A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Synthetic Studies with Bacitracin A and Preparation of Analogues Containing Alternative Zinc Binding Groups.

The growing threat of drug-resistant bacteria is a global concern, highlighting the urgent need for new antibiotics and antibacterial strategies. In this light, practical synthetic access to natural product antibiotics can provide important structure-activity insights while also opening avenues for the development of novel analogues with improved properties. To this end, we report an optimised synthetic route for the preparation of the clinically used macrocyclic peptide antibiotic bacitracin. Our combined solid- and solution-phase approach addresses the problematic, and previously unreported, formation of undesired epimers associated with the stereochemically fragile N-terminal thiazoline moiety. A number of bacitracin analogues were also prepared wherein the thiazoline motif was replaced by other known zinc-binding moieties and their antibacterial activities evaluated.

Determination of Polypeptide Antibiotic Residues in Food of Animal Origin by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

Imaging Peptide and Protein Chirality via Amino Acid Analysis by Chiral × Chiral Two-Dimensional Correlation Liquid Chromatography.

The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (1D) and tert-butylcarbamoyl quinidine CSP in the second dimension (2D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the 1D and 2D chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between 1D and 2D CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed 2D-flow rates which were 60 times faster than the 1D-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in 1D and 2D, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.

Pluronic-based nano-self-assemblies of bacitracin A with a new mechanism of action for an efficient in vivo therapeutic effect against bacterial peritonitis.

BACKGROUND: Although assemblies of hydrophobic-modified bacitracin A with PLGA (Nano-BAPLGA) have demonstrated promising antibacterial activities against both Gram-positive and Gram-negative bacteria, the desirable antibacterial potency has remained challenging due to the low solubility of Nano-BAPLGA. To address this tissue, a series of Pluronic copolymers (Pluronic® F127, Pluronic® P123 and Pluronic® P85) were selected to link the N-terminus of bacitracin A to construct Pluronic-based nano-self assemblies (Nano-BAF127, Nano-BAP123 and Nano-BAP85). RESULTS: Impressively, all the newly designed Pluronic-based Nano-BAs possessed higher solubility and stronger effectiveness against both Gram-positive and Gram-negative bacteria compared with Nano-BAPLGA, especially the modification with Pluronic® P85. Surface tension measurements indicated that Nano-BAP85 was much more tensioactive than Nano-BAPLGA, which usually translated into a good membranolytic effect. Fluorescence spectroscopy and electron microscopy analyses confirmed the speculation that the cell wall/membrane might be the main action target of Nano-BAP85 by permeabilizing the cell membrane and damaging the membrane integrity. In vivo results further demonstrated that Nano-BAP85 significantly suppressed bacterial growth and prolonged survival time in the bacterial peritonitis mouse model with negligible toxicity. CONCLUSIONS: Collectively, the membrane targeting mechanism of action is entirely distinct from those of clinically used antibacterial agents. Furthermore, the new approach of construction nanoantibiotics based on the modification of commercially available antibiotics with Pluronic copolymers is demonstrated to have an efficient therapeutic effect against bacterial infection.

Accelerated Stability Modeling for Peptides: a Case Study with Bacitracin.

The Accelerated Stability Assessment Program (ASAP) was applied for the first time to a peptide, the antibiotic active pharmaceutical ingredient bacitracin. Bacitracin and its complex with zinc were exposed to temperature and relative humidity conditions from 50 to 80°C and from 0 to 63% for up to 21 days. High-performance liquid chromatography was used to analyze the stressed samples for both degradant formation and loss of the active (bacitracin A) and two inactive isoforms, with identities confirmed by mass spectrometry. These data were then analyzed using a humidity-corrected Arrhenius equation and isoconversion approach to create a shelf-life predicting model for typical storage conditions. Model fitting was found to be good with low residuals in both temperature and relative humidity axes for all parameters examined. The generated model's predictions for both the native and zinc complex of bacitracin for both formation of the major degradation product (F) and loss of the active isoform (A) were consistent with longer-term measured values at 30°C/53%RH and 40°C/75%RH, validating this approach for accelerating the determination of long-term stability of a peptide.

Photooxidation of the Antimicrobial, Nonribosomal Peptide Bacitracin A by Singlet Oxygen under Environmentally Relevant Conditions.

Bacitracin is a mixture of nonribosomal peptides (NRPs) that is extensively used as an antibiotic in both human and veterinary medicine. Despite its widespread use over the past six decades, very few studies have addressed the environmental fate of bacitracin and zinc-bacitracin complexes. In this study, the photochemical transformation of bacitracin components (i.e., cyclic dodecapeptides) in the aquatic environment was investigated. A high resolution mass spectrometry (HRMS)-based approach enabled monitoring of the photochemical degradation kinetics of individual bacitracin components, investigation of the relative contribution of reactive oxygen species (e.g., singlet oxygen, (1)O2) in dissolved organic matter-sensitized photoreactions, and identification of oxidative modifications in bacitracin photoproducts. The results of this study support the hypothesis that indirect photochemical oxidation of the histidine (His) residue by (1)O2 is a major degradation pathway for bacitracin A, the most potent congener of the mixture. Furthermore, the photooxidation rate of bacitracin A with (1)O2 decreased upon bacitracin A coordination with Zn(2+), demonstrating that the photochemistry of metal-bound His is different from that of metal-free His. Overall, these results provide insight into the fate of bacitracin components in the aquatic environment and highlight the potential of utilizing this HRMS-based methodology to study transformations of other environmentally relevant NRPs.

High-resolution crystal structure reveals molecular details of target recognition by bacitracin.

Bacitracin is a metalloantibiotic agent that is widely used as a medicine and feed additive. It interferes with bacterial cell-wall biosynthesis by binding undecaprenyl-pyrophosphate, a lipid carrier that serves as a critical intermediate in cell wall production. Despite bacitracin's broad use, the molecular details of its target recognition have not been elucidated. Here we report a crystal structure for the ternary complex of bacitracin A, zinc, and a geranyl-pyrophosphate ligand at a resolution of 1.1 Å. The antibiotic forms a compact structure that completely envelopes the ligand's pyrophosphate group, together with flanking zinc and sodium ions. The complex adopts a highly amphipathic conformation that offers clues to antibiotic function in the context of bacterial membranes. Bacitracin's efficient sequestration of its target represents a previously unseen mode for the recognition of lipid pyrophosphates, and suggests new directions for the design of next-generation antimicrobial agents.

Dereplicating nonribosomal peptides using an informatic search algorithm for natural products (iSNAP) discovery.

Nonribosomal peptides are highly sought after for their therapeutic applications. As with other natural products, dereplication of known compounds and focused discovery of new agents within this class are central concerns of modern natural product-based drug discovery. Development of a chemoinformatic library-based and informatic search strategy for natural products (iSNAP) has been constructed and applied to nonribosomal peptides and proved useful for true nontargeted dereplication across a spectrum of nonribosomal peptides and within natural product extracts.

Antibacterial properties of photo-crosslinked chitosan/methacrylated hyaluronic acid nanoparticles loaded with bacitracin.

The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion.

Sulfonamide Bioisosteres of Niclosamide Enhance Antibacterial Activity of Colistin and Bacitracin.

Multicomponent therapy combining antibiotics with enhancer molecules known as adjuvants is an emerging strategy to combat antimicrobial resistance. Niclosamide is a clinically relevant anthelmintic drug with potential to be repurposed for its inherent antibacterial activity against Gram-positive bacteria and its ability to potentiate the antibacterial activity of colistin against susceptible and resistant Gram-negative bacteria. Herein, sulfonamide analogs of niclosamide were prepared and found to enhance colistin activity against Gram-negative bacteria. The ability of niclosamide and the new sulfonamide analogs to synergize with bacitracin against vancomycin-resistant Enterococcus faecium was also discovered.

Use of mixed micelles for presentation of building blocks in a new combinatorial discovery methodology: proof-of-concept studies.

We describe a new method of combinatorial screening in which building blocks, instead of being linked together chemically, are placed on the surface of nanoparticles. Two- or three-dimensional structures form on the surface of these particles through the close approach of different building blocks, with sufficient flexibility to be able to adapt and interact with putative binding sites in biological systems. The particles assemble without the need for formation of chemical bonds, so libraries comprised of many structures can be prepared rapidly, with large quantities of material available for testing. Screening methods can include solid and solution-phase binding assays, or tissue culture models, for example looking for structures which can change the behaviour of cells in a disease-modifying manner.

Bacitracin-conjugated superparamagnetic iron oxide nanoparticles: synthesis, characterization and antibacterial activity.

Bacitracin-conjugated superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles were prepared by click chemistry and their antibacterial activity was investigated. After functionalization with hydrophilic and biocompatible poly(acrylic acid), water-soluble Fe(3)O(4) nanoparticles were obtained. Propargylated Fe(3)O(4) nanoparticles were then synthesized by carbodiimide reaction of propargylamine with the carboxyl groups on the surface of the iron oxide nanoparticles. By further reaction with N(3)-bacitracin in a Cu(I)-catalyzed azide-alkyne cycloaddition, the magnetic Fe(3)O(4) nanoparticles were modified with the peptide bacitracin. The functionalized magnetic nanoparticles were characterized by powder X-ray diffraction, X-ray photoelectron spectroscopy, TEM, zeta-potential analysis, FTIR spectroscopy and vibrating-sample magnetometry. Cell cytotoxicity tests indicate that bacitracin-conjugated Fe(3)O(4) nanoparticles show very low cytotoxicity to human fibroblast cells, even at relatively high concentrations. In view of the antibacterial activity of bacitracin, the biofunctionalized Fe(3)O(4) nanoparticles exhibit an antibacterial effect against both Gram-positive and Gram-negative organisms, which is even higher than that of bacitracin itself. The enhanced antibacterial activity of the magnetic nanocomposites allows the dosage and the side effects of the antibiotic to be reduced. Due to the antibacterial effect and magnetism, the bacitracin-functionalized magnetic nanoparticles have potential application in magnetic-targeting biomedical applications.

Solution NMR of polypeptides hyperpolarized by dynamic nuclear polarization.

Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300-2000 are obtained in partially deuterated polypeptide when hyperpolarized on (13)C and of 30-180 when hyperpolarized on (1)H. A simulated spectrum is used to identify different resonances in the hyperpolarized (13)C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.

Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.

1H NMR, mechanism, and mononuclear oxidative activity of the antibiotic metallopeptide bacitracin: the role of D-Glu-4, interaction with pyrophosphate moiety, DNA binding and cleavage, and bioactivity.

The peptidyl antibiotic bacitracin (Bc) is one of the most widely used antibiotics which can bind divalent transition metal ions, including Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The metal binding is essential for its antimicrobial activity. Previous analysis of the hyperfine-shifted (1)H NMR signals of Co(II)-Bc A(1) revealed the structure of the metal binding environment and a potential hydrophobic site important for the bioactivity of this antibiotic. Co(II)-Bc in DMSO shows relatively sharper hyperfine-shifted (1)H NMR signals compared with the spectrum acquired in an aqueous solution, allowing more thorough analysis of the signals with 1D and 2D NMR methods. Pyrophosphate and derivatives bind to Co(II)-Bc to form kinetically inert ternary complexes. The coordinated D-Glu-4 is found detached from the metal center of metallobacitracin upon trimetaphosphate binding, implying its role in the antibiotic activity of Bc. We further demonstrate in this report the structure-function relationship on desamido-Bc of low antibiotic activity by the use of NMR, wherein D-Glu-4 is suggested to be important for the bioactivity of Bc. The interaction of the phospho-moiety with Bc is also reflected by DNA binding, wherein metal-free Bc does not bind DNA, whereas various metal complexes of Bc do. Cu(II)-Bc was further demonstrated to bind and oxidatively cleave DNA under reduction conditions in the air. It also exhibited a significant oxidative activity toward catechol oxidation, showing enzyme-like saturation kinetics with k(cat) = 7.0 x 10(-3) s(-1) and k(cat)/K(m) = 2.1 M(-1) s(-1) aerobically and k(cat) = 0.38 s(-1) and k(cat)/K(m) = 14.7 M(-1) s(-1) in the presence of 32 mM of H(2)O(2). The binding of pyrophosphate moiety to metallobacitracin, the detachment of d-Glu-4, and the significant oxidative activity of Cu(II)-Bc provide further insights into the bioactivity of this metallopeptide and Cu-oxygen chemistry.

Preparation of aminated fish scale collagen and oxidized sodium alginate hybrid hydrogel for enhanced full-thickness wound healing.

Acute full-thickness wounds require a more extended healing period, thus increasing the risk of infection. Severe infection frequently resulted in wound ulceration, necrosis, and even life-threatening complications. Here, a hybrid hydrogel comprising aminated collagen (AC), oxidized sodium alginate (OSA), and antimicrobial peptides (polymyxin B sulfate and bacitracin) was developed to enhance full-thickness wound healing. The AC with low immunogenicity and high biocompatibility was made from marine fish scales, which are eco-friendly, low-cost, and sustainable. The cross-linked hydrogel was formed by a Schiff base reaction without any catalysts and additional procedures. As expected, the presented hybrid hydrogel can effectively against E. coli and S. aureus, as well as promote cell growth and angiogenesis in vitro. In addition, the hydrogel can promote full-thickness wound healing in a rat model through accelerating reepithelialization, collagen deposition, and angiogenesis. Our work demonstrated that the hybrid hydrogel has promising applications in the field of wound healing, which would prompt the utilization of marine fish resources during food processing.

PEGylated Self-Assembled Nano-Bacitracin A: Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery in Vivo.

Although nano-self-assemblies of hydrophobic-modified bacitracin A with poly(d,l-lactic- co-glycolic acid) (PLGA) (nano-BAPLGA) have demonstrated promising antibacterial activities, the application of nano-BAPLGA was severely compromised by low water solubility. In this study, a series of PEGylated PLGA copolymers were selected to conjugate with the N-terminus of bacitracin A to construct PEGylated self-assembled nano-BAs and to further develop nano-self-assemblies of bacitracin A with strong antibacterial potency and high solubility. Compared with nano-BAPLGA, all PEGylated nano-BAs, except nano-BA5k, exhibited strong antibacterial efficiency against both Gram-positive and Gram-negative bacteria by inducing loss of cytoplasmic membrane potential, membrane permeabilization, and leakage of calcein from artificial cell membranes. Studies elucidating the underlying mechanism of PEGylated nano-BAs against Gram-negative bacteria indicated that the strong hydrophobic and van der Waals interactions between PLGA and lipopolysaccharide (LPS) could bind, neutralize, and disassociate LPS, facilitating cellular uptake of the nanoparticles, which could destabilize the membrane, resulting in cell death. Moreover, PEGylated nano-BAs (nano-BA12k) with a longer PLGA block were expected to occupy a higher local density of BA mass on the surface and result in stronger hydrophobic and van der Waals interactions with LPS, which were responsible for the enhanced antibacterial activity against Gram-positive and emerging antibacterial activity against Gram-negative bacteria, respectively. In vivo imaging verified that PEGylated nano-BAs exhibited higher inflammatory tissue distribution and longer circulation time than nano-BAPLGA. Therefore, although PEGylation did not affect antibacterial activity, it is necessary for target delivery and resistance to clearance of the observed PEGylated nano-BAs. In vivo, nano-BA12k also showed the highest therapeutic index against infection burden in a mouse thigh infection model among the tested formulations, which showed good correlation with the in vitro results. In conclusion, nano-BA12k showed high efficacy in the treatment of invasive infections. This new approach of constructing nanoantibiotics by modification of commercially available antibiotics with PEGylated copolymers is safe, cost-effective, and environmentally friendly.

Self-assembly of bacitracin-gold nanoparticles and their toxicity analysis.

As the widely use of gold nanoparticles (AuNPs) in drug delivery, the precise control on the size and morphology of the AuNPs is urgently required. In this scenario, traditional synthesis methods cannot meet current requirement because of their inherent defects. We have depicted here a novel method for fabricating monodispersed large size gold nanoparticles, based on the self-assembly of bacitracin. The AuNPs could be facilely, low-cost, and green synthesized with repeatability and controllability in this method. The Bac gold nanoparticles (Bac-AuNPs), composed by bacitracin core and gold shell, exhibited a spherical morphology in TEM and a face-centered cubic crystal structure in X-Ray diffraction and selected area electron diffraction. The mean diameter of the Bac-AuNPs was 89nm. The nanoparticles were mono-dispersed and the zeta potential of the nanoparticles was 4.1±0.64mV. Notably, in cell viability assay, the Bac-AuNPs showed less toxicity to HepG2 cells and HEK293 cells compared to small size AuNPs. Collectively, the size, rheological characteristic and the biocompatibility supported the use of the gold nanoparticles as intracellular delivery vehicles for drug delivery, especially for tumor therapy. And this study could provide a maneuverable, controllable and green strategy for the synthesis of AuNPs, which would be applied in disease diagnosis and therapy with biosafety.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.