Development and Validation of HPTLC Method for the Estimation of Rifaximin
Bulk and in Tablet Dosage Forms
T.Sudha, * J.Saminathan1
*Department of Pharmaceutical Analysis,
The Erode college of Pharmacy, Erode (Dt)
1 Department of Pharmaceutical Analysis,
Adhiparasakthi College of Pharmacy
Melmaruvathur, Kanchipuram (Dt),
TamilNadu,India.
For Correspondence,
*T.Sudha M.Pharm,
Assistant Professor,
Department of Pharmaceutical Analysis,
The Erode College of Pharmacy,
Erode-638112.
E-mail- jvchrsty@yahoo.co.in
Mobile: 9362857380
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ABSTRACT
Rifaximin is a semi synthetic, rifamycin-based non-systemic antibiotic used in the
treatment of traveler's diarrhea and hepatic encephalopathy.The present work describes a
simple, precise and accurate HPTLC method for rifaximin, its estimation as bulk and in
tablet dosage form. Standard and sample solutions of rifaximin was applied to precoated
silica gel G 60 F254 HPTLC plates and the plates were developed with methanol: Acetic
acid: chloroform, 8:1:1 (v/v), as mobile phase. UV detection was performed
densitometrically at 254 nm using Camag TLC Scanner-3 with win CAT 1.3.4 version
software. The retention factors of rifaximin were 0.83±0.2 respectively. The linear range
was 100– 600 ng per spot for rifaximin. The correlation coefficient r2 was found to be
0.9994. The experimental parameters like band size of the spot applied, chamber
saturation time, solvent front migration, slit width were critically studied and optimum
conditions were evolved. The accuracy and reliability of the proposed method was
ascertained by evaluating various validation parameters like linearity (100-600 ng/spot),
precision (intra-day RSD 0.46-0.75%, inter-day RSD 0.46-1.59%), accuracy
(98.72±0.20) and specificity according to ICH guidelines. The proposed method provides
a faster and cost-effective quality control tool for routine analysis of rifaximin as bulk
drug and in tablet formulation.
Key words: Rifaximin, HPTLC, densitometric estimation, method development
and validation
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INTRODUCTION
Rifaximin (Fig. 1) is 2S, 16Z, 18E, 20S, 21S,22R, 23R,24R, 25S, 26S,27S, 28E5,6,21,23,25-pentahydroxy- 27-methoxy-2,4,11, 16, 20,22, 24, 26,-octamethyl-2,.7(epoxy penta deca- [1,11,13] trienimino) benzofuro[4,5-e] pyrido[1,2-a]- benzimidazole1,15(2H)- dione,25 acetate. Rifaximin (Xifaxan tablet information 2008) is a semi
synthetic, rifamycin-based non-systemic antibiotic, meaning that very little of the drug
will pass the gastrointestinal wall into the circulation as is common for other types of
orally administered antibiotics. It is used in the treatment of traveler's diarrhoea and
hepatic encephalopathy. It is one of the non-absorbable antibiotics for the treatment of
Hepatic encephalopathy (HE) because it is not absorbed from the gastroenteric mucosa
and persists longer at higher levels in the lumen of the gastroenteric tract, minimizing the
risk of systemic side effects (DuPont H 2007). Literature survey revealed that very few
methods for determination of rifaximin individually have appeared in the literature.
Several methods including quantification of rifaximin in human plasma by LC-MS,
Rapid determination of rifaximin in rat serum and urine by HPLC, estimation of
rifaximin in bulk and in tablet dosage form by HPLC, UV-Visible (Xianhua Zhang et al,
2007, Sudha T. et al 2009, Sudha T. et al 2010) HPLC coupled with electrochemical
detection. (Nageswara Rao R. et al 2009) and application of derivative resolution of UV
spectra to the quality control of Rifaximin and the possible impurities (Corti P. et al
1992) have been reported for the determination of rifaximin. But these methods are
sophisticated, expensive and time consuming when compared to HPTLC method. RFX is
not official in any Pharmacopoeia. A literature survey has revealed that there is no
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HPTLC method for analysis of rifaximin in pharmaceutical preparations. The purpose of
this research was to establish such a method and, after validation (ICH GUIDELINES) in
accordance with International Conference on Harmonization (ICH) guidelines and the
directives for good laboratory practice, to use the method for analysis of the drug content
of tablets. A silica gel 60 F254 TLC plate (20x20 cm, layer thickness 0.2 mm, E Merck,
Germany) is used as stationary phase. The objective of the present investigation was to
develop an HPTLC method to accurately and rapidly determine the rifaximin.
2. MATERIALS AND METHODS
Chemicals and Reagents
Rifaximin, gift sample was provided by ZhiZang Sixian Pharmaceuticals, China,
Methanol (Fisher Chemical, USA), chloroform (Dikma Technologies Inc., USA) and
acetic acid (Dikma Technologies Inc., USA) of HPLC grade were used. The mobile
phase was filtrated with a 0.45µm filter before use. The water used was distilled and
deionized.
Instrumentation
A Camag HPTLC system (Switzerland) comprising of Camag Linomat IV semiautomatic
sample applicator, Camag TLC Scanner 3, Camag twin-trough chamber (10×10 cm),
Camag CATS 4 software, Hamilton syringe (100 μl), Shimadzu libror AEG- 220
weighing balance, Sonicator (shimadzu) were used during the study.
Preparation of standard solution of rifaximin
About 10 mg of working standard of RFX was accurately weighed and transferred in to
100 ml volumetric flask. About 25 ml of methanol was added and sonicated for about 20
min. Finally the volume was made up to 100ml with methanol to obtain the concentration
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of about 100μg/ml. 0.1 ml was taken from this stock solution and the volume made up to
100 ml to get a concentration of about 100 ng/μl.
Preparation of sample solution
Ten tablets were weighed and finely powdered. The powder which is equivalent to 10 mg
of the rifaximin was weighed, mixed with 25 ml of methanol and sonicated for 15 min.
The solution of tablet was filtered through Whatman filter paper No. 41 and the residue
was thoroughly washed with methanol. The filtrate and washings were combined in a 100
ml volumetric flask and diluted to the mark with the methanol to get the final
concentration of 100 μg/μl of RFX. 0.1 ml was taken from this stock solution and the
volume made up to 100ml to get a concentration of about 100ng/μl. Three micro liters of
sample solution was applied on a TLC plate under a nitrogen stream using a semi
automatic spotter. The amount of RFX present in the sample solution was determined by
fitting the area values of peaks corresponding to RFX into the equation of the line
representing the calibration curve of RFX. All determinations were performed in
triplicate.
Preparation of calibration curve
Aliquots (1, 2, 3, 4, 5 and 6 μl) of standard solution of RFX were spotted on pre coated
TLC plates using semi automatic spotter under nitrogen stream. The plate was dried in air
and developed up to 72 mm at constant temperature with a mixture of methanol: Acetic
acid: chloroform, 8:1:1 (v/v/v) as mobile phase in a CAMAG twin trough chamber which
was previously saturated with mobile phase for about 30 min. The plate was removed
from the chamber and dried in air. Photometric measurements were performed at 254 nm
in absorbance/reflectance mode with the CAMAG TLC scanner 3 using CATS 4 software
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incorporating track optimizing option. The standard plot of RFX was established by
plotting the peak area Vs concentration (ng/ml) corresponding to each spot.
System Precision
The precision study was conducted for RFX standard stock solutions. 3µl concentration
solutions was prepared and assessed by spotting six times on a TLC plate, followed by
development of plate and recording the peak area of 6 spots. The %RSD for peak area
was calculated.
Method Precision
The method reproducibility (The intra-day precision) was determined by analyzing
standard solutions in the concentration range of 300 ng/spot to 400 ng/spot of drug for 3
times on the same day and inter-day precision was determined by analyzing
corresponding standards daily for 3 day over a period of one week. The intra-day and
interday related standard deviations were found in the range of 0.6-3.4% and 0.4-2.4%
respectively. The smaller values of intraday and interday variation in the analysis indicate
that the method is precise.
Accuracy
Accuracy of method was evaluated by calculating recovery studies of addition of
standard drug RFX solution to the prepared sample solution at different concentration
levels (50%, 100% and 150%)(n=3) with in the range of linearity of the drug. The results
analysis of recovery studies were obtained by method validation by statistical evaluation.
Specificity
The method specificity was assessed by comparing the chromatogram obtained from the
drug and most commonly used excipients mixture with those obtained from blank
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(excipients solution in water without drugs). It was observed that there was no
interference of the peak obtained for the chromatogram of blank and placebo with that of
RFX peaks obtained for the chromatograms of sample and standard.
RESULTS AND DISCUSSION
In present work HPTLC method was developed for estimation of RFX pure powder and
its pharmaceutical formulation. HPTLC method is cost effective and less time
consuming. RFX is soluble in methanol; therefore methanol was selected as solvent. The
formulation was dissolved in methanol with sonication for 10 min to assure complete
release of drug from the formulation matrix.
Method optimization
For optimization, different mobile phases and composition were employed to achieve the
good separation. The method development was initiated with using a mobile phase of nhexane –methanol in various proportions. In the above conditions, elution was very broad
for RFX. Introduction of chloroform in the above mobile phase gave sharp peaks, but
poor separation and band broadening was observed. Early elution with a little separation
was observed with the mobile phase consisting of methanol-chloroform (5:5). In the same
mobile phase change proportion of methanol: chloroform (7:3) gave reasonable Rf but
not sharp band. Therefore further optimization if needed on the other hand. Addition of
acetic acid solution to the mobile phase helped in sharpening of the peak. Finally, the
mobile phase consisting of the mixture of methanol: Acetic acid: chloroform (8:1:1 v/v)
could resolve rifaximin spot with better peak shape. Combination of methanol: Acetic
acid: chloroform (8:1:1 v/v) offered optimum migration (Rf = 0.83±0.03) and resolution
of RFX from other components of formulation matrix. Even saturation of TLC chamber
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with mobile phase for 30 min assured better reproducibility and better resolution. The
typical densitogram of RFX was shown in Fig.2. Rifaximin shows significant UV
absorbance at wavelength 254 nm. Hence this wavelength has been chosen for detection
in the analysis of RFX.
Method Development
The method was validated in terms of linearity, inter-day and intra-day precision,
repeatability of measurement of peak area as well as repeatability of sample application,
accuracy and specificity. The limit of detection and limit of quantification were also
determined. A representative calibration curve of RFX was obtained by plotting the mean
peak area of RFX against the concentration over the range of 100 - 600 ng/spot.
Calibration curve was shown in Fig.3. A correlation coefficient was found to be 0.9994
The average linear regression equation was represented as Y=2.5395X+1677.1, where
X=concentration of RFX and Y=peak area. The limit of detection and limit of
quantification for RFX were found to be 2 ng/spot and 6 ng/spot, respectively. Inter-day
and Intra-day variation range for RFX were found to be 0.4% - 2.4% and 0.6% - 3.4%
respectively. Precision of the instrument was checked by repeated scanning of the same
spot (400 ng/spot) of six times without changing position of the plate and % CV for
measurement of peak area was found to be 0.64%. Repeatability of the method was
checked by spotting 4 μl of standard solution six times on TLC plate (n=6) and % CV for
peak area was found to be 1.3%. Both the % CV, for measurement of peak area and
sample applications (less than 1% and 3%, respectively), ensures proper functioning of
HPTLC system.
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Accuracy of method was evaluated by calculating recovery of drug by standard addition
method at 3 levels of the calibration curve (n=3). The percentage recovery was found to
be 100.38 to 101.22% ensuring that the method was accurate. The results indicate that the
recovery of added sample was between 50-100 %. This clearly indicates that the method
is accurate and precise.
The method was found to be specific for RFX. The purity of the peak was determined by
comparing the spectra at three different levels i.e. at peak start(S), peak\apex (M) and
peak end (E). Correlation between the three spectra indicated the purity of peak
(correlation, r(S,M) =0.9999, r(M,E)=0.9993, fig. 1). The spectrum of extracted from
tablet was also compared with the spectrum of standard, which showed correlation
0.9994. It was observed that the excipients present in formulation did not interfere with
the peak of RFX.
Different validation parameters for the proposed HPTLC method for determining RFX
content are summarized in Table 1. This method was applied to determine the content of
RFX in rifaximin tablets. The content and percentage of RFX in market samples were
found to be 100.03 mg & 100.1±2.9% respectively (n=3). The results indicate that the
proposed HPTLC method was found to be simple, specific, rapid, precise and accurate
for estimation of RFX in its formulations.
CONCLUSION
The proposed HPTLC method is precise, accurate and selective. The method is rapid,
sensitive, reproducible and economical. It does not suffer any positive or negative
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interference due to common excipients present in formulations and can be conveniently
used for routine quality control analysis of rifaximin in bulk and its formulations.
Fig.1 Structure of Rifaximin
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Fig. 2: Peaks showing Rifaximin in standard solution at 254 nm
CALIBRATION CURVE
12000
10000
8000
Peak area
6000
4000
2000
0
0
0
100
200
300
400
500
CONCENTRATION ng/ml}
Fig. 3: Calibration curve of Rifaximin
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600
700
S.no
Parameters
Results
1
Linearity range (ng/ml)
100-600 ng/spot
2
Correlation co-efficient
0. 9994
3
Precision
Intra day % CV (n = 3)
Inter day % CV (n =3)
0.6 - 3.4
0.4 - 2.4
4
Repeatability of sample
application (n = 6)
1.3
5
Repeatability of peak
area( n = 6)
0.6
6
Limit of detection
2 ng/spot
7
Limit of quantification
6ng/spot
8
Specificity
Specific
Table-1Summary of validation parameters
Label claim
(mg)
Amount found
Mean
(area)
405
408
Rifaximin
100.1±2.9%
402
100mg
413
407
410
Table 2. Estimation of Rifaximin in tablets
% R.S.D
Label claim
Mg/tab
Amount added
%
Total amount
added (ng)
Amt recovered
%
Rifaximin
100mg
50
75
100
150
225
300
101.22
100.38
100.87

Each value is mean + standard deviation of three determine
Table 3: Recovery study of Rifaximin in pre-analyzed samples
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1.18%
Mean
Recovery
%
100.82
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