Analytica Chimica Acta (v.634, #1)
Editorial Board (iii).
The potential of mass spectrometry to study iron-containing proteins used in clinical diagnosis by M. Estela del Castillo Busto; Maria Montes-Bayón; Alfredo Sanz-Medel (1-14).
Many proteins contain iron as metal ion either within their own structures or bound to their active sites. These iron-containing proteins are involved in numerous biological processes and some of them serve as biomarkers of clinical pathologies, not only related to iron homeostasis but also to other physiological disorders. Thus, a variety of analytical strategies have been developed over the last years in order to conduct studies on Fe-containing proteins. Among them, mass spectrometric (MS) methods still remain as preferred tools since they provide the capabilities of structure elucidation together with quantitative possibilities. Therefore, in this work we have tried to summarize the most recent applications of elemental and molecular mass spectrometric-based methods for the characterization (mostly qualitative but quantitative in some cases) of the high abundant Fe-containing proteins used for clinical diagnosis.
Keywords: Ion; Iron-proteins; Mass Spectrometry; Clinical diagnosis;
Hollow fiber liquid phase microextraction combined with electrothermal atomic absorption spectrometry for the speciation of arsenic (III) and arsenic (V) in fresh waters and human hair extracts by Hongmei Jiang; Bin Hu; Beibei Chen; Linbo Xia (15-21).
A new method of hollow fiber liquid phase microextraction (HF-LPME) using ammonium pyrrolidine dithiocarbamate (APDC) as extractant combined with electrothermal atomic absorption spectrometry (ETAAS) using Pd as permanent modifier has been described for the speciation of As(III) and As(V). In a pH range of 3.0–4.0, the complex of As(III)–APDC complex can be extracted using toluene as the extraction solvent leaving As(V) in the aqueous layer. The post extraction organic phase was directly injected into ETAAS for the determination of As(III). To determine total arsenic in the samples, first As(V) was reduced to As(III) by l-cysteine, and then a microextraction method was performed prior to the determination of total arsenic. As(V) assay was based on subtracting As(III) form the total arsenic. All parameters, such as pH of solution, type of organic solvent, the amount of APDC, stirring rate and extraction time, affecting the separation of As(III) from As(V) and the extraction efficiency of As(III) were investigated, and the optimized extraction conditions were established. Under optimized conditions, a detection limit of 0.12 ng mL−1 with enrichment factor of 78 was achieved. The relative standard deviation (R.S.D.) of the method for five replicate determinations of 5 ng mL−1 As(III) was 8%. The developed method was applied to the speciation of As(III) and As(V) in fresh water and human hair extracts, and the recoveries for the spiked samples are 86–109%. In order to validate the developed method, three certified reference materials such as GBW07601 human hair, BW3209 and BW3210 environmental water were analyzed, and the results obtained were in good agreement with the certified values provided.
Keywords: Hollow fiber liquid microextraction; Electrothermal atomic absorption spectrometry; As(III) and As(V); Fresh water sample; Human hair extracts; Ammonium pyrrolidine dithiocarbamate;
Overcoming interference from the alumina matrix on the determination of arsenic at 189 nanometers using electrothermal atomic absorption spectrometry by Lenka Husáková; Tomáš Černohorský; Jitka Šrámková; Iva Urbanová-Doležalová (22-26).
A method is described enabling to eliminate the spectral interference from alumina matrix onto As determination at the wavelength 189 nm by electrothermal atomic absorption spectrometry with deuterium background correction. Matrix modification was performed by the addition of ammonium fluoride to protect the formation of aluminium oxide implicated in causing spectral interference and to increase volatility of alumina matrix via the formation of AlF3. Pre-treating of the pyrolytic graphite platform with a solution of rhodium and citric acid has enabled to stabilize the analyte up to temperature of 1300 °C at which most of AlF3 could be removed from the graphite furnace. The application of 2 μg of Rh + 20 μg of citric acid + 200 μg of NH4F has enabled an accurate and interference-free determination of As up to 40 μg of Al in the form of AlCl3 as verified by analytical recoveries study and resulted in characteristic mass and LOD value in the original sample 15 pg and 50 ng g−1, respectively (10-μL aliquots of sample).
Keywords: Arsenic analyte; Alumina matrix; Electrothermal atomic absorption spectrometry; Chemical modifiers; Background correction;
QSPR models for half-wave reduction potential of steroids: A comparative study between feature selection and feature extraction from subsets of or entire set of descriptors by Bahram Hemmateenejad; Mahdieh Yazdani (27-35).
Steroids are widely distributed in nature and are found in plants, animals, and fungi in abundance. A data set consists of a diverse set of steroids have been used to develop quantitative structure-electrochemistry relationship (QSER) models for their half-wave reduction potential. Modeling was established by means of multiple linear regression (MLR) and principle component regression (PCR) analyses. In MLR analysis, the QSPR models were constructed by first grouping descriptors and then stepwise selection of variables from each group (MLR1) and stepwise selection of predictor variables from the pool of all calculated descriptors (MLR2). Similar procedure was used in PCR analysis so that the principal components (or features) were extracted from different group of descriptors (PCR1) and from entire set of descriptors (PCR2). The resulted models were evaluated using cross-validation, chance correlation, application to prediction reduction potential of some test samples and accessing applicability domain. Both MLR approaches represented accurate results however the QSPR model found by MLR1 was statistically more significant. PCR1 approach produced a model as accurate as MLR approaches whereas less accurate results were obtained by PCR2 approach. In overall, the correlation coefficients of cross-validation and prediction of the QSPR models resulted from MLR1, MLR2 and PCR1 approaches were higher than 90%, which show the high ability of the models to predict reduction potential of the studied steroids.
Keywords: Quantitative structure–property relationship; Steroid; Electrochemistry; Feature selection; Feature extraction; Reduction potential;
Nano level detection of Cd(II) using poly(vinyl chloride) based membranes of Schiff bases by Vinod K. Gupta; Maysoon Al Khayat; Ashok K. Singh; Manoj K. Pal (36-43).
The construction and performance characteristics of polymeric membrane electrodes based on two neutral ionophores, 2,2′-(1Z,1′Z)-(1E,1′E)-(1,2-phenylenebis(methan-1-yl-1-ylidene))bis(azaan-1-yl-1-ylidene)bis(methylene)bis(azan-1-yl-1-ylidene)bis(methan-1-yl-ylidene)diphenol (L1) and 4,4′-(1E,1′E)-(butane-1,4-diylbis(azan-1-yl-1-ylidene))bis(methan-1-yl-1-ylidene)dinaphthalen-1-ol (L2) for quantification of cadmium ions, are described. The influences of membrane compositions on the potentiometric response of the electrodes have been found to substantially improve the performance characteristics. The best performance was obtained with the electrode having a membrane composition (w/w) of (L1) (2.6%):PVC (31.6%):DOP (63.2%):NaTPB (2.6%). The proposed electrode exhibits Nernstian response in the concentration range 5.0 × 10−9 to 1.0 × 10−1 M Cd2+ with limit of detection 3.1 × 10−9, performs satisfactorily over wide pH range (2.0–8.5) with a fast response time (11 s). The electrode has been found to work satisfactorily in partially non-aqueous media up to 40% (v/v) content of methanol, ethanol and acetonitrile and could be used for a period of 2.5 months. The analytical usefulness of the proposed electrode has been evaluated by its application in the determination of cadmium in cigarette samples. The practical utility of the membrane electrode has also been observed in the presence of surfactants.
Keywords: Ion selective electrodes; Cd(II); Schiff base; Electrodes; Nano level detection;
Label-free monitoring of site-specific DNA cleavage by EcoRI endonuclease using cyclic voltammetry and electrochemical impedance by Yan Jin (44-48).
Cyclic voltammetry (CV) combined with electrochemical impedance spectroscopy (EIS) were proposed to monitor the site-specific DNA cleavage by EcoRI endonuclease without using external label. The alteration of CV and EIS signal demonstrated that double-strands (dsDNA) contain recognition sequence was cleaved by EcoRI endonuclease. Real-time monitoring indicated that the dsDNA was cleaved by EcoRI more than 90% after 2 h of enzyme digestion time. Control experiment showed that the DNA cleavage by EcoRI endonuclease is site-specific for DNA sequence. Experimental results demonstrated that the efficiency of EcoRI cleavage was highly dependent on the concentration of EcoRI concentration in the range from 0.04 to 0.4 U μL−1 with one almost linear relationship.
Keywords: EcoRI endonuclease; Site-specific; DNA cleavage; Label-free; Electrochemical impedance; Cyclic voltammetry;
A detailed investigation for determination of tannic acid by anodic stripping voltammetry using porous electrochemical sensor by Lijian Xu; Nongyue He; Jingjing Du; Yan Deng; Zhiyang Li; Ting Wang (49-53).
The electrochemical responses of tannic acid have been obtained at porous pseudo-carbon paste electrode (PPCPE), polypyrrole modified carbon paste electrode (PCPE), SBA-15 modified CPE (SBA-MCPE) and carbon paste electrode (CPE) under same conditions, respectively. The results show that the sensitivity of PPCPE is the highest among all the checked electrodes. The detection limit at PPCPE is 0.01 μM, which is about 10 times lower than that at CPE and is about 5 times lower than that at PCPE or SBA-MCPE. The developed electrode PPCPE possesses a few obvious advantages and no binding reagents are needed. The surface area of PPCPE is 59.26 m2 g−1 with pores ranging from 2 to 5 μm in diameter. The PPCPE is easy to preserve and has good reusability, which affords a nice electrochemical platform for detecting tannic acid.
Keywords: Porous pseudo-carbon paste electrode; Tannic acid; Electrochemical sensor; Anodic stripping voltammetry;
Rapid Folin–Ciocalteu method using microtiter 96-well plate cartridges for solid phase extraction to assess urinary total phenolic compounds, as a biomarker of total polyphenols intake by Alexander Medina-Remón; Ana Barrionuevo-González; Raúl Zamora-Ros; Cristina Andres-Lacueva; Ramón Estruch; Miguel-Ángel Martínez-González; Javier Diez-Espino; Rosa M. Lamuela-Raventos (54-60).
Nutritional markers have several advantages for epidemiologic and clinical assays, when compared to dietary data obtained by food frequency questionnaires. Few studies have assessed whether total polyphenol (TP) compounds provide a valid biomarker for TP intake. To date, there has been almost no literature describing methods to determine TP in complex matrices such as urine, which have many interfering substances.We report a rapid Folin–Ciocalteu method to determine TP in urine samples using Oasis® MAX 96-well plate cartridges for solid phase extraction. These plates allow analysis of a high number of samples at the same time. We performed a prospective, randomized, crossover trial and one cross-sectional study with 60 volunteers from the PREDIMED trial, seeking to evaluate whether the TP in urine were correlated with polyphenol intake and could, therefore, be considered as a marker of intake of these compounds.The assay was optimized; the sensitivity and the polarity range of urine polyphenols were increased and the detection and quantification limits were significantly reduced. The metabolites in standards solution and urine samples were stable under the storage and handling conditions. In the clinical trial and the cross-sectional study, TP excreted in spot urine samples were positively correlated with TP intake, r = 0.48, P < 0.01 and r = 0.257, P = 0.04, respectively.The methodology described may be used to detect TP in urine samples, employing the high throughput of 96-well microtiter plates and reader. The method is fast and simple and it allows analysis of a large number of samples at the same time.
Keywords: Solid phase extraction; 96-Well plate; Phenols; Folin–Ciocalteu; Biomarker; Urine; PREDIMED; Polyphenols stability;
Microfluidic pool structure for cell docking and rapid mixing by Jun Yang; Jing Yang; Zheng-Qin Yin; Irina Svir; Jing Xu; Hong-Yan Luo; Min Wang; Yi Cao; Ning Hu; Yan-Jian Liao; Xiao-Lin Zheng (61-67).
A microfluidic pool structure for cell docking and rapid mixing is described. The pool structure is defined as a microchamber on one structural layer of a bilayer chip and connects with two or more individual microchannels on the other structural layer. In contrast to the turbulent flow in a macroscale pool, laminar streams enter and exit this microfluidic pool structure with definite and controllable direction that may be influenced by the location and geometry of the pool. A simple microfluidic model was used to validate this hypothesis. In this model, a microscale pool structure was made on the lower layer of a chip and connected with three parallel microchannels in the upper layer. Simulation and experimental results indicated that the flow profile within the pool structure was determined by its geometry and location. This could be used as a flow control method and it was simpler than designs based on microvalve, hydraulic pressure, or electrokinetic force, and has some important applications. For example, controllable streams within this structure were used to immobilize biological cells along the microchannel walls. When different solution streams flowed through the pool, rapid diffusion of analytes occurred for short diffusion distance between vertical flow laminas. Furthermore, desired dilution (mixing) ratio could be obtained by controlling the geometry of the microfluidic pool.
Keywords: Microfluidic; Pool structure; Cell docking; Mixing; Polydimethylsiloxane;
Chromium(III) determination without sample treatment by batch and flow injection potentiometry by Raúl A. Sánchez-Moreno; Ma Jesús Gismera; Ma Teresa Sevilla; Jesús R. Procopio (68-74).
A new and easy device for direct detection of chromium(III) in batch and flow analysis without previous oxidation/reduction or preconcentration steps of samples is designed and evaluated. For this purpose a potentiometric sensor with solid state membrane based on carbon paste matrix is developed. The sensor is modified with di(2-hydroxyphenylimino)ethane and the principal analytical parameters of the potentiometric response in batch and flow analysis are optimized and calculated. Optimal detection limits (1.4 × 10−7 M in static mode and 5.4 × 10−7 M in on-line analysis) and selectivity to trivalent chromium are obtained in both analysis modes. The use of this device to direct detection of chromium(III) in real samples is tested using a sediment Certified Reference Material. Chromium(III) determination is also carried out with successful results in environmental samples such as extracts from soils used as barriers in landfills and industrial samples such as waste waters from electroplating industries.
Keywords: Chromium(III); Chromium potentiometric sensor; Flow injection potentiometry; Di(2-hydroxyphenylimino)ethane;
Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya by Mireia Fernández Ocaña; Paul D. Fraser; Raj K.P. Patel; John M. Halket; Peter M. Bramley (75-82).
The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC–MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study.
Keywords: Genetically modified soya; Enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS); Mass spectrometry; Quantitation; Stable isotope;
Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling by Dafeng Jiang; Lei Wang; Wei Jiang (83-88).
We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin–streptavidin affinity, streptavidin-coated quantum dots were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge-coupled device. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, one by one. The linear range of 8.0 × 10−14 to 5.0 × 10−12 mol L−1 was obtained between the number of single molecules and the sample concentration.
Keywords: Single-molecule counting; Total internal reflection fluorescence microscopy; Quantum dots; Antibody;
Development of enzyme-based bar code-style lateral-flow assay for hydrogen peroxide determination by Ka-Kei Fung; Cangel Pui-Yee Chan; Reinhard Renneberg (89-95).
A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG–horseradish peroxidase conjugate (Gt anti-M IgG–HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3′,5,5′-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H2O2) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H2O2 in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H2O2-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 μM and 100 μM.
Keywords: Hydrogen peroxide; Bar code; Lateral flow; Enzyme-based; Peroxidase;
Activated aluminum oxide selectively retaining long chain n-alkanes. Part I, description of the retention properties by Katell Fiselier; Dennis Fiorini; Koni Grob (96-101).
Aluminum oxide activated by heating to 350–400 °C retains n-alkanes with more than about 20 carbon atoms, whereas iso-alkanes largely pass the column non-retained. Retention of n-alkanes is strong with n-pentane or n-hexane as mobile phase, but weak or negligible with cyclohexane or iso-octane. It is strongly reduced with increasing column temperature. Even small amounts of polar components, such as modifiers or impurities in the mobile phase, cause the retention of n-alkanes to irreversibly collapse. Since n-alkanes are not more polar than iso-alkanes and long chain n-alkanes not more polar than those of shorter chains, retention by a mechanism based on steric properties is assumed. The sensitivity to deactivation by polar components indicates that polar components and n-alkanes are retained by the same sites. The capacity for retaining n-alkanes is low, with the effect that the retention of n-alkanes depends on the load with retained paraffins. These retention properties are useful for the pre-separation of hydrocarbons in the context of the analysis of mineral oil paraffins in foodstuffs and tissue, where plant n-alkanes, typically ranging from C23 to C33, may severely disturb the analysis (subject of Part II).
Keywords: Activated aluminum oxide; Retention of long chain n-alkanes; Dependence on the eluent; Capacity of the retention mechanism; Determination of mineral paraffins in foods;
Activated aluminum oxide selectively retaining long chain n-alkanes: Part II. Integration into an on-line high performance liquid chromatography–liquid chromatography–gas chromatography–flame ionization detection method to remove plant paraffins for the determination of mineral paraffins in foods and environmental samples by Katell Fiselier; Dennis Fiorini; Koni Grob (102-109).
Aluminum oxide activated by heating to 300–400 °C retains n-alkanes with more than about 20 carbon atoms, whereas iso-alkanes largely pass non-retained (with characteristics described in more detail in Part I). This property is useful for the analysis of mineral oil contamination of foods and other matrices: it enables the removal of plant n-alkanes, typically ranging from C23 to C33, when they disturb the analysis of mineral paraffins (usually almost exclusively consisting of iso-alkanes). An on-line HPLC–LC–GC–FID method is proposed in which a first silica gel HPLC column isolates the paraffins from the bulk of edible oils or extracts and is backflushed with dichloromethane. In a second separation step, a 10 cm × 2 mm i.d. column packed with activated aluminum oxide separates the long chain n-alkanes from the fraction of the iso-alkanes which is transferred to GC–FID by the on-column interface and the retention gap technique. The retained n-alkanes are removed by flushing with iso-octane.
Keywords: Activated aluminum oxide; Retention of long chain n-alkanes; Determination of mineral paraffins in foods; On-line high performance liquid chromatography–liquid chromatography–gas chromatography–flame ionization detection;
High-performance liquid chromatographic analysis of amphotericin B in rat plasma using α-naphthol as an internal standard by J.L. Italia; D. Singh; M.N.V. Ravi Kumar (110-114).
A simple, sensitive and accurate reverse phase high-performance liquid chromatographic (RP-HPLC) method with photo-diode array detector (PDA) was developed and validated for the determination of amphotericin B (AMB) in the rat plasma using a new internal standard (IS) α-naphthol. The plasma samples were subjected to protein precipitation with methanol prior to a HPLC analysis. Chromatographic separations were achieved on a Nucleosil® 100-5C18 (150 mm × 4.6 mm) column. The mobile phase consisted of acetonitrile and sodium acetate buffer (pH 4; 10 mM) in a gradient mode. Detection was carried out at a wavelength of 407 and 294 nm for AMB and IS, respectively. The retention times of AMB and IS were about 6.8 and 7.8 min, respectively. The calibration curve was linear in the range of 10–2000 ng mL−1 for AMB (r 2 > 0.998). No significant matrix effect was observed on quantification of AMB or IS. At three quality control concentrations of 20, 500, and 2000 ng mL−1, the intra-day and inter-day relative standard deviation ranged from 1.13% to 4.91%. The limit of detection (LOD) was 5 ng mL−1 and the limit of quantification (LOQ) was 10 ng mL−1 for AMB in rat plasma. This method is simple, sensitive, rapid and does not require any extensive sample purification before injecting into HPLC.
Keywords: Amphotericin B; α-Naphthol; Plasma; Reverse phase;
An investigation of bioactive phytochemicals in the leaves of Melicope vitiflora by electrospray ionisation ion trap mass spectrometry by F. O’Donnell; V.N. Ramachandran; T.J.P. Smyth; W.F. Smyth; P. Brooks (115-120).
The ethyl acetate extract of the leaves of Melicope vitiflora was separated by column chromatography and the resulting fractions tested for their bioactivity towards methicillin-resistant-Staphylococcus aureus (MRSA) and Micrococcus luteus (ML).
Keywords: Bioactive phytochemicals; Melicope vitiflora; Electrospray ionisation ion trap mass spectrometry;
Simple spectrophotometric assessment of the trans-/cis-resveratrol ratio in aqueous solutions by Laurent Camont; Charles-Henry Cottart; Yara Rhayem; Valérie Nivet-Antoine; Raja Djelidi; Fabrice Collin; Jean-Louis Beaudeux; Dominique Bonnefont-Rousselot (121-128).
The solubility and molar absorptivity of trans- and cis-resveratrol isomers in aqueous solvents are poorly described. This study aimed to develop and describe a new simple method for the determination of trans- and cis-resveratrol concentrations in aqueous solutions. Up to 300 μM trans-resveratrol was dissolved in water by sonication for 2 h. Cis-resveratrol was obtained by exposing a 100-μM trans-resveratrol aqueous solution to sunlight for 8 h, followed by HPLC separation and analysis by mass spectrometry (resveratrol oxidation products were absent). Accurate values for UV absorbance in water were ε λ max = ε 304 nm = 30 335 M − 1 c m − 1 , ɛ 286 nm = 23 400 M−1 cm−1 for trans-resveratrol and ε λ max = ε 286 nm = 14 986 M − 1 c m − 1 , ɛ 304 nm = 9515 M−1 cm−1 for cis-resveratrol. These values allowed us to propose formulae to assess the trans-/cis-resveratrol ratio in water, using a simple and reliable UV–vis spectrophotometric method. Statistical analysis revealed no significant difference between our UV method and the commonly used HPLC method. All these data are transferable to 150 mM NaCl and 10 mM phosphate buffer solutions, which could be particularly useful for cell culture, ex vivo and in vivo studies.
Keywords: Resveratrol; Ultraviolet spectrophotometry; High performance liquid chromatography; Mass spectrometry; Stability; Solubility;
Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk by HuiLing Xie; Wei Ma; LiQiang Liu; Wei Chen; Chifang Peng; ChuanLai Xu; Libing Wang (129-133).
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.
Keywords: Immunochromatographic; Colloidal gold; Cephems; Polyclonal antibodies;