Development and validation of spectrophotometric methods for simultaneous

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Int J Pharm Biomed Res 2012, 3(2), 90-93
International Journal of
PHARMACEUTICAL
AND BIOMEDICAL
RESEARCH
ISSN No: 0976-0350
Research article
Development and validation of spectrophotometric methods for simultaneous
estimation of norfloxacin and tinidazole in combined dosage form
A.J. Shinde*, H.N. More, S.R. Jadhav, V.S. Ingole, H.R. Kakade
Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416 013, Maharashtra, India
Received: 11 May 2012 / Revised: 24 May 2012 / Accepted: 26 May 2012 / Online publication: 01 Jun 2012
ABSTRACT
The present work relates with two simple, accurate and reproducible spectrophotometric methods have been developed
for the simultaneous estimation of Norfloxacin and Tinidazole in pharmaceutical dosage forms. The first method involves
determination using the AUC (Area under Curve) method; the sampling wavelengths selected are 272-282nm and 313323nm over the concentration ranges of 2-12μg/mL and 3-18μg/mL for Norfloxacin and Tinidazole, respectively. The second
method involves determination using the Q-analysis method (Absorbance Ratio Method); the sampling wavelengths selected
are 277nm and 318nm over the concentration ranges of 2-12μg/mL and 3-18μg/mL for Norfloxacin and Tinidazole,
respectively. The % recovery was between 98-102% indicating high degree of accuracy of the proposed methods. The results
of the analysis were validated statistically and recovery studies were carried out as per ICH guidelines. The developed
methods are simple, rapid, precise, accurate and can be employed for the routine estimation of NF and TZ in both bulk and
tablet dosage form.
Key words: Norfloxacin, Tinidazole, AUC method, Q-Analysis method
1. INTRODUCTION
Norfloxacin (NF), [1-ethyl-6-fluoro-1, 4-dihydro-4-oxo7-(piperazin-1-yl) quinoline-3 carboxylic acid], is a
fluoroquinolone carboxylic acid derivative used as broadspectrum antibacterial (Fig.1). The mechanism of action of
Norfloxacin is through the inhibition of bacterial gyrase, an
enzyme involved in DNA replication, recombination and
repair. By interfering with gyrase, Norfloxacin arrest
bacterial cell growth. The affinity of quinolones to metal ions
seems to be an important prerequisite of their antibacterial
activity: probably, quinolones bind to the DNA-gyrasecomplex via a magnesium ion [1]. NF is the subject of a
monograph in each of British Pharmacopoeia, (BP) [2] and
the United States Pharmacopoeia, USP [3]. The BP and USP
recommended non aqueous titration for the raw material and
HPLC (High Performance Liquid Chromatography) methods
*Corresponding Author. Tel: +91 9823866196 Fax: +91 231 2638833
Email: ajshinde07@rediffmail.com
©2012 PharmSciDirect Publications. All rights reserved.
for tablets. Because of the therapeutic importance of NF,
numerous analytical methods have been developed for its
determination.
In bulk, pharmaceutical formulations and/or biological
fluids Spectrophotometric technique is the most widely used
in pharmaceutical analysis [4-7]. Literature survey revealed
that a number of methods have been reported for estimation
of Norfloxacin [8] and Tinidazole [9-10] individually or in
combination with other drugs. Other analytical methods have
been used such as, HPLC [11-13], electrochemical analysis
[14-15], Difference spectroscopy [16], and capillary
electrophoresis [17].
Tinidazole (TZ), [1-(2-(ethylsulfonyl) ethyl)-2-methyl-5nitroimidazole], is an effective antiprotozoal and antibacterial
agent (Fig. 1). Tinidazole appears to be a promising agent for
the treatment of trichomoniasis, giardiasis, amebiasis, and
amebic liver abscess. TZ is the subject of monograph in each
of the BP and the USP. The BP and USP recommended non
aqueous titration for determination of TZ. There are several
reports on the determination of TZ, both in formulations and
biological fluids, viz: spectrophotometry [18-19], HPLC [20],
and titrimetric and spectrophotomertic analysis [21],
A.JJ. Shinde et al., Int
In J Pharm Biomed Res 2012, 3(2), 90-93
91
disssolved in aceetate buffer pH
H 4 and soniccated for 10miin and
vo
olume made to
t 100mL w
with acetate buffer.
b
It wass then
filttered throughh Whatmann ffilter paper No.41.
N
The soolution
waas suitably diluted with aacetate bufferr to obtain saample
solutions contaiining NF and TZ in the con
ncentrations raatio of
2:3
3, respectiveely as in the formullation. The final
concentrations are
a 4μg/mL off NF and 6μg//mL of TZ.
Fig.1. Structural formulae of a) Norfloxacin
N
(NF) and b) Tinidazolee (TZ)
2.5
5 Method A (A
AUC method)
pootentiometry [22]. The Norfloxacin
N
a
and
Tinidazoole cofoormulation has as an exxtended rangge of antimiccrobial
sppectrum and is effective inn mixed gut infections, diiarrhea
annd dysentery.. Both drugs were simultaaneously deterrmined
byy spectrophootometry, HP
PLC, electroochemical annalysis,
caapillary electtrophoresis annd differencee spectrophotoometry
sttability indicatting assay meethod [23].
In the pressent work, AUC
A
Method (Area under Curve
M
Method)
and Q-analysis
Q
meethod (Absorbbance Ratio Method)
M
sppectrophotom
metric methodd is describedd for simultaaneous
deetermination of
o NF and TZ
Z in the presennce of each other
o
in
puure form and in pharmaceuutical dosage forms.
f
For the AUC
C Method, 2772-282nm, and 313-323nm
m were
sellected as the two samplingg wavelength
hs. Fig.2 repreesents
thee overlain UV
V spectra of N
NF and TZ. NF
F and TZ exhhibited
lin
nearity with abbsorbances inn the range off 2-12μg/mL and
a 318
8μg/mL at thheir respective selected wavelengths. For
sim
multaneous estimation of NF and TZ
Z, mixed stanndards
containing NF and
a TZ in a cconcentration ratio of 2:3μ
μg/mL
each were preppared by apprropriate dilutiion of the staandard
sto
ock solutions with acetatee buffer pH4.. The areas of
o the
miixed standardd solutions w
were measureed at the selected
waavelengths. A set of twoo simultaneou
us equations were
esttablished usinng the mean absorptivity coefficients of
o NF
an
nd TZ at the seelected waveleength intervals.
2.. MATERIAL
LS AND METHODS
2..1 Instrumentaation
A Jasco UV
V/Visible specctrophotometeer, model V 6330 was
em
mployed withh spectral banndwidth of 2nnm and waveelength
acccuracy of ± 0.5nm, with automatic waavelength corrrection
w employedd. A Shimadzzu electronic analytical balance
was
b
(A
AX-200) was used for weiighing the sam
mple. An ultrrasonic
baath sonicator was
w used for sonication
s
of the
t sample solution.
2..2 Reagents and Chemicalss
Analytical pure sampless of NF and TZ were obbtained
frrom Hindustann Antibiotic Limited,
L
Pimppri, Pune, Indiia. The
gllacial acetic acid
a
was obtaiined from Purre-Chem Lab.. Pune.
Sodium hydroxxide pellets obtained
o
from
m Universal laab. The
phharmaceuticall dosage form
m used in this study
s
was Hinndustan
A
Antibiotic
Lim
mited, Pimpri, Pune, India labelled to contain
c
4000mg NF andd 600mg of TZ
Z.
2..3 Preparationn of standard stock solutionn
Standard sttock solutions (100μg/mL) of NF and TZ
Z were
prrepared by diissolving sepaarately 10 mg
m of drug each
e
in
1000mL acetatee buffer pH 4. The workingg standard solutions
off these drugss were obtainned by dilutioon of the resppective
sttock solution with
w acetate buffer.
b
Fig.2. Overlain
O
spectra of NF and TZ in AUC
A
method
A1
1 = 1.52 CNF + 0.1063 CTZ
Z ---------- Eq
q.(i) at 272-2882nm
(λ1- λ2)
A2
2 = 0.44 CNF + 0.40 CTZ ---------- Eq
q.(ii) at 313-3223nm
(λ3
3- λ4)
Where,
W
1.52 and 0.44 are meean absorbtiviity values of NF at
(λ1-λ2) and (λ3--λ4), respectivvely.
0.1
1063 and 0.400 are mean abbsorbtivity vaalues of TZ at
a (λ1λ2
2) and (λ3- λ44), respectivelyy.
A1
1 and A2 are the
t absorbancce of mixed sttandards at (λ1- λ2)
an
nd (λ3- λ4), resspectively.
CN
NF and CTZ are
a concentrattions of NF an
nd TZ.
Th
he concentratiion of CNF aand CTZ in mixed
m
standarrd and
tab
blet formulatiion can be obbtained by so
olving equatioons (i)
an
nd (ii).
2..4 Preparationn of sample sttock solutions
2.6
6 Method B (A
Absorbance raatio or Q-anallysis method)
An accuratelly weighed poowder sample equivalent too 40mg
off NF was trransferred to a 100L voolumetric flassk and
From the overlain speectrum of NF
N and TZ,, two
waavelengths weere selected; one at 277nm
m λmax of NF
F and
A.JJ. Shinde et al., Int
In J Pharm Biomed Res 2012, 3(2), 90-93
otther is 310nm
m, isoabsorpttive point forr both the drrugs as
shhowed in Figg.3. The standdard and sam
mple solutionss were
prrepared in thee same manneer as in the previous methood and
abbsorbance measured
m
at 277nm
2
and 318nm.
3
The drugs
shhowed lineariity in the conncentration rannges of 2-12μ
μg/mL,
3--18μg/mL forr NF and TZ respectively.
r
S mixed staandards
Six
inn ratio of 2:33μg/mL show
wing linearityy within the Beer’s
cooncentration range of NF
N and TZ were preparred by
apppropriate dillution of standdard stock sollutions (100μg/mL).
T method em
The
mploys Q valuues; the concenntrations of drrugs in
saample solutioon were deteermined by using
u
the folllowing
eqquations.
CNF =
×
----------- Eq.(iii)
CTZ =
×
----------- Eq.(iv)
92
concentrations of each druug in samplle solutions were
M
calculated usingg equations (ii) and (ii) forr the AUC Method
nd equations (iiii) and (iv) foor the Q-analysis method.
an
2.8
8 Validation
The proposeed methods w
were validated
d according too ICH
Q2B guideliness for validation of analyttical procedurres in
ord
der to determ
mine the linearity, sensitiv
vity, precisionn and
accuracy for thee analyte [12]..
2.8
8.1 Accuracy
To ascertainn the accuraacy of the proposed
p
metthods,
reccovery studiees were carrried out by standard adddition
meethod at three different leveels (80%, 100%
% and 120%).
2.8
8.2 Linearity
w
where,
Q0 =
Q1 =
Q2 =
A
A
A
F
A
F
A
Z
A
Z
A = Absorbance of sample at isoabsorpttive point; a1 and a2
abbsorptivities of
o NF and TZ
Z, respectivelly at isoabsorrptivity
pooint.
The linearityy measuremeent was evalu
uated by anallyzing
diffferent concenntration of thee standard so
olution of NR
RF and
TIIZ. For AUC method and Q-analysis, the
t Beer-Lam
mbert’s
concentration raange was founnd to be 2-12
2μg/mL for NF
N and
3-18μg/mL for TZ,
T respectiveely.
2.8
8.3 Precision
Precision was
w studied too find out in
ntra and inteer-day
vaariations in thhe test methood of NF an
nd TZ. Calibration
cu
urves preparedd in medium w
were run in trip
plicate in sam
me day
an
nd for three daays. % RSD (rrelative standaard deviation)) were
calculated whicch should bee less than 2%. The resuults of
staatistical validaation data are given in Table 1. The resuults of
thee analysis andd statistical validation dataa of recovery study
off the tablet form
mulation are ggiven in Tablee 2 and Table 3.
Tab
ble 1
Op
ptical characteristiics and validationn data of NF and TZ
Paarameters
Norffloxacin
Tinidazole
Methhod A Method B Method A Metthod B
277
277
318
318
2-12
2-12
3-18
3-188
Fig.3. Overlain Spectra
S
of NF andd TZ in Q analysiis Method (Absorrbance
Ratiio Method)
2..7 Assay of tabblet formulatiion
Powder equuivalent to 100mg of NF annd 10mg of TZ
T was
w
weighed
and transferred 100mL volumetric flaskk and
diissolved in 500mL acetate buffer
b
pH 4 and
a volume iss made
upp with water upto100mL
u
affter the ultrasoonication for 15min.
T solution was
The
w then filtereed through Whhatmann filterr paper
N
No.41
and dilluted further to obtain finnal concentrattion of
4μg/mL of NF and 6μg/mL of
o TZ. The saample solutionns were
annalyzed as per
p the proceedure for mixxed standards. The
Waavelengths (nm)
Lin
nearity & Range (µg/mL)
Preecision*
Interday (%RSD
D)
0.4677
0.670
0.234
0.3445
Intraday (%RSD
D)
0.1355
0.105
0.458
0.2332
LO
OD (µg/mL)*
2.4800
0.788
2.330
1.4990
LO
OQ (µg/mL)*
7.5400
1.550
1.342
5.5882
Reegression values
Slope*
1.5200
0.155
0.874
0.0445
r2*
0.99999
0.9997
0.9997
0.99998
*D
Denotes average of six estimations;; Method A – AU
UC method ; Methhod B –
Q-aanalysis method (Absorbance
(
ratioo method)
3. RESULTS AND
A
DISCUS
SSION
Under the experimental
e
cconditions described, calibbration
cu
urve, assay off tablet and reecovery studiies was perfoormed.
Th
he developed methods
m
weree validated as per ICH guiddelines
forr linearity, reepeatability, iintermediate precision
p
(inteer-day
A.J. Shinde et al., Int J Pharm Biomed Res 2012, 3(2), 90-93
Table 2
Statistical validation data of tablet formulation and results of commercial
sample analysis (n=6)
Component Method Label claim Amount
(mg/tablet) obtained
(mg/tablet)
NF
A
400
404.25
B
400
393.6
TZ
A
600
600.86
B
600
607.31
%
Amount
found
101.06
98.4
100.14
101.22
SD
%
RSD
0.0239
0.0243
0.0181
0.0391
0.1640
0.1764
0.1168
0.2530
Table 3
Statistical validation of recovery studies (n=3)
Level of %
recovery
Methods
80
A
B
A
B
A
B
100
120
% Mean recovery
NF
TZ
99.54
100.24
99.18
99.82
99.72
99.28
99.59
100.39
100.11
99.81
99.63
100.21
% RSD
NF
0.6248
0.4621
0.3523
0.2951
0.8429
0.3982
TZ
0.8731
0.5425
0.5847
0.5866
0.6825
0.1751
93
List of symbols and Abbreviations:
%
: Percent
Nm
: Nanometer
µg/mL
: Microgram per Mililiter
UV
: Ultraviolet
HPLC
: High Performance Liquid Chromatography
High Performance Thin Layer
HPTLC :
Chromatography
NF
: Norfloxacin
TZ
: Tinidazole
AUC
: Area under curve
ICH
: International conference on Harmonization
SD
: Standard deviation
RSD
: Relative Standard deviation
LOD
: Limit of Detection
LOQ
: Limit of Quantitation
REFERENCES
and intra-day precision studies), LOD, LOQ as shown in
Table 1. Co-efficient of correlation was found to be 0.9998
and 0.9982 for NF and TZ, respectively by AUC method.
The optical characteristics and regression values for the
calibration curves are presented in Table 1. Q-analysis
method shows regression coefficient (r2) values of 0.9978
and 0.9958 for NF and TZ, respectively. The mean % content
of by the developed methods was 100.32% and 100.56%
respectively Table 2. The mean % recoveries of NF and TZ
were found to be 99.62% and 99.96 %, respectively (Table
3). The ruggedness of the developed methods was determined
by evaluating the effect of change in instruments and analysts
on the % mean content of drugs.
4. CONCLUSIONS
The combination of NF and TZ is commercially available
in tablet dosage forms to control gastrointestinal infections
caused by bacterial or amoebic infection, prostatitis and
urinary tract infections due to susceptible uropathogens.
Here, two simple UV spectrophotometric methods AUC
Method and Q-analysis method were developed for their
simultaneous analysis. The standard deviation, RSD and
standard error calculated for the methods are low, indicating
high degree of precision of the methods. The RSD is also less
than 2% as required by ICH guidelines. The % recovery was
between 98-102% indicating high degree of accuracy of the
proposed methods.
ACKNOWLEDGEMENTS
The authors express their gratitude to Dr. H.N. More,
Principal, Bharati Vidyapeeth College of Pharmacy,
Kolhapur, MH, India for providing necessary facilities, and
to Hindustan Antibiotic Limited, Pimpri, Pune, India for the
generous gift samples of pure NF and TZ.
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