Rifaximin, an oral antibiotic, is used for the treatment of hepatic encephalopathy, ulcerative colitis, irritable bowel syndrome, Clostridium difficile, travelers' diarrhea and acute diarrhea. The development of methods which evaluate the potency of antimicrobials is fundamental. So, this work presents the validation of a microbiological method by turbidimetry to evaluate the power of rifaximin tablets, as well as the evaluation of degraded drug in acidic, basic, neutral and photolytic conditions. For this, Escherichia coli ATCC 10536 were used. The method was linear over the concentration range of 50-98 μg mL-1 with correlation coefficients 0.9976 (standard) and 0.9999 (sample), precise (repeatability RSD=4.96 and interday RSD=3.92), exact (recovery 100.70%); robust against small and deliberate variations in method and indicative of stability because it allowed evaluating rifaximin degraded under stress conditions. The results were compared with data obtained by high-performance liquid chromatography. The validated turbidimetric method is very useful to the routine quality control for microbiological evaluation of rifaximin tablets.
Keywords: Rifaximin; Tablets; Antibiotic; Microbiological method; Turbidimetry; Indicative of stability; HPLC
Rifaximin is used for the treatment of various diseases such as hepatic encephalopathy, ulcerative colitis, irritable bowel syndrome, Clostridium difficile, travelers' diarrhea and acute diarrhea [1-9]. This makes rifaximin close to many people and therefore the proper quality control of this type of medicine is extremely important and required.
The question of the use of antimicrobials currently requires caution and, above all, information. The increasing human resistance to antimicrobials can make these drugs ineffective in the near future, leading the world into a period "post-antibiotic" in which, for example, a simple infection can be fatal .
The main cause of bacterial resistance is the abusive and indiscriminate use of antimicrobials, or by bad medical indication or self-medication, which creates an environment extremely favorable to the emergence of resistant bacteria . Another relevant factor, but that is often overlooked is the lack of microbiological quality control of antimicrobials simultaneously with the physico-chemical tests.
Rifaximin does not present microbiological method described neither in official compendia [11-16] nor in the literature and they are extremely important and necessary in the evaluation of power of antimicrobials [17-24]. The physical-chemical methods are unable to provide this type of information. Therefore, the union of these two types of methods is essential in the release of result of antimicrobial product.
The lack of microbiological method to evaluate the power of rifaximin tablets was a gap that propelled us. Another motivation was to make the microbiological method more practical, dynamic, low-cost optimizing equipment and analysts. For this, we chose the microbiological test by turbidimetry.
So, this work presents the validation of a microbiological method by turbidimetry to evaluate the power of rifaximin tablets, as well as the evaluation of degraded drug in acidic, basic, neutral and photolytic conditions. The results were compared with data obtained by highperformance liquid chromatography.
Apparatus Shaker MarconiTM model MA 420, oven OdontobrásTM model ECB2 at 35°C, autoclave PhoenixTM model AV 50 and automatic pipettors with capacity of 200 and 1000 μL were used. A QuimisTM spectrophotometer model Q-798 DRM was used to measure the turbidity of the tubes in absorbance.
LC analysis was performed on a WatersTM LC system equipped with Waters 1525 binary gradient chromatography pump, Rheodyne Breeze 7725i manual injector and Waters 2487 UV-Vis detector, Eclipse PlusTM C18 5 um column (150 mm × 4.6 mm, 5.0 μm particle sizes). Analytical balance model DV215CD ToledoTM, ultrasonic bath Ultrasonic Cleaner UniqueTM, water purification system MilliporeTM and membranes of polytetrafluoroethylene (PTFE) hydrophilic with pore 0.45 im and diameter 47.0 mm were used.
Chemicals and reagents
Rifaximin standard, content of 99.0%, acquired of the company NutraTech Development Limited (China). The pharmaceutical form used was rifaximin 200 mg tablets (labeled content), lot 12927, under the trade name Flonorm® of Laboratory Gonher Pharmaceuticals Ltd.
The turbidimetric method used broth Brain Heart Infusion (BHI) (AcumediaTM), distilled water and formaldehyde 12% (DinâmicaTM).
The chemical used were HPLC grade ethyl alcohol (J.T.BakerTM), HPLC grade glacial acetic acid (SynthTM), analytical reagent grade ethyl alcohol (SynthTM), and deionised Milli Q water (MilliporeTM). Mobile phase was prepared by mixing water + 0.1% glacial acetic acid and ethyl alcohol in the ratio 52:48 (v/v) filtered through 0.45 μm membrane filter. The diluent used was the mobile phase.
Microorganism and inoculums
The strain of Escherichia coli ATCC 10536 were cultivated and maintained on BHI agar medium in the freezer and pealed to BHI broth (24 h before the assay) that was kept at 35 ± 2°C. On the test day, the bacteria, previously incubated in BHI broth, was also diluted with BHI broth to achieve a suspension turbidity of 25 ± 2% (transmittance) using a spectrophotometer and wavelength at 580 nm [11,14].
The microbiological method describe followed the 3×3 assay design (3 doses of standard and 3 doses of sample).
The turbidimetric method consists of 8 tubes:
3 tubes for each standard concentration.
3 tubes for each sample concentration.
1 tube for the positive control.
1 tube for the negative control.
In 10 mL of BHI broth, contained in the tubes, were added 200 μL of each reference solution at concentrations of 50, 70 and 98 μg mL-1 and 200 μL of each sample solution at the same concentrations of 50, 70 and 98 μg mL-1. The negative control contained only 10 mL of BHI and the positive control contained 10 mL of BHI with standardized inoculum to 25 ± 2% of transmittance.
The tubes were incubated at 35 ± 2°C for 4 h. The turbidity of the culture media, after this time, was measured by spectrophotometry.
Chromatographic analysis was carried out at ambient temperature on an Eclipse PlusTM C18 5 um column. The mobile phase was a mixture of water + 0.1% glacial acetic acid and ethyl alcohol in the ratio 52:48 (v/v). Flow rate was 0.9 mL min-1. The detector wavelength was set at 290 nm with injection volume at 20 μL.
Stock and standard: 5 mg of rifaximin standard was weighed into a 25 mL volumetric flask, added 5 mL of ethyl alcohol and 20 mL of distilled water. It was sonicated for 10 min to complete dissolution of the drug. The mixture was diluted to the mark with distilled water. This stock solution (200 μg mL-1) was filtered through filter paper and aliquots were removed and diluted in distilled water immediately before analysis.
Stock and sample: From a pool of 20 tablets of rifaximin pulverized, 9.44 mg (equivalent to 5 mg of standard) was weighed. This amount was transferred to a 25 mL volumetric flask, added 5 mL of ethyl alcohol and 20 mL of distilled water. It was sonicated for 10 min to complete dissolution of the drug. Then the mixture was diluted to the mark with distilled water, obtaining a concentration of 200 μg mL-1. This stock solution was filtered through filter paper and aliquots were removed and diluted in distilled water.
Stock and placebo: The placebo was analyzed along with the standard and the sample to evaluate its influence on the turbidimetric analysis.
In preliminary tests were tested microorganism, culture medium, diluent, inoculum concentration and drug concentration, as can be seen in Table 1.
|Microorganism||Bacillus subtilis ATCC 9372 IAL 1027
Micrococcus luteus ATCC 9341 IAL 636
Staphylococcus epidermidis ATCC 12228 IAL 2150
Escherichia coli ATCC 10536 IAL 2393
|Culture medium||Broth BHI, Broth Casoy, Broth Müller-Hinton|
|Inoculum concentration||0.8; 1.0; 1.5; 1.85; 1.9; 2.0; 3.5;
4.0; 4.5; 6.5; 8.0; 8.5; 10.0%
|Diluent solution||Distilled water
Phosphate buffer pH 6.8
|Drug concentration||40, 60, 90 mg mL-1; 50, 70, 98 mg mL-1; 50, 100, 200 mg mL-1;
25, 50, 100 mg mL-1; 30, 60, 120 mg mL-1
Table 1: Parameters tested during the development of microbiological assay by turbidimetry for analysis of rifaximin tablets.
The analytical parameters of linearity, selectivity, precision, accuracy and robustness were evaluated for validation of the turbidimetric method, according to AOAC , Brazil , ICH  and INMETRO .
The linearity was evaluated by construction of three calibration curves developed on three different days and in triplicate. The concentrations used were 50, 70 and 98 μg mL-1.
The data obtained in the construction of the calibration curve were analyzed to obtain the equation of the line by the least squares method and the check of linearity and parallelism was confirmed by analysis of variance (ANOVA).
The selectivity of the method was confirmed by the forced degradation of rifaximin tablets in HCl 0.01 M subject to 1 h in bath at 80°C, NaOH 0.001 M and distilled water (neutral degradation) subject to 6 h in bath at 80°C and in UV light at 25°C after 1 week of exposure. Degraded samples were analyzed by HPLC method to compare the results.
For evaluation of the intraday precision were performed six stock solutions of rifaximin standard, at concentration of 70 μg mL-1, on the same day, same experimental conditions and same analyst. The evaluation of the results was done by calculating the Relative Standard Deviation (RSD).
For evaluation of interday precision were performed three stock solutions of rifaximin standard, at concentration of 70 μg mL-1, on three different day, but the same experimental conditions and same analyst. The evaluation of the results was done by calculating the Test F (Snedecor) and Test t (Student).
The accuracy of the method was determined by the test of standard addition.
Stock solutions of rifaximin standard and sample were prepared at a concentration of 200 μg mL-1. Of these solutions, aliquots of 1.25 mL of each one were taken and transferred to 5 mL volumetric flasks, obtaining a standard solution and another sample. They were completed with distilled water and obtained solutions with theoretical concentrations of 50 μg mL-1.
From the sample stock solution, aliquots of 1.25 mL were removed and transferred to 5 mL volumetric flask added 150 μL of standard stock solution, completed with purified water to obtain a solution containing theoretical concentration of 56 μg mL-1. Changing only the volume of standard stock solution, was similarly carried out for more two solutions through aliquots of 500 μL (theoretical concentration of 70 μg mL-1) and 850 μL (theoretical concentration of 84 μg mL-1).
The percent of recovered rifaximin was calculated by Equation I .
Cr=Concentration of sample solution added of standard (μg mL-1)
Ca=Concentration of sample solution (μg mL-1)
Cp=Theoretical concentration of standard solution added (μg mL-1)
In parameter robustness the following conditions were changed and tested:
Incubation time of tubes (4 h to 3:40).
Concentration of inoculum (850 μL to 830 μL).
Shaker rotation speed (32 rpm to 30 rpm).
Mark of BHI broth (AcumediaTM to HimediaTM).
Volume of culture medium in the tube (10 mL to 9.5 mL).
The evaluation of the results was done by calculating the Test F (Snedecor) and Test t (Student).
The parameters in Table 2 were established in preliminary tests because they showed better performance in the analysis.
|Microorganism||Escherichia coli ATCC 10536 IAL 2393|
|Culture medium||Broth BHI|
|Diluent solution||distilled water|
|Drug concentration||50; 70 and 98 mg mL-1|
Table 2: Established parameters for the determination of rifaximin tablets by turbidimetry method.
Figure 2 shows the analytical curve for rifaximin standard and sample by turbidimetry.
The equation of the line, determined by the method of least squares, is: y=-0.8143 ln(x) + 4.0717, with coefficient of correlation (r) equal to 0.9994 for rifaximin standard and y=-0.8034 ln(x) + 3.9727, with coefficient of correlation (r) equal to 0.9999 for rifaximin sample. The analysis of variance (ANOVA) of data from the curves with rifaximin is shown in Table 3.
|Linearity deviation||Variation sources||Degrees of freedom||Sum of quadratic||Mean quadratic||Fcal||Ftab (0.05)|
Table 3: Analysis of variance of the absorbances obtained from the analytical curve of rifaximin by turbidimetry. *Significant for p<5%.
The values of absorbances of the adjuvant solution and the diluent solution containing 20% ethyl alcohol were compared to the positive control, not interfering with the method.
The comparison of the results of turbidimetric and HPLC methods after forced degradation test of rifaximin tablets is shown in Table 4.
|Type of degradation||Method|
|HCl 0.01 M||82.25%||86.77%|
|NaOH 0.001 M||77.01%||70.00%|
Table 4: Comparison of results of turbidimetric and HPLC methods for rifaximin tablets after stress test.
Table 5 shows the values obtained in the recovery test for rifaximin using turbidimetric method.
|Standard rifaximin added (µg mL-1)||Standard rifaximin recovered (µg mL-1)||Recovery (%)||Average recovery (%)||RSD (%)|
Table 5: Values obtained in the recovery test of rifaximin using the turbidimetric method.
The intraday and interday precisions were made by repeatability and intermediate precision, respectively. The results are shown in Table 6.
|Intermediate precision||0.553||0.583||0.536||0.569||0.508||0.532||7.05 (n=12)|
Table 6: Values determined for the parameter precision of rifaximin by turbidimetry.
The results of the parameters evaluated in the robustness are in Table 7.
|Parameters||Normal condition||Modified condition||Test t||Robustness|
|Incubation time of tubes||4:00 hrs||3:40 hrs||0.56 (2.57)||Robust|
|Concentration of inoculum||850 µL||830 µL||2.38 (2.31)||No robust|
|Shaker rotation speed||32 rpm||30 rpm||12.91 (2.31)||No robust|
|Mark of BHI broth||AcumediaTM||HimediaTM||11.32 (2.31)||No robust|
|Volume of culture medium in the tube||10 mL||9.5 mL||0.30 (2.31)||Robust|
Table 7: Robustness analysis of the turbidimetric method for rifaximin in tablets.
Potency of the antimicrobial agent
The potency of rifaximin tablets was checked by the method validated in this work and the result is shown in Table 8.
|Day||Potency of rifaximin in tabletsa (%)||Average||RSD (%)|
Table 8: Values determined for the verification of potency of rifaximin in tablets by turbidimetric method. aEach value represents the mean of three determinations.
Microbiological methods have been replaced by physical-chemical tests, which are faster and more practical. However, the replacement of microbiological tests can create serious consequences on the safety and efficacy of antimicrobial [17,18,23].
In some cases, the content obtained by physical-chemical method does not correspond to the potency of the antimicrobial, generating false conclusions about quality of the product. This product, on the market, will not comply with its action and effectiveness and will maintain the individual's disease state, which suffocates the public health system, and additionally contribute to bacterial resistance.
The turbidimetric assay is a tool that combines microbiological test, required for the quality control of antimicrobial agents, and the optimization of time and analysts. It is a fast method, with the release of results in 4 h, and simple to run.
In the turbidimetric method validated for rifaximin in tablets were chosen concentrations 50-98 μg mL-1 and Escherichia coli as microorganism to 0.85%.
The results obtained in validation of the method demonstrated that rifaximin solutions showed linear correlation between the absorbances and concentrations in the range of 50 to 98 μg mL-1 and the data were validated by ANOVA.
The presence of adjuvants in the sample did not interfere in the analysis of rifaximin by turbidimetric method. This was proven by the absorbance values presented by adjuvants solution. They were compared to the positive control, that is, they have no antimicrobial activity.
The purpose of forced degradation test is to validate an indicative method of stability. The turbidimetric method was sensitive to acid, basic, neutral and photolytic degradation. The results of HPLC for acidic, basic and neutral degradation are approximately similar to the microbiological results for power of rifaximin in tablets. However, the results of photolytic degradation by HPLC (93.08%) and turbidimetry (43.18%) showed results significantly different. This is the importance of conducting a microbiological method simultaneously with a physical-chemical method. The portion of the molecule essential for activity cannot be the same detected in the physical-chemical method, generating false conclusions about quality of the product.
The precision of the method was proven on two levels, intraday and interday. Both showed RSD lower than 8%.
In the study of accuracy, determined by the recovery test, the result was 100.70%, which proved the ability of the method to determine levels predefined accurately.
In robustness test was observed that there was no difference statistically significant when small changes are made in the shaker time and in the volume of medium in the tube. However, variations in mark of the culture medium, shaker speed and the concentration of the inoculum, in this case, were not robust. Therefore, care in these parameters is very important.
The potency of rifaximin in the pharmaceutical product analyzed was 106.78%.
This paper proposes a microbiological method for evaluation of rifaximin in tablets that values the quality and safety of pharmaceuticals and optimizes time and analysts.
A microbiological method should always accompany the analysis of an antimicrobial agent in order to present to the population a quality and safe product.
The turbidimetric method presented in this work showed linearity, selectivity, precision, accuracy, robustness and can be easily applied in routine analysis of rifaximin in tablets.
The authors acknowledge CNPq (Brasília, Brazil), FAPESP (São Paulo, Brasil), CAPES (São Paulo, Brasil) and PADC/FCF/UNESP (Araraquara, Brazil).
The authors report no declarations of interest.