Vishwanath Agrahari et al.

/ Journal of Pharmacy Research 2010, 3(11),2747-2749

Research Article
ISSN: 0974-6943

Available online through

www.jpronline.info
Estimation of satranidazole in bulk and tablet dosage form by RP-HPLC
Vishwanath Agrahari *1 , Meenakshi Bajpai 1 , Sanju Nanda 2 , G.N.Singh3 , Robin Kumar3
1
College of Pharmaceutical Sciences, RKGIT, Ghaziabad-201003, Uttar Pradesh, India
2
Department of Pharmaceutical Sciences, M.D. University, Rohtak-124001, Haryana, India
3
Indian Pharmacopoeia Commission, CIPL Campus, Sector-23, Raj Nagar, Ghaziabad-201002 Uttar Pradesh, India

Received on: 15-06-2010; Revised on: 18-08-2010; Accepted on:13-09-2010

ABSTRACT
A simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of satranidazole in tablet dosage form. The separation was
achieved by using an Agilent 1200 Series HPLC System consisting of analytical column Eclipse XDB-C18 (5m; 150x4.6mm) along with guard column and variable
wavelength detector- G1314B was used for analysis. The mobile phase consisting of water:acetonitrile in the ratio of 80:20 (v/v) was used. The flow rate was 1.0
mL/min and the effluents were monitored at 318 nm. The retention time was 6.139 min. The detector response was linear in the concentration of 5-70 mcg/mL.
The respective linear regression equation being y = 39.742x + 45.939.The limit of detection and limit of quantification was 0.034g/ml and 0.106g/ml.,
respectively. The percentage assay of satranidazole was 99.9%. The method was validated by determining its accuracy, precision and system suitability. The results
of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of satranidazole in bulk
drug and in its pharmaceutical dosage form.

Key words: Satranidazole, RP-HPLC, Validation, System suitability tests.

INTRODUCTION
Satranidazole (CG-10213-Go), a novel nitroimidazole possessing a C-N linkage at C2 of the imidazole ring has been examined (during reduction), for its
ability to damage DNA. The drug produces extensive DNA damage characterized by helix destabilization and strand breakage. Its comparison with other 2and 5-nitroimidazoles indicates it may be more active towards anaerobes than
many 5-nitroimidazoles. It is due to its relatively high redox potential which
may make it more resistant to inactivation by oxygen [1]. It is recently
introduced as an anti-protozoal agent in tablet dosage form. It is a highly
potent, well-tolerated, and clinically useful agent against common protozoa.
It is rapidly absorbed and exhibits higher plasma and liver concentration than
metronidazole. Satranidazole (SAT) is not included in any official
pharmacopoeias such as IP, USP and BP. Chemically, it is 3-(1-methyl-5nitroimidazol-2-yl)-1-(methylsulfonyl) imidazolidin-2-one (m.f.C8H11N5O5S;
m.w. 289.26) [2] Fig.1.Literature survey revealed an electron-capture gas chromatographic assays in blood[3], spectrophotometric methods[4-7],
HPTLC[8,9] and HPLC [10-13] methods in pharmaceutical dosage form for
estimation of Satranidazole individually as well as in combination with
Ofloxacin. An attempt has been made to develop a new RP-HPLC method for
its estimation in pharmaceutical dosage form with good accuracy, precision
and simplicity. The method was validated according to the ICH guidelines [1415].
2.0. MATERIALS AND METHODS
2.1. Chemicals
Acetonitrile and methanol used were of HPLC grade from E. Merck, India.
HPLC grade water was obtained using millipore water purification system.
Working standard of satranidazole with potency of 99.67 % was obtained
from Alkem Laboratories Limited, Baddi. Other chemicals were analytical
grade of above 99% purity. All volumetric-ware was pre-calibrated by the
manufacturer (Borosil) and was of grade A. HPLC grade water was obtained
using millipore water purification system. Commercial tablets containing
Satranidazole (Satrogyl-300mg) were procured from the local chemist shop.
2.2. Instrumentation
Agilent 1200 Series HPLC System, consisting of degasser-G1322A, quaternary pump-G1311A, column oven- G1316A, and variable wavelength detector- G1314B was used for analysis. The analytical column Eclipse XDB-C18
(5m; 150x4.6mm) was used along with guard column. The software Agilent
Chemstation ran on HP computer operated with Windows 2003 professional

*Corresponding author.
Vishwanath Agrahari
College of Pharmaceutical Sciences,
RKGIT, Ghaziabad-201003, UP, India
Tel.: + 91-9871229177
E-mail:v09world@gmail.com

Fig.1.3-(1-Methyl-5-nitroimidazol-2-yl)-1-(methylsulfonyl) imidaz
olidin-2-one
used for this method. A rheodyne injector with 20 l loop was used for injecting
the sample. Shimadzu balance, AY-120 was used for weighing purpose in this
method.
2.3. Chromatographic conditions
The analysis was carried out with UV detection at 318 nm using a 20 l
injection volume. Assay was performed using a C18 reversed-phase column
eluted with Acetonitrile and water (20:80, %v/v) at a flow rate of 1.0 ml/ min.
Chromatography was carried out at ambient temperature. The solvents were
mixed, filtered t hrough a membrane filter of 0.45 micron pore and degassed in
ultrasonic bath prior to use.
2.4. Standard solution preparation
Standard stock solutions were prepared by dissolving 10 mg of satranidazole
working standard in 8.0 ml of mobile phase and diluting to 10.0 ml with the
same to obtain concentration of 1000 g/ml. It was filtered through a .22
membrane filter. The stock solution was protected from light using aluminium
foil and stored for 1 week at 40C and was found to be stable during this period.
2.5. Procedure for analysis of tablet formulation
20 Tablets of the product under study were weighed, crushed and mixed in a
mortar and pestle for 20 min. A portion of powder equivalent to the weight of
100.00mg was accurately weighed and transferred to a dry 100 ml A-grade
volumetric flask and 80 ml mobile phase was added. The volumetric flask was
sonicated for 20 min to effect complete dissolution of satranidazole and made
up to the volume with mobile phase. Suitable aliquots of solution were filtered
through a 0.45 m nylon filter. This was further diluted with mobile phase to
yield concentration of satranidazole in the range of linearity (40ppm). Each
of standard and test preparation was injected into the chromatograph and the
responses recorded.
3.0. METHOD VALIDATION
3.1. Linearity
A series of standard curves were prepared over a concentration range of 5 - 70
g/ml by diluting the standard stock solution of SAT (1mg/ml) in mobile phase.

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The data from peak area versus drug concentration plots were treated by
linear least square regression analysis and r2 was found 0.994. The standard
curves were evaluated for intra-day and inter-day reproducibility. Each experiment was repeated in triplicate.

3500

3.2. Precision
Precision was measured in accordance with ICH recommendations. The precision study was carried out by injecting sample preparation of 40g/ml concentration six times.

2500

3000

2000
Area
(mAU x S)

3.3. Accuracy
Recovery studies by the standard addition method were performed with a view
to justify the accuracy of the proposed method. Previously analyzed samples
SAT (40 g/ml) were spiked with known amount of standard so as to get three
different levels (80%, 100% and 120%) and the mixtures were analyzed by the
proposed method. The experiment was performed in triplicate. Recovery
(%), RSD (%) were calculated for each concentration.
3.4. Limit of detection and limit of Quantitation
In order to estimate the limit of detection (LOD) and limit of quantitation
(LOQ) values, the blank sample was injected six times and the peak area of
this blank was calculated as noise level. The LOD was calculated as three times
the noise level (S/N = 3:1) while ten times the noise level gave the LOQ (S/
N=10:1).

y = 39.742x + 45.939
R2 = 0.9986

1500
1000
500
0
0

10

20

30

40

50

60

70

80

Concentration ( g/ml)

Fig. 2 Linearity curve of Satranidazole

3.5. Ruggedness
The ruggedness of the method was demonstrated by analysis of the samples as
for precision study by a second analyst. The RSD of the two sets of data
indicates the ruggedness of the method. Further, the t-test was performed on
the data and the difference was found to be not significant.
3.6. Robustness
The robustness of the method was determined to assess the effect of small but
deliberate changes of the chromatographic conditions on the determination
of SAT. The different variations are in flow rates by 0.1 mL/min, in wavelength by 2 nm and in temperature by 5 C. The concentration of the
solution analyzed was 40 g/mL.
3.7. System suitability tests
The chromatographic systems used for analyses must pass the system suitability limits before sample analysis can commence. The capacity factor (K),
injection repeatability, tailing factor (T), theoretical plate number (N) and
resolution (Rs) for the principal peak were the parameters tested on a 40 g/
mL sample of SAT to assist the accuracy and precision of the developed HPLC
system.
3.8. Specificity
The specificity of the method was tested by chromatographing a mixture of
commonly used tablet excipients, for example starch, microcrystalline cellulose, lactose, talc, magnesium stearate, colloidal silicon dioxide, sodium starch
glycollate and comparing the chromatogram with that obtained from a mixture of drug and the same additives.
4.0. RESULTS
4.1. Linearity
Peak area versus drug concentration wa s plotted to construct a standard curve
for SAT and linearity was shown in concentration range of 5 g/ml to 70g/
ml. The polynomial regression for the calibration plots showed good linear
relationship with coefficient of correlation, r = 0.9986; slope = 39.742 and
intercept = 45.939 over the concentration range studied. Fig.2
4.2. Precision
The % assay for tablet was calculated and % RSD was found to be 0.48%.which
proved that the method was precise, as depicted in Table 1.
4.3. Accuracy
The % recovery was calculated for triplicate samples and for all levels and
mean recovery was calculated. The mean recovery was well within the acceptance limit hence the method was accurate, as depicted in Table 2.
4.4. Limit of detection and LOQ
The LOD was calculated to be .034g/ml and the LOQ was calculated to be
.106g/ml.

Fig.3 HPLC Chromatogram of Standard Satranidazole

Table1: Precision of developed method at working level (n=6)
Injection no.

% Assay

1
2
3
4
5
6
Mean
SD
RSD

99.48
100.10
99.23
99.11
99.85
100.35
99.68
0.48
0.48

Table 2: Recovery studies of Satranidazole

Amount (%)
of drug added
to analyte

Theoretical
content (g/ml )

Conc. Found
(g/ml ) SD *

Recovery
(%)

RSD
(%)

80
100
120

72
80
88

71.93 0.245
80.02 0.340
87.95 0.315

99.8
100.5
99.9

0.12
0.14
0.13

*n=3
Table 3: Ruggedness Analysis
Analyst 1
Sample

% Assay

Analyst 2
Sample
% Assay

1
2
3
4
5
6
Mean
SD
RSD

100.04
100.22
99.84
100.35
100.67
100.41
100.25
.289
.29

1
2
3
4
5
6
Mean
SD
RSD

99.89
100.06
100.45
99.78
100.07
99.49
99.95
.476
.48

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4.5. Ruggedness
The % assay and RSD for samples prepared by second analyst was calculated
and found within limit. Then RSD of analyst 1 and analyst 2 was calculated and
found within limit. This proved that the method is rugged, as depicted in Table
3.
4.6. Robustness
The results of the analysis (% RSD ranged from 0.078 to 1.286 %) of the
samples under the conditions of the above variations indicated the nature of
robustness of the method.
4.7. System suitability tests
The results of the system suitability tests assure the adequacy of the proposed
HPLC method for routine analysis of SAT. The capacity factor (k) was found
to be 2.405, indicating that the SAT peak is well resolved with respect to the
void volume. The RSD of six consecutive injections performed under the
precision test (Table 1) was found to be 0.48% and thus shows good injection
repeatability. The tailing factor (T) for SAT peak was found to be 0.9, reflecting good peak symmetry. The theoretical plate number (N) was found to be
8466, thus demonstrating good column efficiency.
4.8. Specificity
The chromatograms obtained showed separation of the analyte from the
excipients was complete, i.e. there was no interference from the excipients
under the chromatographic conditions used for the analysis. No interference
of the placebo mixtures with the peak of satranidazole was observed.

three levels (80, 100 and 120 %) showed below 2.0% and precision was found
to be 0.48. The method was also found to be robust as there was no significant
change in the peak area, peak shape and retention time of SAT. Furthermore,
the low values of LOD and LOQ indicate that the method can be employed
over a wide concentration range for linearity. The system suitability tests
performed verified the resolution, column efficiency and repeatability of the
chromatographic system
6.0. CONCLUSION
The HPLC method developed is accurate, precise, reproducible and specific.
The method is linear over a wide range, economical and utilizes a mobile phase
which can be easily prepared. All these factors make this method suitable for
quantification of SAT in bulk drugs and in pharmaceutical dosage forms. The
method developed was then subjected to validation as per ICH guidelines and
showed that method is linear, precise, accurate and rugged.
ACKNOWLEDGEMENT
The authors are thankful to M/s Alkem Laboratories Limited, Baddi, India, for
the gift of SAT.

REFERENCES
1.
2.
3.
4.
5.

5.0. DISCUSSION
Satranidazole, a weak acid, is sparingly soluble in water. The final decision on
mobile phase composition and flow rate was made on the basis of peak shape,
peak area, tailing factor, baseline drift, ease of preparation, use of readily
available cost-effective solvents and time required for analysis. Initial trial
experiments were conducted, with a view to select a suitable solvent system
for the accurate estimation of the drug. These included methanolwater,
methanolacetonitrile-water and acetonitrilewater in different ratio. Flow
rates between 0.5 and 1.2ml/min were studied. A mobile phase system comprising of acetonitrile-water (20:80 % v/v) was found to be optimum and a flow
rate of 1.0 ml/min gave an optimal peak shape and was selected. The same
solvent mixture was used for the extraction of the drug from the formulation
containing excipients. No internal standard was used because no extraction or
separation step was involved. The solvents were mixed, filtered through a
membrane filter of 0.45 micron pore and degassed in ultrasonic bath prior to
use. Using a reversed-phase C18 column, the retention times for satranidazole
was observed to be 6.13 min. Total time of analysis was kept 8.0 min. The
maximum absorption of satranidazole was detected at 318 nm and this wavelength was chosen for the analysis. (Fig3) The developed method was linear
showing the coefficient of correlation of 0.9986. % RSD of accuracy study for

6.
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10.
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 11.November 2010

2747-2749