Development of a novel method based on liquid chromatography-evaporative light scattering detection for the direct determination of streptomycin and dihydrostreptomycin in raw materials, pharmaceutical formulations, culture media and plasma.

A novel method for the non-derivatization liquid chromatographic determination of streptomycin (STR) and dihydrostreptomycin (DHSTR) was developed and validated based on evaporative light scattering detection (ELSD). Utilizing a ThermoHypersil BetaBasic C18 analytical column, evaporation temperature of 50 degrees C and pressure of nebulizing gas (nitrogen) of 3.5 bar, the optimized mobile phase was 1.25 mL L(-1) TFA aqueous solution, in an isocratic mode at a rate of 1.0 mL min(-1). STR was eluted at 5.6 min and DHSTR at 7.8 min with a resolution of 4.4. Linear calibration curves were obtained from 2 to 120 microg mL(-1) (r > 0.9990) for STR and 2-75 microg mL(-1) (r > 0.9994) for DHSTR, with a LOD equal to 0.7 and 0.5 microg mL(-1), respectively. The developed method was applied for the assay of STR and DHSTR (sulfate) in pharmaceutical raw materials and formulations, while the simultaneous direct determination of sulfate was feasible (tR = 2.5 min, LOD = 1.4 microg mL(-1), double logarithmic calibration curve in the range of 4-50 microg mL(-1), r > 0.9998). Modified isocratic mobile phase (H2O-ACN, 90:10, v/v, containing 1.25 mL L(-1) TFA), was used for the determination of streptomycin B impurity in STR sulfate raw material and a gradient mobile phase (H2O-ACN containing TFA) was used for the determination of DHSTR in the presence of penicillinG procaine. The developed method was also applied for the assay of commercial formulations (STR powder and DHSTR injection solution and suspension) (%recovery 98-102, %RSD < 1.3, n = 3 x 3), for the determination of STR in bacteria culture medium (%recovery 99.6, %RSD = 0.8, n = 3 x 3), and for the determination of DHSTR in human plasma (2.0-23.0 microg mL(-1)) after solid phase extraction using carboxylate cartridges (%recovery 98.4-101.8, %RSD = 3.2, n = 3 x 3).

Whole-body autoradiography of [3H]dihydrostreptomycin in guinea pigs and rats: the labelling of the inner ear in relation to other tissues.

[3H]Dihydrostreptomycin was given intramuscularly to young pigmented guinea pigs and rats. Whole-body autoradiography, combined with densitometric measurement of the blackening of the autoradiograms, and liquid scintillation counting were used to determine the levels of radioactivity in the inner ear in relation to blood and other tissues. It was found that there was an accumulation of radioactivity in the perilymph of both the cochlear and vestibular parts of the labyrinth. The labelling of the endolymph was weak. The levels of radioactivity in the membranous linings of the labyrinth including the areas covered with the neuroepithelial structures, were about the same as in the perilymph. The general distribution pictures were characterized by a localization of radioactivity in extracellular tissues, such as cartilages and connective tissues and by strong labelling of the kidney cortex. The preferential uptake in the perilymph indicates a route by which the inner ear hair cells can be exposed to high levels of aminoglycoside antibiotics.

Determination of streptomycin in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for monitoring the serum concentration of streptomycin. The method includes clean-up using a Sep-Pak C18 cartridge and quantitation using dihydrostreptomycin as an internal standard. Streptomycin and dihydrostreptomycin were separated by reversed-phase ion-pair chromatography on LiChrosorb RP-18 and detected by UV absorption (195 nm). The calibration graph of serum streptomycin concentration was linear over the range 5-50 micrograms/ml. Streptomycin was added to serum at the level of 20.0 micrograms/ml and its concentration was determined to be 18.9 micrograms/ml with a coefficient of variation of 2.07% (n = 5). The clinical application of this method was confirmed by comparison with fluorescence polarization immunoassay.

Bioavailability in the rabbit of penicillin and dihydrostreptomycin from three commercial penicillin/aminoglycoside fixed combination products for intramuscular injection.

The bioavailability of penicillin and dihydrostreptomycin from three penicillin/ aminoglycoside fixed combination products for intramuscular injection was investigated in a four-way, randomized, crossover experiment in rabbits. Attention is focused on bioequivalence based on plasma concentration vs. time profiles to study whether the rabbit is a good model to detect differences in in vivo delivery of penicillin and/or dihydrostreptomycin after intramuscular administration of different products. In all products, penicillin was present as a suspension. Although the extent of absorption of penicillin did not differ between the three products, large differences in the rates of absorption were observed. With respect to dihydrostreptomycin, no significant differences were observed between the products. The results from this study demonstrate that the rabbit is a good model to detect differences in bioavailability of suspended penicillin from penicillin /dihydrostreptomycin fixed combination products for intramuscular injection. A study with the same products is presently being carried out in calves to investigate whether bioequivalence studies in rabbits could replace studies in the target animals.

Bioequivalence study in calves of three commercial penicillin/dihydrostreptomycin fixed combination products for intramuscular injection.

A bioequivalence study with three penicillin/dihydrostreptomycin fixed combination products for intramuscular administration was performed in dairy calves. In addition to plasma concentrations of penicillin and dihydrostreptomycin, creatine phosphokinase concentrations were determined during a period of 72 h after administration of the drug products. Considerable differences were observed in the pharmacokinetics of penicillin from the three products. Although the extent of absorption was similar for all products, one product showed a significantly slower release from the site of injection. Except for the AUC, the 90% confidence intervals for these parameters exceeded the acceptable range of 0.80-1.20. Therefore, these products are not bioequivalent with respect to the rate of absorption of penicillin. Concerning the pharmacokinetics of dihydrostreptomycin in calves, it could not be concluded that the products were bioequivalent, since the 90% confidence intervals of the ratios for Cmax, tmax and MRT exceeded the range of 0.80-1.20. From this study in calves, it was also found that the product with the slowest release of penicillin from the injection site caused the most severe tissue damage, based on plasma creatine phosphokinase concentrations. Comparing the results from this study in calves with those from a previous study in rabbits, it can be concluded that the rabbit is a good animal model that could substitute for large animals, at least calves, in bioequivalence studies for penicillin/dihydrostreptomycin fixed combination products.

Distribution of penicillin G, dihydrostreptomycin, oxytetracycline, and chloramphenicol in serum and subcutaneous chamber fluid.

Penetration of penicillin G, dihydrostreptomycin, oxytetracycline, and chloramphenicol into interstitial fluid of calves was estimated using subcutaneously implanted, multiple perforated spherical polypropylene capsules as a model. Antibiotic concentrations were determined in simultaneously withdrawn serum and capsular fluid (CF) samples at intervals after single and multiple intramuscular injections of antibiotics at recommended dose schedules. Peak concentrations of penicillin G in CF were 57% of those in serum, and the drug was eliminated from CF at a slower rate than from serum. Dihydrostreptomycin diffused into CF to a limited degree and was eliminated from CF much more slowly than from serum leading to gradual drug accumulation in CF upon repeated dosing. Multiple injections of oxytetracycline resulted in CF drug levels comparable with those in serum. Concentrations of chloramphenicol in CF were generally similar to free (non-protein bound) serum drug levels. CF concentrations of penicillin G were within the range of the minimal inhibitory concentrations of the drug for pathogenic gram positive micro-organisms and CF levels of dihydrostreptomycin, oxytetracycline, and chloramphenicol were apparently sufficient to inhibit the majority of gram negative pathogens involved in bovine injections. Advantages and limitations of the tissue cage model are briefly discussed.