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.

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry.

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.

Determination of antibiotics from soil by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry.

A method for the analysis of several macrolide and ionophore antibiotics, as well as tiamulin, from soil was developed using pressurized liquid extraction (PLE), reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (LC-APCI+-MS-MS). The analytes were extracted from soil by PLE in 30 min and the extracts were cleaned up by solid-phase extraction (SPE) on a diol SPE cartridge. Liquid chromatographic (LC) separation of the antibiotics was achieved in 35 min. Recovery experiments were performed using spiked soil and concentrations varying from 1 to 2000 microg/kg. By using a macrolide internal standard the recovery rates for the macrolides erythromycin and roxithromycin ranged from 43 to 94% (RSD 20-23%), for the ionophore salinomycin the recovery rate was 76% (RSD 29%), while the pleuromutilin tiamulin was completely recovered. The limits of detection ranged from 0.2 to 1.6 microg/kg. In soil samples a maximum concentration of 0.7 microg/kg tiamulin was found.

Determination of five macrolide antibiotic residues in honey by LC-ESI-MS and LC-ESI-MS/MS.

Liquid chromatography-electrospray ionization mass spectrometry methods (LC-ESI-MS and LC-ESI-MS/MS) for the determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in honey are presented. Macrolides were protonated to form singly and/or doubly charged pseudomolecular ions, depending on their chemical structures, in an electrospray positive ionization mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity. The recoveries, that is, determined by LC-ESI-MS/MS, of the five macrolides at fortified levels of 6, 16, 40, and 80 microg/kg ranged from 75.5 to 135.7% in light honey and from 42.1 to 111.0% in dark honey. The ion ratios obtained under MS/MS were key criteria to confirm the identity of macrolides in incurred samples. LC-ESI-MS/MS method detection limits of the five macrolides were <0.1 microg/kg.

Simple methods for the qualitative identification and quantitative determination of macrolide antibiotics.

Pyrolysis-gas chromatography is shown to be a rapid straightforward method for the qualitative differentiation of the macrolide antibiotics erythromycin, oleandomycin, troleandomycin, spiramycin and tylosin. Organic salts do not interfere and identification of erythromycin and troleandomycin in commercial products is viable. Spectrophotometric quantitation of these same five antibiotics after reaction with concentrated sulphuric acid is studied at about 470 nm. Reaction conditions such as acid concentration, time and temperature are provided. The sugar moieties of the antibiotics are proposed as the reactive sites. Detection limits are about 0.2-1.0 microg ml-1 [corrected] and analysis of pharmaceutical products should be possible.

[Oleandomycin and its natural and synthetic analogs. I. Dissociative ionization of oleandomycin and its derivatives]. / Oleandomycin, ego prirodnye i sinteticheskie analogi. I. Dissotsiativnaia ionizatsiia oleandomitsina i ego proizvodnykh.

Regularities of dissociative ionization of compounds belonging to the oleandomycin group were studied. Mass spectra of oleandomycin and some of its derivatives including anhydrooleandomycin, oleandomycin chlorhydrine, desoleandomycin, oleandomycin oxide, trimethylsilyl and acetyl derivatives were analyzed comparatively. Directions of disintegration with breakage of the glycoside bonds, macrolactone and carbon cycles were detected. The data are useful in structural analysis of not described oleandomycin-related compounds formed during biosynthesis and isolation of the main product.

[Study of the process of inactivating oleandomycin phosphate preparations]. / Izuchenie protsessa inaktivatsii preparatov oleandomitsina fosfata.

Ageing of oleandomycin preparations was studied. Certain biologically inactive components accumulating on storage of the preparations were isolated and investigated. A scheme for ageing of oleandomycin phosphate preparations is described. The scheme includes cleaving up of the water molecule at C11 and cleaving by the epoxide ring at the early stages and hydrolysis by the glycoside bonds at the later stages.

[Oleandomycin and its natural and synthetic analogs. II. Structure and properties of oleandomycin B, a new compound of the oleandomycin group]. / Oleandomitsin, ego prirodnye i sinteticheskie analogi. II. Struktura i svoistva novogo soedineniia oleandomitsinovoi gruppy oleandomitsina B.

The revealed regularities of mass spectroscopic disintegration of oleandomycin and its derivatives made it possible to determine analytic criteria for identification of compounds related by their structure to oleandomycin. Analysis of the extracts from oleandomycin fermentation broth filtrates on the basis of the selected group of diagnostic ions showed that along with the main antibiotic there formed during the biosynthesis oleandomycin B, a structurally close minor component. The structure of the substance was assigned and its physico-chemical and biological properties were studied.

[Determination of five macrolide antibiotic residues in royal jelly samples by using high performance liquid chromatography tandem mass spectrometry].

The macrolides are lipophilic molecules having a central lactone ring bearing 12 to 20 atoms to which several amino and/or neutral sugars are bound. They are broad spectrum antibiotics active against Gram-positive bacteria and mycoplasmas, as well as some Gram-negative organisms and members of the chlamydia group. Macrolides are a group of antibacterial compounds that have been widely used in medical and veterinary practices. A method of high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was developed for the confirmation of five macrolide antibiotic residues (spiramycin, oleandomycin, tylosin, roxithromycin, josamycin) in royal jelly samples. Trichloroacetic acid solution was used to precipitate the protein in the sample. The upper layer solution was extracted with acetonitrile. Then it was cleaned up with a C18 column. The one precursor/two product ion transitions for each macrolide antibiotics were monitored. The results show that the working curves for five macrolide antibiotics were linear in the range of 0.002 - 0.05 mg/L by HPLC-MS/MS in selective ion monitoring model. The limits of quantitation of the antibiotics in royal jelly were all 20 microg/kg. The recoveries were between 73.0% -90.2% at three spiked levels (20, 100 and 200 microg/kg for each macrolide antibiotic), and the relative standard deviations were between 5.6% - 10.5%.

[Factors determining the stability of oleandomycin phosphate]. / O faktorakh, opredeliaiushchikh stabil'nost' oleandomitsina fosfata.

Comparative stability of crystalline and amorphous oleandomycin phosphate was studied. It was shown that the structure and humidity of oleandomycin phosphate were among the main factors determining its stability. Crystalline oleandomycin phosphate was highly stable and the effect of humidity on its stability was relatively low. A significant effect of humidity on the stability of amorphous oleandomycin phosphate was observed.

[Polarographic method of determining oleandomycin]. / Poliarograficheskii metod opredeleniia oleandomitsina.

It was shown with the use of classical and differential polarography that oleandomycin in aqueous solutions undergoes electrolytic reduction on the mercury-dropping electrode. The polarographic wave of the reduction was distorted with adsorption phenomena. Its height was proportional to the analytical concentration of oleandomycin in the solution. The polarographic study on the process of acid and alkaline hydrolysis showed a satisfactory correlation between the height of the oleandomycin peak on the differential polarogramme [Formula: see text] and the biological activity of the antibiotic in the solution. The quantitative analysis implies recording of the data of the differential polarogramme of the test-solution and measurement of the antibiotic concentration with the calibration graph. Comparison of the results of determination of the oleandomycin levels in oleandomycin phosphate drugs and fermentation broth filtrates with the polarographic and micro-biological methods revealed no systematic deviations and showed that the average casual deviation between the results was due only to the errors of the methods reproducibility.

[Epoxy ring scission in the oleandomycin molecule during aging]. / O raskrytii époksidnogo kol'tsa v molekule oleandomitsina v protsesse stareniia.

Opening of the epoxide ring in the molecule of oleandomycin phosphate in the process of its aging was shown to be possible by the data of the analysis. Certain correlation between the "glycol" content and drug activity was detected.