Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Determination of beta-lactam antibiotics in milk using micro-flow chemiluminescence system with on-line solid phase extraction

In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K₃Fe(CN)₆ CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C₁₈-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL⁻¹ of penicillin, 0.04 μg mL⁻¹ of cefradine, 0.08 μg mL⁻¹ of cefadroxil and 0.1 μg mL⁻¹ of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.     

Resonance Rayleigh scattering, frequency doubling scattering and second-order scattering spectra of the heparin-crystal violet system and their analytical application

In pH 6.0-11.2 Britton-Robinson buffer solution, binding of heparin with crystal violet (CV) can result in a significant enhancement of resonance Rayleigh scattering (RRS) and resonance non-linear scattering, such as frequency doubling scattering (FDS) and second-order scattering (SOS). Their maximum scattering wavelengths, λ_ex/λ_em, appear at 492 nm/492 nm for RRS, 984 nm/492 nm for FDS and 492 nm/984 nm for SOS, respectively. The optimum conditions of the reaction, the influencing factors and the relationship between the three scattering intensities and the concentration of heparin have been investigated. New methods for the determination of trace amounts of heparin based on the RRS, FDS and SOS methods have been developed. The methods exhibit high sensitivities, the detection limit for heparin is 2.9 ng ml⁻¹ for the RRS method, 3.5 ng ml⁻¹ for the FDS method and 3.3 ng ml⁻¹ for the SOS method. The methods have good selectivity and were applied to the determination of heparin in heparin sodium injection samples with satisfactory results.     

Room-temperature luminescence optosensings based on immobilized active principles actives: Application to nafronyl and naproxen determination in pharmaceutical preparations and biological fluids

This paper presents two easy and selective methods for determining the active principles nafronyl (NFL) and naproxen (NAP), using a flow-through fluorescence optosensor based on the on-line immobilization on a nonionic-exchanger (Silica Gel, Davisil™ and Amberlite XAD 7, respectively) solid support. The determination was performed in 5×10⁻³ M HAc/NaAc buffer solution at pH 5 for NFL and 15×10⁻³ M glycine/HCl buffer solution at pH 2.5 for NAP at a working temperature of 20 °C. The fluorescence intensities were measured at λ_ex/em=294/336 nm and λ_ex/em=332/354 nm for NFL and NAP, respectively. The response time for these optosensors were practically instant, obtaining a linear concentration range between 0 and 700.0 ng ml⁻¹ with a detection limit of 20.8 ng ml⁻¹, an analytical sensitivity of 10.1 ng ml⁻¹ and a standard deviation of 1.27% at a 500 ng ml⁻¹ concentration level for NFL and a linear concentration range between 0 and 200.0 ng ml⁻¹ with the detection limit of 13.3 ng ml⁻¹, an analytical sensitivity of 6.0 ng ml⁻¹ and a standard deviation of 3.52% at a 100 ng ml⁻¹ concentration level for NAP. The proposed methods were satisfactorily applied to real samples (three commercial formulations and urine samples). The effects of the possible interferences were evaluated in all cases.     

Column preconcentration of mercury as HgI4 using methyltrioctylammonium chloride-naphthalene adsorbent with subsequent anodic stripping-differential pulse voltammetric determination

A sensitive and selective preconcentration method has been developed for mercury using naphthalene-methyltrioctylammonium chloride (Aliquat 336s) as an adsorbent. Mercury as HgI₄²⁻ was retained by the adsorbent in the column at a flow rate of 1 ml min⁻¹. The column was washed by a solution of sodium tetraphenylborate and sodium iodide to elute the adsorbed mercury. The eluents were collected in a 10 ml volumetric flask and diluted to the mark with water, transferred to a voltammetric cell and anodic stripping-differential pulse voltammetry was performed. Preconcentration factors of 40 and 80 could be achieved when using a 10 and 5 ml voltammetric cell, respectively. The calibration graph was linear in the range of 1.2-8.7 ng ml⁻¹ Hg(II) in the initial solution with r=0.9998 (n=6) and the 3 s detection limit was 0.13 ng ml⁻¹ when using a 10 ml cell. The relative standard deviation for eight replicate measurements of 1.2, 5.0 and 8.7 ng ml⁻¹ of Hg(II) in the initial solution was 0.51, 0.71 and 0.80%, respectively. The proposed method was successfully applied to determination of mercury in natural waters, wastewater and synthetic samples.     

Determination of cadmium by flame atomic absorption spectrometry after preconcentration on naphthalene-methyltrioctylammonium chloride adsorbent as tetraiodocadmate (II) ions

A sensitive and simple solid-phase preconcentration procedure for enrichment of cadmium prior to analysis by flame atomic absorption spectrometry (FAAS) is described. The method is based on the adsorption of cadmium as CdI₄²⁻ on naphthalene-methyltrioctylammonium chloride adsorbent, elution by nitric acid and subsequent determination by FAAS. The effect of pH, iodide concentration, sample flow rate, volume of the sample and diverse ions on the recovery of the analyte was investigated and optimum conditions were established. A preconcentration factor of 40 was achieved using the optimum conditions. The calibration graph was linear in the range 1-100 ng ml⁻¹ cadmium in the initial solution. The detection limit based on the 3S_b criterion was 0.6 ng ml⁻¹ and the relative standard deviations (RSD) were 3.9 and 1.05% for 5 and 40 ng ml⁻¹, respectively (n=8). The method was successfully applied to the determination of cadmium added to river, tap and Persian Gulf water samples.     

Highly sensitive catalytic determination of molybdenum

A novel, highly sensitive, selective, and simple kinetic method was developed for the determination of Mo(VI) based on its catalytic effect on the oxidation of 1-amino-2-naphthol-4-sulfonic acid (ANSA) with H₂O₂. The reaction was followed spectrophotometrically by tracing the oxidized product at 465 nm after 30 min of mixing the reagents. The optimum reaction conditions were: 10 mmol l⁻¹ ANSA, 50 mmol l⁻¹ H₂O₂, 100 mmol l⁻¹ acetate buffer of pH 5.0 ± 0.05 and at 40 °C. Addition of 200 μg ml⁻¹ diethylenetriaminepentaacetic acid (DTPA) conferred high selectivity for the proposed method. Following the recommended procedure, Mo(VI) could be determined with a linear calibration graph up to 2.5 ng ml⁻¹ and a detection limit, based on the 3S_b-criterion, of 0.027 ng ml⁻¹. The unique sensitivity and selectivity of the implemented method allowed its direct application to the determination of Mo(VI) in natural and industrial waste water. The method was validated by comparison with the standard ETAAS method. Moreover, published catalytic-spectrophotometric methods for the determination of molybdenum were reviewed.     

Direct spectrophotometric determination of calcium in clinical samples with carboxyazo-p-CH3

A highly sensitive and relatively interference-free spectrophotometric method for determination of calcium is described. The method is based on the reaction between calcium ions and carboxyazo-p-CH₃ in aqueous citrate medium of pH 7, to form a blue complex with maximum absorption at 716 nm. The calibration is linear up to 0.12 μg ml⁻¹ calcium with a repeatability (R.S.D.) of 1.0% at a concentration of 0.04 μg ml⁻¹ (n=5). The molar absorptivity of the complex and Sandell’s sensitivity are 3.5×10⁵ l mol⁻¹ cm⁻¹ and 0.11 ng cm⁻², its 10σ limit of quantification and the 3σ limit of detection were found to be 0.3 ng ml⁻¹ and 0.09 ng ml⁻¹ respectively. The influence of reaction variables and the effect of interfering ions are studied; no interference was observed in clinical samples. The proposed method has been applied directly to the determination of calcium in clinical samples without the need for pre-concentration, masking metal ions and digesting samples.     

Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy)₃³⁺) chemiluminescence (CL) reaction. Ru(bpy)₃³⁺ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38-38 μg ml⁻¹ and 8.7-1300 ng ml⁻¹ for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml⁻¹ for citrate and 0.3 ng ml⁻¹ for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.     

Hydride generation atomic fluorescence spectrometric determination of ultratraces of selenium and tellurium in cow milk

A sensitive procedure has been developed for selenium and tellurium determination in milk by hydride generation atomic fluorescence spectrometry (HG-AFS) after microwave-assisted sample digestion. The method provides sensitivity values of 1591 and 997 fluorescence units ng⁻¹ ml⁻¹ with detection limits of 0.005 and 0.015 ng ml⁻¹ for Se and Te, respectively. The application of the developed methodology to the analysis of cow milk samples of the Spanish market evidenced the presence of concentration ranges from 11.1 to 26.0 ng ml⁻¹ for Se, and from 1.04 to 9.7 ng ml⁻¹ for Te having found a good comparability with data obtained after dry-ashing of samples.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

Synthesis of amidinothioureido-silica gel and its application to flame atomic absorption spectrometric determination of silver, gold and palladium with on-line preconcentration and separation

A simple and highly selective flow injection (FI) on-line preconcentration and separation flame atomic absorption spectrometric (FAAS) method was developed for the determinations of trace amounts of silver, gold and palladium. The selective preconcentration of the noble metals was achieved in a wide range of sample acidity (0.1-6 M HNO₃ or HCl) on a microcolumn packed with amidinothioureido-silica gel (ATuSG). The analytes retained on the column were effectively eluted with 5.0% thiourea solution. The analytical procedure was optimized for sample acidity, elution, interferences, flow rate of sampling and eluting, and concentration of sample. Common co-existing cations and anions did not interfere with the preconcentration and determination of the three metals. At a sample loading flow rate of 4.5 ml min⁻¹ with 60 s preconcentration, detection limits (3σ) of 1.1 ng ml⁻¹ Ag, 13 ng ml⁻¹ Au and 17 ng ml⁻¹ Pd were obtained. The precisions (R.S.D., n=11) were 1.2% for Ag, 1.2% for Au and 1.7% for Pd, respectively. The detection limits can be further improved by increasing sample volume. The analytical results obtained by the proposed method for a number of standard reference materials were in good agreement with the certified values.     

Application of generalized artificial neural networks coupled with an orthogonal design to optimization of a system for the kinetic spectrophotometric determination of Hg(II)

A simple and sensitive kinetic method for the determination of traces of mercury (70-760 ng ml⁻¹) based on its inhibitory effect on the addition reaction between methyl green and sulfite ion is proposed. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of methyl green at 596 nm between 2 and 4 min using a fixed time method. Artificial neural networks with back propagation algorithm coupled with an orthogonal array design were applied to the modeling of the proposed kinetic system and optimization of experimental conditions. An orthogonal design was utilized to design the experimental protocol, in which pH, concentration of sulfite, temperature, and concentration of methyl green were varied simultaneously. Optimum experimental conditions in term of sensitivity were generated by using ANNs. The rate of decrease in absorbance is inversely proportional to the concentration of Hg(II) over entire concentration range tested (100-550 ng ml⁻¹) with a detection limit of 45 ng ml⁻¹ and a relative standard deviation at 200-400 ng ml⁻¹ Hg(II) of 3.2% (n=5). A simple preconcentration step improved the limit of detection and linear dynamic range of the method to about 8 and 12-760 ng ml⁻¹, respectively, by about 10 times enrichment of mercury between 12 and 75 ng ml⁻¹. The method was based on enrichment of Hg(II) from dilute samples on an anionic ion exchanger fixed on a plastic strip and was applied to the determination of Hg(II) in environmental samples with satisfactory results.     

High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of honokiol and magnolol in rat plasma. The plasma was deproteinized with acetonitrile which contained an internal standard (diphenyl) and was separated from the aqueous layer by adding sodium chloride. Honokiol and magnolol are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC and ultraviolet detection. The limits of quantitation for honokiol and magnolol were 13 and 25 ng ml⁻¹ in plasma, respectively, and recovery of both analytes was greater than 93%. The assay was linear from 20 to 200 ng ml⁻¹ for honokiol and from 40 to 400 ng ml⁻¹ for magnolol. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of honokiol and magnolol in the plasma following rectal administration of Houpo extract at a dose of 245 mg kg⁻¹, equivalent to 13.5, 24.4 mg kg⁻¹ of honokiol and magnolol, respectively.     

A novel spectrofluorimetric method for the ultra trace analysis of nitrite and nitrate in aqueous medium and its application to air, water, soil and forensic samples

A highly sensitive and virtually specific method has been developed for the trace and ultra trace 5 ng ml⁻¹-1 μg ml⁻¹ fluorimetric analysis of nitrite. The method is based on the quenching action of nitrite on the native fluorescence of murexide (ammonium purpurate) [λ_ex=349.0 nm, λ_em=444.5 nm] in the acid range of 0.045-0.315 (M) H₂SO₄. The method is very precise and accurate (S.D.=±0.4877 and R.S.D.=0.4878% for the determination of 0.1 μg ml⁻¹ of nitrite in 11 replicates). Relatively large excesses of over 35 cations and anions do not interfere. The proposed technique has been successfully applied for the determination of nitrite and nitrate in ground water, surface water and sea water, nitrite in soil and nitrate in forensic samples. The method has also been extended for the analysis of NO_x in air.     

Partial least squares multicomponent fluorimetric determination of fluoroquinolones in human urine samples

Ternary mixtures of fluoroquinolones, with a 7-piperazinyl substituted group have been simultaneously determined in human urine samples by application of a multivariate calibration partial least squares (PLS) model. The calibration set was designed with 15 urine samples containing different concentrations of the three fluoroquinolones and 16 blank urine samples. The concentration range for the fluoroquinolones were up to 25 ng ml⁻¹ for norfloxacin (NOR), 80 ng ml⁻¹ for ofloxacin (OFLO) and 300 ng ml⁻¹ for enoxacin (ENO). The method is based on the native fluorescence emission of these compounds in sodium dodecyl sulfate (SDS) medium, at pH 4.0, when exciting at 277 nm. A selection of the emission wavelength range used for the analysis was made for each component. Intraday and interday precision values were determined. Figures of merit as selectivity, sensitivity, limit of detection (LOD) and analytical sensitivity were also calculated. Using the standard addition methodology, five urine samples from five different persons, fortified with three concentration levels of the fluoroquinolones, were analyzed. The limits of detection in urine were 10.0, 0.5 and 0.8 ng ml⁻¹ for ENO, NOR and OFLO, respectively.     

Determination of carbendazim, fuberidazole and thiabendazole by three-dimensional excitation-emission matrix fluorescence and parallel factor analysis

We simultaneously determined carbendazim, fuberidazole and thiabendazole by excitation-emission matrix (EEM) fluorescence in combination with parallel factor analysis (PARAFAC). Three-way deconvolution provided the pure analyte spectra from which we estimated the selectivity and sensitivity of the pesticides, and the relative concentration in the mixtures from which we established a linear calibration. Special attention was given calculating such figures of merit as precision, sensitivity and limit of detection (LOD), derived from the univariate calibration curve. The method, which had a relative precision of around 2-3% for the three pesticides, provided limits of detection of 20 ng ml⁻¹ for carbendazim, 4.7 ng ml⁻¹ for thiabendazole and 0.15 ng ml⁻¹ for fuberidazole. The accuracy of the method, evaluated through the root mean square error of prediction (RMSEP), was 27.5, 1.4, and 0.03 ng ml⁻¹, respectively, for each of the pesticides.     

Kinetic method for determination of nanogram amounts of copper(II) by its catalytic effect on hexacynoferrate(III)-citric acid indicator reaction

This paper describes a highly sensitive, selective catalytic-kinetic-spectrophotometric method for the determination of copper(II) concentration as low as 6 ng ml⁻¹. The method is based on the catalytic effect of copper(II) on the oxidation of citric acid by alkaline hexacyanoferrate(III). The reaction was followed by measuring the decrease in absorbance of hexacyanoferrate(III) at 420 nm (λ_max of [Fe(CN)₆]³⁻, ∈ = 1020 dm³ mol⁻¹ cm⁻¹). The dependence of rate of the indicator reaction on the reaction variables has been studied and discussed to propose a suitable mechanism to get a relation between the reaction rate and [Cu²⁺]. A fixed time procedure has been used to obtain a linear calibration curve between the initial rate and lower [Cu²⁺] or log[Cu²⁺] in the range 1 × 10⁻⁷ to 4 × 10⁻⁴ mol l⁻¹ (6.35-25,400 ng ml⁻¹). The detection limit has been calculated to be 4 ng ml⁻¹. The maximum average error is 3.5%. The effect of the presence of various cations, commonly associated with copper(II) and some anions has also been investigated and discussed. The proposed method is sensitive, accurate, rapid and inexpensive compared to other techniques available for determination of copper(II) in such a large range of concentration. The new method has been successfully applied for the determination of copper(II) in various samples.     

An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates

Based on direct hapten coated format a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for bisphenol A (BPA) was developed. Polystyrene surface was modified by 3-Aminopropyltriethoxysilane (APTES) to produce amino groups after H₂SO₄/HNO₃-pre-treatment. 4,4-bis (4-hydroxyphenyl) valeric acid (BVA) which is analogue of BPA, was successfully immobilized on the surface of microtiter plates by N,N′-dicyclohexylcarbodiimide (DCC) method. The essential steps of the assay were optimized, especially blocking procedure which is key step to prevent unspecific binding of antibody. The results indicated that compared with hapten-protein coated format (IC₅₀ = 176.67 ng ml⁻¹, LOD = 15.90 ng ml⁻¹), the direct hapten coated format (IC₅₀ = 23.50 ng ml⁻¹, LOD = 0.27 ng ml⁻¹) could improve assay sensitivity and the detection ranges were 2.30 ng∼157.60 ng ml⁻¹ with good signal reproducibility (P value > 0.05) after careful optimization of assay conditions. Tap water samples and seawater samples were spiked with a known amount of BPA and measured by ciELISA. The average recoveries were between 70 and 142%. As far as we are aware this is the most sensitive ELISA for BPA yet reported.     

Determination of vanadium, molybdenum and tungsten in complex matrix samples by chelation ion chromatography and on-line detection with inductively coupled plasma mass spectrometry

In this work, two kinds of chelating resin, bis(2-aminoethylthio)methylated resin (BAETM) and γ-aminobutyrohydroxamate resin (γ-ABHX) were synthesized. Of these, the former has a hydrophobic skeleton, and the latter a hydrophilic skeleton. The functionalities of each were 0.91 and 2.21 mmol g⁻¹, respectively. The chelating behavior of these resins towards vanadium, molybdenum and tungsten as a function of pH was studied. To perform trace metals analysis in complex matrices, a hyphenated method-chelation ion chromatography (CIC) coupled on-line detection with inductively coupled plasma mass spectrometry (ICP-MS) was developed. With a BAETM resin column (5×0.4 cm i.d.) as the separator, a sample volume of 20 μl, nitric acid (pH 1.5) as the eluent and a flow rate of 1 ml min⁻¹, the detection limits for the determination of vanadium, molybdenum and tungsten were lower than 0.05 ng ml⁻¹and the linear ranges were up to 100 ng ml⁻¹ for each element. By increasing the injected sample volume to 250 μl, the resin concentrator improved the detection limit to 0.01 ng ml⁻¹. For the determination of these elements (5 ng ml⁻¹ for each) spiked in artificial sea water samples, γ-ABHX resin column (3×0.6 cm i.d.) demonstrated well resolved peak separation between the analytes and the matrix elements—calcium and magnesium, by using sodium nitrate (10 ml, 10⁻⁴ M) as the eliminator.