Determination of medroxyprogesterone acetate residues by CE immunoassay with chemiluminescence detection

A rapid and simple method is developed for the determination of medroxyprogesterone acetate (MPA) by CE immunoassay with chemiluminescence (CL). This method is based on the competitive reactions between horseradish peroxidase (HRP)-labeled MPA (MPA-HRP) and free MPA with anti-MPA antiserum. The influencing factors on the electrophoresis and CL detection were studied completely and the optimal conditions of separation and determination were obtained. The linear range was 2.0-50 nmol/L and the LOD for MPA was 0.9 nmol/L. The present method was applied to the analysis of pork tissues.     

Multi-residue analysis of traces of pesticides and antibiotics in honey by HPLC-MS-MS

The aim of this work was to develop an analytical method for simultaneous assay of residues of two families of antibiotics, and three pesticides, in honey. The assays involved a mixture of five tetracyclines, four sulfamides, and the pesticides coumaphos, carbendazim, and amitraz (two metabolites). All the compounds were extracted from honey and pre-concentrated by optimised solid-phase extraction (SPE). Analysis was by high-performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS-MS) using a triple-quadrupole spectrometer in multiple reaction monitoring (MRM) mode in order to identify and quantify the compounds present (Sheth et al J Agric Food Chem 38:1125–1130, 1990). During development of the analytical method a strong matrix effect was found that depended on the floral origin of the honey. This led to the development of a standard additions method to quantify the contaminants sought.     

Simplified liquid-chromatographic determination of residues of tetracycline antibiotics in eggs

Summary A high-performance liquid chromatographic (HPLC) procedure is described for the identification and quantification of residues of tetracycline antibiotics (TCA) (oxytetracycline, tetracycline, chlortetracycline, and doxycycline), in eggs. Spiked and blank samples were prepared by homogenization with 1∶1 (v/v) acetonitrile-mixed Mcllvaine buffer and EDTA solution (pH 4.0) then centrifugal ultrafiltration. HPLC was performed on a reversed-phase column with acetonitrile-5% (v/v) aqueous acetic acid, 35∶65 (v/v), as mobile phase and photo-diode array detection. Average recoveries (each drug spiked at 0.1, 0.2, 0.3, 0.5 and 1.0 μg g⁻¹) were >-77% with standard deviations (SD) between 1.5 and 3.5%. The inter-assay variabilities and theirSD were <3.4% and <0.7%, respectively, and intra-assay variability was between 2.0 and 3.9%. The limits of quantitation (LOQ) were 0.064 0.087, 0.121, and 0.131 μg g⁻¹ for OTC, TC, CTC, and DC, respectively. The total time required for the analysis of one sample was less than 30 min.     

Determination of tetracyclines residues in honey by on-line solid-phase extraction high-performance liquid chromatography

An automated system using on-line solid-phase extraction (SPE) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of tetracyclines (TCs), such as tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), metacycline (MC), and doxycycline (DC) in honey. One milliliter diluted honey sample was injected into a conditioned C18 SPE column and the matrix was washed out with water for 3 min. By rotation of the switching valve, TCs were eluted and transferred to the analytical column by the chromatographic mobile phase. Chromatographic conditions were optimized. TCs were separated in less than 8 min with a gradient elution using a mixture of 0.8% formic acid and acetonitrile. The UV detection was performed at 365 nm. The conditions for on-line SPE, including solvent and total time for loading sample and washing matrix were also optimized. Time for extraction and separation decreased greatly. For the five kinds of TCs, the limits of detection (LODs) at a signal-to-noise of 3 ranged from 5 to 12 ng g⁻¹. The relative standard deviations (R.S.D.) for the determination of TCs ranged from 3.4 to 7.1% within a day and ranged from 3.2 to 8.9% in 3 days, respectively.     

超高效液相-串联质谱法同时测定蜂蜜中15种喹诺酮类药物残留

建立了蜂蜜样品中15种喹诺酮类兽药残留的超高效液相色谱-串联质谱检测方法。蜂蜜样品用磷酸盐缓冲溶液溶解提取后,用Oasis HLB固相萃取柱净化,超高效液相-电喷雾串联四级杆质谱检测,外标法定量。测定时用Acquity UPLC BEHC18色谱柱(50 mm×2.1 mm,1.7μm)分离,体积分数0.1%甲酸溶液-乙腈系统梯度洗脱,质谱测定采用多重反应监测(MRM)模式。15种喹诺酮类兽药的检出限均低于或等于1.0 ng/mL,回收率均在78.6%～112.9%范围内,相对标准偏差均在10%范围内。该方法各项指标均能满足国内外各项法规的要求,可用于蜂蜜样品中喹诺酮类药物残留的定量和定性检测。     

Liquid chromatography with ultraviolet absorbance detection for the analysis of tetracycline residues in honey

The separation of tetracyclines (TCs) using reversed-phase liquid chromatography (LC) is proposed. The use of an amide-based stationary phase prevents the interaction of tetracyclines with the residual silanol groups and thus avoids the appearance of tailed peaks. Detection was based on using an UV spectrophotometer and gradient elution with acetonitrile-oxalic acid as mobile phase permitted good separation of all the peaks. Specificity was demonstrated by the retention characteristics, UV spectra and peak purity index. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of tetracycline residues (tetracycline, oxytetracycline (OTC), chlortetracycline (CTC), doxycycline (DC), minocycline (MINO) and methacycline (MTC)) in honey of different types. Extraction involved using a mild acidic solvent containing EDTA to release protein-bound or sugar-bound tetracyclines. For the clean-up step, solid phase extraction using phenyl cartridges was applied. Detection limits in the honey using the proposed procedure are between 15 and 30 ng g(-1), depending on the tetracycline.     

A sensitive and rapid immunoassay for quantification of CA125 in human sera by capillary electrophoresis with enhanced chemiluminescence detection

In this paper we have presented a sensitive and rapid immunoassay (IA) method by capillary electrophoresis with an enhanced chemiluminescence detection system (CE-CL) based on the catalytic effects of horseradish peroxidase (HRP) on the luminol-hydrogen peroxide reaction. The conditions for the CL reaction and electrophoresis were systematically investigated using HRP as a model sample. The linear range from 2.5 x 10(-11) to 1.0 x 10(-9) mol/L (R = 0.999), and the detection limit of 1.0 x 10(-12) mol/L (signal-to-noise ratio = 3) for HRP were achieved using para-iodophenol as CL enhancer. The relative standard deviations of the migration time and peak area for 5.0 x 10(-10) mol/L HRP (n = 7) were 0.26 and 4.8%, respectively, using a CE system with a home-built CL detector. Under the optimal condition, the HRP-labeled CA125 antibody (Ab) and the Ab-antigen complex were well separated within 4 min by CE using a high-pH buffer (pH 10.20). The assay was successfully used for quantification of CA125 in human sera from health controls and patients associated with ovarian cancer, and the recoveries of the standard addition experiments were 93-109%. Our primary results demonstrated that IA based on CE-CL detection is a powerful tool for clinical diagnosis combined with these commercial IA kits.     

Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection

This paper describes a rapid, sensitive and specific method for determination of free amino acids in honey involving a new reaction of derivatization and gas chromatography (GC) with flame ionization (FID) and mass spectrometric (MS) detection. The method allows the determination of 22 free amino acids in honey samples in a short time: 8 and 5 min for GC-FID and GC-MS, respectively. Quantitation was performed using Norvaline as internal standard, with detection limits ranging between 0.112 and 1.795 mg/L by GC-FID and between 0.001 and 0.291 mg/L by GC-MS in the selected-ion monitoring mode. The method was validated and applied to a set of 74 honey samples belonging to four different botanical origins: eucaliptus, rosemary, orange and heather. The statistical treatment of data shows a correct classification of different origins over 90%.     

A sensitive immunoassay based on direct hapten coated format and biotin-streptavidin system for the detection of chloramphenicol

A novel enzyme liked immunosorbent assay (ELISA) was developed for the detection of chloramphenicol (CAP). In this assay, the small molecular hapten (Hap) was directly coated on the surface of microtiter plates and biotin-streptavidin system (BSAS) was employed to improve the sensitivity of immunoassay (BSAS-direct Hap coated ELISA). The surface of microtiter plates was treated with glutaraldehyde (GA) polymer network to introduce aldehyde group, which was used to cross-link with amino group of CAP. Compared with conventional ELISA (the plates were coated with Hap-carrier protein conjugates), the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and HRP-labeled streptavidin were employed in this assay for amplification of signals. The sensitivity of BSAS-direct Hap coated ELISA was increased by approximately 20-folds and the stability was also improved greatly compared to conventional ELISA. Its 50% inhibition concentration (IC₅₀) for CAP was 10.5 ng mL⁻¹ and the limit of detection (LOD) was 0.2 ng mL⁻¹ after optimization of reaction conditions. To our knowledge, this was one of the most sensitive immunoassay for CAP yet reported. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and high performance liquid chromatography (HPLC). Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.     

A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe

Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad action spectrum against Gram-positive and Gram-negative bacteria, as well as anaerobes. The use of CHL is strictly restricted in poultry because of its toxic effect. However, CHL is still illegally used in animal farming because of its accessibility and low cost. Therefore, sensitive methods are highly desired for the determination of CHL in foodstuffs. The immunoassays based on labeling as an important tool have been reported for the detection of CHL residues in food-producing animals. However, most of the labeling procedures require multi-step reactions and purifications and thus they are complicated and time-consuming. Recently, in our previous work, luminol functionalized silver nanoparticles have been successfully synthesized, which exhibits higher CL efficiency than luminol functionalized gold nanoparticles. In this work, the new luminol functionalized silver nanoparticles have been used for the labeling of small molecules CHL for the first time and a competitive chemiluminescent immunoassay has been developed for the detection of CHL. Owing to the amplification of silver nanoparticles, high sensitivity for CHL could be achieved with a low detection limit of 7.6 × 10⁻⁹ g mL⁻¹ and a wide linear dynamic range of 1.0 × 10⁻⁸–1.0 × 10⁻⁶ g mL⁻¹. This method has also been successfully applied to determine CHL in milk and honey samples with a good recoveries (92% and 102%, 99% and 107% respectively), indicating that the method is feasible for the determination of CHL in real milk and honey samples. The labeling procedure is simple, convenient and fast, superior to previously reported labeling procedures. The immunoassay is also simple, fast, sensitive and selective. It is of application potential for the determination of CHL in foodstuffs.     

Development of a simple extraction procedure for chlorpyrifos determination in food samples by immunoassay

The suitability of immunoassay methodology for rapid and accurate determination of chlorpyrifos in vegetables was tested. The optimised ELISA detection limit was 0.32 ng/ml, with a working range from 0.69 to 6.21 ng/ml and an immunoassay test-mid point (IC₅₀) of 2.08 ng/ml. A rapid sample preparation procedure considering different parameters such as the amount of sample, volume of extractant, extraction time and dilution factor was optimised. The developed direct extraction (DE) and multiresidue (ME) standard procedures were performed in different fortified fresh and processed vegetable samples (tomato, bonnet pepper, bean, pea, asparagus, broccoli, watermelon, melon, lettuce, cucumber, celery and red pepper). Recoveries were in all cases in the whole range 85.2-108.9% for both DE and ME extracts. Also, the comparison of the results obtained by both immunochemical and chromatographic methods for spiked fruits and vegetables were good with a correlation coefficient (r) of 0.97.     

高效毛细管电泳-电导检测对四环素衍生物分离测定的研究

运用毛细管电泳-电导检测方法对4种四环素衍生物——土霉素(OTC)、金霉素(CTC)、强力霉素(DOC)和四环素(TC)的分离进行了研究。在3．5mmol/L三羟基氨基甲烷(Tris)-7．5mmol/L柠檬酸(Cit)pH4．0的运行缓冲液中，4种四环素衍生物在15min内获得完全分离。四环素衍生物的线性范围分别为5．0-500μg/mL OTC，3．6-420μg/mL CTC，4.5-470μg/mL DOC和2.5-400μg/mL TC。检测限(S／N＝3)分别为OTC2．0μg/mL，CTC 1．8μg／mL，DOC2．5μg/mL和TC1．0μg/mL。采用本法对实际样品强力霉素片中强力霉素和土霉素片中土霉素进行测定，回收率分别为97．2％和96.4％。     

Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis

A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8 mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD = 3 pg/l, IC₅₀ = 37 pg/l, DR = 10-1000 pg/l).     

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL⁻¹ Cry1Ab (3 or 5 pg mL⁻¹, depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.     

Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk

Tetracycline (TC) antibiotics are widely used for prevention and control of disease because they inhibit the growth of bacteria. However, the presence of TC antibiotics residues in food causes harmful effects on consumer's health such as allergic reactions, liver damage and gastrointestinal disturbance, so that many countries have set MRLs (maximum residue levels). Therefore, it is necessary to detect tetracycline residues, to develop suitable analytical techniques to be used as routine screens and field detection.A new approach to the biotin-avidin mediated competitive ELISA is developed to determine tetracycline residues in milk. After optimization, the LOD and LOQ were 1.0 × 10^− 10 M (0.048 μg/L) and 1.0 × 10^− 9 M, respectively, and the working range from 3.16 × 10^− 10 M to 3.16 × 10^− 7 M toward TC in milk. No cross-reactivity was observed with the structurally similar compounds; chlortetracycline (13.7%), oxytetracycline (10%) and doxytetracycline (< 1%). Additionally percent recoveries of TC spiked in milk were quite satisfactory (∼ 90%). Comparing our results obtained in this work with others, it shows with the capability to detect TC ranging in MRLs (100 μg/L in milk) sufficiently with highly sensitivity in milk, and with simple pre-treatment. In addition, this method can apply to developing useful ELISA test kit for determination of TCs in milk.     

Determination of antibiotic residues in honey

Honey produced by honeybees is a valuable food product. The presence of xenobiotics in honey may harm its quality and constitute a danger to human health. Antibiotics are commonly applied by beekeepers to eliminate disease among honeybees. Moreover, ubiquitous administration of antibiotics may cause bacteria to become resistant to many drugs and spread antibiotic-resistant strains of bacteria. Appropriate sample preparation and determination of antibiotics at very low concentrations in foodstuffs are real analytical challenges. This article reviews analytical methods used for determination of residues of different sorts of antibiotic in honey and other honeybee products.     

A novel flow-through fluorescence optosensor for the sensitive determination of tetracycline

A flow-through fluorescence optosensor with Sephadex G-50 microbeads as solid support is developed for the sensitive determination of tetracycline (TC). The fluorescent TC derivative encapsulated in CTAB micelle structures is retained onto the surface of the microbeads packed into a fluorescent flow cell in a flow system, followed by measurement of the native fluorescence of the TC derivative on the bead surface. The retained TC derivative is easily stripped off with DI water from the bead surface by breaking-down the micelle structure. This offers a convenient and effective way for the regeneration of the used solid support with DI water as a carrier. Under the optimal conditions, a linear calibration graph is obtained within a range of 3-500 μg L⁻¹, along with a detection limit of 1.0 μg L⁻¹. The present solid surface fluorescence optosensor provides a 22-fold improvement on the detection sensitivity for TC in comparison with that derived by fluorescence detection in aqueous medium. The feasibility of this flow-through fluorescence optosensor is evaluated by analyzing TC in a commercial drug tablet and surface water samples.     

A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey

Summary Results are presented that were obtained independently by two laboratories employing solid-phase micro extraction (SPME) for the analysis of pesticide residues in honey. The compounds Amitraz, Coumaphos, Cymiazole, Bromopropylate and Fluvalinate, used for chemitherapic treatment of beehives and other widely used agricultural insecticides, were extracted using a polydimethylsiloxane fiber of 100 μm thickness. The precision, accuracy, intervals of linearity and detection limits of the methodare discussed. Considering the rapidity and simplicity of this technique and, on the other hand, the difficulties arising from its application to a very complex matrix, SPME can certainly be considered a useful tool for the rapid screening of residues in honey.     

Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I

Photon upconverting nanophosphors (UCNPs) have a unique capability to produce anti-Stokes emission at visible wavelengths via sequential multiphoton absorption upon infrared excitation. Since the anti-Stokes emission can be easily spectrally resolved from the Stokes' shifted autofluorescence, the upconversion luminescence (UCL) is a highly attractive reporter technology for optical biosensors and biomolecular binding assays – potentially enabling unprecedented sensitivity in separation-based solid-phase immunoassays.     

线性扫描极谱法检测环境水样中四环素

基于四环素在pH=5.5的柠檬酸-柠檬酸钠缓冲溶液中可产生一灵敏的还原峰,建立了一种测定四环素的极谱新方法。在优化条件下,于起始电位-0.9 V(vs.SCE),峰电位-1.4V(vs.SCE),四环素浓度分别在0.1~30、30~90μg/mL范围内与峰电流呈现良好的线性关系,检出限为0.0024μg/mL。该方法直接用于环境水样中四环素的检测,回收率为97.8%~102.9%。用线性扫描极谱法研究了检测体系的电化学行为,证明了极谱波为不可逆还原吸附波,并讨论了电极反应机理。