A specific stability indicating high-performance thin-layer chromatographic method for analysis of rifaximin both as a bulk drug and in formulations was developed and validated. The method employed HPTLC aluminium plates precoated with silica gel 60 F254 as the stationary phase. The optimized mobile phase system consisted of n-hexane : 2-propanol : acetone : ammonia (5 : 4.1 : 1, v/v/v/v), which gave compact spots for rifaximin at
Rifaximin, a benzimidazole derivative, is a structural analogue of rifampicin. Chemically, it is a 2S,16Z,18E,20S,21S,22R,23R,24R,25S,26S,27S,28E-5,6,21,23,25-pentahydroxy-27-methoxy-2,4,11,16,20,22,24,26-octamethyl-2,7-epoxypentadeca-[1,11,13] trienimino) benzofuro [4,5-e] pyrido [1,2 benzimidazole 1,15(2H)-dione,25-acetate (Figure
Chemical structure of rifaximin.
Rifaximin is a newer antibiotic, used for the treatment of patients having more than 12 years of age with traveller’s diarrhoea caused by noninvasive strains of
Literature reports various analytical methods like spectrophotometric [
An ideal stability indicating analytical method is one that quantifies the drug and also resolves its degradation products. The ICH guideline explicitly states the requirement of stability indicating methodology by conducting forced degradation studies under a variety of conditions, like pH, light, oxidation, dry heat, and so forth, and for separating drug from its degradation products, [
Although stability indicating methods have been reported for assay of various drugs in drug products, chromatographic methods have taken precedence over the conventional methods of analysis, showing greater accuracy and sensitivity for small amounts of degradation products generated [
The aim of the present work hence was to develop an economic, accurate, specific, and reproducible and stability-indicating HPTLC method for the determination of rifaximin in the presence of its degradation products and related impurities from pharmaceutical formulation. The proposed method was validated as per ICH guideline. Furthermore, the developed HPTLC method was also used to investigate the kinetics of the acidic degradation process by quantitation of drug at different temperatures and to calculate the activation energy and half-life for rifaximin degradation.
Rifaximin was received as a gift sample from Lupin Pharma Ltd., Mumbai, India. All solvents and chemicals used were of analytical grade, purchased from Merck Specialities Pvt. Ltd., India. Marketed tablet formulations used in the study were RCIFAX 400 and 200 mg (Lupin Pharma Ltd.), RIFAGUT (Sunpharma) TORFIX (Torrent Pharma), and RIXMIN 200 mg (Cipla) procured from local market.
Hamilton microlitre syringe (Linomat syringe 659.0014, Hamilton-Bonaduz Schweiz, Camag, Switzerland), precoated silica gel aluminium plate 60 F254, (
Suitable volume of standard and sample solution was spotted in the form of bands having band width of 6 mm on precoated silica gel 60 F254 HPTLC plate, 8 mm from the bottom and 15 mm from the side edges in the form of bands. Linear ascending development was carried out and the optimized mobile phase consisted of n-hexane : 2-propanol : acetone : ammonia (5 : 4 : 1 : 1 v/v/v/v). The optimized chamber saturation time before chromatographic development was 20 min and the length of chromatographic run was 8 cm. Subsequent to the development, HPTLC plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed and all measurements were made in the reflectance absorbance mode at 443 nm, with slit dimension (
A standard stock solution was prepared by dissolving accurately weighed 10 mg of rifaximin in methanol in 10 mL volumetric flask, to obtain a concentration of 1000
To determine the amount of rifaximin in various tablet dosage forms (label claim: 400 and 200 mg per tablet), the contents of 20 tablets were weighed, their mean weight was determined, and they are finely powdered. An accurately weighed powder sample equivalent to 10 mg of rifaximin was transferred into a 100 mL volumetric flask containing 50 mL methanol, followed by sonication for 30 min and further dilution up to the mark with methanol. The resulting solution was filtered through Whatman filter paper no. 42. Eight microlitres of the filtered solution (800 ng per band) was applied on the HPTLC plate followed by development and scanning as per optimized chromatographic conditions.
The method was validated in accordance with ICH guideline [
Precision of the developed method was studied by performing repeatability and intermediate precision studies. Repeatability was carried out by performing three replicates of three different concentrations (800 ng, 1600 ng, and 3200 ng) and peak area measured was expressed in terms of percent relative standard deviation. The intermediate precision study was performed on different days. The intermediate precision was assessed by studying three different concentrations (800 ng, 1600 ng, and 3200 ng) for three different days.
Accuracy of method was ascertained by performing recovery at three concentration level of 50%, 100%, and 150%, by spiking rifaximin standard (500 ng, 1000 ng, and 1500 ng) to the dosage form (1000 ng/band). Recovery studies were performed in triplicate.
As per ICH guideline, limit of detection and quantitation of the developed method were calculated from the standard deviation of the
The effect of deliberate variations on method parameters like the composition of the mobile phase saturation time, development distance, spot scanning time interval, wavelength scan, time from spotting to chromatography, and mobile phase volume was evaluated. The effect of these changes on both the
The specificity of the method was ascertained by comparing both the chromatogram and spectra of samples of marketed formulation and degradation sample with standard drug. The spot for rifaximin in both samples was confirmed by comparing the
To evaluate the stability indicating property of the developed HPTLC method, standard drug was subjected to acid/base hydrolysis, oxidation, wet heat, and photodegradation. 10 mg of accurately weighed rifaximin was transferred to 10 mL volumetric flask and diluted with methanol to obtain a final concentration of 1000
To 5 mL of the above standard drug solution, 5 mL of 0.1 M hydrochloric acid was added and refluxed at 80°C for 30 min. Neutralized solution was directly applied to HPTLC plate followed by development and scanning under optimized chromatographic conditions.
To 5 mL of above methanolic drug solution, 5 mL of 0.1 M sodium hydroxide was added and refluxed at 80°C for 30 min. Neutralized solution was directly applied to HPTLC plate followed by development and scanning under optimized chromatographic conditions.
To 5 mL of methanolic drug solution, 5 mL of 3% hydrogen peroxide solution was added and refluxed at 80°C for 30 min. The resulting solution was directly applied to HPTLC plate and the chromatogram was recorded under optimized chromatographic condition.
For wet heat degradation study, the methanolic standard solution was refluxed at 80°C for 30 min. The resulting solution was directly applied to HPTLC plate followed by development and scanning under optimized chromatographic conditions.
The rifaximin standard, 10 mg was exposed to UV light in a UV chamber and sunlight for 24 hours and appropriate dilutions were made in methanol to obtain final concentration of 1000
To study the acid induced degradation kinetics of rifaximin, 5 mL of the above methanolic drug solution (1 mg/mL) was refluxed at 313, 323, 333, and 343 K, after addition of 5 mL of 0.1 M hydrochloric acid. Three microlitres (750 ng) of neutralized solution was applied on HPTLC plate at various time intervals up to 90 min and chromatograms were recorded under optimized chromatographic conditions. Each experiment was repeated three times at each temperature and time interval and the concentration of the remaining drug was calculated. Further degradation kinetics was evaluated by plotting
Statistical parameters like standard deviation (SD) and percent relative standard deviation (% RSD) were computed by using MS Excel (Microsoft Corporation, USA). Bartlett’s test was applied on calibration data for evaluation of homoscedasticity of variance.
Both the pure drug and the degraded products were spotted on the HPTLC plates and run in different solvent systems and ratios, n-hexane, toluene, 2-propanol, methanol, and ethyl acetate. From these, combination of n-hexane : 2-propanol gave good results and hence further trials were initiated for different ratios of n-hexane : 2-propanol. The mobile phase n-hexane : 2-propanol (5 : 4, v/v) gave good resolution with
Chromatogram of rifaximin standard (
Rifaximin showed good correlation over a concentration range of 400–3200 ng/band with respect to peak area (Figure
Linear regression parameters for rifaximin by HPTLC method.
Parameters | Rifaximin |
---|---|
Linearity rangea (ng/band) | 400–3200 |
Correlation coefficient ( |
|
Slope ± SD ( |
|
Confidence limit of slopec | 4.76–4.91 |
Intercept ± SD ( |
|
Confidence limit of interceptc | 197.40–420.18 |
Limit of detection (ng/band) | 61.25 |
Limit of quantitation (ng/band) | 185.59 |
Bartlett’s testb ( |
0.267 |
3D densitogram for calibration curve linearity of rifaximin standard.
The tablets formulations, RCIFAX 400 mg, RCIFAX 200 mg, RIFAGUT 200 mg, RIXMIN 200 mg, and TORFIX 200 mg when analyzed in triplicate using the developed method, showed only one peak at
Applicability of the proposed HPTLC method for determination of rifaximin in pharmaceutical formulation.
Drug | Brand name | Amount of rifaximin present (mg per tablet) | % amount of rifaximin founda | SD | % RSD |
---|---|---|---|---|---|
Rifaximin | RCIFAX | 400 | 98.26 | 1.58 | 1.61 |
RCIFAX | 200 | 96.69 | 1.20 | 1.24 | |
RIFAGUT | 200 | 95.59 | 1.68 | 1.76 | |
TORFIX | 200 | 98.02 | 1.66 | 1.69 | |
RIXMIN | 200 | 97.22 | 1.01 | 1.04 |
3D densitogram of pharmaceutical formulations of rifaximin
Repeatability and intermediate precision expressed in terms of % RSD reveal that the proposed method provides an acceptable intraday and interday variation as shown in Table
Intra- and interday precision of HPTLC method.
Amount (ng/band) | Intraday precisiona | Interday precisiona | ||
---|---|---|---|---|
SD | % RSD | SD | % RSD | |
800 | 1.06 | 1.03 | 1.37 | 1.31 |
1600 | 0.17 | 0.16 | 0.42 | 0.41 |
3200 | 1.59 | 1.63 | 1.62 | 1.64 |
The proposed method when used for evaluation of recovery at three concentrations levels, 50%, 100%, and 150% after spiking with standard, showed percentage recovery between 100.35% and 103.12%, with acceptable % RSD, less than 2 (Table
Recovery study for determination of rifaximin.
Recovery level (%) | Initial amount (ng/band) | Amount added (ng/band) | % recoverya | SD | % RSD |
---|---|---|---|---|---|
50 | 1000 | 500 | 103.12 | 0.12 | 0.12 |
100 | 1000 | 1000 | 100.35 | 0.28 | 0.28 |
150 | 1000 | 1500 | 101.10 | 1.39 | 1.38 |
Limit of detection and limit of quantitation were found to be 61.24 ng/band and 185.59 ng/band, indicating good sensitivity of the method.
The
Robustness study of developed HPTLC method.
Parameters | SDa | (%) RSD |
---|---|---|
Mobile phase composition (4.9 : 3.9 : 1 : 1, 5.1 : 4.1 : 0.9 : 0.9) | 1.31 | 1.36 |
Saturation time ( |
0.50 | 0.50 |
Development distance ( |
1.18 | 1.16 |
Scanning time interval (1 h, 2 h, 3 h, 4 h, 5 h, and 6 h) | 0.15 | 0.15 |
Wavelength change ( |
0.72 | 0.73 |
Time from spotting to chromatography (0, 20, 40, and 60 min) | 0.52 | 0.52 |
Mobile phase volume ( |
1.58 | 1.58 |
The chromatogram of the pharmaceutical formulation and degradation samples using the developed method showed only one peak at
Overlain spectra of five pharmaceutical tablet formulations with rifaximin standard showing peak purity.
Overlain spectra of acid degradation sample with rifaximin standard showing peak purity.
The densitogram of the forced degradation sample (acid, base, hydrogen peroxide, sunlight, UV light, and wet heat) showed well-separated peak of rifaximin standard from degradation peak at different
Degradation study of rifaximin in various conditions.
Exposure conditions | Degradation products ( |
Recovery (%)a |
---|---|---|
None (control sample) | Not detected | 100 |
0.1 M HCl, refluxed (30 min) | 0.44, 0.49 | 55.39 |
0.1 M NaOH, refluxed (30 min) | 0.46, 0.51 | 81.94 |
3% Hydrogen peroxide, refluxed (30 min) | 0.47 | 94.95 |
Wet heat, refluxed (30 min) | — | 100 |
Sunlight (24 h) | 0.67 | 89.72 |
UV light, 254 nm (24 h) | 0.49 | 98.34 |
Recovery of rifaximin at the level of 55.39% and 81.34% from the acid and base stressed samples, respectively, suggests significant degradation of rifaximin in acid degradation compared to alkaline degradation (Table
Chromatogram of rifaximin standard drug subjected to acid degradation (rifaximin = 0.63,
Chromatogram of rifaximin standard subjected to base degradation (rifaximin = 0.63,
The chromatogram of rifaximin standard when treated with 3% v/v hydrogen peroxide showed additional peak at
Chromatogram of rifaximin standard subjected to 3% hydrogen peroxide induced degradation (rifaximin = 0.56,
Rifaximin solution when heated at 80°C for 30 min showed no additional peak of degradation product suggesting stability of rifaximin in methanolic solution (Figure
Chromatogram of rifaximin standard subjected to wet degradation (rifaximin = 0.60).
Both UV and sunlight degraded samples of rifaximin showed additional peak at
Chromatogram of rifaximin standard subjected to UV degradation (rifaximin = 0.57,
Chromatogram of rifaximin subjected to sunlight degradation (rifaximin = 0.57,
At the selected temperatures of 313, 323, 333, and 343 K, the degradation process followed first-order kinetics, revealing a decrease in concentration of drug with increasing time (Figure
First-order plot for degradation of rifaximin at various temperatures:
First-order rate constant
Degradation rate constant (
Temp. (K) |
|
|
|
---|---|---|---|
313 | 0.00208 | 5.55 | 0.84 |
323 | 0.00524 | 2.20 | 0.33 |
333 | 0.01 | 1.05 | 0.15 |
343 | 0.03 | 0.41 | 0.06 |
Arrhenius plot for the degradation of rifaximin in presence of 0.1 M HCl and their extrapolation for predicting degradation at room temperature (25 ± °C).
Degradation rate constant of acidic degradation process at room temperature (25 ± °C) was obtained by extrapolation from Arrhenius plot. The first-order
Degradation kinetic profile for rifaximin in acidic media at
Parameters | 0.1 M HCl |
---|---|
Ea (kcal mol−1) | 18.45 |
|
7.4 × 10−6 |
|
25.89 |
|
3.92 |
A stability indicating HPTLC method was developed and validated for the determination of rifaximin. The developed method was found to be simple, specific, sensitive, and suitable for the determination of rifaximin. The proposed method was applied for the analysis of rifaximin in tablet formulation, where the results were statistically compared with the reported HPLC method [
Statistical comparison of the results obtained by the proposed HPTLC method in comparison to the reported HPLC method.
Parameter | HPTLC | HPLC* |
---|---|---|
Mean % found for analysis of |
96.69 | 99.59 |
TORFIX tablet formulationa |
98.02 |
98.48 |
Formulation analysisa | 1.15 | |
Intraday precisiona | 3.87 | |
Interday precisiona |
3.61 |
|
Formulation analysisa | 2.38 | |
Intraday precisiona | 0.32 | |
Interday precisiona | 0.22 |
The authors are highly grateful to Lupin Pharma Ltd., Mumbai, India, for providing gratis sample of rifaximin.