Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

Jelikić-Stankov, Milena; Veselinović, Dragan; Malešev, D.; Radović, Z.

doi:10.1016/0731-7085(89)80166-9

Cited by 16 publications

(8 citation statements)

References 9 publications

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“…The E. coli feoB, tonB, and fur mutants exhibited OTC fluorescence similar to that of the wild type (results not shown), indicating that the effect of external Fe could not be attributed to an incidental effect of Fe on the fluorescence properties of OTC. Moreover, we validated the above findings with a quantitative spectrophotometric assay of OTC (3,12). OTC uptake in non-Fe-supplemented medium continued up to 60 min, giving ϳ13 g of OTC 10 9 cells Ϫ1 (Fig.…”

Section: Vol 48 2004mentioning

confidence: 95%

“…1), could be due to inhibition of antibiotic uptake, we exploited the availability of convenient OTC assays (9,12) to examine OTC accumulation. Fluorescence due to the antibiotic (9) was readily observable in cells incubated for 15 min with 64 g of OTC ml Ϫ1 (Fig.…”

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confidence: 99%

“…Supernatants were retained at 4°C until analysis. OTC in the supernatants was determined spectrophotometrically (3,12). Cellular OTC accumulation was calculated by subtraction from OTC determinations for control incubations lacking cells.…”

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confidence: 99%

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Iron Blocks the Accumulation and Activity of Tetracyclines in Bacteria

Avery

Goddard

Sumner

et al. 2004

Antimicrob Agents Chemother

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The apparent sensitivities of several bacterial pathogens to tetracyclines varied by up to 128-fold with the medium content of Fe, but not of other metals. The effect of Fe was independent of superoxide dismutase activity and of intracellular Fe, but accumulation of tetracyclines was blocked in high-Fe medium. Thus, synergistic suppression of bacterial growth in the presence of a low Fe concentration and tetracyclines arises because of elevated antibiotic accumulation.The tetracyclines (TCs) are broad-spectrum bacteriostatic antibiotics that inhibit the binding of aminoacyl-tRNA to the ribosomal A site, blocking protein synthesis (8). Environmental parameters may modulate the activity of the TCs. For example, MICs are influenced by pH, oxygen concentration, and external cations (1,11,16,19,21,23). Such factors are an important consideration when predicting MICs for bacteria isolated from human infections: laboratory-derived MICs may not faithfully reflect antibiotic concentrations that will be effective in vivo. There have been almost no studies to explore the mechanisms by which certain external parameters affect the activity of TCs.It has been established recently that Cu,Zn-superoxide dismutase (Sod1p)-defective mutants of the yeast Saccharomyces cerevisiae are susceptible to oxytetracycline (OTC) via a mechanism that is dependent on oxidative damage (2, 3, 5). This susceptibility could be suppressed with Cu in a Sod1p-independent manner (2). We initiated the present work to investigate whether similar effects occur in prokaryotic cells that are normally sensitive to TCs.To test whether the sensitivity of Escherichia coli to TCs is copper dependent, the MICs of OTC and TC against E. coli were determined at a range of Cu(NO 3 ) 2 concentrations by the agar dilution method (17). The MICs (ϳ4 g ml Ϫ1 for both antibiotics) were unaffected at up to 3 mM Cu, the highest Cu concentration tested (data not shown).Since OTC can promote oxidative stress (3) and Cu is a Fenton catalyst, we also tested whether Fe influences the sensitivity of E. coli to TCs. The MICs of TC, OTC, and minocycline (MC) were increased markedly by increasing concentrations of FeSO 4 in the medium (Fig. 1). Thus, 25 M FeSO 4 rescued E. coli growth at 4 g of OTC ml Ϫ1 and the MIC was increased to 64 g ml Ϫ1 at 400 M Fe and to 128 g ml Ϫ1 at 1.6 mM Fe (Fig. 1A). The effect of Fe supplementation on the MICs of TC and MC was less marked than for OTC. Nevertheless, eightfold higher TC and MC concentrations were required to inhibit E. coli growth at 1.6 mM Fe than in Feunsupplemented Luria-Bertani (LB) medium (Յ3 M Fe) (Fig. 1A). In medium supplemented with the Fe chelator ferrozine (0.5 mM), the MIC of OTC was lowered to 1 g ml Ϫ1(giving a 128-fold full-range variation in the MIC of OTC in this study) and the MIC of TC was lowered to 2 g ml Ϫ1 (data not shown). Furthermore, ferrozine at 0.5 mM reversed the protective effect of 25 M FeSO 4 against OTC, and protection with 100 M FeSO 4 was reversed at 2.5 mM ferrozine. The effects were relatively spec...

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Section: Vol 48 2004mentioning

confidence: 95%

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Iron Blocks the Accumulation and Activity of Tetracyclines in Bacteria

Avery

Goddard

Sumner

et al. 2004

Antimicrob Agents Chemother

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“…Supernatants were retained at 4°C until analysis. OTC in the supernatants was determined as described previously (17). Briefly, 100 l of sample was mixed with 0.9 ml of a reaction solution, giving final concentrations of the reaction components of 5 mM Na 2 MoO 4 ⅐ 2H 2 O, 100 mM NaNO 3 , and 10 mM sodium acetate buffer.…”

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Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of Saccharomyces cerevisiae

et al. 2000

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Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1⌬ mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 g ml ؊1 , whereas concentrations of up to 1,000 g ml ؊1 had no effect on the growth of the wild type. OTC resistance was restored in the sod1⌬ mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1⌬ (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1⌬ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1⌬ mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.Reactive oxygen species (ROS) are generated during normal cellular respiratory metabolism, but their damaging effects are generally suppressed by antioxidant defenses. Protective enzymes operating in the budding yeast Saccharomyces cerevisiae are well characterized (30); these include superoxide dismutases (SODs) and catalases, which specifically protect against O 2 ⅐ Ϫ and H 2 O 2 , respectively. S. cerevisiae mutants lacking the principal cellular SOD (Cu/Zn SOD; encoded by SOD1) display certain aerobic growth defects, e.g., reduced growth rate, and methionine and lysine auxotrophies (4). Moreover, sod1⌬ strains are hypersensitive to several types of stress, including oxidative stress (16), metal toxicity (6, 36), prolonged stationary incubation (24), and freeze-thaw stress (28). Such evidence has underscored the central role of ROS in mediating various stresses. However, there is presently no evidence to suggest that antioxidant defenses play a role in the insensitivity of eukaryotes, such as S. cerevisiae, to the action of prokaryotespecific antibiotics.The tetracyclines (e.g., tetracycline, doxycycline, and oxytetracycline [OTC]) are classic broad-spectrum bacteriostatic antibiotics. They are commonly thought to act by inhibiting protein synthesis, through inhibition of binding by aminoacyltRNA to the ribosomal A site (22). Although binding of the tetracyclines to eukaryotic ribosomes occurs in vitro, the in vivo insensitivity of eukaryotes to these antibiotics is usually considered a reflection of the inaccessibility of tetracyclines to the eukaryotic intracellular environment (19); genes conferring OTC resistance to prokaryotes generally encode OTC export proteins (18, 32). The generality of fungal tetracycline resistance is exemplified by the use of OTC in fungiselective growth media (Difco manual, Difco, Detroit, Mich.) and the opportunistic yeast infections that commonly arise following tetracycline administration to humans (22).Inhib...

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“…UV-Visible spectrophotometry is still considered to be as a convenient and low cost method for the determination of pharmaceuticals in bulk and dosage forms. Several spectrophotometric and colorimetric methods have been reported for the determination of tetracyclines in bulk material and dosage forms based on their reaction with different reagents such as ammonium vanadate [21] , cupric chloride [22] , Diphenyl-1-Picrylhydrazyl (DPH) [23] and sodium molybdate [24] . The charge transfer reactions of certain -acceptors have been utilized successfully in pharmaceutical analysis.…”

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confidence: 99%

Analysis of Certain Tetracyclines and Oxytetracyclines through Charge Transfer Complexation

Fahelelbom¹

2008

American J. of Pharmacology and Toxicology

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Problem statement: Several studies were carried out to develop and validate a simple, rapid and accurate spectrophotometric method for the analysis of tetracycline (TC) and oxytetracycline (OTC) in the pure form and in pharmaceutical dosage forms, despite the varieties of these analytical techniques, the literature revealed that no spectrophotometric method for determination of tetracycline and oxytetracycline using charge transfer complexation with chloranilic acid has been reported. Approach: In the present study, a new and simple spectrophotometric method is described for the determinations of tetracycline (TC) and oxytetracycline (OTC), the method is based on the molecular interaction between the tetracyclines and Chloranilic Acid (CA), to form a charge-transfer complex in which the drugs acts as n-donor and chloranilic acid as -acceptor in acetonitrile. The spectra, various experimental parameters for the reaction, the stability of the complex and the validty of the method were investigated. Results: The formed complex was found to absorb maximally at 540nm, Beer's law is obeyed in the concentration ranges 2.5-30 µg mL −1 and 5.0-40 µg mL −1 for TC and OTC, respectively. The limits of detection (S/N = 3) were 0.40 µg mL −1 for tetracycline and 0.50 µg mL −1 for oxytetracycline. The molar ratio was found to be approximately 1:1, confirming that one molecule of tetracycline or oxytetracycline combines with one molecule of chloranilic acid. Conclusions and Recommendations: The proposed method was found to be rapid, accurate and sensitive and may be applied for estimation of named drugs in pharmaceutical dosage forms without interferences from the common additives encountered. Percentage recoveries ranged from 99.17%±0.95-100.38%±0.82. From the statistical analysis of the obtained results one can conclude that no significant difference between the proposed method and other official methods as evident from the t-test and variance ratio. These findings recommend the utility of the proposed method in the quality control analysis of TC and OTC in the pure form and pharmaceutical dosage forms.

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Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

Cited by 16 publications

References 9 publications

Iron Blocks the Accumulation and Activity of Tetracyclines in Bacteria

Iron Blocks the Accumulation and Activity of Tetracyclines in Bacteria

Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of Saccharomyces cerevisiae

Analysis of Certain Tetracyclines and Oxytetracyclines through Charge Transfer Complexation

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