Search

This article described the development and validation of a method for spiramycin related substances based on hybrid particle column. The chromatographic conditions were as follows: water - 0.2 mol/L dipotassium hydrogen phosphate (the pH value adjusted to 9.5 using a 1 mol/L KOH solution) - acetonitrile - methanol (10: 60: 28.5: 1.5, v/v/v/v) as mobile phase A, water - 0.2 mol/L dipotassium hydrogen phosphate (pH 9.5) - acetonitrile - methanol (10: 30: 57: 3, v/v/v/v) as mobile phase B and gradient elution was performed. Compared with previous analytical methods, this method has strong specificity, excellent sensitivity and stability, which could be used for the daily testing of related substances of spiramycin. Furthermore, impurities above 0.1 % were characterized using two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (2D LC-QTOF-MS/MS) and there were 6 impurities reported for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bile acid (BA) is one of the main active components of bile and has multiple isomers, the structure or content of its isomers often changes due to diseases and other health problems; thus, the accurate detection of BA isomers is very important. In this study, two groups of BA isomers of glycine-conjugated BAs and taurine-conjugated BAs were simultaneously separated and quantitatively analyzed by ion mobility mass spectrometry (IM-MS). Especially, baseline mobility separation between the isomers was achieved by the formation of binary complexes via simple interaction with spiramycin (SPM), for which a separation resolution (R p-p ) of 1.96 was reached. Moreover, BA isomers were quantitatively analyzed, and the limit of detection (LOD) of absolute quantification for TCDCA/TUDCA and GUDCA/GCDCA/GHDCA was 0.514 and 0.611 ng∙mL -1 , respectively; the LODs for molar ratio ranges of relative quantification for TCDCA/TUDCA, GUDCA/GHDCA, and GCDCA/GHDCA were 1:18-30:1, 1:18-21:1, and 1:19-21:1, respectively. Additionally, BA isomers analyzed in pig bile powder and bear bile powder were measured, which were in good consistency with those labeled, revealing the differences in BA composition and content between the two powders. Finally, BA detection and recovery analyses were performed on serum samples, with a recovery rate of ≥73.69%, RSD of ≤6.8%, and S R (standard deviation of recoveries, the degree of difference between measured values and average recovery) of ≤1.27. Due to the simple, rapid, and lack of need for complex sample preparation and chromatographic separation, the proposed method can be an effective method for BA detection in practical samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3 mL/minute. Separation and analysis were performed on X-bridge C18 (150 mm × 4.6 mm × 3.5 μm) column with diode array detector set at 230 nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125 μg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62 μg/mL for MTR and 0.92 and 2.83 μg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time., (© 2023 Wiley-VCH GmbH.)

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of residues of spiramycin, a macrolide antibiotic, and its active metabolite neospiramycin in cow's milk as well as in minor species 'milk, goat and ewe. Spiramycin-d3 was used as internal standard for quantification of both analytes. This analytical method was validated using a global accuracy profile as a graphical decision tool built according to the trueness and the precision of the method. A unique and optimal linear model with logarithm transformation (with a determination coefficient of 0.9991) allowed the measurement of both analytes in the milks of the three animal species, in a wide range from 0.2 to 10 times the Maximal Residue Limit (MRL) (40-2000 µg.kg -1 ). The limits of detection and quantification were 13 µg.kg -1 and 40 µg.kg -1 , respectively. The accuracy profile was established to get 80% of future measurements in routine assays that will fall within the acceptance limits. Trueness of the method, expressed as relative bias, was comprised between -1.6% and 5.7% over the whole range of concentrations. The mean relative standard deviation for repeatability and intermediate precision were comprised between 1.1% and 2.7%; 2.5 and 4.2%, respectively, in all levels of concentration for the three milks. Moreover, a two-order polynomial function was used to model the relative expanded uncertainty with a determination coefficient of 0.834. This function aimed to determine the uncertainty of the future quantifications within the validated dosing range. Overall, the global accuracy profile highlighted the reliability of the method for the routine assays of spiramycin and neospiramycin even in milk from minor species (goat, ewe) by using the most accessible milk (often from cow), while guaranteeing a very high proportion of samples within the fixed acceptance limits. The applicability of this method was tested during a depletion study of spiramycin and neospiramycin in the milk of cow, goat and ewe. The developed analytical method will be useful to assess the distribution profile of the antibiotic and its metabolite in milk of minor species where few studies are available., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 × 10 10 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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Breaking down the structural bonds and eliminating the functional groups are more efficient than destroying the whole molecule in antibiotic production wastewater (APW) pretreatment before further biotreatment. Two sulfated titania (TiO 2 /SO 4 ) solid superacids, SSA1 and SSA2 were synthesized, characterized and used for hydrolytic pretreatment of spiramycin in APW. Spiramycin removal followed an order of SSA2>SSA1>TiO 2 ≈pH = 3>control. The hydrolytic efficiencies increased at elevated temperature from 25 °C to 65 °C. The hydrolytic kinetics followed a first-order model and SSA2 performed the fastest. The performances were positively correlated with both the total acidity determined by n-butylamine titration and the strength of acid sites measured by NH 3 -temperature-programmed desorption (TPD). The residual solution for SSA2 presented the least antibacterial potency and anaerobic inhibition among all treatments. The hydrolyzed product was identified as the m/z 699.4321 fragment using UPLC-Q/TOF-MS, which was formed after losing a functional mycarose moiety from the parent molecular. The solid superacids were effective in selectively eliminating 433 mg/L of spiramycin and the antibacterial potencies of the spiramycin production wastewater, which contained very high concentrations of COD (33,000 mg/L). This hydrolytic method avoids using and handling hazardous and corrosive mineral acids on site. It is attractive as a selective catalytic pretreatment method to cleave antibiotics' functional groups and to reduce its inhibitory effects before sequential biotreatments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study aimed to evaluate the feasibility of hyperthermophilic anaerobic digestion at 70 °C in the pretreatment of spiramycin fermentation residue. By feeding municipal excess sludge under a solid retention time of 5 days, the hyperthermophilic digester was successfully started up within 3 days from mesophilic digestion by a one-step temperature increase from 35 to 70 °C. MiSeq sequencing showed the fast establishment of thermophilic fermenting bacterial communities in 3 days immediately after the temperature increase, with increases in abundance of Coprothermobacter, Spirochaetaceae&#95;uncultured and Fervidobacterium from <0.001%, 1.06% and <0.001% to 33.77%, 11.65% and 3.42%, respectively. The feasibility of hyperthermophilic digestion for spiramycin residue was evaluated in batch experiments for 7 days. Hyperthermophilic digestion considerably reduced antibiotic concentrations, with removal efficiencies of 55.3% and 99.0% for the spiramycin residue alone and its mixture with hyperthermophilic sludge, respectively. At the same time, the abundances of four macrolide-lincosamide-streptogramin resistance genes were also reduced within 7 days, due to the decrease of their corresponding hosts. These results suggest that hyperthermophilic digestion could easily be started up from mesophilic digestion and might be a suitable pretreatment approach for spiramycin residue.

Spiramycin is a 16-membered macrolide antibiotic produced by Streptomyces ambofaciens and used in human medicine for the treatment of various respiratory tract and genital infections. Several impurities were detected in spiramycin-fermentation broth, especially impurities D and F, which decreased the separation-extraction yield and increased production cost. Dextrins, as the main carbon source, influence the accumulation of spiramycin and impurities. In this work, two types of dextrin from vendor Y and Z were compared to study their influences on spiramycin production. Our results showed that final spiramycin production with dextrin Z was enhanced twofold as compared with dextrin Y; however, the content of impurities F and D were higher with dextrin Z relative to dextrin Y. Several parameters (adenosine triphosphate, total sugar, reducing sugar, and reducing sugar to total sugar) were analyzed to reveal differences in the fermentation process. In vitro dextrin hydrolysis by amylase revealed structural differences in the two types of dextrin, and real-time quantitative polymerase chain reaction analyses showed that the transcription of srm7 and srm21 (involved in forosaminyl methylation) was enhanced and potentially related to the reduced formation of impurity F with dextrin Y. Furthermore, the srm20/srm33 ratio, representing flux balance of forosaminyl and mycarosyl, was ~ 1, implying that forosaminyl and mycarosyl biosynthesis were well balanced, resulting in reduced production of impurity D with dextrin Y.

A series of molecularly imprinted polymers (MIPs) comprising reactionary sites which are complementary to macrolide antibiotic spiramycin (SPI) were synthetized by noncovalent bulk polymerization technique. MIPs were synthesized under different polymerization process and their recognition efficiency was evaluated in binding studies in comparison with non-imprinted polymers. The best MIP was morphologically characterized and equilibrium assays were carried out. The MIP was evaluated as a sorbent for extraction and preconcentration of SPI from aqueous and sheep milk samples, and an off-line MISPE method followed by high-performance liquid chromatography with UV diode-array detection was established. Good linearity were obtained for SPI in a range of 24-965μgkg -1 and the average recoveries at three spiked levels in milk samples were higher than 90% (RSD<5%). Limit of quantification was 24.1μgkg -1 . Cross-reactivity studies from other macrolides with similar structure were tested. The optimum imprinted polymer showed a good selectivity and affinity for SPI, demonstrating the potential of the proposed MISPE for rapid, sensitive and effective sample pretreatment for selective determination of SPI in sheep milk samples., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Research on the combined effects of antibiotic contaminants and environmental factors in cyanobacteria is still limited. This study focused on the action and its mechanism of spiramycin combined with changes in nitrogen and phosphorus level in Microcystis aeruginosa at environmentally relevant concentrations. Though photosynthetic activity was stimulated by spiramycin at a high nutrient level, no significant correlation (p>0.05) was found between photosynthesis-related proteins and growth-related proteins, and the growth rate was inhibited by 200ngL -1 of spiramycin. At low nitrogen and low phosphorus levels, up-regulated photosynthesis-related proteins were closely correlated with (p<0.05) stress response-related, transcription-related and cell division-related proteins, which consequently led to stimulated growth of M. aeruginosa under spiramycin exposure. Spiramycin exposure also regulated the production of microcystins (MCs) and the expression of two microcystin synthetases (mcyB and mcyC). The spiramycin-induced protein secretion process and the up-regulation of ATP binding cassette transporters might contribute to the increased MC release. Enolase, superoxide dismutase, protein GrpE, DNA-directed RNA polymerase subunit alpha and serine protease were candidate target proteins of spiramycin in M. aeruginosa under different nutrient conditions. Coexisting spiramycin mitigated the threat of cyanobacteria to aquatic environments at a high nutrient level but aggravated cyanobacterial bloom at a low nitrogen level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A new simple, sensitive, rapid and accurate gradient reversed-phase high-performance liquid chromatography with photodiode array detector (RP-HPLC-DAD) was developed and validated for simultaneous analysis of Metronidazole (MNZ), Spiramycin (SPY), Diloxanidefuroate (DIX) and Cliquinol (CLQ) using statistical experimental design. Initially, a resolution V fractional factorial design was used in order to screen five independent factors: the column temperature (°C), pH, phosphate buffer concentration (mM), flow rate (ml/min) and the initial fraction of mobile phase B (%). pH, flow rate and initial fraction of mobile phase B were identified as significant, using analysis of variance. The optimum conditions of separation determined with the aid of central composite design were: (1) initial mobile phase concentration: phosphate buffer/methanol (50/50, v/v), (2) phosphate buffer concentration (50 mM), (3) pH (4.72), (4) column temperature 30°C and (5) mobile phase flow rate (0.8 ml min -1 ). Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9999. Limits of detection for all of the analyzed compounds ranged between 0.02 and 0.11 μg ml -1 ; limits of quantitation ranged between 0.06 and 0.33 μg ml -1 The proposed method showed good prediction ability. The optimized method was validated according to ICH guidelines. Three commercially available tablets were analyzed showing good % recovery and %RSD., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Antibiotics that enter the environment can present human and ecological health risks. An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence as biologically active contaminants. In this study, hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for two amphenicol (chloramphenicol and florfenicol) and two macrolide (spiramycin and tylosin) antibiotics. Antibiotic hydrolysis rates in pH 4-9 buffer solutions at 25°C, 50°C, and 60°C were quantified, and degradation products were characterized. All of the antibiotics tested remained stable and exhibited no observable hydrolysis under ambient conditions typical of aquatic ecosystems. Acid- and base-catalyzed hydrolysis occurred at elevated temperatures (50-60°C), and hydrolysis rates increased considerably below pH 5 and above pH 8. Hydrolysis rates also increased approximately 1.5- to 2.9-fold for each 10°C increase in temperature. Based on the degradation product masses found, the functional groups that underwent hydrolysis were alkyl fluoride, amide, and cyclic ester (lactone) moieties; some of the resultant degradation products may remain bioactive, but to a lesser extent than the parent compounds. The results of this research demonstrate that amphenicol and macrolide antibiotics persist in aquatic systems under ambient temperature and pH conditions typical of natural waters. Thus, these antibiotics may present a risk in aquatic ecosystems depending on the concentration present., (Copyright © 2015. Published by Elsevier Ltd.)

To evaluate the potential effects of antibiotics on ammonia-oxidizing microbes, multiple tools including quantitative PCR (qPCR), 454-pyrosequencing, and a high-throughput functional gene array (GeoChip) were used to reveal the distribution of ammonia-oxidizing archaea (AOA) and archaeal amoA (Arch-amoA) genes in three wastewater treatment systems receiving spiramycin or oxytetracycline production wastewaters. The qPCR results revealed that the copy number ratios of Arch-amoA to ammonia-oxidizing bacteria (AOB) amoA genes were the highest in the spiramycin full-scale (5.30) and pilot-scale systems (1.49 × 10(-1)), followed by the oxytetracycline system (4.90 × 10(-4)), with no Arch-amoA genes detected in the control systems treating sewage or inosine production wastewater. The pyrosequencing result showed that the relative abundance of AOA affiliated with Thaumarchaeota accounted for 78.5-99.6% of total archaea in the two spiramycin systems, which was in accordance with the qPCR results. Mantel test based on GeoChip data showed that Arch-amoA gene signal intensity correlated with the presence of spiramycin (P < 0.05). Antibiotics explained 25.8% of variations in amoA functional gene structures by variance partitioning analysis. This study revealed the selection of AOA in the presence of high concentrations of spiramycin in activated sludge systems.

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram-negative organisms and some anaerobes. The components of macrolide antibiotics are generally complicated. Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control. Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles. In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16-membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, β-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CCβ) for tylosin was determined as 0.2 mg kg(-1) in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg(-1) of ≤13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37°C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust.

Spiramycin (SPY) is a medium-spectrum antibiotic with high effectiveness against Gram-positive bacteria. The voltammetric behaviour of spiramycin was studied using differential pulse polarography (DPP) and square wave polarography (SWP). The drug in Britton-Robinson buffer (pH 11.5) is reduced at - 1.45 V, giving rise to a well-defined cathodic peak using hanging mercury drop electrode (HMDE) versus Ag/AgCl electrode. This peak is attributed to the reduction of the aldehyde group. The results proved that the reduction of SPY is an irreversible diffusion-controlled process. The diffusion current-concentration relationship was shown to be rectilinear over the range of 20-80 and 0.8-80 µg ml(-1) using DPP and SWP modes, respectively, with detection limit of 8.5 µg ml(-1) (1.01 × 10(-5) M) and 0.46 µg ml(-1) (5.46 × 10(-7) M) for DPP and SWP modes, respectively. A mechanism is postulated for the reduction of SPY. The proposed techniques were successfully applied to the determination of the studied compound either in pure form or in its formulation., (Copyright © 2010 John Wiley & Sons, Ltd.)

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC-DAD-UV-ESI-MS(n) analysis played a crucial role, and stop-flow LC-(1)H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development., (Copyright 2009 Elsevier B.V. All rights reserved.)

Spiramycin, a widely used veterinary macrolide antibiotic, was found at traceable levels (nanograms per litre range) in Po River water (N-Italy). The aqueous environmental fate of this antibiotic compound was studied through drug decomposition, the identification of the main and secondary transformation products (TPs), assessment of mineralisation and the investigation of drug TPs toxicity. Initially, laboratory experiments were performed, with the aim of stimulating the antibacterial transformation processes followed in aquatic systems. The TPs were identified through the employment of the liquid chromatography (LC)-mass spectrometry technique. Under illumination, spiramycin degraded rapidly and transformed into numerous organic (intermediate) compounds, of which 11 could be identified, formed through five initial transformation routes. These laboratory simulation experiments were verified in situ to check the mechanism previously supposed. Po River water was sampled and analysed (by LC-high-resolution mass spectrometry) at eight sampling points. Among the previously identified TPs, five of them were also found in the river water. Three of them seem to be formed through a direct photolysis process, while the other two are formed through indirect photolysis processes mediated by natural photo sensitisers. The transformation occurring in the aquatic system involved hydroxylation, demethylation and the detachment of forosamine or mycarose sugars. Toxicity assays using Vibrio fischeri proved that even if spiramycin did not exhibit toxicity, its transformation proceeded through the formation of toxic products.

Metronidazole (MZ) is an anti-infective drug used in the treatment of anaerobic bacterial and protozoa infections in humans. It is also used as a veterinary antiparasitic drug. Spiramycin (SP) is a medium-spectrum antibiotic with high effectiveness against Gram-positive bacteria. Three simple, sensitive, selective and precise spectrophotometric methods were developed and validated for the simultaneous determination of MZ and SP in their pure form and in pharmaceutical formulations. In methods A and B, MZ was determined by the application of direct spectrophotometry and by measuring its zero-order (D(0)) absorption spectra at its λ(max) = 311 nm. In method A, SP was determined by the application of first derivative spectrophotometry (D(1)) and by measuring the amplitude at 218.3 nm. In method B, the first derivative of the ratio spectra (DD(1)) was applied, and SP was determined by measuring the peak amplitude at 245.6 nm. Method C entailed mean centering of the ratio spectra (MCR), which allows the determination of both MZ and SP. The methods developed were used for the determination of MZ and SP over a concentration range of 5-25 µg ml(-1). The proposed methods were used to determine both drugs in their pure, powdered forms with mean percentage recoveries of 100.16 ± 0.73 for MZ in methods A and B, 101.10 ± 0.90 in method C, 100.09 ± 0.70, 100.02 ± 0.88 and 100.49 ± 1.26 for SP in methods A, B and C, respectively. The proposed methods were proved using laboratory-prepared mixtures of the two drugs and were successfully applied to the analysis of MZ and SP in tablet formulation without any interference from each other or from the excipients. The results obtained by applying the proposed methods were compared statistically with a reported HPLC method and no significant difference was observed between these methods regarding both accuracy and precision., (© 2010 John Wiley & Sons, Ltd.)

The paper is to report the establishment of an HPLC method for determination of the components of bitespiramycin and its products, and to evaluate the components profile of bitespiramycin and its related products. Liquid chromatography combined with mass spectrometry (LC-MS) was used to identify the nine major components of the reference substance of bitespiramycins. The nine components of bitespiramycins and its products have been quantified by HPLC with gradients elution methods. The content of the component of 4"-O-isovalerylspiramycin III was not less than 35%, the content of the components of 4"-O-isovalerylspiramycin (I + II + III) was not less than 60%, and the contents of the nine components of bitespiramycin were not less than 80%, separately. In addition to the components mentioned above, there also exists some other impurities, however, with lower contents. This gradient elution HPLC method reported by this paper is considered to be suitable for the quality control of bitespiramycin.

An efficient multiresidue method for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in tilapia fish muscle, based on high performance liquid chromatography with UV detection (HPLC-UV), has been developed. The drugs were extracted with 0.2% orthophosphoric acid-methanol (6:4), and the extracts were cleaned up on a solid phase extraction cartridge, C18 Sep-Pak light column. The LC separation was performed on a RP stainless-steel C-18 analytical column (150 mm x 4.6 mm, 5 microm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 2.4-acetonitrile as the mobile phase at the flow rate of 1.0 ml min(-1). A wavelength programming was applied for the UV detection of the analytes. The method not only enabled the determination of the parent drugs, MET and SPY, but also permitted the determination of their metabolites, hydroxymetronidazole (HMET) and neospiramycin (NSPY). The calibration graphs for each drug were rectilinear in the range of 0.005-1.000 microg g(-1) for MET and HMET and 0.025-1.000 microg g(-1) for SPY and NSPY. With this method, the cited drugs with their metabolites were determined in fortified fish muscle tissues at levels of 0.025, 0.1 and 1.0 microg g(-1) with good accuracy and precision. LOD and LOQ obtained for each drug were as follows: 0.002 and 0.005 microg g(-1) for MET and HMET and 0.005 and 0.025 microg g(-1) for SPY and NSPY. Utilization of the method to successfully analyze tilapia fish muscle samples incurred with MET and SPY was described.

A microbiological screening method (three-plate) for the detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin, and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg(-1) (false-positives, 2%; false-negatives, 3, 0, and 6%, respectively). Avoparcin could be detected at 1 mg kg(-1) in feed in general (false-positives, 2%; false-negatives, 0%). However, in calf feed the sensitivity was lower. The percentages of false-negatives were found to be 12%, 7%, and 0% at 1, 3, and 5 mg kg(-1), respectively (false-positives, 4%). The limit of detection for zinc bacitracin was 3-5 mg kg(-1) (false-positives, 5-10%; false-negatives, 77% at 1 mg kg(-1), 45% at 2 mg kg(-1), 12% at 3 mg kg(-1), and 4% at 5 mg kg(-1)). The method allowed for a distinction to be made between the groups of antibiotics: avoparcin/zinc bacitracin versus tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis.

Multivariate calibration problems often involve the identification of a meaningful subset of variables, from a vast number of variables for better prediction of output variables. A new graph theoretic method based on partial correlations (variable interaction network-VIN) is proposed. Many well studied representative calibration datasets spanning different application domains are selected for investigating the performance. Partial least squares (PLS) regression models combined with variable selection techniques are employed for benchmarking the performance. Subsets of variables with different number of variables are retained for the final analysis after VIN selection and progressive prediction accuracies are used for comparison. VIN-PLS results show significant improvement in prediction efficiencies and variable subset optimization. Improvement of up to 45% over existing methods with significantly fewer variables is achieved using the new method. Advantages of VIN based variable selection are highlighted.

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%.

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.

An inter-laboratory validation was carried out to determine the performance characteristics of an analytical method based on thin-layer chromatography (TLC) coupled to microbiological detection (bio-autography) for screening feed samples for the presence of spiramycin, tylosin and virginiamycin. Twenty-four samples including blank samples and samples with concentrations of the target analytes ranging between 1 and 5 mg kg(-1) (expressed in microbiological activity) were analysed by seven laboratories participating in the study. The required detection limit was 1 mg kg(-1) (expressed in microbiological activity). For spiramycin, acceptable values for the sensitivity (at least 95%) indicating the rate of correct positive results were obtained for samples containing this substance at or above 2 mg kg(-1), whereas at 1 mg kg(-1), the sensitivity rate dropped to about 70%. Therefore, it was concluded that the detection limit was 2 mg kg(-1). For tylosin and virginiamycin, acceptable values of the sensitivity were obtained for all concentrations including 1 mg kg(-1). Therefore, the method fulfils the criterion regarding the required sensitivity at the target detection limit for tylosin and virginiamycin.

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.

Aim: To propose a novel polarographic method for the determination of acetylspiramycin (ASPM) is proposed., Methods: In 0.1 mol x L(-1) NH4Cl-NH3 x H2O (pH 8.9) buffer containing dissolved oxygen, ASPM yielded a sensitive parallel catalytic hydrogen wave with the peak potential of -1.63 V (vs SCE) by single sweep polarography., Results: The 2nd order derivative peak currents (i(p)") of the parallel catalytic hydrogen waves of ASPM showed a linear relationship with its concentrations in the range from 1.74 x 10(-3) microg x mL(-1) to 3.84 microg x mL(-1) (r = 0.9979, n=13). Its detection limit was 5.80 x l0(-4) microg x mL(-1) (3sigma) and RSD (n=13) was 1.24% at the concentration level of 0.871 microg x mL(-1)., Conclusion: The proposed method could be applied to the determination of ASPM in ASPM tablets.

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.

Bitespiramycin is a novel antibiotic containing a number of 4''-acylated spiramycin derivatives (isovalerylspiramycins I-III, butanoylspiramycin III, propanoylspiramycin III and acetylspiramycin III) as major components. These spiramycin derivatives are susceptible to degradation in acid solution. Liquid chromatography-ion trap mass spectrometry (LC/MS(n)) was used to study the degradation of these spiramycin derivatives in simulated gastric fluid at 37 degrees C. All derivatives degraded by first-order reactions for which rate constants (k) and half-lives (t(1/2)) were calculated. Acyl groups at position 3 had less effect on acid-stability of spiramycin derivatives than acyl groups at position 4''. The introduction of 4''-acyl groups enhanced the acid-stability of spiramycin derivatives and altered the degradation pathway in simulated gastric fluid such that loss of forosamine rather than loss of mycarose becomes the major degradation pathway.

The use of the two macrolides antibiotics Spiramycin (S) and Tylosin (T) as growth promoters in animal feeding has been recently withdrawn in the European Union due to a concern about the outbreaks of farmacoresistance fenomena as a possible hazard for humans. For feed additives monitoring purposes, an analytical method has been developed for their extraction, purification and identification in different animal feedingstuffs (pelleted beef, pig, poultry feeds and calves milk replacer) at a minimum performance required limit (MRPL) of 1 microg g(-1) (ppm). Such limit has been established according to the lowest dosage of additives still able to elicit an appreciable growth promoting effect. Blank feeds were spiked at two concentration levels, 1.0 and 2.5 ppm in six replicates. After methanolic extraction, samples were cleaned up on SPE CN columns and extracts analysed in HPLC-UV/DAD, using a gradient elution. Detection limits, calculated as the tree time mean noise of 20 blank feeds, were 176 and 118 ng g(-1) for S and T, respectively. Results show good repeatability (CV% not exceeding the value of 15) and mean recovery in the range of 99-74% and 81-53% for S and T, respectively, at 1 ppm. When the standards were injected up to 250 ng the chromatographic method can resolve the components of analytes (Spiramycin I, II and III; Tylosin A and B) but can not resolve the components on real feed samples at the spiked levels considered. For this reason the identification and quantification of analytes on matrix were carried out considering the main compound of the drugs (Spiramycin I and Tylosin A). As a verification, the overlapping of UV spectra in the range 220-350 nm between analytical standards and the compounds in the matrix were considered.

A microbiological method for screening of residual benzylpenicillin (PCG), oxytetracycline (OTC) and spiramycin (SPM) in meat using a single mixed mode, reversed-phase and cation-exchange cartridge was developed. A meat sample was extracted with 0.1 mol/mL pH 4.5 phosphate buffer and the extract was applied to a MCX cartridge. The cartridge was washed, and adsorbed antibiotic residues were eluted with acetonitrile for acidic fractions and acetonitrile containing 5% ammonia solution-0.1 mol/mL pH 4.5 phosphate buffer (9:1, v/v) for basic fractions. Each eluate was evaporated to dryness and the residue was dissolved in phosphate buffer to prepare test solutions for microbiological assay. When the diameter of the inhibition zone was more than 12 mm, the result was considered positive. In this method, the average recoveries of PCG at 0.05 microg/g, OTC at 0.1 microg/g and SPM at 0.2 microg/g were 70%, 92% and 84%, respectively. Tolerances of the three antibiotics were detected. All the results demonstrate that this method is simple, rapid and useful for screening of these three antibiotic residues in meat.

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.

Aim: To establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM)., Methods: ASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K3Fe (CN)6 system., Results: The decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) microgram.L-1, the detection limit was 40 ng.L-1 (3 sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n = 5)., Conclusion: The FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.

A simple and reliable method using liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI-MS) has been developed for the determination of the macrolide antibiotics spiramycin and tilmicosin in meat and fish. The drugs were extracted from meat and fish with 0.2% metaphosphoric acid-methanol (6:4), and the extracts were cleaned up on an Oasis HLB cartridge (60 mg). Positive ionization produced the molecular related ions, (M + 2H)2+, at m/z 350.2, 422.3 and 435.3 for neospiramycin I, spiramycin I and tilmicosin, respectively. The LC separation was performed on a Capcell Pak MG-C18 column (150 x 2 mm i.d.) with a gradient system of 0.02% formic acid-acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. The molecular-related ions of the drugs were very clear under this condition. The calibration graph for each drug was rectilinear from 0.05 to 5 ng with selected ion monitoring (SIM). The recoveries of the drugs from meat and fish fortified at the level of 0.2 microgram/g was 73.2-89.2%, with high precision. The limits of detection of the drugs in meat and fish were 0.01 microgram/g.

Three plate systems (combinations of indicator organism and growth medium) were evaluated for the detection of analytical standards of the banned feed additives avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin. When authorized in the EU, the previously recommended minimum inclusion rate (MIR) for each compound was 5 mg kg(-1). One of the plate systems (Micrococcus luteus ATCC 10240, nutrient agar) detected all five additives. This plate was used in a further study that evaluated the suitability of accelerated solvent extraction (ASE) as a first step in the development of a rapid single-plate screening assay. A drug-free (negative control) feedingstuff was fortified with the compounds (0-50 mg kg(-1)), extracted by ASE and the extracts applied to the plate at each of three pH ranges - unadjusted extract (pH 5.7-5.9), pH 6.5 and 8.0. At pH 6.5, sub-MIR concentrations of virginiamycin and tylosin were detectable. Avoparcin was detectable at 6.3 mg kg(-1). The detection of zinc bacitracin was#10; pH-independent (10 mg kg(-1)). At pH 8.0, spiramycin was detectable at 5.4 mg kg(-1). Mean +/- SD analytical recoveries from fortified feedingstuffs (n = 10) ranged from 57 +/- 1.5% for avoparcin to 96 +/- 4% for virginiamycin. The five additives were also detectable following ASE extraction from a range of different feedingstuffs fortified with each of the drugs. A further 24 compounds permitted for use in animal feeds were tested. Of these, nine were detectable at their recommended MIR. It is concluded that ASE is a versatile technique suitable for the automated extraction of a range of antimicrobials from animal feedingstuffs. Employing ASE with this single-plate detection system permits the rapid antimicrobial screening of animal feedingstuffs and allows the detection of the banned additives. Whilst the method is applicable as a screening test, more specific postscreening methods would be necessary for subsequent identification (and quantification) of antimicrobials in screening-positive samples.

A method for determining organic acids in spiramycin fermentation broth by high performance liquid chromatography is described. The operating conditions were Zorbax 300-SB C18 column (5 microns, 4.6 mm i.d. x 15 cm) at 35 degrees C, 0.01 mol/L phosphoric acid buffer solution (pH 2.32) and methanol as mobile phase at a flow rate of 0.6 mL/min and UV detection at 210 nm. The relative standard deviations were 0.33%-0.10% and the recoveries were 99.95%-100.08%. It's a simple, rapid and accurate method.

Shengjimycin is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycin as the major component, produced by recombinant S. spiramyceticus F21 harboring a 4"-O-acyltransferase gene from S. mycarofaciens 1748. A stable bioengineered strain of Streptomyces spiramyceticus WSJ-1 was constructed by integrating the 4"-O-acyltransferase gene (ist) by homologous recombination into the chromosome of the spiramycin-producing strain S. spiramyceticus F21. In this construction, a Streptomyces/E. coli shuttle plasmid pKC1139 (AmR) was used as the vector with the tsr gene used as selection marker for homologous recombination. The constructed strain, S. spiramyceticus WSJ-1,was genetically stable in production titer and proportion of components of shengjimycin as well as in maintaining the tsr selective marker when grown without selection. Southern hybridization confirmed the integrated status of the ist gene in the host genome. The production and the proportion of major component of 4"-isovalerylspiramycin of S. spiramyceticus WSJ-1 was also improved comparing with the strain harboring an autonomous plasmid -S. spiramyceticus F21/pIJ680(311) as shown by HPLC analysis. Physiological studies indicated that increase of the VDH ( valine dehydrogenase ) and LDH ( leucine dehydrogenase ) activities of WSJ-1 may be involved in this improvement.

Spiramycin (SP), oxytetracycline (OTC) or sulphamonomethoxine (SMM) was fed to laying hens at a dietary level of 400 p.p.m. for 7 successive days. After 7 days of medicated feed, the concentrations of SP, OTC and SMM were determined in the blood, liver, ovary, oviducts (magnum and isthmus plus shell gland) and eggs (albumen and yolk) by high-performance liquid chromatography. Of the three drugs, OTC showed the lowest content in the above tissues and eggs, while the reverse was true for SMM. Low concentrations of SP were measured in the blood, whereas contents in the liver and the oviducts were relatively much higher.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.

Monographs of the European Pharmacopoeia describe in the LC-test for related substances usually a system suitability test in order to ensure the adequate separation of impurities. Since the reference substances required are often not available a recent approach to avoid this problem is the generation of the required impurity by 'in situ degradation' of the active principle. This paper describes some typical applications of this technique as well as recent examples, such as the controlled degradation of cefalotin sodium, imipenem and spiramycin.

The aim of this study was to use statistical tools, especially the analysis of variance (ANOVA), to improve knowledge of the characteristics of the dispersion of results in high-performance liquid chromatography (HPLC) methods for quantitative analysis. It is in this regard that two interlaboratory studies have been carried out in collaboration with Rhône-Poulenc Rorer. The first concerned the analysis of a single drug product (ketoprofen) and was typically a "simple analysis". The second one involved a complex mixture of drug products and related substances (spiramycin), requiring far more constraining analysis conditions. Preliminary studies of the analyses were carried out to develop an optimized protocol. Statistical exploitation of the data for ketoprofen showed that there was no significant influence of the factors "laboratory" and "preparation", under the conditions of the study. On the other hand, in the case of spiramycin, a significant influence of the factors "laboratory" and "preparation" was observed under the conditions of the collaborative study, indicating that the latter factor must be taken into account to establish certified assays. Results of these two studies will help to determine the factors that have a significant influence, depending on the product and the chromatographic method used. By completing the statistical data base, interlaboratory studies will also contribute in the near future to the elaboration of more rigorous protocols for analytical transfers.

A particle beam/liquid chromatographic/mass spectrometric (PB/LC/MS) method capable of determining 5 macrolides in bovine muscle is described. Spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in bovine muscle are confirmed by reversed-phase LC/MS incorporating gradient elution. Macrolides are extracted from tissue with chloroform, and the extract is purified with a diol solid-phase extraction cleanup. The 5 macrolides are identified by negative and positive chemical ionization with selective ion monitoring of 2 ions in each mode. The procedure confirms the presence of each macrolide at 50 micrograms/kg in spiked samples.

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.

After chloroform extraction, the rapid and sensitive determination of spiramycin and neospiramycin can be performed with AASP-diol clean-up cartridges prior to reversed-phase C18 high-performance liquid chromatography. The limits of quantification of spiramycin in plasma and milk are 0.023 and 0.013 microgram/ml, respectively, and those of neospiramycin, are 0.058 and 0.006 microgram/ml, respectively. Application of the method to the analysis of plasma and milk samples obtained from pharmacokinetic studies is described. Spiramycin has a terminal half-life of 14.27 h in plasma and 34.59 h in milk, while neospiramycin has a half-life of 25.62 h in plasma and 105.85 h in milk.

To ensure that residues of veterinary drugs, above their respective maximum residue limits, do not reach the human food supply, European Community regulations specify requirements for detection, quantification and confirmation analytical methods and control procedures. The European Community member states base meat controls on these protocols. A liquid chromatographic/mass spectrometric analysis of spiramycin in calf muscle is presented as a confirmatory method for this compound. A particle beam interface was used, with negative ion chemical ionization mass spectrometry, using methane as the reagent gas. Samples (2 g muscle were prepared by liquid/liquid extraction followed by solid-phase extraction clean-up. On-line liquid chromatography/mass spectrometry of extracts was carried out on a C-18 bonded silica column. The specificity required for a regulatory confirmation procedure was achieved by monitoring five fragment ions with m/z 304, 330, 475, 683 and 684. Variation of the relative ion abundances was less than 20% at the maximum limit of residue, 50 micrograms kg-1. The method specificity was tested for three related compounds: neospiramycin, erythromycin and tylosin. The detection limit based on ion chromatogram peaks areas obtained with control samples was determined to be 20 micrograms kg-1.

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.