[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%.

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 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.

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.

Analysis of erythromycin and oleandomycin residues in food by high-performance liquid chromatography with fluorometric detection.

A reliable and sensitive procedure is presented for the analysis of erythromycin (ERY) and oleandomycin (OLE) in food of animal origin, such as meat, liver, kidney, raw milk and egg. The method is based on a solid-phase extraction clean-up with a cation exchange cartridge, a 9-fluoromethylchloroformate (FMOC) precolumn derivatization and a separation by HPLC with fluorometric detection. The selectivity is satisfactory enough to control ERY and OLE residues as not many interfering peaks are observed for various food matrices. The macrolides recoveries of the total procedure were low, although >50%. However, addition of an internal standard (roxithromycin) corrected for recovery to give satisfactory quantitative results for repeatability, linearity, detection and quantification limits and mainly accuracy.

Coupling of a supercritical fluid chromatography system to a hybrid (Q-TOF 2) mass spectrometer: on-line accurate mass measurements.

This note describes a simple and economical method to couple a supercritical fluid chromatography (SFC) system (Berger Instruments, US) with a high-resolution hybrid mass spectrometer (Q-TOF 2; Micromass, UK). This experimental arrangement has three distinct advantages: (1) coupling between the two systems can be effected without the need for an interface or hardware modifications of either system, (ii) this experimental arrangement provides on-line accurate mass SFC/MS measurements which are indispensable for the characterisation of new chemical entities and unknown metabolites, and (iii) the characteristically fast spectral acquisition rate of the time-of-flight (TOF) analyser renders the present arrangement an important contribution to future semipreparative fraction collection setups which use mass spectrometry as a detector.

Analysis of erythromycin by liquid chromatography/mass spectrometry using involatile mobile phases with a novel atmospheric pressure ionization source.

A critical limitation of electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) sources is the susceptibility to blockage of interface orifices due to the deposition of involatile components from the sample and/or mobile phase. These components, including salts, buffers, and ion-pairing agents, can be essential to the performance of the chosen analytical method. We report here the performance enhancements provided by a novel atmospheric pressure ionization (API) source in the analysis of erythromycin A (ERY) using mobile phases that contain involatile components. The enhanced robustness of the new source is derived from the use of a continuous flow of aqueous solvent at the sampling cone orifice that maintains unobstructed ion transmission. The ESI mass spectral responses measured for ERY, using an LC separation that incorporates 10 mM sodium phosphate with and without 10 mM octane sulfonate, were monitored by repeated injections over 13-15 h total analysis time. Minimal effects on ESI mass spectral responses (integrated peak area) or chromatographic performance (peak shape, retention time) were observed during these studies. In the absence of the aqueous cleaning flow, complete loss of mass spectral responses and total blocking of the sampling cone was observed in less than 30 min. Responses for ERY spiked into chicken and beef liver, and catfish muscle at or below the regulatory level of interest (100 ppb), were quantified by internal standard calibration using this procedure. These results demonstrate the ability of a novel API-MS ion source to perform analyses that require the use of involatile mobile phase additives.

Analysis of macrolide antibiotics by capillary electrophoresis.

Capillary electrophoresis was utilized in the study of the macrolide antibiotics (i.e. pharmaceutical glycoconjugates) clarithromycin, erythromycin, oleandomycin, troleandomycin, and spiramycin. In order to assist in analyte solubilization, two buffer systems using acetonitrile were developed. The first system involved 30 mM sodium cholate and 20% acetonitrile in 80 mM sodium phosphate, pH 6. This buffer permitted the baseline resolution of all five glycoconjugated antibiotics. In addition, erythromycin was separated from its derivatives estolate and ethylsuccinate. In the absence of surfactants, a higher acetonitrile quantity, 65%, was used in the second buffer system, with 35 mM sodium phosphate, pH 6. Selectivity between oleandomycin and clarithromycin was reversed in this system compared to the cholate buffer, indicating solute interaction with the cholate micelles in the previous system. Calibration linearity and detection sensitivity were improved in the high acetonitrile buffer, due to decreased background absorbance. It was demonstrated that both buffer systems can be utilized for the visualization of minor components that may be present in bulk pharmaceuticals.

Determination of erythromycin in gastric juice and blood plasma by liquid chromatography and electrochemical detection.

A liquid chromatographic method for determination of the antibiotic erythromycin in biological samples is described. Erythromycin and the internal standard, oleandomycin, were extracted from alkalinized samples with a mixture of 1-hexane and 2-butanol. After evaporation and reconstitution of the sample, separation was performed on a base-deactivated octadecylsilica column. The effects of pH in the mobile phase and of column temperature on the chromatographic performance were studied. Multiple and irregularly shaped peaks were obtained for some chromatographic systems, but by choosing appropriate conditions erythromycin could be eluted as a single symmetric peak. The absolute recovery was above 90% for erythromycin from blood plasma and above 85% from gastric juice. The limits of quantitation were 20 nM and 100 nM, respectively. Comparison of analytical results for a series of authentic samples with a microbiological assay showed excellent agreement.

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.

[The interrelation between the formation of oleandomycin and the resistance to it in different strains of Streptomyces antibioticus]. / Vzaimosviaz' mezhdu obrazovaniem oleandomitsina i ustoichivost'iu k nemu u razlichnykh shtammov Streptomyes antibioticus.

Strains, producers of oleandomycin, with different level of antibiotic-formation have been studied for their resistance to their own antibiotic. The obtained highly active strain possesses double resistance to oleandomycin and 50% higher activity. Identity of oleandomycin phosphate substances synthesized by initial and produced highly active strains is shown by the HELC method.

[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. 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.

[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.

[Polysaccharide composition of the cell wall of Streptomyces antibioticus RIA-594(39), a producer of the antibiotic oleandomycin]. / Polisakharidnyi sostav kletochnoi stenki Streptomyces antibioticus RIA-594(39), produtsenta antibiotika oleandomitsina.

The main polysaccharide components of the cell wall in S. antibioticus RIA-594 (39) i.e. peptidoglycan, teichoic acid and polysaccharide were studied. Peptidoglycan consists of the polysaccharide fraction containing equimolar quantities of N-acetylglucosamine and muramic acid and the peptide subunits including alanine, glutamic and L,L-diaminopimelic acids and glycine at a ratio of 1.4:0.9:1:0.9. It is characteristic that certain peptide subunits of the streptomycete contain no alanine. A polysaccharide differing from glycerol teichoic acid and containing galactose and N-acetylglucosamine was isolated from the cell wall. During the streptomycete development the quantity of peptidoglycan remained constant, the quantity of teichoic acid lowered and the quantity of polysaccharide increased. Correlation between the presence of aminosugars in the composition of teichoic acid and polysaccharide specific of the streptomycete cell wall and the presence of aminosugars in the structure of oleandomycin was shown. This is probably connected with characteristic features of the organism physiology.

[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.

[Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture]. / Tsiklicheskaia adenozin-3',5-monofosfornaia kislota u Strptomyces antibioticus i ee vozmozhnaia rol' v reguliatsii biosinteza oleandomitsina i rosta kul'tury.

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.

High-performance liquid chromatographic analysis of oleandomycin in serum and urine.

The determination of oleandomycin in serum and urine by high-performance liquid chromatography using erythromycin as internal standard is described. The separation was achieved on a reversed-phase C18 column employing acetonitrile-0.05 M phosphate buffer (30:70), adjusted to pH 7.0, as the mobile phase with UV detection at 200 nm. A solid-phase extraction procedure, combined with a simple phase-separation step was used prior to chromatographic analysis. Linear calibration curves were obtained in the concentration ranges 0.25-5.0 micrograms/ml (serum) and 1.0-25.0 micrograms/ml (urine). Precise quantitative analysis has been achieved at these levels with relative standard deviations of less than 5%.