liquid microextraction for selective extraction and preconcentration of triazine herbicides from aqueous samples

In this study, a sensitive and developed method based on the use of molecularly imprinted-solid phase extraction along with dispersive liquid–liquid microextraction has been reported for selective extraction and pre-concentration of triazine pesticides from aqueous samples. Molecularly imprinted microspheres (template, atrazine) were synthesized using precipitation polymerization and used as sorbent in SPE procedure. A model solution containing the studied pesticides was slowly passed through the atrazine-MIP cartridge. The adsorbed analytes were eluted with methanol, mixed with carbon tetrachloride (as extraction solvent) and rapidly injected into deionized water. In this process, the analytes were extracted into fine droplets of carbon tetrachloride and the fine droplets were sedimented in bottom of the conical test tube by centrifugation. Finally, GC-FID was used for the separation and determination of analytes in the sedimented phase. Some important parameters affecting the performance of developed method were completely investigated. The linear ranges of calibration curves were wide and limits of detection and limits of quantification were between 0.2–7 and 0.5–20 ng mL^?1, respectively. The relative standard deviation obtained for six repeated experiments of atrazine (10 ng mL^?1) was 3.1 %. The relative recoveries obtained for the atrazine in the spiked samples were within in the range of 92–98 %.     

Determination of phthalate esters in edible oils by use of QuEChERS coupled with ionic-liquid-based dispersive liquid–liquid microextraction before high-performance liquid chromatography

A selective and low organic-solvent-consuming method of sample preparation combined with high-performance liquid chromatography with diode-array detection is introduced for analysis of phthalic acid esters in edible oils. Sample treatment involves initial liquid–liquid partitioning with acetonitrile, then QuEChERS cleanup by dispersive solid-phase extraction with primary secondary amine as sorbent. Preconcentration of the analytes is performed by ionic-liquid-based dispersive liquid–liquid microextraction, with the cleaned-up extract as disperser solvent and 1-hexyl-3-methylimidazolium hexafluorophosphate as extraction solvent. Under the optimized conditions, correlation coefficients (r) were 0.998–0.999 and standard errors (S _y/x ) were 2.67–3.37?×?10³ for calibration curves in the range 50–1000 ng g^?1. Detection limits, at a signal-to-noise ratio of 3, ranged from 6 to 9 ng g^?1. Intra-day and inter-day repeatability, expressed as relative standard deviation, were in the ranges 1.0–6.9 % and 2.4–9.4 %, respectively. Recovery varied between 84 % and 106 %. The developed method was successfully used for analysis of the analytes in 28 edible oils. The dibutyl phthalate content of four of the 28 samples (14 %) exceeded the specific migration limit established by domestic and international regulations.

Determination of Biogenic Amines in Cheese Using Simultaneous Derivatization and Microextraction Method Followed by Gas Chromatography–Mass Spectrometry

Microwave-assisted extraction and dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry as a sensitive and efficient method was applied to extract and determine four biogenic amines (BAs) in Iranian Lighvan cheese samples. Carrez solutions were used for the sedimentation of proteins. Effective factors on the performance of microextraction were studied and optimized. The proposed method showed good linear ranges from 5 to 500 ng mL^?1, with the coefficients of determination higher than 0.9929. Average recoveries were between 97 and 103%. Limits of detection for all analyzed BAs ranged from 5.9 to 14.0 ng g^?1, and limits of quantitation ranged between 19.7 and 46.2 ng g^?1. Compared with previous methods, the proposed method is simple, fast, accurate, and precise and gives low detection limits for investigating trace amounts of BAs in Iranian Lighvan cheese samples. The levels of four BAs were determined in five Lighvan cheese samples. Cadaverine was found as prevailing amine in the cheese samples. Putrescine, tyramine, and histamine were present at the second, third, and fourth highest levels, respectively.     

Two-phase/Three-phase Hollow Fibre Liquid-Phase Simultaneous Microextraction Combined with HPLC for Analysis of Phenolic Acids and Flavonoids in Traditional Chinese Medicine

A two-phase/three-phase hollow fibre liquid-phase simultaneous microextraction (2p/3p-HF-LPSME) method, coupled with high-performance liquid chromatography and ultraviolet detection, was developed and introduced for simultaneous extraction and determination of phenolic acids and flavonoids in Lonicera japonica, Herba Taraxaci and Cortex Eucommiae. Several factors affecting performance were investigated and optimized, including the type of hollow fibre liquid-phase microextraction, extraction solvent, pHs of the sample and acceptor phases, extraction time, stirring rate, salt concentration in the sample solution and volume of sample phase. Under optimised conditions, the enrichment factors of 2p/3p-HF-LPSME for analytes ranged from 9 to 171, and good linearities were obtained for all analytes with regression coefficients of between 0.9939 and 0.9996. In addition, the limits of detection were between 0.3 and 4.0 ng·mL⁻¹, and satisfactory recoveries (90.0–106.3 %) and precisions (RSD 2.3–10.4 %) were also achieved. The simultaneous microextraction mechanism of the approach was also analysed and described. Experimental results show that the method is simple, sensitive, practical and effective, and it can be used for simultaneous preconcentration and determination of phenolic acids and flavonoids in traditional Chinese medicines.

     

Determination of Trace Amounts of Chlorogenic Acid and Three of Its Metabolites Using Time-Resolved LPME and LC-UV Detection in Biological Specimens

Chlorogenic acid (CGA) is an effective antitumor, anti-inflammatory and antimicrobial agent. Since the absorption and metabolism of CGA remains controversial, time-resolved binary-solvent synergy liquid-phase microextraction (TRBSS-LPME) using hollow fiber was developed for the extraction of CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid, from biological specimens. In this technique, the target drugs were extracted into a binary-solvent immobilized in the wall pores of hollow fiber. The extraction occurred due to a pH gradient between the two sides of the fiber. After extraction, an aliquot was analyzed by LC. Under the optimal conditions, the CGA, caffeic acid, p-hydroxycinnamic acid and ferulic acid had good correlation of determination values (R > 0.97) and the detection limits (LODs) were 1.0, 1.0, 2.0, and 5.0 ng mL^?1 in plasma; and 1.0, 50, 10, and 50 ng mL^?1 in urine. The mean recoveries in plasma were 90.8–119.8% for CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid evaluated and the mean recoveries of caffeic acid and p-hydroxycinnamic acid in urine were 81.6–111.6%. Finally, TRBSS-LPME was successfully used for the determination of target drugs in biological specimens. It not only extended the linear range of CGA determination in biological samples and improved the sensitivity, but also eliminated interferences from complex constituents in the biological specimens and reduced the LOD.     

Determination of Anilines and Toluidines in Water by Salt-Assisted Dispersive Liquid–Liquid Microextraction Combined with GC-FID

Dispersive liquid–liquid microextraction (DLLME) assisted with salting-out was applied for the determination of five aromatic amines in water samples by using gas chromatography with flame ionization detection. In this extraction method, several factors influencing the extraction efficiency of the target analytes, such as extraction and disperser solvent type and their volume, salt addition and amount, and pH, were studied and optimized. Under the optimal DLLME conditions, good linearity was observed in the range of 4–1,000 ng mL^?1 with the RSDs from 1.2 to 7.9 %. The LODs based on S/N of 3 ranged from 0.2 to 3.4 ng mL^?1 and the enrichment factors ranged from 207 to 4,315. The proposed method was successfully applied to the water samples collected from the tap and the lake, and the relative recoveries were in the range of 87.7–108.4 %.     

Liquid Chromatographic Determination of NSAIDs in Urine After Dispersive Liquid–Liquid Microextraction Based on Solidification of Floating Organic Droplets

An environmentally benign method of sample preparation based on dispersive liquid–liquid microextraction and solidification of floating organic droplets (DLLME-SFO) coupled with high-performance liquid chromatography with ultraviolet detection has been developed for analysis of non-steroidal anti-inflammatory drugs (NSAIDs) in biological fluids. A low-toxicity solvent was used to replace the chlorinated solvents commonly used in conventional DLLME. Seven conditions were investigated and optimized: type and volume of extraction solvent and dispersive solvent, extraction time, effect of addition of salt, and sample pH. Under the optimum conditions, good linearity was obtained in the range 0.01–10 µg mL⁻¹, with coefficients of determination (r ²) >0.9949. Detection limits were in the range 0.0034–0.0052 µg mL⁻¹ with good reproducibility (RSD) and satisfactory inter-day and intra-day recovery (95.7–115.6 %). The method was successfully used for analysis of diclofenac, mefenamic acid, and ketoprofen in human urine. Analysis of urine samples from a patient 2 and 4 h after administration of diclofenac revealed concentrations of 1.20 and 0.34 µg mL⁻¹, respectively.