Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

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.

Effectively remediating spiramycin from production wastewater through hydrolyzing its functional groups using solid superacid TiO2/SO4.

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 (TiO2/SO4) 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>TiO2≈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 NH3-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.

Pretreatment of spiramycin fermentation residue using hyperthermophilic digestion: quick startup and performance.

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_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.

High Concentrations of the Antibiotic Spiramycin in Wastewater Lead to High Abundance of Ammonia-Oxidizing Archaea in Nitrifying Populations.

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.

[Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry].

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.

A liquid chromatography coupled to tandem mass spectrometry method for the quantification of spiramycin and its active metabolite neospiramycin in milk of major and minor species: Validation using the accuracy profile.

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.

Fate of antibacterial spiramycin in river waters.

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.

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

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

[Determination of the components of bitespiramycin by HPLC].

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.

Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection.

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.

Influence of dextrins on the production of spiramycin and impurity components by Streptomyces ambofaciens.

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.

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.

Synthesis and characterization of a molecularly imprinted polymer for the determination of spiramycin in sheep milk.

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.

iTRAQ-based quantitative proteomic analysis of Microcystis aeruginosa exposed to spiramycin at different nutrient levels.

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.

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.

Development and Optimization of HPLC Analysis of Metronidazole, Diloxanide, Spiramycin and Cliquinol in Pharmaceutical Dosage Forms Using Experimental Design.

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.

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.

[Parallel catalytic hydrogen wave of acetylspiramycin caused by dissolved oxygen and its application].

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.

Hydrolysis of amphenicol and macrolide antibiotics: Chloramphenicol, florfenicol, spiramycin, and tylosin.

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.

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.