A novel high selective and sensitive metronidazole voltammetric sensor based on a molecularly imprinted polymer-carbon paste electrode

The design and construction of a highly selective voltammetric sensor for metronidazole by using a molecularly imprinted polymer (MIP) as recognition element were introduced. A metronidazole selective MIP and a nonimprinted polymer (NIP) were synthesized and then incorporated in the carbon paste electrodes (CPEs). The sensor was applied for metronidazole determination using cathodic stripping voltammetric method. The MIP-CP electrode showed very high recognition ability in comparison to NIP-CPE. Some parameters affecting the sensor response were optimized and then the calibration curve was plotted. Two dynamic linear ranges of 5.64 × 10⁻⁵ to 2.63 × 10⁻³ mg L⁻¹ and 2.63 × 10⁻³ to 7.69 × 10⁻² mg L⁻¹ were obtained. The detection limit of the sensor was calculated as 3.59 × 10⁻⁵ mg L⁻¹. This sensor was used successfully for metronidazole determination in biological fluids.     

Sensitive and selective electrochemical determination of quinoxaline-2-carboxylic acid based on bilayer of novel poly(pyrrole) functional composite using one-step electro-polymerization and molecularly imprinted poly(o-phenylenediamine)

A facile and efficient molecularly imprinted polymer (MIP) recognition element of electrochemical sensor was fabricated by directly electro-polymerizing monomer o-phenylenediamine (oPD) in the presence of template quinoxaline-2-carboxylic acid (QCA), based on one-step controllable electrochemical modification of poly(pyrrole)-graphene oxide-binuclear phthalocyanine cobalt (II) sulphonate (PPY-GO-BiCoPc) functional composite on glassy carbon electrode (GCE). The MIP film coated on PPY-GO-BiCoPc functional composite decorated GCE (MIP/PPY-GO-BiCoPc/GCE) was presented for the first time. The synergistic effect and electro-catalytic activity toward QCA redox of PPY-GO-BiCoPc functional composite were discussed using various contrast tests. Also, the effect of experimental variables on the current response such as, electro-polymerization cycles, template/monomer ratio, elution condition for template removal, pH of the supporting electrolyte and accumulation time, were investigated in detail. Under the optimized conditions, the proposed MIP sensor possessed a fast rebinding dynamics and an excellent recognition capacity to QCA, while the anodic current response of square wave voltammetry (SWV) was well-proportional to the concentration of QCA in the range of 1.0 × 10⁻⁸–1.0 × 10⁻⁴ and 1.0 × 10⁻⁴–5.0 × 10⁻⁴ mol L⁻¹ with a low detection limit of 2.1 nmol L⁻¹. The established sensor was applied successfully to determine QCA in commercial pork and chicken muscle samples with acceptable recoveries (91.6–98.2%) and satisfactory precision (1.9–3.5% of SD), demonstrating a promising feature for applying the MIP sensor to the measurement of QCA in real samples.     

A novel high selective and sensitive para-nitrophenol voltammetric sensor, based on a molecularly imprinted polymer-carbon paste electrode

By using a molecularly imprinted polymer (MIP) as a recognition element, the design and construction of a high selective voltammetric sensor for para-nitrophenol was formed. Para-nitrophenol selective MIP and a non-imprinted polymer (NIP) were synthesized, and then used for carbon paste (CP) electrode preparation. The MIP-CP electrode showed greater recognition ability in comparison to the NIP-CP. It was shown that electrode washing after para-nitrophenol extraction led to enhanced selectivity, without noticeably decreasing the sensitivity. Some parameters affecting sensor response were optimized and a calibration curve was plotted. A dynamic linear range of 8 × 10⁻⁹ to 5 × 10⁻⁶ mol L⁻¹ was obtained. The detection limit of the sensor was calculated as 3 × 10⁻⁹ mol L⁻¹. Thus, this sensor was used successfully for the para-nitrophenol determination in different water samples.     

A highly selective molecularly imprinted electrochemiluminescence sensor for ultra-trace beryllium detection

A new molecularly imprinted electrochemiluminescence (ECL) sensor was proposed for highly sensitive and selective determination of ultratrace Be²⁺ determination. The complex of Be²⁺ with 4-(2-pyridylazo)-resorcinol (PAR) was chosen as the template molecule for the molecularly imprinted polymer (MIP). In this assay, the complex molecule could be eluted from the MIP, and the cavities formed could then selectively recognize the complex molecules. The cavities formed could also work as the tunnel for the transfer of probe molecules to produce sound responsive signal. The determination was based on the intensity of the signal, which was proportional to the concentrations of the complex molecule in the sample solution, and the Be²⁺ concentration could then be determined indirectly. The results showed that in the range of 7 × 10⁻¹¹ mol L⁻¹ to 8.0 × 10⁻⁹ mol L⁻¹, the ECL intensity had a linear relationship with the Be²⁺ concentrations, with the limit of detection of 2.35 × 10⁻¹¹ mol L⁻¹. This method was successfully used to detect Be²⁺ in real water samples.     

Molecular imprinting solid phase extraction for selective detection of methidathion in olive oil

A specific adsorbent for extraction of methidathion from olive oil was developed. The design of the molecularly imprinted polymer (MIP) was based on the results of the computational screening of the library of polymerisable functional monomers. MIP was prepared by thermal polymerisation using N,N’-methylene bisacrylamide (MBAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. The polymers based on the itaconic acid (IA), methacrylic acid (MAA) and 2-(trifluoromethyl)acryl acid (TFMAA) functional monomers and one control polymer which was made without functional monomers with cross-linker EGDMA were also synthesised and tested. The performance of each polymer was compared using corresponding imprinting factor. As it was predicted by molecular modelling the best results were obtained for the MIP prepared with MBAA. The obtained MIP was optimised in solid-phase extraction coupled with high performance liquid chromatography (MISPE-HPLC-UV) and tested for the rapid screening of methidathion in olive oil. The proposed method allowed the efficient extraction of methidathion for concentrations ranging from 0.1 to 9 mg L⁻¹ (r² = 0.996). The limits of detection (LOD) and quantification (LOQ) in olive oil were 0.02 mg L⁻¹ and 0.1 mg L⁻¹, respectively. MIPs extraction was much more effective than traditional C18 reverse-phase solid phase extraction.     

Construction of a novel molecularly imprinted sensor for the determination of O,O-dimethyl-(2,4-dichlorophenoxyacetoxyl)(3′-nitrophenyl)methinephosphonate

A novel voltammetric sensor for O,O-dimethyl-(2,4-dichlorophenoxyacetoxyl)(3′-nitrophenyl)methinephosphonate (Phi-NO₂) based on molecularly imprinted polymer (MIP) film electrode is constructed by using sol-gel technology. The sensor responds linearly to Phi-NO₂ over the concentration range of 2.0 × 10⁻⁵ to 1.0 × 10⁻⁸ mol L⁻¹ and the detection limit is 1.0 × 10⁻⁹ mol L⁻¹ (S/N = 3). This sensor provides an efficient way for eliminating interferences from coexisting substances in the solution. The high sensitivity, selectivity and stability of the sensor demonstrates its practical application for a simple and rapid determination of Phi-NO₂ in cabbage samples.     

Chromatographic characterisation, under highly aqueous conditions, of a molecularly imprinted polymer binding the herbicide 2,4-dichlorophenoxyacetic acid

The affinity of a 2,4-dichlorophenoxyacetic acid (2,4-D) molecularly imprinted polymer (MIP), which was synthesised directly in an aqueous organic solvent, for its template (2,4-D) was studied and compared with the affinity exhibited by two other reference (control) polymers, NIPA and NIPB, for the same analyte. Zonal chromatography was performed to establish the optimal selectivity, expressed as imprinting factor (IF), under chromatographic conditions more aqueous than those described so far in the literature. Frontal analysis (FA) was performed on columns packed with these polymers, using an optimized mobile phase composed of methanol/phosphate buffer (50/50, v/v), to extract adsorption isotherm data and retrieve binding parameters from the best isotherm model. Surprisingly, the template had comparable and strong affinity for both MIP (K = 3.8 × 10⁴ M⁻¹) and NIPA (K = 1.9 × 10⁴ M⁻¹), although there was a marked difference in the saturation capacities of selective and non-selective sites, as one would expect for an imprinted polymer. NIPB acts as a true control polymer in the sense that it has relatively low affinity for the template (K = 8.0 × 10² M⁻¹). This work provides the first frontal chromatographic characterization of such a polymer in a water-rich environment over a wide concentration range. The significance of this work stems from the fact that the chromatographic approach used is generic and can be applied readily to other analytes, but also because there is an increasing demand for well-characterised imprinted materials that function effectively in aqueous media and are thus well-suited for analytical science applications involving, for example, biofluids and environmental water samples.     

Molecularly imprinted polymer based potentiometric sensor for the determination of hydroxyzine in tablets and biological fluids

Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. In this work, a biomimetic potentiometric sensor, based on a non-covalent imprinted polymer, was fabricated for the recognition and determination of hydroxyzine in tablets and biological fluids. The molecularly imprinted polymer (MIP) was synthesized by precipitation polymerization, using hydroxyzine dihydrochloride as a template molecule, methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensor showed a high selectivity and a sensitive response to the template in aqueous system. The MIP-modified electrode exhibited a Nernstian response (29.4 ± 1.0 mV decade⁻¹) in a wide concentration range of 1.0 × 10⁻⁶ to 1.0 × 10⁻¹ M with a lower detection limit of 7.0 × 10⁻⁷ M. The electrode demonstrated a response time of ∼15 s, a high performance and a satisfactory long-term stability (more than 5 months). The method has the requisite accuracy, sensitivity and precision to assay hydroxyzine in tablets and biological fluids.     

Molecularly imprinted polymer for selective extraction of domoic acid from seafood coupled with high-performance liquid chromatographic determination

In this work, a highly selective sample cleanup procedure that combining molecular imprinting technique (MIT) and solid phase extraction (SPE) was developed for the isolation of domoic acid (a fascinating marine toxin) from seafood samples. The molecular imprinting polymer (MIP) for domoic acid was prepared using 1,3,5-pentanetricarboxylic acid as the template molecule instead of domoic acid. 4-Vinyl pyridine was used as the functional monomer and ethylene glycol dimethacrylate was used as the cross-linking monomer. The obtained imprinted polymer showed high affinity to domoic acid and was used as selective sorbent for the SPE of domoic acid from seafood samples. An off-line molecularly imprinted solid phase extraction (MISPE) method followed by high-performance liquid chromatography (HPLC) with diode-array detection for the detection of domoic acid was also established. Good linearity was obtained from 0.5 mg L⁻¹ to 25 mg L⁻¹ (R² > 0.99) with a quantitation limit of 0.1 mg L⁻¹, which was sufficient to determine domoic acid at the maximum level permitted by several authorities. The mean recoveries of domoic acid from mussel extracts were 93.4-96.7%. It was demonstrated that the proposed MISPE-HPLC method could be applied to direct determination of domoic acid from seafood samples.     

Synthesis and application of a carbamazepine-imprinted polymer for solid-phase extraction from urine and wastewater

A molecularly imprinted polymer (MIP) designed to enable the selective extraction of carbamazepine (CBZ) from effluent wastewater and urine samples has been synthesised using a non-covalent molecular imprinting approach. The MIP was evaluated chromatographically in the first instance and its affinity for CBZ also confirmed by solid-phase extraction (SPE). The optimal conditions for SPE consisted of conditioning of the cartridge using acidified water purified from a Milli-Q system, loading of the sample under basic aqueous conditions, clean-up using acetonitrile and elution with methanol. The attractive molecular recognition properties of the MIP gave rise to good CBZ recoveries (80%) when 100 mL of effluent water spiked with 1 μg L⁻¹ was percolated through the polymer. For urine samples, 2 mL samples spiked with 2.5 μg L⁻¹ CBZ were extracted with a recovery of 65%. For urine, the linear range was 0.05-24 mg L⁻¹, the limit of detection was 25 μg L⁻¹ and precision, expressed as relative standard deviation at 0.5 mg L⁻¹ (n = 3), was 3.1% and 12.6% for repeatability and reproducibility between days, respectively.     

Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples

Molecularly imprinted (MIP) and blank polymers with affinity for nonylphenol were designed using computational modelling. Chromatographic tests demonstrated higher affinity of imprinted polymers towards the template nonylphenol as compared with blank polymers. The performance of both polymers in solid-phase extraction was however very similar. Both blank and imprinted polymers appeared to be suitable for the removal and pre-concentration of nonylphenol from contaminated water samples with 99% efficiency of the recovery. The commercial resins PH(EC) (Biotage) and C18 (Varian) tested in the same conditions used for comparative purposes had recovery rate <84%. The polymer capacity for nonylphenol was 231 mg g⁻¹ for blank and 228 mg g⁻¹ for MIP. The synthesised materials can have significance for sample pre-concentration and environmental analysis of this class of compounds.     

A microflow chemiluminescence system for determination of chloramphenicol in honey with preconcentration using a molecularly imprinted polymer

A novel chemiluminescence (CL) microfluidic system incorporating a molecularly imprinted polymer (MIP) preconcentration step was used for the determination of chloramphenicol in honey samples. The MIP was prepared by using chloramphenicol as the template, diethylaminoethyl methacrylate (DAM) as the function monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linking monomer, 2, 2′-dimethoxy-2-phenylacetophenone (DMPA) as the free radical initiator and toluene and dodecanol as the solvent. The MIP was pre-loaded into a 10 mm long, 2 mm wide and 150 μm deep channel in a planar glass microfluidic device. When the sample containing chloramphenicol was introduced into the microfluidic device it was first preconcentrated on the MIP then detected by an enhancement effect on the chemiluminescence reaction of tris(2, 2′-bipyridyl) ruthenium(II) with cerium(IV) sulphate in sulphuric acid. A micro-syringe pump was used to pump the reagents. The CL intensity was linear in relationship to the chloramphenicol concentrations from 1.55 × 10⁻⁴ to 3.09 × 10⁻³ μmol L⁻¹ (r² = 0.9915) and the detection limit (3σ) and the quantitation limit (10σ) were found to be 7.46 × 10⁻⁶ and 2.48 × 10⁻⁵ μmol L⁻¹, respectively. This method offered a high selectivity and sensitivity for quantitative analysis of chloramphenicol in the honey samples.     

Molecularly imprinted nano particles combined with miniaturized homogenous liquid–liquid extraction for the selective extraction of loratadine in plasma and urine samples followed by high performance liquid chromatography-photo diode array detection

In this work a molecularly imprinted polymer was developed as a selective sorbent for extraction of loratadine (as a model) in complex matrices followed by miniaturized homogeneous liquid–liquid extraction (MHLLE) for the first time. The molecularly imprinted polymer (MIP) which is based on loratadine as the template was synthesized successfully by precipitation polymerization and was used as a selective sorbent. This technique was applied for preconcentration, sample preparation, and determination of loratadine using high performance liquid chromatography-photo diode array detection (HPLC-PDA). Optimization of various parameters affecting molecular imprinted solid phase extraction (MISPE), such as pH of adsorption, composition and volume of eluent, adsorption and desorption times were investigated. Besides, in the subsequent stage (MHLLE) the type and volume of extraction solvent, sodium hydroxide amount, surfactant concentration, and extraction time were investigated and optimized. Under the optimal condition, maximum enrichment capacity and Langmuir constant were 91 mg g⁻¹ and 0.014 L mg⁻¹, respectively. Furthermore, enrichment factor and extraction recovery of MIP-MHLLE method were 30 and 90%, respectively. The LOD of the proposed method was 0.2 μg L⁻¹ and a linear dynamic range of 1–1000 μg L⁻¹ was obtained with correlation coefficient of greater than 0.998. The present method was applied for extraction and determination of loratadine in plasma and urine samples in μg L⁻¹ levels and satisfactory results were achieved (RSD <8% based on three replicate measurements).     

Molecularly imprinted solid-phase extraction for the selective determination of bromhexine in human serum and urine with high performance liquid chromatography

In this work, a novel method is described for the determination of bromhexine in biological fluids using molecularly imprinted solid-phase extraction as the sample cleanup technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and bromhexine as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of bromhexine from human serum and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for molecularly imprinted solid-phase extraction (MISPE) consisted of conditioning 1 mL methanol and 1 mL of deionized water at neutral pH, loading of 5 mL of the water sample (25 μg L⁻¹) at pH 6.0, washing using 2 mL acetonitrile/acetone (1/4, v/v) and elution with 3× 1 mL methanol/acetic acid (10/1, v/v). The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of bromhexine. Results from the HPLC analyses showed that the calibration curve of bromhexine using MIP from human serum and urine is linear in the ranges of 0.5-100 and 1.5-100 μg L⁻¹ with good precisions (3.3% and 2.8% for 5.0 μg L⁻¹), respectively. The recoveries for serum and urine samples were higher than 92%.     

Synthesis by precipitation polymerisation of molecularly imprinted polymer microspheres for the selective extraction of carbamazepine and oxcarbazepine from human urine

Two molecularly imprinted polymers (MIPs), in the physical form of well-defined polymer microspheres, were synthesised via precipitation polymerisation (PP) using an antiepileptic drug, carbamazepine (CBZ), as template molecule, methacrylic acid as functional monomer and either divinylbenzene 80 (DVB-80) or a mixture of DVB-80 and ethylene glycol dimethacrylate (EGDMA) as crosslinking agents. The MIP obtained using DVB-80 alone as crosslinking agent (MIP A) had a narrow particle size distribution (9.5 ± 0.5 μm) and a well-developed permanent pore structure (specific surface area in the dry state = 758 m² g⁻¹), whereas when a mixture of DVB-80 and EGDMA (MIP B) were used as crosslinking agents, the polymer obtained had a broader particle size distribution (6.4 ± 1.8 μm) and a relatively low specific surface area (23 m² g⁻¹). The molecular recognition character of both polymers was evaluated by means of LC and then a molecularly imprinted solid-phase extraction (MISPE) protocol; CBZ was recognised by both polymers, and useful cross-selectivity for oxcarbazepine (OCBZ), which is the main metabolite of CBZ, also observed. In a detailed bioanalytical study, MIP A was selected in preference to MIP B since MIP A enabled a high volume of sample to be extracted such that lower limits of detection were achievable using this polymer. High recoveries of CBZ and OCBZ were obtained in a MISPE protocol when 50 mL of human urine spiked at 0.2 mg L⁻¹ were percolated through MIP A (90% and 83%, respectively).     

Swellable molecularly imprinted polyN-(N-propyl)acrylamide particles for detection of emerging organic contaminants using surface plasmon resonance spectroscopy

Lightly crosslinked theophylline imprinted polyN-(N-propyl)acrylamide particles (ca. 300 nm in diameter) that are designed to swell and shrink as a function of analyte concentration in aqueous media were spin coated onto a gold surface. The nanospheres responded selectively to the targeted analyte due to molecular imprinting. Chemical sensing was based on changes in the refractive index of the imprinted particles that accompanied swelling due to binding of the targeted analyte, which was detected using surface plasmon resonance (SPR) spectroscopy. Because swelling leads to an increase in the percentage of water in the polymer, the refractive index of the polymer nanospheres decreased as the particles swelled. In the presence of aqueous theophylline at concentrations as low as 10⁻⁶ M, particle swelling is both pronounced and readily detectable. The full scale response of the imprinted particles to template occurs in less than 10 min. Swelling is also reversible and independent of the ionic strength of the solution in contact with the polymer. Replicate precision is less than 10⁻⁴ RI units. By comparison, there is no response to caffeine which is similar in structure to theophylline at concentrations as high as 1 × 10⁻² M. Changes in the refractive index of the imprinted polymer particles, as low as 10⁻⁴ RI units could be readily detected. A unique aspect of the prepared particles is the use of light crosslinking rather than heavy crosslinking. This is a significant development as it indicates that heavy crosslinking is not entirely necessary for selectivity in molecular imprinting with polyacrylamides.     

Electrochemical sensor based on chlorohemin modified molecularly imprinted microgel for determination of 2,4-dichlorophenol

A newly designed molecularly imprinted polymer (MIP) was synthesized and successfully utilized as a recognition element of an amperometric sensor for 2,4-dichlorophenol (2,4-DCP) detection. The MIP with a well-defined structure could imitate the dehalogenative function of the natural enzyme chloroperoxidase for 2,4-DCP. Imprinted sensor was fabricated in situ on a glassy carbon electrode surface by drop-coating the 2,4-DCP imprinted microgel suspension and chitosan/Nafion mixture. Under optimized conditions, the sensor showed a linear response in the range of 5.0–100 μmol L⁻¹ with a detection limit of 1.6 μmol L⁻¹. Additionally, the imprinted sensor demonstrated higher affinity to target 2,4-DCP over competitive chlorophenolic compounds than non-imprinted sensor. It also exhibited good stability and acceptable repeatability. The proposed sensor could be used for the determination of 2,4-DCP in water samples with the recoveries of 96.2–111.8%, showing a promising potential in practical application.     

Solid-phase molecularly imprinted pre-concentration and spectrophotometric determination of isoxicam in pharmaceuticals and human serum

A selective molecularly imprinted polymer (MIP) has been synthesized for isoxicam pre-concentration, followed by its spectrophotometric determination based on hydrogen bonding interactions between examined drug and alizarin yellow GG. This method is able to evaluate isoxicam in range of 1.0 × 10⁻³ to 20.0 μg mL⁻¹, with a limit of determination of 1.0 ng mL⁻¹. The retention capacity and pre-concentration factor of prepared sorbent are 18.5 mg g⁻¹ and 200, respectively; and the prepared MIPs can be reused at least for five times. The MIP capability for isoxicam selection and extraction from the solution is higher than non-imprinted polymer (NIP). Under optimum conditions, this procedure can be successfully applied to assay trace amounts of isoxicam in pharmaceutical and biological samples.     

Monolithic molecularly imprinted polymer for sulfamethoxazole and molecular recognition properties in aqueous mobile phase

A monolithic molecularly imprinted polymer (monolithic MIP) for sulfamethoxazole (SMO) was prepared by in situ polymerization method as the HPLC stationary phase. By optimizing the polymerization conditions, the monolithic MIP showed highly specific recognition for the template SMO over its three structurally related analogs. As shown by SEM and the pore size distribution profile, the resultant MIP monolith showed a main pore diameter of 594 nm and a large specific surface area of 124 m² g⁻¹, this allowed the mobile phase to flow through the column with low backpressure. Furthermore, the recognition abilities of the monolithic MIP in aqueous and organic media were studied. The results exhibited that the monolithic MIP possessed excellent recognition ability in aqueous media. Hydrophobic interactions, in addition to shape recognition, were the dominant effect for recognition in the mobile phase with high water content. Moreover, the binding sites and the dissociation constant were also determined by frontal chromatography as 122 μmol g⁻¹ and 1.88 × 10⁻⁵ mol L⁻¹, respectively, which demonstrated that the obtained SMO-MIP monolith had a high binding capacity and strong affinity ability to the template molecule. Furthermore, the resultant SMO-MIP monolith was used as HPLC column directly to determine the SMO contents in three kinds of pharmaceutical tablets with the optimized aqueous mobile phase.     

Molecularly imprinted solid-phase extraction for the selective determination of 17β-estradiol in fishery samples with high performance liquid chromatography

A molecularly imprinted polymer (MIP) has been synthesized by a thermo-polymerization method using methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as cross-linker, acetonitrile as porogenic solvent, and 17β-estradiol as template. The MIP showed obvious affinity for 17β-estradiol in acetonitrile solution, which was confirmed by absorption experiments. After optimization of molecularly imprinted solid-phase extraction (MISPE) conditions, three structurally related estrogenic compounds (17β-estradiol, estriol, and diethylstilbestrol) were used to evaluate the selectivity of the MIP cartridges. The MIP cartridges exhibited highly selectivity for E₂, the recoveries were 84.8 ± 6.53% for MIPs and 19.1 ± 1.93% for non-imprinted polymer (NIP) cartridges. The detection and quantification limits correspond to 0.023 and 0.076 mg L⁻¹. Furthermore, the MISPE methods were used to selectively extract E₂ from fish and prawn tissue prior to HPLC analysis. This MISPE-HPLC procedure could eliminate all matrix interference simultaneously and had good recoveries (78.3-84.5%).