Latin American Journal of Pharmacy Short communication

(formerly Acta Farmacéutica Bonaerense) Received: February 15, 2010

Revised version: March 25, 2010
Lat. Am. J. Pharm. 29 (8): 1455-8 (2010) Accepted: April 3, 2010

HPTLC Method for Quantitative Determination

of Granisetron Hydrochloride in Bulk Drug and in Tablets

S. LAKSHMANA PRABU*, P. SELVAMANI, S. LATHA

Dept. of Pharm. Technology, Anna University Tiruchirappalli,

Tiruchirappalli-620 024. Tamil Nadu, India.

SUMMARY. A new simple, rapid and reproducible high performance thin layer chromatographic method
has been developed and validated for the analysis of granisetron hydrochloride (GSH) in bulk drug and
from pharmaceutical formulation. The chromatographic separation was achieved on HPTLC aluminium
plates precoated with silica gel 60F254 as the stationary phase with chloroform: methanol (80:20 %v/v) as
mobile phase. The method gives a compact band for GSH (Rf value of 0.45 ± 0.02). Densitometric analysis
of GSH was carried out in the absorbance mode at 301 nm. The linear regression analysis data for the cali-
bration plots showed good linear relationship with the correlation coefficient of 0.9980 with respect to
peak area in the concentration range of 400-1600 ngband–1. The method was validated for accuracy, preci-
sion, recovery studies, specificity, sensitivity, limit of detection and limit of quantification. Statistical anal-
ysis proved the method was precise, reproducible, selective, specific, and accurate for analysis of GSH.
The wide linearity range, sensitivity, accuracy, and simple mobile phase composition imply the method is
suitable for routine quantification of GSH with high precision and accuracy in bulk drug and marketed
oral solid dosage form.

INTRODUCTION spectrometry 14-16 detector. But there is no assay

Granisetron hydrochloride (GSH) is chemi-
cally endo-1-methyl-N-[9-methyl-9-azabicyclo
(3.3.1) non-3-yl]-1H-indazole carboxamide hy-
drochloride, which is a selective 5-HT3 receptor
antagonist which may have beneficial therapeu-
tic effects in the treatment of vomiting and nau-
sea resulting from cancer therapy 1-3. It has an
improved side effect and tolerability profile, a
lower risk of drug interactions and a longer du-
ration of action than other 5-HT3 receptor an-
tagonists. GSH is an effective and well-tolerated
agent in the management of chemotherapy-in-
duced, radiotherapy-induced and post-operative
nausea and vomiting in adults and children 4.
A survey of literature revealed that the fol-
lowing analytical methods were reported for de-
termination of GSH in biological samples such
as chromatographic methods like high perfor-
mance liquid chromatography with fluorescence
detector 5-12, UV detector 13 and tandem mass
procedure has been reported for its determina-
tion in pharmaceutical formulation by HPTLC.
Nowadays, HPTLC has become a routine an-
alytical technique due to its advantages of relia-
bility in quantitation of analytes at micro and
nanogram levels and its cost effectiveness. The
major advantage of HPTLC is that several sam-
ples can be analyzed simultaneously using a
small quantity of mobile phase. It is a simple
micro analytical separation technique where a
large number of samples can be handled at a
time. The technique is economical, as the con-
sumption of solvent is low and there is virtually
no solvent disposal problem. This reduces the
time and cost of analysis and possibilities of
pollution of the environment. HPTLC also facili-
tates repeated detection (scanning) of the chro-
matograms with same or different parameters.
The uniform particle size of precoated HPTLC
plates enables the achievement of greater reso-

KEY WORDS: Bulk drug, Granisetron hydrochloride, HPTLC, Tablets.

* Author to whom correspondence should be addressed. E-mail: slaxmanvel@gmail.com

ISSN 0326-2383 1455

LAKSHMANA PRABU S., SELVAMANI P., & LATHA S.
lution and easily reproducible separation. The
method of detection does not place any restric-
tion on the choice of the mobile phase. Simulta-
neous assay of several components in a multi-
component formulation is possible. Because
there is no reported HPTLC method for analysis
of GSH in pharmaceutical formulations, the aim
of this research is to develop a simple and spe-
cific HPTLC method for quantitative determina-
tion of GSH in tablets
EXPERIMENTAL
Chemicals
GSH (Purity 99.93 % w/w) was procured as a
gift sample from Natco Pharmaceuticals Ltd.,
Hyderabad, India. All chemicals and reagents
used were of analytical grade and purchased
from Qualigens Fine Chemicals, Mumbai, India.
Preparation of Stock Solution from the Bulk
Drug
GSH (25 mg, accurately weighed) was dis-
solved in 25 ml of methanol. A series of work-
ing standard solutions containing 40, 60, 80,
100, 120, 140 and 160 µg.ml–1 were prepared by
dilution of the stock solution.
Sample Preparation
To determine the concentration of GSH in
tablets (labeled claim: 2.24 mg per tablet), 20
tablets were weighed, their mean weight deter-
mined and then were finely powdered. The
powder equivalent to 25 mg of GSH was
weighed and the drug from the powder was ex-
tracted with methanol. Complete extraction of
the drug was ensured by sonication for 30 min.
The volume was then diluted to 100 ml with
methanol, the resulting solution was allowed to
settle for about 1 h, and the supernatant was
suitably diluted to give the desired concentra-
tion (80 µg.ml-1). Ten µl of the above solution
(800 ngband–1) was applied on TLC plate fol-
lowed by development and analyzed. The anal-
ysis was made in triplicate. The possibility of
excipient interference in the analysis was stud-
ied.
HPTLC Instrumentation
The chromatographic estimation was per-
formed by spotting standards of GSH on pre-
coated silica gel aluminium plates 60F254 (20 x
10 cm with 200 µm thickness, E. Merck, Darm-
stadt, Germany). Samples were applied to the
plates as 6 mm bands, 8 mm apart, 15 mm from
both the bottom and the left edge of the plates,
1456
by use of a CAMAG (Muttenz, Switzerland)
Linomat IV sample applicator fitted with a 100
µl Hamilton syringe. A nitrogen aspirator was
used and the constant application rate was 100
nLs –1. Linear ascending development of the
plates, to a distance of 7 cm, with chloroform:
methanol (8:2 v/v) as mobile phase, was per-
formed in a 20 cm x 10 cm twin through glass
chamber (CAMAG, Muttenz, Switzerland) previ-
ously equilibrated with mobile phase vapor.
Subsequently to the development, TLC plates
were dried in a current of air with the help of
an air-dryer. Densitometric scanning was per-
formed on Camag TLC scanner III in the ab-
sorbance mode at 301 nm (λ max) using deu-
terium lamp as radiation source.
Method Validation
Linearity
A stock solution of GSH (1000 µg.ml–1) was
prepared in methanol. Different volume of stock
solution, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml
was taken and volume made up to 10 ml by
methanol, to get 40, 60, 80, 100, 120, 140 and
160 µg.ml–1 solution, respectively. The 10 µl of
each above solution were spotted in triplicate
on TLC plate to obtain concentrations of 400,
600, 800, 1000, 1200, 1400 and 1600 ng per
band of GSH, respectively. After chromatogra-
phy, peak areas versus drug concentration were
treated by linear least square regression analysis.
Precision
Repeatability of sample application and mea-
surement of peak area were assessed for six
replicate applications of 800 ngband–1 GSH. The
intra and inter-day variation in analysis of GSH
were assessed at three different concentration
levels of 800, 1000 and 1200 ngband–1.
Robustness of the method
Small deliberate changes in the mobile phase
composition (± 5 %), volume of mobile phase
(± 1 ml) and wavelength (± 5 nm) were intro-
duced and the effects on the results were exam-
ined. Robustness of the method was done at
800 ngband–1.
Sensitivity
The sensitivity of the method was measured
at the limit of detection (LOD) and limit of
quantification (LOQ), calculated as three and
ten times the noise level, respectively.
Specificity
The specificity of the method was assessed
by analyzing standard drug, pharmaceutical
product and placebo and comparing the Rf of
the standard with that of the sample to deter-
 Latin American Journal of Pharmacy - 29 (8) - 2010

Actual conc. Intra-day precision Inter-day precision

(ngband–1)
Mean area# S.D. % RSD Mean area# S.D. % RSD

800 8735 107.74 1.23 8659 77.42 0.89

1000 10825 146.02 1.35 10720 89.95 0.84
1200 12622 100.38 0.80 12734 70.09 0.55

Table 1. Intra and inter day precision of HPTLC method. # n=6.

mine whether the pharmaceutical product and
placebo led to interfere.
Recovery studies
Previously analyzed samples were spiked
with known concentration of GSH standard and
the mixtures were re-analyzed by the proposed
method to check the recovery of different
amounts of the drug from the formulation. At
each level of the amount, six determinations
were performed.
RESULT AND DISCUSSION
Selection of Mobile Phase
Since there are no literature reports of
HPTLC method of analysis for GSH for routine
analysis, the selection of the mobile phase was
carried out on the basis of polarity. i.e., choice
of a solvent system that would give dense and
compact spot Rf value for GSH. The composi-
tion of the mobile phase for development of
chromatographic method was optimized by test-
ing different solvent mixtures of varying polari-
ty. Mobile phase composition chloroform :
methanol (8:2 % v/v) was found to give a sharp
and well defined peak at Rf value of 0.45 ± 0.02
(Fig. 1). Well defined spots were obtained when
the chamber was saturated with the mobile
phase for 30 min at room temperature.
Method Validation
Linearity
The calibration curve of amount of analyte
against average response (peak area) was linear
over the concentration range 400-1600 ngband–1
with the correlation coefficient of 0.9980.
Precision
The repeatability of sample application and
measurement of peak area were expressed in
the terms of % RSD and results are depicted in
Table 1, which revealed intra and inter day vari-
ation of GSH at three different concentration
levels of 800, 1000 and 1200 ngband–1.
Robustness of the method
Small deliberate changes in the chromato-
graphic conditions did not affect the method.
The low values of %RSD (Table 2) indicated the
robustness of the method.
Parameter S.D. of peak area # % RSD
Mobile phase composition 85.88 0.98
Amount of mobile phase 94.43 1.11
Wavelength 97.43 1.13
Table 2. Robustness of the method. #: n= 6.

Figure 1 . Typical HPTLC chromatogram of
granisetron hydrochloride. Experimental condition:
Temperature at 25 °C. Concentration : 1000 ngband–1.
Sensitivity
Under the experimental conditions used, the
smallest amount of drug which could be detect-
ed was 50 ngband–1 and the lowest amount of
drug which could be quantified was 10
ngband–1.
Specificity
The specificity of the method was confirmed
by comparing the Rf value of standard with that
of GSH in the marketed formulation. There is no
interference from the excipients commonly pre-
sent in the tablets. The blank chromatogram ob-
tained from the placebo is shown in Figure 2.
Hence the developed method is specific and se-
lective.

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LAKSHMANA PRABU S., SELVAMANI P., & LATHA S.

analysis of GSH in pharmaceutical dosage

forms.

Figure 2. Chromatogram obtained from placebo. Ex-
perimental condition: temperature at 25 °C.
CONCLUSIONS
The developed HPTLC method combined
with densitometry was found suitable for deter-
mination of GSH as bulk drug and in marketed
solid dosage formulation without any interfer-
ence from the excipients. Statistical analysis
proves that, the method is repeatable and selec-
tive for the analysis of GSH. Its advantages are
low cost of reagents, speed and simplicity of
sample treatment, satisfactory precision and ac-
curacy.

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