Surveillance study of novobiocin and phenylbutazone residues in raw bovine milk using liquid chromatography-tandem mass spectrometry.

A simple method permitting the simultaneous determination of trace residues of novobiocin and phenylbutazone in raw milk samples using liquid chromatography-tandem mass spectrometry was developed. Raw milk samples were mixed with acetonitrile to facilitate the concurrent precipitation of milk proteins and extraction of both veterinary drugs. Without additional clean-up or concentration of the resulting extract, the analytes could be quantified at concentrations as low as 0.0025 and 0.001 microg ml(-1) for phenylbutazone and novobiocin, respectively. The analysis of a series of fortified raw milk samples at analyte concentrations ranging from 0.005 to 0.1 microg ml(-1) and from 0.01 to 0.2 microg ml(-1) for phenylbutazone and novobiocin, respectively, yielded average recoveries ranging from 89.2% to 104.3% with standard deviations below 7%. The analytical method was applied to the analysis of raw milk samples collected from transport trucks upon delivery at dairy-processing plants throughout Alberta, Canada. Novobiocin was detected in 13 of 1072 samples tested at concentrations ranging from 0.001 to 0.007 microg ml(-1). Phenylbutazone was not detected in any of the samples tested.

LC-MS/MS and centrifugal ultrafiltration method for the determination of novobiocin in chicken, fish tissues, milk and human serum.

We present a rapid and simple method for detecting novobiocin in biologic samples using a methanol-based extraction of the tissue matrix and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. The sample, prepared using centrifugal ultrafiltration with 5.0% SDS, was directly injected into the LC-MS/MS. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.5% formic acid in water/methanol. The method was validated according to the Japanese Maximum Residue Limits recommendations. Detection was linear over a range of 5-100 ppb matrix solution (r>0.998). Novobiocin recovery values from chicken (0.05 ppm) and fish tissues (0.05 ppm), milk (0.08 ppm), and human serum (0.05 and 0.01 ppm) samples ranged from 71+/-1 to 95+/-2%.

Mass spectrometric pathway monitoring of secondary metabolites: systematic analysis of culture extracts of Streptomyces species.

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects. The combination of full scan mass spectrometry (MS), parent ion scan MS, product ion scan MS, and in-source collision-induced fragmentation prior to product ion scans (pseudo-MS(3) scan), using characteristic fragmentation of the central aminocoumarin unit, was employed for the detection and structural interpretation of expected and new intermediates. We were able to show the applicability of this methodology to the three culture extracts, where the main intermediates could be found, and to demonstrate its use for interpretation of secondary metabolite biosynthesis. Some new compounds were discovered, including bis-carbamoylated novobiocin, hydroxylated clorobiocin, and several structurally and not yet fully elucidated coumermycin derivatives or precursors.

Determination of pharmaceuticals in aqueous samples using positive and negative voltage switching microbore liquid chromatography/electrospray ionization tandem mass spectrometry.

Analytical methods were developed for atorvastatin, novobiocin and roxithromycin using microbore liquid chromatography/electrospray ionization tandem mass spectrometry (microbore LC/ESI-MS/MS) in positive and negative voltage switching mode. Atorvastatin and roxithromycin require the positive-ion mode, whereas the negative-ion mode is required for the determination of novobiocin. Using the positive and negative voltage switching function, the three analytes were determined with one injection, and the time required was half that required using separately run positive- and negative-ion modes, without any reduction in sensitivity. A microbore LC column (100 x 1.0 mm i.d.) was chosen for chromatographic separation with mobile phase solvents acetonitrile and 10 mM aqueous ammonium acetate. The flow-rate was 0.1 ml min(-1) and the injection volume was 1 micro l. The analytes were quantified in the multiple reaction monitoring mode with external standards. By switching the positive and negative voltage, the three analytes were determined with a 4 min chromatographic run and with instrumental detection limits of 1-3 pg. This analytical method, using a microbore LC column combined with solid-phase extraction, was applied successfully to the determination of trace levels of the above pharmaceuticals in aqueous samples. Atorvastatin was detected in a sewage treatment plant final effluent.

A scintillation proximity assay amenable for screening and characterization of DNA gyrase B subunit inhibitors.

DNA gyrase is the target of coumarin and cyclothialidine antibacterials, which bind to the B subunit of the enzyme (GyrB). Currently available GyrB inhibitors have not been clinically successful, but their high in vitro potency against DNA gyrase has raised interest in the development of novel noncoumarin antibacterials acting at the same site. We report the development of a simple scintillation proximity assay (SPA) for the study of binding interactions between coumarin or noncoumarin antibacterials and GyrB, which prevents the needs of separation steps and can be run in microtiter plate formats. The assay is based on the detection of the binding of a radioligand, [3H]dihydronovobiocin, to a biotin-labeled 43-kDa fragment of GyrB (biotin-GyrB43), which is captured by streptavidin-coated SPA beads. The typical assay was conducted in 96-well microtiter plates, with final concentration of 10 nM for biotin-GyrB43, 20 nM for [3H]dihydronovobiocin, and 33 microg of SPA beads/well. From saturation experiments, an equilibrium dissociation constant (K(d)) for dihydronovobiocin of 8.10 nM was found. Displacement studies gave 50% inhibitory concentrations (IC(50)) of 42, 64, and 11 nM for novobiocin, dihydronovobiocin, and the cyclothialidine analogue GR122222X, respectively, consistent with previous findings. The assay was found to be robust to dimethyl sulfoxide up to 5% (v/v) and can be used for high-throughput screens of large chemical collections in the search of novel DNA gyrase inhibitors.

Liquid chromatographic procedure for the determination of novobiocin residues in bovine milk: interlaboratory study.

Novobiocin is used for the treatment of mastitis in dairy cattle. In 1982, the tolerance was set at 0.1 ppm in milk from dairy animals. A laboratory trial has been completed for a liquid chromatographic procedure that can quantitate novobiocin residues in bovine milk at tolerance level. In this procedure the milk is diluted with buffer, the proteins are precipitated with methanol, and the solution is filtered. Novobiocin is determined after separation of milk components using reversed-phase chromatography with UV detection at 340 nm. The participating laboratories analyzed 2 concentrations of biologically incurred residues as well as control milk and control milk fortified at 0.05, 0.1, and 0.2 ppm. Recoveries of novobiocin reported by the participating laboratories were 89 to 99% at 0.05 ppm; 93 to 101% at 0.1 ppm; and 89 to 100% at 0.2 ppm. Coefficients of variation (CVs) ranged from 2.0 to 6.2%. The average concentrations for the low levels of incurred novobiocin in milk samples were 0.073, 0.072, and 0.081 ppm, with intralaboratory CVs of 3.3, 7.2, and 2.4%, respectively. The samples with high levels of incurred novobiocin averaged 0.139, 0.121, and 0.144 ppm, with CVs of 6.2, 0.7, and 4.7%, respectively.

Microbial O-carbamylation of novobiocin.

Novobiocin was inactivated by Streptomyces niveus US 2094 in fermentation. The inactivation product was isolated and characterized by NMR and MS as 2"-O-carbamylnovobiocin. The MICs of novobiocin and 2"-O-carbamylnovobiocin were determined for S. niveus strains.

Electrocatalytic detection of streptomycin and related antibiotics at ruthenium dioxide modified graphite--epoxy composite electrodes.

The application of ruthenium dioxide (RuO2) modified electrodes to the electrocatalytic detection of the saccharide-related antibiotics streptomycin, novobiocin and neomycin, at low fixed potentials, was investigated. The RuO2-modified graphite - epoxy composite electrodes give extremely stable and reproducible catalytic oxidation currents for these antibiotics at potentials as low as +0.2 V (versus Ag - AgCl). Rapid quantification at the micromolar level is therefore possible. Standard calibration graphs for streptomycin and neomycin yielded slopes of 4.43 and 0.08 nA microM-1 over the linear ranges of 1.5 x 10(-6) - 2.5 x 10(-4) and 1 x 10(-5) - 2 x 10(-3) M, respectively. Owing to its catalytic oxidation by the RuIII - RuIV couple, rather than the RuIV - RuVI transition (which catalyses the oxidation of streptomycin and neomycin), novobiocin could be detected at a lower (+0.2 V) potential, with a sensitivity of 1.31 nA microM-1. Detection limits of 1.5, 6.0 and 10 microM were obtained for streptomycin, novobiocin and neomycin, respectively. These catalytic surfaces can be renewed (by polishing), with a surface-to-surface reproducibility of 6.5% for the detection of 5 x 10(-5) M streptomycin. The analytical application of RuO2-modified carbon paste electrodes to the analysis of these antibiotics by flow injection was investigated, with a view to liquid chromatographic separation with electrochemical detection applications.

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography.

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.

Dye marked antibiotics for lactating cow mastitis therapy.

Dye markings of intramammary antibiotic infusions could give a dairy farmer immediate visual warning that milk contains antibiotic residue. Six dye and antibiotic preparations for lactating cows were studied for rates of dye and antibiotic milk-out. Albacillin containing 1 X 10(5) IU of penicillin plus 150 mg of novobiocin combined with 25, 125, or 250 mg of Food, Drug, and Cosmetic Blue No. 1 or No. 2 per infusion was used. Thirty cows with healthy udders producing 13.6 to 22.7 kg milk per day were treated in all quarters twice in 24 h (0500 and 1500 h). Milk samples from 14 posttreatment twice per day milkings (0500 and 1500 h) were tested for dye and antibiotic residue. Dye content was determined by a visual method and subvisually by an ion exchange resin method. No antibiotic residues were found by the cylinder plate method after the second to fourth posttreatment milkings. Antibiotic residue was detected up to the sixth milking by Delvotest-P. Depending on the dye type and its concentration, milk was visibly blue for one to four milkings. Subvisual quantities for dye were detectable by the ion exchange resin method for three to five milkings. The preparation showing the most promise for farm use contained 250 mg Blue No. 1 per infusion. Milk from cows treated with this preparation contained visually and subvisually detectable dye through three or four and five milkings, respectively. The dye persistence exceeded or coincided with the maximum antibiotic persistence in nearly all cows treated regardless of dye formulation or method of antibiotic detection.

[Structure of carminomycins II and III]. / Struktura karminomitsinov II i III.

Carminomycins II and III, the main components of the carminomycin complex were isolated in pure state. Their crystalline exalates and acetate of cardminomycin II were prepared. The PMR spectra of both carminomycins and the 13C-NMR spectra of the oxalates were obtained. The molecular weights of the antibiotics were determined by mass-spectrometry. On the basis of the PMR spectra it was shown that carminomycins II and III had similar structures and differed in the stereoisomerism of the nitrogen-free fragment linked to the amino sugar. This was confirmed by the 13C-NMR spectra. The above fragment (C7H15O3) is analogous to the fragment of baumycins A1 and A2 described earlier.

Improvement of the efficiency of ion-exchange cellulose columns and their application.

Packing procedures and operating conditions for microparticulate ion-exchange cellulose columns were investigated. The efficiencies of ion-exchange cellulose and mixed-bed ion-exchange cellulose columns were compared. The mixed-bed columns contained ion-exchange cellulose and diatomite, and those with a 5:1 volume ratio of ion-exchange cellulose (average particle size 7 micronm) and diatomite were found to be superior in almost every respect. Medical, pharmaceutical, biochemical and environmental applications of this type of column are shown.

Application of dynamic calorimetry for monitoring fermentation processes.

The rate of heat evolution (kcal/liter-hr) in mycelial fermentations for novobiocin and cellulase production with media containing noncellular solids was measured by an in situ dynamic calorimetric procedure. Thermal data so obtained have proved significant both in monitoring cell concentration during the trophophase (growth phase) and in serving as a physiological variable in the fermentation process. The validity of this technique has been demonstrated by closing the overall material and energy balances. The maintenance energy in a batch fermentation can be calculated by integrating heat evolution data. This integration method is applicable to a fermentation lacking a precise cell growth curve. The maintenance coefficient, obtained for the novobiocin fermentation by Streptomyces niveus, is equal to 0.028 g glucose equivalent/g cell-hr. The production of novobiocin in the idiophase (production phase) also correlates well with the amount of energy catabolized for maintenance and this results in an observed conversion yield of glucose to novobiocin of 11.8 mg of novobiocin produced per gram of glucose catabolized. A new physiological variable, kilocalories of heat evolved per millimole of oxygen consumed, has been proposed to monitor the state of cells during the fermentation. This method may provide a simple way to monitor on-line shifts in the efficiency of cell respiration and changes in growth yields during a microbial process.

The C-13 NMR spectrum of novobiocin.

The carbons of novobiocin have been assigned to peaks in the CMR spectrum on the basis of chemical shifts and off-resonance experiments on novobiocin and related compounds.

Analysis of tetracycline in pharmaceutical preparations by improved high-performance liquid chromatographic method.

The analysis of tetracycline in pharmaceutical preparations by an improved high-performance liquid chromatographic (HPLC) method is described. The improved method uses a 30-cm long stainless steel column packed with octadecylsilane bonded on 10-mum silica gel, with a linear gradient from 10 to 60% acetonitrile in pH 2.5, 0.02 M phosphate buffer in 11 min at a flow rate of 1.0 ml/min (68 atm). The resolution functions obtained between 4-epitetracycline and tetracycline and between 4-epianhydrotetracycline and anhydrotetracycline were improved 150 and 250%, respectively. The analysis of a tetracycline sample takes approximately 16 min; the original method required more tan 25 min. The relative standard deviation for the analysis of tetracycline powder was 0.66%, and the recovery of 4-epianhydrotetracycline added in tetracycline was linear over the 0.3-100% range. Recovery of tetracycline from products was better than 99.6% at label concentration. The drug content of products as calculated from the HPLC data agreed well with those of the microbiological assay methods.