The fluoroquinolones are a class of compounds that comprise a large and expanding group of synthetic antimicrobial agents. Orbifloxacin (Figure 1) (1-cyclopropyl-5,6,8-trifluoro-1,4-dihydro-7-(cis-3,5-dimethyl-1-piperazinyl)-4-oxoquinoline-3-carboxylic acid) is a new third-generation fluoroquinolone antibacterial drug, developed exclusively for the treatment of gastrointestinal and respiratory infections in animals [1-3], but it is also used for treatment of skin, soft tissue, and urinary tract infections (in dogs and cats, especially) [4,5]. In addition, research is still being carried out on the antimicrobial effects of this analyte. Recent studies have focused on the effectiveness of orbifloxacin in the treatment of staphylococcal pyoderma both superficial and deep [6], the antimicrobial activity of orbifloxacin against Pseudomonas aeruginosa isolated from canine otitis [7] and activity against Staphylococcus intermedius isolated from canine skin and ear infections [8]. Research is also being conducted on the possibility of widening the scope of orbifloxacin applications and using it for treatment of other animals, such as horses or rabbits [4,5,9].

Fluoroquinolones have become an important group, particularly for the treatment of infections caused by antibiotic-resistant bacteria [9].

Antimicrobial resistance of bacteria is a phenomenon that has been in constant evolution since the introduction of antibiotics. Several factors are known to promote bacterial resistance, including poor compliance with treatment regimen [10,11], prophylactic use of antibiotics [12] and the use of antibiotics as growth promoters [13,14], thus emphasizing the importance of dosage and appropriate treatment.

For an appropriate dosage of the pharmaceutical form orbifloxacin analytical methods should be developed and validated. To date, all methods described in the scientific literature for determination of this analyte in biological and others materials involve liquid chromatography, with UV detection [2,4,15-19], fluorimetry detection [5,19-27] or mass spectrometry [21-24,28] and a spectroscopic technique coupled to a flow system [29] as detection techniques.

There have been few reported methods for orbifloxacin, nor is there a method described in official compendia and no method for determination of orbifloxacin in dosage forms. Hence, an attempt has been made to develop a simple, sensitive, accurate and reproducible method for the determination of orbifloxacin in pharmaceutical dosage forms along with method validation.

The development and validation of analytical methods for the potency determination has received considerable attention in recent years, mainly from regulatory agencies, because of their importance in pharmaceutical analysis [36-39].

In this case, a microbiological assay was proposed as a suitable method for the determination of orbifloxacin in tablet forms.

The experimental conditions were tested and adjusted to accurately determine the performance of the assay. The strain of Staphylococcus aureus ATCC 25923 was found to be an appropriate test microorganism because of its sensitivity to orbifloxacin and its capacity to form sharply defined inhibition growth zones, allowing measurements with precision.

The potency of an antibiotic may be demonstrated under suitable conditions by comparing the growth inhibition of sensitive microorganisms induced by known concentrations of the antibiotic to be examined and a reference standard [30,31].

In this experimental work of 3 × 3 design (Figure 2), 3 dose levels for each standard and sample, were respectively used following the procedures described in the Brazilian Pharmacopoeia [30]. The calculation procedures normally assume a direct relationship between the observed zone diameter and the logarithm of the applied dose. The results of growth inhibition zone diameter of orbifloxacin reference substance are shown in Table 1.

The calibration curve for orbifloxacin was constructed by plotting zone diameter (mm) versus log of concentrations (μg/mL) and showed good linearity in the 16–64 μg/mL range.

The representative linear equation for orbifloxacin was y = 8.5263 Ln(x) + 9.2756 (n = 3, r = 0.9996), where x is log dose and y is zone diameter. The experimental values obtained for the determination of orbifloxacin in samples are presented in Table 1. According to the Brazilian [30] and The United States [31] Pharmacopeias, if a parallel-line model is chosen, the 2 log dose-response lines of the preparation to be examined and the reference preparation must be parallel, and they must be linear over the range of doses used in the calculation. These conditions must be verified by validity tests for a given probability, usually P = 0.05. The assays were validated by means of the ANOVA, as described in those official codes. There were no deviations from parallelism and linearity with the obtained results (P < 0.05).

The method precision in terms of repeatability (intra-assay) was evaluated by analyzing, on the same day, seven samples of orbifloxacin reference substance with same theoretical concentration. The intermediate precision was determined by analyzing the same sample on 3 days (between-day) with obtained RSD values of 2.88 and 3.33%, respectively. The orbifloxacin activity ranged from 99.18 to 101.84%, with a RSD value of 1.42%. The lower RSD values achieved confirm that the proposed method has capacity to generate, for the same sample, reproducible results with low response variation between independent assays.

The accuracy of the method was evaluated at 80, 100 and 120% of the nominal analytical concentration in the specified range of 16.0–64.0 μg/mL. The mean accuracy was 100.31% and RSD was 1.04% (Table 2), which confirms the ability of the method to determine with accuracy the orbifloxacin concentration within the range of 80–120% and, in the same way, shows that the results obtained from the bioassay were close to the true concentration values of the samples.

The quantification of antibiotic components by chemical methods, such as HPLC and UV spectrophotometry, although precise, cannot provide a true indication of biological activity. Attempts to correlate antibiotic bioassay results with those from chemical methods have proved disappointing. Therefore, bioassays continue to play an essential role in manufacturing and quality control of antibiotic medicines, and still demand considerable skill and expertise to assure success [40]. Although the biological assays can have a high variability, the analysis of the obtained results demonstrated that the proposed method might be very useful for determination of this drug in pharmaceutical dosage forms.

The results obtained in this study were very satisfactory, and the performed validation proved that microbiological assay is a good alternative methodology for pharmaceutical analysis of orbifloxacin in tablets. It is a useful analytical tool as a supplement or substitution for the physicochemical method.

The results indicated that the microbiological cylinder plate assay demonstrated good linearity, precision and accuracy at concentration ranging from 16.0 to 64.0 μg/mL, therefore, being an acceptable alternative method for the routine quality control of orbifloxacin in pharmaceutical forms. The method uses simple reagents, with minimum sample preparation procedures, and no toxic residues, encouraging its application in routine analysis.