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A one-step chemiluminescence immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single chain Fv–alkaline phosphatase fusion protein
Abstract
An one-step generic chemiluminescent competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv-AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv-AP fusion protein in combination with norfloxacin-ovalbumin conjugate (NOR-OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg-1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating the seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv-AP), and the linear response range of the assay extended from 0.04 to 1.08 μg kg-1. The limit of detections (LODs) of the assay for NOR were 0.017 μg kg−1 in shrimp and 0.018 μg kg−1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg−1 for ciprofloxacin (CIP) to 4.19 μg kg−1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv-AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) comfirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp.
... Fluoroquinolones (FQs, Fig. 1a) and sulfonamides (SAs, Fig. 1b) are two families of synthetic antibiotics that have broad-spectrum antimicrobial activity and used extensively to treat and prevent various infections in animals and humans and to promote growth in low dosage animal husbandry (Chen et al. 2014b;Yu et al. 2015). β-Lactams (Fig. 1c) are a broad class of antibiotics containing β-lactam nucleus in their molecular structure and have been widely used for the treatment of diseases in livestock animals, such as bovine mastitis, pneumonia, and so on (Goto et al. 2005). ...
... Hence, the simultaneous detection of four kinds of veterinary drugs in one well was achieved through the sequential reaction combination of four ligand-receptor couples. (Yu et al. 2015). PBP 2×* and AMP-EDC-HRP were prepared in previous study (Zeng et al. 2013). ...
... The CR values for 20 FQs, 15 β-lactams, 15 SAs, and CAP were all consistent with our previous studies (Zeng et al. 2013;Yu et al. 2015;Wang et al. 2013;Tao et al. 2014) (Table 1). No significant CR of four types of receptors (scFv, PBP2×*, MAb, and PAb) to RAC, IVE, GEN, FF, and TAP was observed. ...
In this paper, a novel dual-label time-resolved chemiluminescent multiplexed immunoassay (DLTRC-MIA) based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) with approximate estimation approach for simultaneous determination of 20 fluoroquinolones (FQs), 15 β-lactams, 15 sulfonamides (SAs), and chloramphenicol (CAP) in milk was developed. The strategy integrated a single-chain variable fragment–alkaline phosphatase fusion protein (scFv-ALP), a recombinant penicillin-binding protein (PBP) 2×*, a monoclonal antibody (MAb), and a polyclonal antibody (PAb) in one immunoassay and in a single well together to fulfill the simultaneous detection of 51 low-molecular weight contaminants (20 FQs, 15 β-lactams, 15 SAs, and CAP). The limits of detection for FQs, β-lactams, SAs, and CAP range from 0.29 μg L⁻¹ for ciprofloxacin (CIP) to 81.6 μg L⁻¹ for trovafloxacin (TRO), 0.27 μg L⁻¹ for ceftiofur (CEF) to 44.1 μg L⁻¹ for cephalexin (CEL), 0.089 μg L⁻¹ for sulfadimethoxine (SDM) to 2.7 μg L⁻¹ for sulfadiazine (SDZ), and 0.028 μg L⁻¹ for CAP, respectively. The results demonstrated that the detection limits of DLTRC-MIA meet the requirement of detection levels for 51 drug residues in milk, suitable for high-throughput screening of low-molecular weight contaminants.
... Furthermore, rAbs can be genetically engineered into bifunctional specific reagents with various detector labels, e.g., alkaline phosphatase (ALP) or different antibody fragments, retaining the activity of each fusion monomer. Our group designed a homogeneous and stable bifunctional scFv-ALP fusion protein to QNs, which could realize the detection of target QNs within 40 min with high sensitivity and robustness [74]. In order to detect multianalytes simultaneously, our group successfully constructed a bispecific single-chain diabody (scDb) comprising two scFvs, which could recognize 20 FQs and 14 SAs simultaneously [75]. ...
... Yu et al. proposed a CL-ELISA based on the HRPluminol for highly sensitive detection of ENR with a LOD of 0.03 ng/mL [101]. In another report, a one-step CL-ELISA employing scFv-ALP-AMPPD was established for 20 QNs detection with LOD of 0.017 mg/kg in shrimp, which was 7-fold as sensitive as the corresponding ELISA with the colorimetric substrate p-nitrophenyl phosphate [74]. Yu et al. compared two CL-ELISAs for the detection of ENR with HRP-luminol and ALP-AMPPD, showing both systems possessed similar detection performances and the choice for which reporter depends on availability [102]. ...
... Many factors can influence the performance of an immunoassay [25]. We evaluated 9 parameters to fully improve the performance of multianalyte ELISA for multi-SAs residue detection (from coating plates to color development; Table S3 in the Supporting Information). ...
The development of multianalyte immunoassays with an emphasis on food safety has attracted increasing interest, due to its high target throughput, short detection time, reduced sample consumption, and low overall cost. In this study, a superior polyclonal antibody (pAb) against sulfonamides (SAs) was raised by using a bioconjugate of bovine serum albumin with a rationally designed hapten 4-[(4-aminophenyl) sulfonyl-amino]-2-methoxybenzoic acid (SA10-X). The results showed that the pAb could recognize 19 SAs with 50% inhibition (IC50) below 100 µg L−1 and a recognition profile for SAs containing, either a five-atom ring or a six-atom ring, with highly uniform affinity. A three-dimensional quantitative structure-activity relationship analysis indicated that the electrostatic features of SAs play a considerably important role, during recognition with pAb than stereochemical effects. Skimmed milk samples were directly diluted five times before analysis. After optimization, the limit of detection for sulfamonomethoxine, sulfamethoxazole, sulfaquinoxaline, sulfadimethoxine, and sulfamethazine were 1.00, 1.25, 2.95, 3.35, and 6.10 µg L−1, respectively. The average recoveries for these 5 SAs were 72.0–107.5% with coefficients of variation less than 14.1%. The established method, based on pAb, with broad specificity and uniform affinity, offered a simple, sensitive, and high-throughput screening tool for the detection of multi-SAs in milk samples.
... [7][8][9][10][11][12] Numerous methods have been developed for the determination of FQs in environmental water, bio-uids, and foodstuffs etc., including electrochemical methods, 13 liquid chromatography tandem mass spectrometry (LC-MS/MS), 14,15 high performance liquid chromatography with ultraviolet detection (HPLC-UV), 16,17 high-performance liquid chromatography coupled to photodiode array (HPLC-PDA), 18 and enzyme immunoassays. 19,20 However, laborious sample pretreatments (e.g., centrifugation and chemical pre-extraction, etc.) and large volumes of organic solvents are usually required in the analysis of complex environmental water samples. Therefore, a highly sensitive, efficient, and simple method is needed. ...
In this study, a facile method based on molecularly imprinted polymers (MIPs) combined with internal extractive electrospray ionization tandem mass spectrometry (iEESI-MS/MS) was developed for the quantitative analysis of fluoroquinolones (FQs) in environmental water samples. FQ molecules in water samples were captured by the MIPs, which was retained on a 0.22 μm syringe filter. Then, an electrospray solution selected as the elution solution was employed to extract the FQs from the MIPs, getting an eluate of FQs for mass spectrometric interrogation. Under the optimized experimental conditions, low limits of detection (LODs, 0.015–0.026 μg L⁻¹), with relative standard deviations (RSDs) less than 8.81% (n = 6) were obtained. The present method also provides good recoveries (91.14–103.60%) with acceptable precision (RSDs < 6.18%) and have no serious matrix effects for environmental water samples. The experimental results demonstrated that MIPs-iEESI-MS/MS has advantages including easy use, high speed (less than 3 min per sample) and high sensitivity for the analysis of FQs in environmental water samples, showing potential application in environmental science and water safety control.
... The IC 50 values of 0.053 ng mL À1 for NOR and 0.21 ng mL À1 for SMZ were slightly higher than the values obtained for BL-cdELISA FQs-scFv-Rluc (IC 50 ¼ 0.031 ng mL À1 ) and BL-cdELISA SAs-scFv-Fluc (IC 50 ¼ 0.203 ng mL À1 ). However, the DBL-cdELISA was more sensitive than the chemiluminescence competitive direct enzyme immunoassay (IC 50 ¼ 0.15 ng mL À1 ) [38] and chemiluminescence competitive indirect enzyme immunoassay (IC 50 ¼ 0.12 ng mL À1 ) [32] evaluated by the IC 50 for NOR. It was also six times more sensitive than the traditional ELISA method with colorimetric detector (IC 50 ¼ 0.32 ng mL À1 ) [29] based on the same scFv. ...
The enzyme-linked immunosorbent assay (ELISA) has become the most important and widely used rapid detection technology for food safety because of its simple operation, fast speed and high sensitivity. Multiplex synchronous detection is the goal of ELISA that is always pursuing for. However, the reported multiplex ELISAs have not truly realized synchronous detection because of the complex signal generation and collection procedures. Here, we developed a dual-luciferases competitive direct bioluminescent immunoassay (DBL-cdELISA) with only one substrate addition step followed immediately by simultaneous signal acquisition. It is the first report of simultaneous multiplex analysis of small molecules based on microtiter plates and enzymes without any additional steps. The IC50 values for norfloxacin (NOR) and sulfamethazine (SMZ) were 0.051 ng mL-1 and 0.211 ng mL-1, respectively. The results demonstrated that the application of different luciferases and substrates simplified the signal generation and collection procedures and enabled simultaneous detection of small molecules with a simple procedure, high throughput and fast speed, that will be of great significance for the development of multiple assays.
... The sample preparation procedure for the reference method was adopted from Ref. [30]. The shrimp sample of 1 g was accurately weighed and mixed with 4 mL of ethanol-acetic acid mixture (99:1, v/v) in a 10 mL flask. ...
A switchable hydrophilicity solvent membrane-based microextraction (SHS-MME) strategy for simple and highly-available sample pretreatment of complex matrices has been proposed. The SHS-MME procedure based on extraction of target analytes from an aqueous sample into a porous hydrophobic membrane impregnated with a switchable hydrophilicity solvent (SHS) followed by SHS ionization and back-extraction of the analytes in alkaline acceptor solution. The medium-chain fatty acids were investigated as SHS for the SHS-MME of fluoroquinolones (fleroxacin, lomefloxacin, norfloxacin and ofloxacin). The SHS-MME was successfully coupled with a HPLC-FLD for the determination of fluoroquinolones in shrimp samples with no further sample pretreatment. The calibration graphs were linear over the concentration ranges of 3–1500 μg L⁻¹ for ofloxacin, 10–1000 μg L⁻¹ for norfloxacin, 15–1500 μg L⁻¹ for lomefloxacin and fleroxacin. The limits of detection, calculated from a blank test based on 3σ, were 1 μg L⁻¹ for ofloxacin, 3 μg L⁻¹ for norfloxacin and 5 μg L⁻¹ for lomefloxacin and fleroxacin. The SHS ionization can be considered as new potential fields in analytical application of membrane-based liquid phase microextraction.
Quinolones (QNs), used extensively as therapeutic agents and growth promoters worldwide, have significantly residued in food and environment, causing serious threat to human health through food chain. There is a high and urgent demand for easy, rapid, sensitive, and economical analytical methods for QNs residues monitoring. Immunoassays are recommended as the first-rate choice in the rapid screening of QNs residues in food and environmental samples due to various advantages. Great progress has been achieved in recent researches of immunoassays for screening QNs. This comprehensive review systematically presents specific recognition elements production, immunoassay formats construction and improvement, outlook and challenges of immunoassays for QNs. The review provides new information and insightful commentary, critically important to the development and innovation of further immunoassay for QNs detection.
Considering processing concerns regarding emerging food safety incidents and large-scale food testing, efficient and straightforward screening tools are urgently needed. The requirements for this technology include effective sample pretreatment, sensitive assay, and sample-to-answer solution that enable the detection of trace amounts of contaminants in complex food matrices. Here, we developed a chemiluminescence- and optical fiber-based fully automated immunosensing (COFFAI) system that conducts chemical contaminant food screening in a truly “sample-to-result” manner. Instead of utilizing laborious benchtop sample purification techniques followed by a time-consuming manual ELISA procedure, the user simply injects the sample of interest, and the whole procedure is automatically processed. We demonstrated that the utility of our COFFAI system can be used to detect trace amounts of chemical contamination in various milk samples within approximately 75 min. This method showed an ultrahigh sensitivity with a quinolone detection limit in the 0.022-0.065 µg/L range for skim milk, whole milk, and raw milk samples. The integration of pretreatment and assay into one automated system is an effective method for food testing. We believe that this platform provides a general method for efficient contaminant screening in foodstuffs.
Levofloxacin (LVFX) is a fluoroquinolone antibacterial agent and widely used in the treatment of bacterial infections. However, the abuse of LVFX will do great harm to human being, and alkaline urine (pH > 7.0) with decreased solubility of LVFX will further cause adverse side effects. There is an urgent demand and great challenge to construct a mutlifunctional nano-system for the detection of LVFX and pH. We report a novel water-dispersible europium (Eu)-grafted covalent organic framework nanoplates ([email protected]) with luminescent characteristics of Eu³⁺. [email protected] shows high selective and sensitive luminescence sensing for LVFX with a limit of detection (LOD) at 0.51 μM. A visual test strip has been further designed and fabricated for on-site and rapid pH measurement with a detection range from 1 to 4 and 11 to 14. This proof-of-concept study not only reports a new type of luminescent LVFX and pH sensor, but also provides a promising strategy for the design of water-dispersible lanthanide-grafted luminescent CONs to further extends its application in chemo-biosensing.
A simple, highly available and cheap approach for effective pretreatment of meat samples, based on the formation of deep eutectic solvent (DES), was proposed for the first time. The procedure assumed dissolution of a hydrogen bond acceptor of DES (quaternary ammonium salt) in homogenized meat sample, followed by heating with a hydrogen bond donor of DES (medium-chain alcohol). Heating the mixture promoted DES formation and separation of analytes from the meat sample into the DES phase formed. The developed approach was proven to be highly effective for ofloxacin and fleroxacin separation from meat samples (chicken and beef). It was shown, that the developed procedure provided higher extraction efficiency of analytes (extraction recovery 98-100 %) in comparison with conventional extraction in pre-synthesized viscous DES, composed of tetrabutylammonium bromide and heptanol (extraction recovery 33-67 %). The developed approach was coupled with high-performance liquid chromatography with fluorometric detection for the determination of ofloxacin and fleroxacin in real and spiked meat samples. The limits of detection obtained from peaks 3 times the signal-to-noise ratio were 10 μg kg⁻¹ for ofloxacin and 15 μg kg⁻¹ for fleroxacin.
For the multiple and rapid detection of β-lactams, the broadly specific penicillin-binding protein (PBP) that recognizes the β-lactam structure was prepared. A chromatographic strip based on europium chelate-loaded fluorescent microspheres was assembled with a goat anti-mouse antibody (C line) and penicillin coating (T line). The penicillin coating competes with free β-lactam antibiotics to bind PBP-labeled fluorescent microspheres. The strip can theoretically detect all kinds of β-lactams, including amoxicillin, ampicillin, oxacillin, ceftazidime, lenampicillin, cefoperazone, sultamicillin, cefotaxime, ceftriaxone, sulbenicillin, piperacillin, cephalothin, flucloxacillin, and mezlocillin, in samples within 10 min. The europium chelate-labeled lateral flow assay does not cross-react with other antibiotics, including chloramphenicol, tetracycline, sulfamethazine, enrofloxacin, gentamicin or lincomycin. In short, we developed a very useful method for preliminary screening of β-lactams.
A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.
In this paper, a novel combination of quadruple-labeling luminescence strategy multiplexed immunoassay (QLL-MIA) and automated pretreatment system for simultaneous extraction and determination of 20 fluoroquinolones (FQs), 15 β-lactams, 15 sulfonamides (SAs), and chloramphenicol (CAP) in milk was developed. The strategy integrated 2 fluorescence probes, 2 chemiluminescence probes to achieve signal separation, and a new combination of nucleic acid extraction system and immunomagnetic beads (IMBs) as automated pretreatment system to achieve simultaneous determination of 51 drugs. Taking the advantage of automated pretreatment system for saving extraction time and concentration sample with 5 times, the limits of detection (LOD) for FQs, β-lactams, SAs, and CAP in QLL-MIA range from 29.1 ng/L for ciprofloxacin (CIP) to 8000.0 ng/L (8.0 μg/L) for trovafloxacin (TRO), 20.0 ng/L for ceftiofur (CEF) to 3409.1 ng/L (3.4 μg/L) for cephalexin (CEL), 8.6 ng/L for sulfadimethoxine (SDM) to 328.2 ng/L (0.3 μg/L) for sulfadiazine (SDZ), and 6.0 ng/L for CAP. Recoveries ranged from 80.6% to 105.5% at fortified concentration of LOD and 2LOD, with coefficient of variation <15%. Analysis of field milk samples by the combination of QLL-MIA and automated pretreatment system (the developed QLL-MIA) was in accordance with that of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The above results demonstrated that the developed QLL-MIA could simultaneously screen trace FQs, β-lactams, SAs, and CAP in filed samples, suitable for high-throughput screening of low-molecular weight contaminants.
In this study, heterologous competitive antigens (HCAs) suitable for improving the sensitivity of ELISA were successfully screened based on their cross-reactivities (CRs) with 19 quinolone analogues; each containing the norfloxacin amino derivative (NOR0) coupled with bovine serum albumin (BSA) as a coating antigen. HCAs prepared with hapten analogues (CRs of 0.77%-49.92%) remarkably enhanced the sensitivity of the subsequent ELISA. ELISA sensitivity for NOR detection improved 26-fold when moxifloxacin-BSA was used as a heterologous coating antigen relative to when NOR0-BSA was used as a homologous coating antigen. This work, therefore, represents a detailed screening method to select suitable heterologous competitive antigens that improve ELISA sensitivity. Secondly, we present new theoretical tools to estimate hapten structures for use in the method, which may also be applied to improve the sensitivity of other immunoassays.
Ochratoxin A (OTA) is a common food contaminant that threatens the consumers’ safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC50) of 0.14 ng mL-1 and the limit of detection (LOD = IC10) of 0.028 ng mL-1 for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 μg kg-1 in barley, 0.56 μg kg-1 in oats, and 0.84 μg kg-1 in rice for OTA. The average recovery percent was in a range of 84-137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system.
An automated magnetic dispersive micro-solid phase extraction procedure in a fluidized reactor was developed for the determination of fluoroquinolone antimicrobial drugs (fleroxacin, norfloxacin and ofloxacin) in meat-based baby food samples. A stepwise injection system was successfully combined with afluidized reactorand applied for the magnetic dispersive micro-solid phase extraction procedure automation. The developed automated procedure involved injection of the sample solution into the fluidized reactor followed by the on-line separation of the analytes from the sample matrix based on fluidized beds strategy using magnetic nanoparticles, elution and determination of the analytes using a high performance liquid chromatography system with fluorescence detection. The floating of the magnetic nanoparticles in a liquid sample phase was accomplished by air-bubbling. In the developed method Zr-Fe-C magnetic nanoparticles were used as an efficient sorbent for the determination of fleroxacin, norfloxacin and ofloxacin. Under the optimal conditions, the calibration graphs were linear over the concentration ranges of 10-1000 μg L⁻¹ for fleroxacin (R² = 0.996), 5-1000 μg L⁻¹for norfloxacin (R² = 0.998) and ofloxacin (R² = 0.998). The limits of detection, calculated from the blank tests based on 3σ, were 3.0 μg L⁻¹forfleroxacin, 1.5 μg L⁻¹for norfloxacin and ofloxacin. The limits of quantification, calculated from the blank tests based on 10σ, were 10 μg L⁻¹ forfleroxacin, 5 μg L⁻¹for norfloxacin and ofloxacin. The method was applied for the determination of fluoroquinolonesin meat-based baby food samples and the results were compared with those obtained by the reference method. The recovery values for all analytes were within of 86-122% range.
Here we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and Renilla luciferase (Rluc) as the donor (QD-BRET) for the determination of small molecules. The ratio of the donor-acceptor that could occur energy transfer varied in the presence of different concentrations of free enrofloxacin (ENR) - an important small molecule in food safety. The calculated Förster distance (R0) was 7.86 nm. Under optimized conditions, the half-maximal inhibitory concentration (IC50) for ENR was less than 1 ng/mL and the linear range covered four orders of magnitude (0.023 to 25.60 ng/mL). The cross-reactivities (CRs) of seven representative fluoroquinolones (FQs) were similar to the data obtained by an enzyme-linked immunosorbent assay (ELISA). The average intra-and inter-assay recoveries from spiked milk of were 79.8 - 118.0%, and the relative standard deviations (RSD) was less than 10%, meeting the requirement of residue detection, which was a satisfactory result. Furthermore, we compared the influence of different luciferase substrates on the performance of the assay. Considering sensitivity and stability, coelenterazine-h was the most appropriate substrate. The results from this study will enable better-informed decisions on the choice of Rluc substrate for QD-BRET systems. For the future, the QD-BRET immunosensor could easily be extended to other small molecules, and thus represents a versatile strategy in food safety, environment, clinical diagnosis and other fields.
We report on the time-resolved detection of the three fluoroquinolone (FQs) antibiotics ciprofloxacin (CIP), enrofloxacin (ENR) and flumequine (FLU). On addition of terbium(III) ions, the terbium(III)-FQs chelates are formed in-situ in an on-capillary derivatization reaction of a microfluidic system. The laser-induced terbium(III)-sensitized luminescence of the chelates is measured at excitation/emission wavelengths of 337/545 nm. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis. The analytical ranges of the calibration graphs for CIP, ENR and FLU are from 10.6 to 60.0, 10.3 to 51.0, and 11.5 to 58.8 ng mL−1, respectively, and the detection limits are 3.2, 3.1 and 3.6 ng mL−1, respectively. The precision was established at two concentration levels of each analyte and revealed relative standard deviations in the range from 3.0 to 10.2 %. The method was applied to the analysis of FQ-spiked water samples.
Figure
We report on the time-resolved detection of the three fluoroquinolone antibiotics. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis.
Polyclonal antibodies were raised against pefloxacin, and their specificity to different quinolones was investigated and validated.
The results of the competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) demonstrated that the mice anti-pefloxacin
antibody could efficiently bind with at least nine quinolones, and to six of them, the cross-reactivity was calculated higher
than 65%. Exploiting these antibodies as receptors, six quinolones in fortified eel samples were effectively detected by both
ci-ELISA and dot immunogold filtration assay, in which the least detection limit was estimated as ≤ 10 μg/kg and ≤ 20 μg/kg,
respectively. These results fit well with molecular modeling studies based on field-overlapping and allow us to suggest possible
applications of anti-pefloxacin antibody as a new broad selective receptor for generic assay of quinolones.
A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1. Such an antibody has major potential applications in therapy and diagnostics. To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent. The performance of this reagent was further characterized, demonstrating its efficacy. The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form. The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries. Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets. Equally, the approach demonstrated should also be applicable for other haptens and larger antigens.
Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.
In this study, ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM][PF6]) was used to develop a dispersive liquid-liquid microextraction method for the extraction of four fluoroquinolone drugs in meat followed by determination with high performance liquid chromatography. During the experiments, some important parameters (ionic liquid and its volume, disperser solvent and its volume, extraction and centrifuge time, pH and salt addition) were investigated and optimized. Under the optimal conditions, the method achieved different enrichment factors (11-42 folds) for four fluoroquinolones (norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin). Results showed that the limits of detection for the four drugs were in the range of 0.5-1.1 ng mL-1 and their recoveries from the standards fortified blank meat (chicken, pork and fish) were in the range of 60.4%-96.3% with coefficients of variation lower than 11.5%. Among 60 real meat samples, seven samples were determined to contain the residues of norfloxacin, ciprofloxacin and enrofloxacin, but the residual levels were lower than their maximum residue levels (100 ng g-1).
A rapid, sensitive and reliable analytical method has been developed for the simultaneous determination of 16 fluoroquinolones (FQs) in cosmetic samples by use of ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The employment of ultrasound-assisted extraction (UAE) and solid-phase extraction (SPE) enabled exhaustive extraction of target analytes and effective clean-up of sample matrices prior to instrumental measurement. The UPLC separation was performed on an ACQUITY UPLC BEH Shield RP18 column (100 mm × 2.1 mm, 1.7 μm), preceded by a VanGuard pre-column (5 mm × 2.1 mm, 1.7 μm) of the same packing material with gradient elution. The MS/MS detection was carried out using electrospray positive ionization under the selected reaction monitoring (SRM) mode. The established method was validated with satisfactory sensitivity and specificity, and can be applied for the monitoring of FQ antibiotics residues in cosmetic products to ensure consumer health and protection.
This paper describes the synthesis of novel molecularly imprinted polymer (MIP) micro-beads for the selective extraction (MISPE) of six fluoroquinolone (FQ) antibiotics (enrofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, sarafloxacin and norfloxacin) from chicken muscle samples and further analysis by high-performance liquid chromatography (HPLC) with fluorescence (FLD) or mass spectrometry (MS) detection. A combinatorial screening approach has been applied to select the optimal functional monomer and cross-linker formulation for polymer synthesis. The MIP prepared using enoxacin (ENOX) as the template - a mixture of methacrylic acid (MAA) and trifluoromethacrylic acid (TFMAA) as functional monomers and ethylene glycol dimethacrylate (EDMA) as the cross-linker - showed superior FQ recognition properties than the rest of the materials generated. MIP spherical particles were prepared using silica beads as sacrificial scaffolds. The polymers were packed in solid phase extraction (SPE) cartridges. The optimized MISPE-HPLC method allows the extraction of the antimicrobials from aqueous samples followed by a selective washing with acetonitrile/water (0.005% TFA, pH=3.0), 20:80 (v/v) and elution with 5% trifluoroacetic acid in methanol. Optimum MISPE conditions led to recoveries of the target FQs in chicken muscle samples ranging between 68 and 102% and precisions in the 3-4% range (RSD, n=18). The method has been validated according to European Union Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCβ) by HPLC-FLD and HPLC-MS/MS. The limits of detection were improved using HPLC-MS/MS analysis and ranged between 0.2 and 2.7μgkg(-1) (S/N=3) for all the FQs tested.
Screening of a group of antibiotics from foodstuffs has traditionally relied on sophisticated chemical or physical analysis methods, such as liquid chromatography and mass spectrometric applications. The equipment for these techniques is expensive and not always applicable for high throughput screening. There is a need for an easy and cost efficient detection method for simultaneous screening of structurally similar compounds. Here we describe the engineering of a recombinant antibody which was subjected for oligonucleotide targeted random mutagenesis to emphasize the generic specificity of fluoroquinolone binding. Phage display together with small sized fluoroquinolone derivatives was used to find antibodies of high affinity and generic specificity. The most improved antibody was used to develop a time-resolved fluorescence immunoassay which was further optimized and applied for the detection of fluoroquinolone residues from spiked whole milk samples. The assay can be used to efficiently screen all EMEA controlled fluoroquinolones from whole milk samples with detection levels ranging from 0.2-68 µg L-1.
A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin-ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC50) at 0.12 μg kg(-1) for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg(-1) and the LODs for CIP and ENR were all <0.2 μg kg(-1). Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg(-1) for PEF to 2.1 μg kg(-1) for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg(-1)) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.
A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety.
Fluoroquinolones (FQs) are a group of synthetic, broad-spectrum antibacterial agents. Due to its extensive use in animal industry and aquaculture, residues of these antibiotics and the emergence of bacteria resistant to FQs have become a major public health issue. To prepare a generic antibody capable of recognizing nearly all FQs, a single-chain variable fragment (scFv) was generated from the murine hybridoma cells C49H1 producing a FQ-specific monoclonal antibody. This scFv was characterized by indirect competitive enzyme-linked immunosorbent assay (ciELISA), and it showed identical binding properties to parental monoclonal antibody: it was capable of recognizing 17 of 20 targeted FQs below maximum residue limits, except for sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO) which are highly concerned members in the FQs family. In order to broaden the specificity of this scFv to SAR and its analogues (DIF and TRO), protein homology modeling and antibody-ligands docking analysis were employed to identify the potential key amino acid residues involved in hapten antibody. A mutagenesis phage display library was generated by site directed mutagenesis randomizing five aminoacid residues in the third heavy-chain complementarity determining region. After one round of panning against biotinylated norfloxacin (NOR) and four rounds of panning against biotinylated SAR, scFv variants we screened showed up to 10-fold improved IC(50) against SAR, DIF, and TRO in ciELISA while the specificity against other FQs was fully retained.
Antibodies with a wide recognition profile of fluoroquinolone antibiotics have been produced based on chemical criteria, theoretical studies, and molecular modeling assisted hapten design. The immunizing hapten preserves the most important and characteristic epitopes of this antibiotic family. The studies have taken into consideration the zwitterionic character of most of the fluoroquinolones and the relative concentration of the different species in equilibrium at physiologic pH. The hapten is prepared in the form of a stable prehapten through a 5 step synthetic pathway. Immediately before conjugation, the immunizing hapten is obtained by removing the diphenylmethane protecting group. The specificity of the antibodies obtained is directed toward the common area defined by the fluorine atom at position 6 and the β-ketoacid moiety. The ELISA developed is able to recognize with very good detectability important fluoroquinolones used in the veterinary field such as ciprofloxacin (CPFX, IC(50), 0.35 μg L(-1)), enrofloxacin (ERFX, IC(50), 0.65 μg L(-1)), danofloxacin (DNFX, IC(50), 7.31 μg L(-1)), difloxacin (DFX, IC(50), 0.91 μg L(-1)), sarafloxacin (SRFX, IC(50), 0.96 μg L(-1)), norfloxacin (NRFX, IC(50), 0.78 μg L(-1)), ofloxacin (OFX, IC(50), 1.84 μg L(-1)), flumequine (Flume, IC(50), 3.91 μ gL(-1)), marbofloxacin (MBFX, IC(50), 4.30 μ gL(-1)), and oxolinic acid (OXO, IC(50), 23.53 μg L(-1)). The results presented here demonstrate that the antibody affinity is strongly affected by the presence of divalent cations, owing to their complexation with the fluoroquinolone molecules. Moreover, the outcome from the effect of the pH on the immunochemical assays suggests that the selectivity could be modulated with the pH due to the zwitterionic character of the fluoroquinolones and as a function of their different pK(a) values.
Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider specificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody.
A novel rapid multiresidue/multiclass procedure with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection has been developed to screen for the presence of veterinary drug residues in animal tissues. The method uses a new sample preparation procedure loosely based on QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) methodology. Validation to date has been restricted to chicken muscle and has been performed according to European Commission guidelines [COMMISSION DECISION of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results] for nitroimidazoles, sulphonamides, fluoroquinolones, quinolones, ionophores and dinitrocarbanilide. Recent work has shown that the method is also applicable to macrolide and lincosamide antibiotics, benzimidazoles, levamisole, avermectins and tranquillisers.
1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.
Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making.
A comprehensive review on the analysis of quinolone antibacterials is presented. The review covers most of the methods described for the determination of quinolone residues in edible animal products. Sample handling, chromatographic conditions and detection methods have been discussed. A summary of the most relevant information about the analytical procedures has been included.
A reversed-phase high-performance liquid chromatographic method with tandem mass-spectrometric detection was developed and validated for the simultaneous analysis of eight quinolones and fluoroquinolones (oxolinic acid, flumequine, piromidic acid, enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin and orbifloxacin) in trout tissue, prawns and abalone. The analytes were extracted from homogenised tissue using acetonitrile and the extracts subjected to an automated two-stage solid-phase extraction process involving polymeric reversed-phase and anion-exchange cartridges. Good recoveries were obtained for all analytes and the limit of quantification was 5 microg/kg (10 microg/kg for ciprofloxacin). The limit of detection was 1-3 microg/kg, depending on the analyte and matrix. Confirmation of the identity of a residue was achieved by further tandem mass-spectrometric analysis. A procedure for estimating the uncertainty associated with the measurement is presented.
Several quinolone and fluoroquinolone haptens have been used to raise polyclonal antibodies exhibiting both specific and generic properties for these classes of antimicrobial compounds. The antisera have been assessed in rapid enzyme immunoassays (ELISAs) designed to exploit the specificities obtained. A direct generic ELISA for both the quinolones and fluoroquinolones has been developed that uses the cross-reactivity of an antibody raised against norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) linked to ovalbumin via a secondary amine group on the piperazinyl moiety to detect nine different drugs in these classes. Specific ELISAs to ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid), enrofloxacin (1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid), flumequin (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo(ij)quinolizine-2-carboxylic acid) and nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) have also been developed with a high degree of specificity to the individual compounds. The assays measure drug residues in bovine milk and ovine kidney with an interassay relative standard deviation (s(r)) of 10.5% or less and intra-assay s(r) of 11.2% or less. Sensitivity is less than 4 microg x kg(-1) for both the generic and specific assays for all but one of the compounds tested. (Pipemidic acid (8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido(2,3-d)pyrimidine-6-carboxylic acid) is detectable at 6 microg x kg(-1) in kidney.)
To measure changes in the rate and type of fluoroquinolones prescribed in the United States from 1995 to 2002.
We performed a longitudinal analysis of the National Ambulatory Medical Care Survey and the National Hospital Ambulatory Medical Care Survey of adult visits to physicians in ambulatory clinics and emergency departments throughout the United States from 1995 to 2002. The main outcomes were fluoroquinolone prescribing rates and prescribing in accordance with Food and Drug Administration approval as of December 2002.
Between 1995 and 2002, fluoroquinolones became the most commonly prescribed class of antibiotics to adults in the United States. Fluoroquinolone prescribing rose threefold, from 7 million visits in 1995 to 22 million visits in 2002 (P < 0.0001). Fluoroquinolone prescribing increased as a proportion of overall antibiotic prescribing (from 10% to 24%; P < 0.0001) and as a proportion of the U.S. population (from 39 to 106 prescriptions per 1000 adults; P < 0.001). These increases were due to the use of newer fluoroquinolones with activity against Streptococcus pneumoniae. Forty-two percent of fluoroquinolone prescriptions were for nonapproved diagnoses. Among patients receiving antibiotics, nonapproved fluoroquinolone prescribing increased over time (odds ratio = 1.18 per year; 95% confidence interval: 1.13 to 1.24).
Fluoroquinolone prescribing increased threefold in outpatient clinics and emergency departments in the United States from 1995 to 2002. Fluoroquinolones became the most commonly prescribed class of antibiotics to adults in 2002. Nonapproved fluoroquinolone prescribing was common and increased over time. Such prescribing patterns are likely to be followed by an increasing prevalence of fluoroquinolone-resistant bacteria.
A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect a broad range of (fluoro)quinolones in various matrices. In the optimized generic test, anti-sarafloxacin antibodies in combination with norfloxacin conjugate showed 50% binding inhibition at 0.21 ng mL(-)(1) for sarafloxacin in buffer. Screening for this class of antibiotics is accomplished using a simple, rapid extraction carried out with a 1:1 mixture of methanol and phosphate-buffered saline adjusted to pH 7.4. This common extraction was able to detect 15 (fluoro)quinolone residues such as sarafloxacin, norfloxacin, difloxacin, ciprofloxacin, pefloxacin, ofloxacin, cinoxacin, danofloxacin, enrofloxacin, marbofloxacin, lomefloxacin, enoxacin, flumequine, oxolinic acid, and nalidixic acid in pig kidney, poultry muscle, egg, fish, and shrimp. The assay's detection capabilities (CCbeta) for most of these compounds were <10 microg kg(-)(1) except for the sarafloxacin-, oxolinic acid-, flumequine-, and cinoxacin-spiked matrices, the estimated CCbeta values of which were <4, <25, <100, and <200 microg kg(-)(1), respectively.
A new analytical method based on capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is proposed and validated for the identification and simultaneous quantification of eight quinolones for veterinary use in bovine raw milk. The studied quinolones include danofloxacin, sarafloxacin, ciprofloxacin, marbofloxacin, enrofloxacin, difloxacin, oxolinic acid, and flumequine, whose contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. Different parameters (i.e., separation buffer composition and electrospray conditions) were optimized in order to obtain both an adequate CE separation and a high sensitivity, using experimental design methodology to consider the interactions among the studied variables. MS/MS experiments using an ion trap as analyzer operating in the multiple reaction monitoring mode were carried out to achieve the minimum number of identification points according to the 2002/657/EC European Decision. For the quantification in bovine raw milk samples, a two-step solid-phase extraction procedure was developed using Oasis MAX and HLB cartridges without protein precipitation. Satisfactory results were obtained in terms of linearity (r2 between 0.989 and 0.992) and precision (RSD below 18%). The limits of detection and quantification (below 6 and 24 ppb, respectively) were in all cases lower than the maximum residues limits tolerated for these compounds in milk, the recoveries ranging from 81 to 110%, indicating the potential of the CZE-MS/MS for the analysis of regulated quinolone antibiotics in the food quality and safety control areas.
An analytical method for the simultaneous determination of seven quinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, flumequine, oxolinic acid and sarafloxacin) in egg samples of laying hens was developed. Their use is totally prohibited in animals from which eggs are produced for human consumption. Protein precipitation was achieved by addition of acetonitrile and ammonia, removal of acetonitrile with dichloromethane, the quinolones remaining in the basic aqueous extract. The aqueous extract was analysed by liquid chromatography with fluorescence detection (LC-FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found were 4-12 ng g(-1). These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in different tissues of eggs-producing animals.
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