Development of sensitive benzofurazan-based spectrometric methods for analysis of spectinomycin in vials and human biological samples.

Spectinomycin hydrochloride (SPEC) is an aminoglycoside antibiotic that has a broad spectrum against several bacterial strains of humans and veterinary infections. However, SPEC lacks a fluorophore or chromophore and this lack makes its analysis a challenge. Our study provides a simple, sensitive and low-cost spectrofluorimetric/spectrophotometric method based on the reaction between secondary amine groups and a benzofurazan reagent using borate buffer (pH 9.2). The yielding compound was measured fluorimetrically at 530 nm (excitation at 460 nm) with colorimetry at 410 nm. The calibration curves ranged from 30 to 400 ng ml-1 and from 0.2 to 6 µg ml-1 for spectrofluorimetric and spectrophotometric analyses, respectively. The limits of detection were calculated to be 4.15 ng ml-1 and 0.05 µg ml-1 for spectrofluorimetric and spectrophotometric processes, respectively. The ultra-affectability and high selectivity of the proposed method permitted analysis of SPEC in the dosage form and in human plasma samples, with good recoveries of about 101.19 and 97.11%, respectively, without any matrix interference. The proposed strategy was validated using International Conference on Harmonization standards and validated bioanalytically using USFDA recommendations.

HPLC-ELSD analysis of spectinomycin dihydrochloride and its impurities.

A simple, rapid and reliable reversed-phase ion-pair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODS-100V. The mobile phase was ACN/aqueous solution of 15 mM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50°C and the nebulizing gas flow rate of air was 3.5 L/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025-3 mg/mL, r=0.9993), sensitivity (LOD: 100 ng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.

Lincomycin and spectinomycin concentrations in liquid swine manure and their persistence during simulated manure storage.

Antimicrobials administered to livestock can be excreted up to 75% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to crop and pasture land. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. In the prairie region of Canada, lincomycin and spectinomycin are two antimicrobials that are frequently administered to pigs for prevention of post-weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in liquid manure from a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage was investigated. LC-MS/MS analysis of manure extracts showed that concentrations of lincomycin and spectinomycin in the accumulating liquid manure at the end of the study were equivalent to 32 and 3%, respectively, of the doses administered to weanling pigs in their feed. In a laboratory study in which lagoon storage was simulated at room temperature using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during the first 6 days, followed by a much slower dissipation, over a period of 5 months. Such persistence indicates that lincomycin and spectinomycin may be present in lagoon manure when applied as an amendment to agricultural land since many lagoons are emptied every 6 months (early spring and late fall).

Sterilization of Mycobacterium tuberculosis infected samples using methanol preserves anti-tuberculosis drugs for subsequent pharmacological testing studies.

Pharmacokinetic/pharmacodynamic studies of anti-tuberculosis agents in animal models of tuberculosis are hampered by the frequent necessity to perform sample bioanalysis outside the biosafety level-3 environment. Thus, each specimen has to undergo tedious and time-consuming sample sterilization procedures that may also affect drug stability. Here, we tested treatment of Mycobacterium tuberculosis (Mtb) infected samples with methanol to sterilize samples while preserving drug integrity for further pharmacokinetic/pharmacodynamic evaluations. Tissue samples harvested from Mtb infected mice were homogenized, incubated in methanol, and tested for sterility. Once sterility was confirmed, the samples were used to determine concentrations of the anti-tuberculosis drug spectinamide-1599 in lung homogenates using liquid chromatography coupled with mass spectrometry. The results demonstrate that methanol sterilizes tissue samples harvested from Mtb infected mice without altering the integrity of the drug in the tissue.

Simultaneous analysis of spectinomycin, halquinol, zilpaterol, and melamine in feedingstuffs by ion-pair liquid chromatography-tandem mass spectrometry.

A method for the simultaneous analysis of veterinary drug residues (spectinomycin, halquinol, and zilpaterol) and contaminants (melamine) in feedingstuffs by liquid chromatography-tandem mass spectrometry was developed. Method performance for all analytes was evaluated by reversed-phase liquid chromatography, reversed-phase with altered chemical equilibrium, and hydrophilic interaction (HILIC) as chromatographic modes. Validation was in accordance to Commission Decision 657/2002/CE, by considering the best chromatographic approach. Ion-pair liquid chromatography with C18 as stationary phase led to the lowest random uncertainties, effective analyte separation and shorter time of analysis. Low precision deviations and good recovery rates were obtained and thus method reliability and sensitivity could be consolidated. Method applicability was evaluated by the analysis of samples of feedingstuffs, such as cattle, pig, and poultry feeds, feed ingredients of both animal and vegetable origins, and mineral feeds. Some samples showed quantifiable concentrations of halquinol and zilpaterol, reinforcing the importance of this new analytical control method.

Development of a hydrophilic interaction chromatography-mass spectrometry assay for spectinomycin and lincomycin in liquid hog manure supernatant and run-off from cropland.

A specific and sensitive analytical method was developed to extract and quantify spectinomycin and lincomycin in liquid hog manure supernatant and simulated rainfall run-off from manure-treated cropland. Sample extracts were prepared using solid-phase extraction (SPE) employing a weak cation-exchange resin (Oasis WCX) for extraction of spectinomycin. An Oasis HLB cartridge was used for extraction of lincomycin. Hydrophilic interaction chromatography (HILIC) was used to obtain the necessary separation of the two antibiotics from interfering compounds and to provide baseline separation. Analytes were detected using atmospheric pressure chemical ionization tandem mass spectrometry. Extraction recoveries were 77% for lincomycin and 84% for spectinomycin in liquid hog manure supernatant, and 89% for lincomycin and 95% for spectinomycin in run-off water. The corresponding limits of quantitation were 6.0 and 0.040 microg l(-1) for spectinomycin and lincomycin, respectively, in liquid hog manure supernatant and 0.2 and 0.008 microg l(-1) for spectinomycin and lincomycin, respectively, in run-off from manure treated cropland. The method is suitable for monitoring the environmental fate and transport of these two antibiotics in both liquid hog manure and agricultural field run-off.

Determination of spectinomycin hydrochloride and its related substances by HPLC-ELSD and HPLC-MSn.

A new and simple high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C(18) (250 mm x 4.6 mm, 5 microm). The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 degrees C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07-3.8 mg/ml (r = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 microg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC-MS(n) method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC-MS(n) data.

Organization and structure of the Methanococcus transcriptional unit homologous to the Escherichia coli "spectinomycin operon". Implications for the evolutionary relationship of 70 S and 80 S ribosomes.

By means of an immunological approach and a subsequent chromosome-walking strategy a chromosomal region encoding ribosomal proteins in the archaebacterium Methanococcus vannielii was cloned. The determination of the nucleotide sequence of the 7.8 x 10(3) base DNA fragment revealed the existence of 14 putative ribosomal protein genes and two unidentified open reading frames. They are organized in a transcriptional unit that is very similar to the Escherichia coli "spectinomycin operon" in respect of both gene composition and gene order. The Methanococcus transcriptional unit contains, in addition to those genes whose products have a homologue in the E. coli operon, three genes whose products share sequence similarity with eukaryotic 80 S but not with eubacterial ribosomal proteins. The Methanococcus ribosomal proteins almost exclusively exhibit a higher sequence similarity to eukaryotic 80 S ribosomal proteins than to those of eubacteria and many of them have a size intermediate between those of their eukaryotic and eubacterial homologues. These results are discussed in terms of a hypothesis that implies that the recent eubacterial ribosome developed by a "minimization" process from a more complex organelle and that the archaebacterial ribosome has maintained features of this ancestor.

Development of high performance liquid chromatography methods with charged aerosol detection for the determination of lincomycin, spectinomycin and its impurities in pharmaceutical products.

Novel and simple liquid chromatography methods with charged aerosol detection (LC-CAD) for simultaneous quantitation of lincomycin and spectinomycin and its related substances have been developed and tested. This type of analysis is complicated due to the different chromatographic behavior of these two agents and the lack of chromophores in spectinomycin complex. CAD seems to be a promising alternative to overcome these difficulties. It shows a consistent inter-analyte response, independent of chemical structure of an analyte. It also enables the direct quantification of related substances for which no reference standards were available, with good accuracy and precision. Chromatographic separations were achieved using a C18 Hypersil(®) Gold column, with mobile phases consisting of water, acetonitrile and trifluoroacetic acid. All impurities were identified using time-of-flight mass spectrometry with electrospray ionization. The developed methods have been successfully used in the routine quality control analysis of pharmaceutical preparations.

Quantitation of spectinomycin residues in bovine tissues by ion-exchange high-performance liquid chromatography with post-column derivatization and confirmation by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry.

Determinative and confirmatory methods of analysis for spectinomycin residue in bovine kidney, liver, muscle and fat have been developed. The determinative method is a single-column HPLC ion-exchange procedure that incorporates a two-step post-column oxidation of the secondary amines to primary amines followed by derivatization with o-phthalaldehyde. The method was validated in all tissues to a low-end concentration of 0.10 micrograms/g (limit of quantitation) and to a high-end of 10 micrograms/g for kidney, which is the rate-limiting tissue for residues of spectinomycin. The recovery of spectinomycin from all tissues was > 80% and the variability (R.S.D.) was generally < 10%. For liver, an alternative reversed-phase HPLC separation was required for incurred-residue samples. The confirmatory method employed an atmospheric pressure chemical ionization-MS-MS approach utilizing a rapid reversed-phase HPLC system with a mobile phase of methanol and 1% acetic acid. The protonated molecular ion for spectinomycin at m/z 333 produced four diagnostic reaction-product ions at 98, 116, 158 and 189 for confirmation. The method was validated to a lower limit of confirmation of 0.10 micrograms/g.

Analysis of spectinomycin by liquid chromatography with pulsed electrochemical detection.

Until now, no LC method is described to determine the purity and content of spectinomycin without prior derivatization. A reversed-phase ion-pair LC method using a base deactivated column and pulsed electrochemical detection is described. The mobile phase consisted of an aqueous solution containing 5.8 g/l pentafluoropropionic acid, 1.25 g/l potassium dihydrogen phosphate and 5.5 ml/l tetrahydrofuran. The pH was adjusted to 6.25 using dilute NaOH solution. An experimental design was used to optimize the chromatographic parameters and to check the robustness. The quality of separation was investigated on different stationary phases. The method allows the separation of spectinomycin from its related substances as well as some other components of unknown identity. The total time of analysis is 65 min. A number of commercial samples were examined using this method.

Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic.

There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay was designed using reagents developed for this assay. The spectinomycin antibody was produced in sheep, and the immunoglobulin (IgG) was purified through a Protein G affinity column and was immobilized onto latex particles. Spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). The optimum assay conditions consisted of preincubating the latex-IgG with spectinomycin in buffer solutions or in bovine kidney extracts. DTAF-spectinomycin was added and was further incubated. The bound spectinomycin-DTAF/IgG-latex complex was separated by centrifugation at 4000 g for 10 min. The fluorescence signals of the unbound spectinomycin-DTAF in the supernatant were measured at 485/535 nm excitation/emission. The measured signals were directly proportional to the concentration of spectinomycin in the samples, and spectinomycin was detected at 0-100 ppb with minimum detectability of 5 ppb. The mean regression correlation of four trials in buffer was 0.936 when the % bound complex vs spectinomycin concentration was plotted. Analysis of the kidney extract spiked with 0-100 ppb spectinomycin had a regression correlation of 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin.

Analysis of a formulation containing lincomycin and spectinomycin by liquid chromatography with pulsed electrochemical detection.

A reversed phase ion-pair liquid chromatographic method using a base deactivated column and pulsed electrochemical detection on a gold electrode is described. It allows the separation of a mixture of spectinomycin sulfate, lincomycin hydrochloride and their related substances. A step gradient was necessary to obtain a good separation together with a reasonable analysis time of 40 min. The mobile phases consisted of an aqueous solution of 3.3 or 0.55 g/l pentanesulfonic acid, 10 mM acetic acid and 20 ml/l tetrahydrofuran. Both mobile phases were adjusted to pH 4.0 with diluted sodium hydroxide. The influence of the different chromatographic parameters on the separation was investigated. Two commercial samples were analyzed using the described method. In total 12 components could be separated.

Identification and determination of oxytetracycline, tiamulin, lincomycin, and spectinomycin in veterinary preparations by thin-layer chromatography/densitometry.

A thin-layer chromatographic/densitometric method was developed for the identification and quantitation of oxytetracycline, tiamulin, lincomycin, and spectinomycin in veterinary preparations. Silica gel-coated thin layer chromatography plates and 2 mobile phases were used to separate these constituents. The appropriate compositions of the suitable mobile phases were established: 10% citric acid solution-n-hexane-ethanol (80 + 1 + 1, v/v) and n-butanol-ethanol-chloroform-25% ammonia (4 + 5 + 2 + 5, v/v). Along with Rf values and spot colors, direct UV and visual densitometric measurements were used for identification. Similar measuring ranges were used for quantitative analysis to obtain repeatable and reliable results for the preparations examined. The results of the quantitative analysis are characterized by a small confidence interval and are close to the declared contents of active constituents: oxytetracycline 30.01 +/- 0.38 g at lambda = 350 nm and 30.24 +/- 0.86 g at lambda = 430 nm; tiamulin, 10.19 +/- 0.86 g at lambda = 450 nm; lincomycin, 2.27 +/- 0.08 g at lambda = 278 nm; and spectinomycin, 2.18 +/- 0.07 g at lambda = 421 nm. The recoveries for all antibiotics ranged from 100.01 to 102.54%.

Application of microbial receptor assay to quantitation of residues of spectinomycin in bovine milk and comparison with liquid chromatographic analysis.

Spectinomycin-contaminated bovine milk samples were assayed by liquid chromatographic (LC) and microbial receptor methods. LC involved a newly developed analytical method to quantitate the concentration of spectinomycin in the contaminated milk samples. The receptor assay used reagents and the reaction system used for the Charm II spectinomycin assay. Three standard curves (selected range, full range, and second-order polynomial) were plotted for the receptor assay and used to quantitate spectinomycin in contaminated milk samples. The levels of spectinomycin obtained by the receptor assay, using only the standard curve in the selected range, were comparable to the results obtained by LC analysis.

Drug residues in milk after intrauterine injection of oxytetracycline, lincomycin-spectinomycin, and povidone-iodine in cows with metritis.

A study was conducted to document the maximum retention times of antimicrobial residues in milk after their use in intrauterine treatment of metritis in lactating cows and to evaluate several risk factors hypothesized to influence the retention time of these drugs. Oxytetracycline (3 g), lincomycin-spectinomycin (2 g of one-third lincomycin and two-thirds spectinomycin), or povidone-iodine (6 g) were given to cows with metritis by intrauterine route. The Bacillus stearothermophilus var calidolactis disk assay was performed on each milk sample. Of the 61 cows treated with oxytetracycline, 30 had residues in their postinjection milk for variable periods (range, 12.5 to 44.0 hours; mean, 26.6 +/- 10.3). Of the 47 cows treated with lincomycin-spectinomycin, 17 had residues in their postinjection milk for various periods (range, 14.5 to 24 hours; mean, 19.5 +/- 8.9). Povidone-iodine was not detected in milk. Because a high number of cows (n = 61) were treated with oxytetracycline, only data from these cows were used in testing the influence of 3 factors (severity of metritis, time after parturition when cows with metritis were treated, and parity) on maximum retention of the drug in milk. Severity of metritis did not have a significant influence (P greater than or equal to 0.1) on the maximum retention time of the drug. The retention time decreased linearly with the increase of time after parturition when the cow with metritis was treated. First lactation cows had a significantly (P less than or equal to 0.01) shorter retention time than did older cows.

[Residue analysis of spectinomycin in tissues of chicken and swine by HPLC].

A reversed-phase HPLC method with ultraviolet detection using p-nitrophenyl hydrazine as a pre-column derivatizing reagent was investigated for the determination of the antibiotic spectinomycin (SPCM) in muscle, liver, kidney and fat of chicken and swine. SPCM was extracted from samples with 10% trichloroacetic acid saturated with EDTA-2Na, and then cleaned up with coupled Sep-Pak Plus PS-2 cartridges. The detection limit of SPCM was 0.02 microgram (potency)/g. Recoveries of SPCM ranged from 77.4 to 97.4% for chicken tissues and from 74.5 to 91.8% for swine tissues. The present method was used for the analysis of chicken tissues after the 11th day of withdrawal (SPCM-medicated drinking water: 500 mg (potency)/L, for 7 days), and swine tissues after the 14th day of withdrawal (SPCM-medicated feed: 100 mg (potency)/kg, for 7 days). Results showed that SPCM concentrations were lower than the MRLs in all tissues.

Graphene coated silica applied for high ionization matrix assisted laser desorption/ionization mass spectrometry: A novel approach for environmental and biomolecule analysis.

The integration of nanotechnology with mass spectrometry for sensitive and selective detection of molecules is a hot/important field of research. Synthesis of graphene (G) coated with mesoporous silica (SiO2, G@SiO2) for mass spectrometric application has been demonstrated. For the first time, we proposed the significant role of surfactant that used during the synthesis of mesorporous silicate (SiO2) in mass spectrometry. It was noticed that G could initiate SiO2 via surfactants which work as initiators for further ionization. The porosity of SiO2 trapped the analytes that was released and ionized with the surfactant fragments. Undoubtedly, strong background interferences were present in the case of organic matrix, which greatly obscured the detection of low molecular weight compounds. G@SiO2 nanocomposite affords several advantages, such as the ability to detect small molecules (<500Da), high sample localization through silica mesoporosity, and high ionization efficiency over than G or conventional matrices. The high performance of G@SiO2 is not only due to the large surface area but also due to high desorption/ionization efficiency of inevitably surfactant (cetyltrimethylammonium chloride, CATB). Unlike the conventional MALDI-MS, the G@SiO2-MS is capable of generating multiply charged polysaccharides. The present method was validated to detect surfactants with low limits of detection.

Furoic and mefenamic acids as new matrices for matrix assisted laser desorption/ionization-(MALDI)-mass spectrometry.

The present study introduces two novel organic matrices for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of small molecules. The first matrix is "2-amino-4,5-diphenylfuran-3-carboxylic acid" (also called furoic acid, FA) which was synthesized and then characterized by ultraviolet (UV), infrared (FTIR), nuclear magnetic resonance NMR ((1)H and (13)C) and mass spectrometry. The compound has organic semiconductor properties and exhibits intense UV-absorption which is suitable for the UV-MALDI laser (N2 laser, 337 nm). The second matrix is mefenamic acid (MA). The two matrices can be successfully applied for various classes of compounds including adenosine-5'-triphosphate (ATP, 0.5 µL(10.0 nmol)), spectinomycin (spect, 0.5 µL(14.0 nmol)), glutathione (GSH, 0.5 µL(9.0 nmol)), sulfamethazole (SMT, 0.5 µL(2.0 nmol)) and mixture of peptides gramicidin D (GD, 0.5µL (9.0 nmol)). The two matrices can effectively absorb the laser energy, resulting in excellent desorption/ionization of small molecules. The new matrices offer a significant enhancement of ionization, less fragmentation, few interferences, nice reproducibility, and excellent stability under vacuum. Theoretical calculations of the physical parameters demonstrated increase in polarizability, molar volume and refractivity than the conventional organic matrices which can effectively enhance the proton transfer reactions between the matrices with the analyte molecules. While the reduction in density, surface tension and index of refraction can enhance homogeneity between the two new matrices with the analytes. Due to the sublimation energy of mefenamic acid is (1.2 times) higher than that of the DHB, it is more stable to be used in the vacuum.

Determination of water content by capillary gas chromatography coupled with thermal conductivity detection.

This article presents some experience obtained by applying capillary gas chromatography coupled with thermal conductivity detection (GC/TCD) to the determination of water in substances for pharmaceutical use. This technique represents a useful, orthogonal tool complementary to water determination methods based on volumetric or coulometric titration. It can also represent an alternative technique when such titrations are not applicable. This article presents the preliminary results obtained in a number of case studies where a GC/TCD procedure was applied in comparison with pharmacopoeial methods to substances with different water contents.