Development of a Monoclonal Antibody-Based ELISA for the Detection of Sulfaquinoxaline in Chicken Tissues and Serum

Abstract

A monoclonal antibody (Mab) was produced by using sulfaquinoxaline-human serum albumin (SQX-HSA) conjugate as immunogen. The anti-SQX Mab exhibited negligible cross reactivity with other commonly used sulfonamides. Using this Mab, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to detect SQX in chicken tissues and serum. The ciELISA showed a 50% inhibition (IC₅₀) value of 2.60 ng/mL. The recoveries of SQX from spiked chicken muscle, liver, and serum at levels of 5–50 µg/kg were 82.6–96.5%, 75.3–94.5%, and 69.7–89.3%, respectively. The coefficient variations (CVs) were 6.22–7.17%, 4.9–8.9%, and 1.20–10.15%, respectively. Detection limits were 1.29 µg/kg in muscle, 1.32 µg/kg in liver, and 2.44 µg/kg in serum.     

Simultaneous Determination 9 Anabolic Steroids Residues in Animal Muscle Tissues by Gas Chromatography‐Mass Spectrometry

Abstract

A method was developed for determining 9 anabolic steroids (ASs) residues in animal muscle tissues by gas chromatography‐mass spectrometry (GC/MS). After undergoing enzymolysis, being homogenized, and then being picked‐up by ultrasound the sample was extracted with tert‐Butyl methyl ether, cleaned up through solid‐phase extraction (SPE) based on the reverse phase (RP) principle, and after derivatization of the analyst of interest, analyzed by GC/MS under selective ion monitoring (SIM). The limits of the detection (LOD) of the GC/MS method used for testing epitestosterone (ETS), nandrolon (17β‐NT), 17α‐methyl‐testosterone (MTS), testosterone 17‐propionate (PTS), 17β‐estradiol 3‐benzoate (BES), estrone (ESN), 17β‐estradiol (17β‐ES), 17α‐ethynylestradiol (EES), and estriol (EST) in animal muscle ranged from 0.10 to 0.33 µg/kg and the limits of quantification (LOQ) were from 0.24 to 0.82 µg/kg. The experiments using spiked samples, such as pork, beef, chicken, and fish, made it clear that at addition level of 2.0 µg/kg, the average recovery of the ASs ranged from 62.5% to 80.5%, and the coefficient of variation ranged from 12.5% to 26.8%, while at an addition level of 5.0 µg/kg, the average recovery was from 70.8% to 86.4%, and the coefficient of variation was from 8.8% to 18.4%.     

Comparison of Different Elisa Formats for the Detection of Cephalexin and Application Validation

Four ELISA formats, antigen-coated indirect, antigen-coated direct, antibody-coated, and the secondary antibody-coated, were developed using monoclonal antibody to determine cephalexin. Results showed that the secondary antibody-coated method of ELISA had a better performance in the establishment of standard curves. The optimized secondary antibody-coated ELISA was used to determine cephalexin spiked in pig muscle, pig kidney, pig liver, chicken muscle, chicken liver, and cow's milk. The limits of detection were 0.09 ng/g, 0.15 ng/g, 0.26 ng/g, 0.13 ng/g, 0.19 ng/g, and 0.08 ng/mL in pig muscle, pig kidney, pig liver, chicken muscle chicken liver, and cow's milk, respectively. A mean recovery of 77.2–128.5% and coefficient of variation of 2.6–14.7% were obtained. The results given by the ELISA method were in agreement with those of the LC-MS/MS method, which confirmed the potential of the ELISA method for the monitoring of cephalexin in milk and animal tissues.     

Comparative Analysis of Medroxyprogesterone Acetate Residue in Animal Tissues by ELISA and GC‐MS

Abstract

Medroxyprogesterone acetate is a xenobiotic growth promoter of meat‐producing animals. In this study, the liver, kidney, and muscle of rabbits were obtained after the medroxyprogesterone acetate was administrated successively for 5 days at 0.8 mg/kg and withdrawn for 7 days before slaughter. The medroxyprogesterone acetate residues were determined by ELISA method. The average residue in muscle, liver, and kidney, as 7, 6, and 23 µg/kg, respectively. Residues were confirmed by GC/MS by monitoring four ions, 373, 283, 265, and 145 m/z. A good correlation was observed between the GC/MS and ELISA methods.     

A Monoclonal Antibody-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Olaquindox in Animal Feed

A reliable indirect competitive enzyme-linked immunosorbent assay (ELISA) based on a new specific monoclonal antibody was developed to determine olaquindox in animal feed. The influence of several physicochemical factors (nonfat dried milk solution, organic solvent, incubation time) on the immunoassay was investigated. In the optimized system, the 50% inhibition concentration was 9.66 ± 1.81 µ g L^?1. The limits of detection for porcine, chicken, and fish feed were 0.28, 0.46, and 0.48 µg kg^?1. The limits of quantification were 1.00 µg kg^?1 for the feed samples. The recoveries from porcine, chicken, and fish feed spiked with olaquindox were 90–104%, 77–103%, and 78–107%, respectively, with coefficients of variation (CVs) between 3.8 and 14.1%. The cross-reactivity was less than 2.08% with four structurally related compounds and no recognition of five other restricted or forbidden drugs was observed. Parallel analysis of the three spiked feed samples showed comparable results between the indirect competitive ELISA and the standard high-performance liquid chromatography method in China (R² = 0.9985 for porcine feed, R² = 0.9896 for chicken feed, and R² = 0.9987 for fish feed). These data suggest that the developed indirect competitive ELISA is a specific and convenient method and is suitable for olaquindox determination in animal feed.     

Determination of Carbamate and Benzoylurea Insecticides in Peach Juice Drink by Floated Organic Drop Microextraction–High Performance Liquid Chromatography

Abstract

Floated organic drop microextraction (FDME)–high performance liquid chromatography (HPLC) has been developed for extraction and determination of carbamate and benzoylurea insecticide residues from peach juice drink samples. The components were separated by a Diamonsil C18 instrument (5 µm, 250 mm × 4.6 mm i.d.). The mobile phase was a mixture of acetonitrile, methanol, and water (the ratio of volume was 18:70:12). Factors relevant to the extraction efficiency were studied and optimized. The methodology exhibited good linearity between 0.01 and 10.0 µg/mL with a correlation coefficiency of 0.9999. The average recovery of pesticides ranged from 88.49% to 101.86%. The relative standard deviations (RSDs) (n = 6) were 1.99–3.47%.     

Development of a Monoclonal Antibody-Based ELISA for the Detection of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Chicken Tissues

In this study, a specific monoclonal antibody (Mab) against oxytetracycline (OTC) and its metabolite 4-epi-Oxytetracycline (4-epi-OTC) was produced. Based on this Mab, a sensitive and reliable method indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the detection of OTC and 4-epi-OTC from chicken meats. The ic-ELISA showed a 50% inhibition (IC₅₀) value of 2.01 ± 0.16 ng/ml and a detection limit of 0.13 ± 0.03 ng/ml. The recoveries from chicken muscles and livers spiked with OTC of 50–600 ng/g were 83.33–88.25% and 84.62–86.12%, respectively. The intra-assay coefficients of variation (CVs) were 4.73–9.31%, and the inter-assay CVs were 6.44–11.01%. The method showed a positive correlation with the traditional method HPLC (R² = 0.997) within a certain concentration of OTC used in this assay. The method developed in this study was simple and independent of specific expensive equipment. Thus, it could be useful as a convenient method to detect OTC residues.     

β-Cyclodextrin-Cross-Linked Polymer as Solid Phase Extraction Material Coupled Graphite Furnace Atomic Absorption Spectrometry for Separation/Analysis of Trace Copper

Abstract

The β-cyclodextrin cross-linked polymer (β-CDCP) was synthesized by the reaction of β-cyclodextrin with epichlorohydrin in NaOH and characterized by fourier transform infrared spectroscopy and thermoanalysis. The β-CDCP as a solid phase extraction (SPE) material was used to preconcentrate/separate trace copper using l-(2-pyridylazo)-2-naphthol (PAN) as a complexing agent for Cu-(II)-PAN coupled with a graphite furnace atomic absorption spectrometry (GFAAS) for analysis. The optimized experiment conditions and adsorption capacity were investigated. The detection limit (DL) was 1.11 µg.L^?1 with RSD 4.27% (n = 5, c = 25.0 µg L^?1). The linear range is 6.25–31.25 µg L^?1. Moreover, the β-CDCP could be used repeatedly and offered better a recovery and estimation of trace copper. In order to verify the accuracy of the method, a certified reference water sample was analyzed and the results obtained were in agreement with the certified values. The recovery for copper was 103.6%. The proposed method was applied to the analysis of copper in lake water samples.     

Quantification of 8-α-hydroxy-mutilin as marker residue for tiamulin in rabbit tissues by high-performance liquid chromatography-mass spectrometry

For the first time, a sensitive and specific method was developed and fully validated for the quantification of the EU marker residue of tiamulin, 8-α-hydroxy-mutilin, in rabbit muscle and liver tissues using liquid chromatography combined with positive heated electrospray ionization triple quadrupole mass spectrometry. The mass spectrometer was operated in the selected reaction monitoring (SRM) mode with selection of the [M + H]⁺ ion in both quadrupoles 1 and 3, resulting in the SRM transition m/z 337.25?>?337.25 for quantification. Chromatography was performed using a Hypersil Gold C18 column using a gradient elution program with water and methanol as mobile phases. The sample preparation procedure for the analysis of 8-α-hydroxy-mutilin in liver and muscle samples consisted of three main steps: (1) extraction of the tissue matrix using 0.1 N hydrochloric acid/acetone (50/50, v/v), (2) hydrolysis of tiamulin and metabolites to 8-α-hydroxy-mutilin in alkaline medium at 45 °C, and (3) liquid–liquid extraction in acidic medium using ethyl acetate. This is the first method presenting fully validated results, encompassing a linearity of 50 to 2,000 μg/kg, within-run and between-run accuracy and precision, limit of quantification (50 μg/kg for both muscle and liver tissues), limit of detection (muscle, 11.9 μg/kg; liver, 20.6 μg/kg), extraction recovery (muscle, 66.2 %; liver, 75.5 %), signal suppression and enhancement (muscle, 51.7 %; liver, 43.3 %), carryover, applicability and practicability, and stability during storage and analysis. This novel method is therefore sensitive enough to be used for residue depletion studies of tiamulin in rabbits and for food safety monitoring with respect to MRL compliance of residues.     

Liquid Chromatographic Method for Determination of Free and Niosome‐Entrapped Nimodipine in Mouse Plasma and Different Tissues

Abstract

A rapid HPLC method for the quantification of nimodipine in mouse plasma and tissues has been developed in this study, with simple procedure of sample preparation by one‐step protein precipitation. The results of HPLC analysis indicated that linear calibration curves were obtained over the concentration range 0.10–10.00 µg/ml for plasma, 0.10–20.00 µg/g for heart, liver, spleen, kidneys, and brain, and 1.00–200.00 µg/g for lung, respectively. The desirable precision and accuracy were achieved, both intraday and interday for plasma and tissue homogenates. Thus, this newly developed procedure was successfully applicable for determination of nimodipine in mouse plasma and tissues following intravenous administration of free and novel niosome‐entrapped nimodipine.     

Multiresidue GC-MS/MS pesticide analysis for evaluation of tea and herbal infusion safety

ABSTRACT

A fast, simple, low-cost and high-throughput multiresidue pesticide analysis method was developed and validated for 300 pesticides in herbal and fruit infusion samples based on modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure combined with gas chromatography coupled with tandem mass spectrometry method (GC-MS/MS). The objectives were to develop low cost GC-MS/MS method, validate the method in accordance to SANTE/11,813/2017 guidance document and application in routine. The results obtained using different GC and MS/MS parameters were evaluated in order to develop quick, robust, accurate and effective multiresidue method. Total analysis time was 28 min with 0.6 µL injection volume. For accurate quantification, matrix-matched calibration (MMC) curves (in range of 10 µg/kg – 250 µg/kg) were applied to compensate matrix effect. The limits of quantification (LOQ) were ranged between 0.06 µg/kg and 135 µg/kg, and for the majority of the pesticides the LOQ were below the regulatory maximum residue limits. Most recoveries at 10 µg/kg and 100 µg/kg were in the range 70%–120% indicating satisfactory accuracy. The validated method was applied to commercial herbal and fruit infusion products detecting chlorpyriphos, DEET, tebuconazole, terbuthylazine, piperonyl butoxide, biphenyl, pendimethalin, pirimiphos-methyl and p,p’-DDE in more than 100 samples from 1,466 so risk assessment on human health was calculated specially for those pesticides.     

Determination of Mycophenolic Acid (MPA) and Its Acyl and Phenol Glucuronide Metabolits Simultaneously in Human Plasma by a Simplified HPLC Method

Abstract

A simple HPLC method with ultraviolet detection for simultaneous determination of Mycophenolic acid (MPA), its phenol glucuronide metabolite (MPAG) and acyl‐MPAG (AcMPAG) in human plasma was established. The plasma samples were prepared with protein‐preciptaing reagent, and the supernatant was eluted on Zorbax column (250 mm×4.6 mm i.d, 5 µm) with 20 mmol/l NaH₂PO₄ buffer (pH 3.0, adjusted with 20% phosphoric acid) and methanol (45:55, v/v) at 304 nm. The column temperature was 45°C, and the flow rate was 1.2 ml/min. The assay was linear within the range of 0.2–50 µg/L for MPA (r=0.9997), 2.8–531 µg/L for MPAG (r=0.9999), and 0.3–24 µg/L for AcMPAG (r=0.9994). Mean absolute recovery of MPA and its metabolites and internal standard was >80%. The average recoveries of MPA, MPAG, and AcMPAG were 94.0–101.4, 98.4–101.9, and 96.1–104.2%, respectively. The RSD of within‐day and between‐day were all lower than 15%. The method described is sensitive, reproducible, and will be useful in TDM or pharmacokinetic studies of MPA.     

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Abstract

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T _max  = (0.22 ± 0.06) h, C _max = (3 ± 1) µg/mL, AUC = (32 ± 6) h?µg/mL, and K _a  = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.     

Chemiluminometric and Fluorimetric Determination of Folic Acid

Abstract

Folic acid was found to inhibit the chemiluminogenic reaction of N‐bromosuccinimide with dichlorofluorescein in alkaline medium. The analyte has also been determined after generation of a fluorescing compound by the action of hexacyanoferrate(III) or N‐bromosuccinimide in alkaline medium. Both procedures were further investigated and an attempt to propose the corresponding mechanisms was also made. The chemiluminogenic procedure allows the determination of folic acid within the range 6.0 to 114 µg/ml with limits of detection and quantification equal to 2.0 and 6.0 µg/ml, respectively and rsd at 11.4 µg/ml equal to 1.0% (n=12) while the fluorogenic procedure allows the determination of folic acid within the range 0.022–1.10 µg/ml with limits of detection and quantification equal to 0.002 and 0.005 µg/ml, respectively and rsd at 0.022 µg/ml equal to 0.7 (n=10). The methods are compared and successfully applied to commercial preparations containing folic acid.     

Ultrasonic-assisted supramolecular solvent-based liquid phase microextraction of mercury as 1-(2-pyridylazo)-2-naphthol complexes from water samples

A separation-preconcentration method based on supramolecular solvent ultrasonic-assisted liquid-phase microextraction (Ss-USA-LPME) for spectrophotometric determination of mercury as 1-(2-pyridylazo)-2-naphthol (PAN) chelates has been established. Red coloured Hg(II)-PAN hydrophobic complex was extracted into the supramolecular phase (1-decanol/THF) at pH 9.5. The extract was separated from aqueous phase by centrifugation, diluted with ethanol and determined by UV–Vis spectrophotometer at λ_max = 560 nm. The influences of important analytical parameters such as pH, amount of PAN, 1-decanol and THF, sample volume and matrix effects for the quantitative recoveries were examined and optimised. Under the optimised experimental conditions, the amount of ligand, 1-decanol and THF were 1.0 × 10^–4 M, 200 µL and 300 µL, respectively. The optimum time of ultrasonic bath and centrifugation were found as 2 min and 5 min. A linear calibration graph was obtained linearly in the concentration ranges of 8.3–1000 µg L^?1. The preconcentration factor was obtained as 20. The limit of detection (LOD) was 2.6 µg L^?1 with the relative standard deviation (RSD) of 2.4% for mercury (C = 100 µg L^?1, n = 7). The validity of the developed Ss-USA-LPME technique was checked with a certified reference material of NIST 1641d. The presented method has been successfully applied to the determination of mercury in water samples.     

Optimization of a Direct Competitive Enzyme-Linked Immunoassay for Carbofuran and Application to Water Samples

Abstract

A direct competitive enzyme-linked immunosorbent assay (ELISA) for carbofuran was developed, which was based on the anti-BFNB IgG monoclonal antibody (McAb) and a homologous enzyme tracer (BFNB-HRP). The influence of several physicochemical factors (salt, pH, detergent, and solvent) on the immunoassay was studied. For the standard curve, an I₅₀ of 2.98 µg/l and a limit of detection (I₁₀) of 0.27 µg/l was obtained in a high salt concentration buffer (0.08 M PBS, pH 7.0) with 0.03% BSA. A common challenge for immunoassay, time-dependent drift, was effectively circumvented in our study. The optimized ELISA has been used to quantify carbofuran in water samples spiked at different amounts. The excellent recoveries (71% to 130%) achieved confirmed the potential of the immunoassay for monitoring of carbofuran in waters without purification steps.     

Simultaneous Determination of Zoalene and Its Metabolite in Chicken Muscle and Liver by SPE and UPLC–MS-MS

This paper presents an analytical method for the simultaneous determination of zoalene and its metabolite 3-amino-5-nitro-o-toluamide (3-ANOT) in chicken muscle and liver by solid phase extraction and UPLC–MS-MS operated in the positive and negative ionization switching mode. Samples were extracted with phosphate buffer solution and purified with OASIS^™ HLB cartridge after pH adjustment. The determination was carried out using UPLC–MS-MS on a Waters Acquity BEH C₁₈ column with 0.1% formic acid in water/acetonitrile as mobile phase with gradient elution. The linearity of the analytical response across the studied range of concentrations (2.0–1,000 μg L⁻¹) was excellent, obtaining correlation coefficients higher than 0.999. Matrix effects had been investigated for zoalene and 3-ANOT. Recovery studies were carried out on spiked chicken muscle and liver blank samples, at four concentration levels (50, 1,500, 3,000, and 4,500 μg kg⁻¹ for chicken muscle and 50, 3,000, 6,000, and 9,000 μg kg⁻¹ for chicken liver) performing six replicates at each level. Mean recoveries of 77.9–94.2% with CVs of 3.2–8.7% were obtained. The method demonstrated to be suitable for the simultaneous determination of zoalene and 3-ANOT in chicken tissues.

     

Simultaneous Determination of Zoalene and Its Metabolite in Chicken Muscle and Liver by SPE and UPLC�CMS-MS

This paper presents an analytical method for the simultaneous determination of zoalene and its metabolite 3-amino-5-nitro-o-toluamide (3-ANOT) in chicken muscle and liver by solid phase extraction and UPLC?CMS-MS operated in the positive and negative ionization switching mode. Samples were extracted with phosphate buffer solution and purified with OASIS^? HLB cartridge after pH adjustment. The determination was carried out using UPLC?CMS-MS on a Waters Acquity BEH C₁₈ column with 0.1% formic acid in water/acetonitrile as mobile phase with gradient elution. The linearity of the analytical response across the studied range of concentrations (2.0?C1,000 ??g L^?1) was excellent, obtaining correlation coefficients higher than 0.999. Matrix effects had been investigated for zoalene and 3-ANOT. Recovery studies were carried out on spiked chicken muscle and liver blank samples, at four concentration levels (50, 1,500, 3,000, and 4,500 ??g kg^?1 for chicken muscle and 50, 3,000, 6,000, and 9,000 ??g kg^?1 for chicken liver) performing six replicates at each level. Mean recoveries of 77.9?C94.2% with CVs of 3.2?C8.7% were obtained. The method demonstrated to be suitable for the simultaneous determination of zoalene and 3-ANOT in chicken tissues.     

Determination of Manganese in Cassava Leaves by Slurry Sampling Flame Atomic Absorption Spectrometry

Abstract

Cassava, Manihot esculenta crantz, constitutes one of the main foods consumed by most of the population of developing countries, mainly in the population of low income. It is largely used for its low cost and the diversity of its root and leaves products. In this paper, a slurry sampling flame atomic absorption spectrometric method for the determination of manganese in cassava leaves is proposed. The slurry sampling was used in order to shorten the analysis time and to minimize the problems associated with solid sample treatment such as wet acid digestion, dry-ashing, sample contamination, and analyte loss. The samples of cassava leaves were washed with extran 10% (v/v), soon afterwards they were dried in an oven to 60°C, triturated in a mill of spheres, and the obtained material was sifted in a mesh of 100 micrometers. Then 0.1 g of the sample was weighed in a balloons volumetric flask, the volume was completed with 25 mL of acid nitric 2,0 mol L^?1, and followed by 20 minutes in an ultrasonic bath and subsequent reading in FAAS. The analyses of the certified reference material of spinach and apple leaves evidenced the accuracy of the method.

The proposed method was applied for the determination of manganese in four samples of cassava leaves acquired in markets from Feira de Santana City, Brazil. The concentration of manganese found in cassava leaves varied from 202.7 to 181.3 µg g^?1, with detection and quantification limits of 0.17 and 0.56 µg g^?1, respectively. The precision expressed as relative standard deviation (RSD) was 1.7 and 3.2% (n = 10) for 100 and 12 µg g^?1, respectively.     

Determination of Indole-3-acetic Acid and Abscisic Acid by Double-Direction Dispersive Liquid–Liquid Microextraction Coupled with High-Performance Liquid Chromatography

A method for the separation and purification of indole-3-acetic acid and abscisic acid based on double-direction dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography was developed. The reverse-direction dispersive liquid–liquid microextraction procedure was very efficient in the purification of the hormones. The forward-direction dispersive liquid–liquid microextraction procedure was very efficient in the enrichment of the analytes. Under optimum conditions, a linear response was observed over the ranges of 0.2 µg/g to 200.0 µg/g for indole-3-acetic acid and 0.1 µg/g to 200.0 µg/g for abscisic acid, with correlation coefficients of 0.9967 and 0.9983. The limits of detection for indole-3-acetic acid and abscisic acid were 0.02 µg/g and 0.01 µg/g. The recoveries by standard addition were 91.4% and 88.6% for indole-3-acetic acid and abscisic acid with corresponding relative standard deviations of 4.5% and 6.1% (n = 5). The developed method was used for the preconcentration and determination of indole-3-acetic acid and abscisic acid in Arabidopsis thaliana, and satisfactory results were obtained.