Gold Immunochromatography Assay for the Rapid Detection of Spiramycin in Milk and Beef Samples Based on a Monoclonal Antibody.

Spiramycin (SP) residues in food do harm to human health. It is necessary to establish rapid detection method for SP. In this work, a monoclonal antibody (mAb)-based gold immunochromatography assay (GICA) is developed for the rapid detection of SP. Under optimum conditions, the half-maximal inhibitory concentration of SP-mAb is 0.43 ng mL-1 . The subtype of SP-mAb is IgG2b. This antibody has no cross-reactivity with other analogues and has high affinity (4.52 × 1010 L mol-1 ). Qualitative results can be visualized with the naked eye, with a visual detection limit of 1.0 ng mL-1 and cut-off value of 10 ng mL-1 . A hand-held strip scanner is used for the quantitative analysis, with LOD 0.43 ng mL-1 in assay buffer. The recoveries of SP ranged from 72.3% to 112% in milk and 98.5% to 115% in beef, with variable coefficient ranging from 9.4% to 11.7% in milk and 8.14% to 15.4% in beef. Besides, the proposed GICA method for SP is confirmed by LC-MS/MS in SP-spiked milk and beef samples. Overall, the developed GICA can be a useful tool for SP residues on-site screening in milk and beef samples.

Spiramycin adsorption behavior on activated bentonite, activated carbon and natural phosphate in aqueous solution.

Efficacy of activated bentonite, activated carbon, and natural phosphate under experimental conditions was tested as low-cost adsorbents for spiramycin antibiotic removal from aqueous solution. Equilibrium kinetic and isotherm adsorption process are well described by pseudo-second order and Langmuir isotherm models for activated bentonite and activated carbon, while natural phosphate follows pseudo-first order and Freundlich models, respectively. Obtained results revealed that activated bentonite has the highest adsorption capacity (260.3 mg/g) as compared to activated carbon (80.3 mg/g) and natural phosphate (1.7 mg/g). The adsorption capacity decreases for all adsorbents in the presence of NaCl. The adsorption processes are facilitated in the alkaline pH range for activated bentonite and activated carbon, whereas, for natural phosphate, the acidic pH range is favorable. They are involving ion exchange and hydrogen bond mechanisms as well as Van der Waals forces and also π interactions for activated carbon. Thermodynamic calculation shows that spiramycin adsorption is endothermic and spontaneous on all adsorbents. The activated bentonite reusability is more efficient by more than 95% in two-step desorption using NaOH and HCl eluents compared to activated carbon. Thus, activated bentonite is a promising adsorbent for spiramycin removal from aqueous solution.

Municipal wastewater spiramycin removal by conventional treatments and heterogeneous photocatalysis.

This study assessed the effects and removal options of the macrolide spiramycin, currently used for both in human and veterinary medicine- with a special focus on advanced oxidation processes based on heterogeneous TiO2_assisted photocatalysis. Spiramycin real concentrations were investigated on a seasonal basis in a municipal wastewater treatment plant (up to 35µgL-1), while its removal kinetics were studied considering both aqueous solutions and real wastewater samples, including by-products toxicity assessment. High variability of spiramycin removal by activated sludge treatments (from 9% (wintertime) to >99.9% (summertime)) was observed on a seasonal basis. Preliminary results showed that a total spiramycin removal (>99.9%) is achieved with 0.1gL-1 of TiO2 in aqueous solution after 80min. Integrated toxicity showed residual slight acute effects in the photocatalytic treated solutions, independently from the amount of TiO2 used, and could be linked to the presence of intermediate compounds. Photolysis of wastewater samples collected after activated sludge treatment during summer season (SPY 5µgL-1) allowed a full SPY removal after 80min. When photocatalysis with 0.1gL-1 of TiO2 was carried out in wastewater samples collected in winter season (SPY 30µgL-1) after AS treatment, SPY removal was up to 91% after 80min.

Identification of 4″-isovaleryl-spiramycin III produced by Bacillus sp. fmbJ.

The production of secondary metabolites with antibiotic properties is a common characteristic to Bacillus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities with medicinal and agricultural interests such as antibiotic. Bacillus sp. fmbJ has been found to produce lipopeptides fengycin and surfactin in accordance with our previous report. In this study, another antimicrobial substance was separated and purified from the culture supernatant of strain fmbJ using the silica gel column chromatography and preparative reversed-phase high-performance liquid chromatography. By means of electrospray ionization mass spectroscopy, infrared spectroscopy, and nuclear magnetic resonance, the antagonistic compound was determined to be 4″-isovaleryl-spiramycin III with the molecular weight of 982 Da. This report is the first to introduce the finding of spiramycin produced from Bacillus sp. The study provides a novel source for the production of spiramycin in pharmaceutical industries.

Determination of spiramycin and neospiramycin antibiotic residues in raw milk using LC/ESI-MS/MS and solid-phase extraction.

A simple confirmatory method for the determination of spiramycin and its metabolite neospiramycin in raw milk using LC ESI MS/MS is presented. Macrolide residues in raw milk were extracted by ACN, and sample extracts were further cleaned up and concentrated using SPE cartridges. Both spiramycin and neospiramycin were protonated in electrospray positive ion mode to form singly and/or doubly charged pseudomolecular ions. Data acquisition was achieved using multiple reaction monitoring, i.e., two transitions, for quantification and confirmation. Matrix-matched standard calibration curves were utilized to achieve the best accuracy for the method. The method performance was evaluated according to both a conventional validation procedure and a designed experimental result. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of an overall recovery, was from 82.1 to 108.8%, and its intermediate precision was less than 20%. LC/ESI-MS/MS method LODs (S/N greater, not dbl equals 3:1) of spiramycin and neospiramycin were less than 1.0 microg/kg.

Sample preparation strategy for the simultaneous determination of macrolide antibiotics in animal feedingstuffs by liquid chromatography with electrochemical detection (HPLC-ECD).

A novel and suitable clean-up method that allows, for the first time, the simultaneous determination of a rather large number of macrolide antibiotics (erythromycin, rosamicin, spiramycin, tylosin, kitasamycin and josamycin in feedingstuffs by high performance liquid chromatography with electrochemical detection (HPLC-ECD) is presented in this work. The effectiveness of the developed clean-up method allows the quantification of the target macrolides in poultry feed using standard calibration curves instead of matrix matched standards, which overcomes the general problem of finding representative blanks. Furthermore an additional back extraction included in the sample preparation procedure allows the determination of an additional macrolide (oleandomycin) with detection limits, expressed as apparent concentration in poultry feed, ranging from 0.04 to 0.22 mg kg(-1) and relative standard deviation values ranging from 3.6 to 10.1% depending on the target analyte. Moreover, this additional step has been proven to enlarge the scope of the method by the extension of its applicability, at the target level of concentration, to other animal feedingstuffs such as pig and cattle. The analysis of real feedingstuffs containing macrolides demonstrated the fitness for purpose of the whole analytical procedure as well as a good fitting between real and spiked samples. The proposed methods appeared therefore as a sound alternative in the frame of control (e.g. for post-screening purposes) and/or monitoring surveillance programmes at the target level of 1.0 mg kg(-1) established according to the reported lowest dosage of additive needed to lead a growth promoting effect.

Liquid chromatography-UV diode-array detection method for multi-residue determination of macrolide antibiotics in sheep's milk.

A rapid, simple and sensitive liquid chromatography-UV diode-array detection method was developed for the simultaneous determination of seven macrolides (erythromycin, oleandomycin, roxithromycin, josamycin, spiramycin, tylosin and ivermectin) in sheep's milk. The column, mobile phase, temperature and flow rate were optimised to provide the best resolution of these analytes. The extraction of the antibiotic residues involves the treatment of protein-free samples with a combination of concentrated sodium hydroxide and ethyl acetate. Necessary defatting is achieved by alkaline hydrolysis. The recovery of each antibiotic was between 55% and 77%, with relative standard deviations ranging from 1% to 6.5%. The limit of quantification was 72.4 microg/kg for ivermectin, 48.3 microg/kg for roxithromycin, and 24.1 microg/kg for erythromycin, oleandomycin, spiramycin, josamycin and tylosin. The procedure was successfully used in the multi-residue determination of these macrolides at levels below the maximum concentrations legally allowed in milk samples.

[LC-ESI/MS identification and reduction of impurities in spiramycin fermentation].

There are several impurities in the spiramycin fermentation broth which leads to a lower yield and lower quality of the product. Four impurities in spiramycin broth have been simultaneously separated and identified by LC-ESI/MS. The generation of these impurities was attributed to the fluctuation of glucosylation in spiramycin biosynthesis. Nitrogen sources, ammonium in particular, were found to play an important role at the glucosylation. Aided with the information of LC-ESI/MS analysis and subsequent optimization of the culture medium, better culture medium of shake flask was designed, which leads to reduction of impurities by 22% - 88%.

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection.

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.

Separation of spiramycin components using high-speed counter-current chromatography.

High-speed counter-current chromatography was successfully applied to the separation of the main components of spiramycin. A 25-mg quantity of sample was separated using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:6:5:5), and the fractions were analyzed by high-performance liquid chromatography and fast atom bombardment mass spectrometry. The separation yielded 13.4, 0.7 and 1.7 mg of spiramycins I, II, and III with purities of 98.2, 92.3 and 97.4%, respectively.