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DEVELOPMENTOFCOLORIMETRICMETHODFORDETERMINATIONOFNITRAZEPAM
INTABLETSANDBULKS
RAKESHKUMARTEKADE*123,VIRENDRAGAJBHIYE1,KAVITARAI1ANDMUKTIKASHARMA2
PharmaceuticsResearchLaboratory,DepartmentofPharmaceuticalSciences,Dr.HariSinghGourUniversity,SAGAR470003,INDIA2Bhopal
InstituteofTechnologyandSciences,Bangarasia,Bhopal(M.P),INDIA.*3SchoolofPharmacyandPharmaceuticalSciences,UniversityofCentral
Lancashire,PrestonPR12HE,ENGLAND(UnitedKingdom).Email:rakeshtekade@yahoo.com;rtekade@uclan.ac.uk
ABSTRACT
Asimple,economical,preciseandreproduciblecolorimetricmethodwithhighersensitivityaswellaslower
limitofdetection(1.26g/ml)fortheroutineestimationofNitrazepamhasbeendeveloped.Themethodis
based on the formation of a yellow coloured complex by Nitrazepam in presence of FeCl3 and NaOH. The
developed coloredcomplex showed max at 490nm corresponding to Filter no.3 of colorimeter, and obeyed
LambertBeers law in the concentration range of 424 g/ml. Results of analysis were authenticated
statistically as well as by recovery studies, which gave mean recovery between 98.8053.242 to
100.804.628, % standard deviation (SD). The method was successful in determining Nitrazepam in
commercialformulationsviz:Nitravet5mg(AngloFrench);Nitrosun10mg(SunPharma)andNipam5mg
(LaPharma),withanaveragerecoveryof101.163.725,98.121.012and98.553.145;%SD,respectively.
Theproposedmethodcouldfindapplicationtoproductdevelopmentscientistsinongoingresearch;aswell
provide an additional tool for routine analysis of Nitrazepam in academia practical based on Nitrazepam
formulations.
Keywords:Nitrazepam,Colorimetry,Accurate,Precise,Reproducible,LowSD
INTRODUCTION
Benzodiazepines
are
commonly
prescribed as anticonvulsant, muscle
relaxant,anxiolyticandsedativeandhave
been considered largely free of neuro
endocrine effect [12]. Most of the
pharmacological actions of Benzodiazepines
areconsideredtobemediatedviabinding
to BZ receptors, and the consequent
increase in the effects of aminobutyric
acid(GABA)[3].Moreover,Benzodiazepines
are suggested to affect the monoamine
transmitters, such as serotonin (5HT)
[4], noradrenaline [5], and dopamine
(DA) [6], directly or by enhancing the
GABA system. They represent a class of
veryusefulmedicines.Nitrazepam,which
is 1,2 dihydro7nitro2Oxo5phenyl
3H1,4benzodiazepine is one of the
efficient member of this class [7]. A
number of methods available to quantify
Nitrazepaminformulationsunfortunately
demand employment of costlier
chemicals. In order to minimize the
research and development costs, the
techniques employed for preliminary
analysis must be simple, precise,
reproducible, and moreover should be
economical.
Colorimetric determination refers to an
analytical technique best suited for
preliminaryconfirmations.Thistechniqueis
concerned about the measurement of
lightinthevisibleregion.Weseeobjects
becausetheytransmitorreflectacertain
portion of light in this region. When an
ordinary light is passed through the
object it absorbs certain wavelength,
leaving certain unabsorbed wavelength
to be transmitted. The transmitted light
orwecansaythetransmittedwavelength
47
48
Fig.4:StabilityprofileofNitrazepam
complexathightemperature(502C)
Fig.1:ScanofvariousNitrazepam
solutionsbetweenbetween420700
nM.
NR2,NR3andNR4havingstrengthof6,10,
12 and 24 g/ml, respectively, were
randomly prepared in triplicate and the
concentrations corresponding to these
solutions were determined with the help
of developed method. Statistical analysis
was performed with Graph Pad Instat
Software (version 3.0, Graph Pad
SoftwareSanDiego,California,USA)using
oneway ANOVA followed by Tukey
Kramer multiple comparison test.
Difference with P>0.05 was considered
statistically
insignificant,
whereas
P<0.001wasconsideredaverysignificant
difference. The results obtained in each
instance were compared with theoretical
value of 100.0% and represented as
meanSD;SEM(Table2).
Fig.2:StabilityprofileofNitrazepam
complexatlowtemperature(81C)
Accuracyandprecision
Fig.3:StabilityprofileofNitrazepam
complexatRoomtemperature
(252C)
RESULTSANDDISCUSSION
The motif behind the project was to
develop an accurate, precise, sensitive
and
reproducible
method
for
determination of Nitrazepam. For this,
colored complex of Nitrazepam was
formedwiththehelpofFeCl3inpresence
of NaOH, and its application in analytical
detection was explored. This formed
complex was intense yellow in color and
showed wavelength of maximum
absorbance (max) at 492 nm (Figure 1).
The stability of color as well as the
developed complex is a prerequisite
towardssuchmotif.Hence,thestabilityof
this complex was assessed under
differentconditions,whereinitwasfound
in
Report
Absorptionmaxima(max,nm)
492
LambertBeerslawrange(g/ml)
424
Regressionvalues:
Slope(m)
0.04
Intercept(c)
0.039
Coefficientofdetermination(R2)
0.999
NormalitytestKSvalue
0.1269
FischersextracttestPvalue.
0.6728
Table2:RecoverystudiesofstandardlaboratorysamplesofNitrazepam
Actualconcentrationof
druginsolution(g/ml)
Drugconcentration
recoveredafteranalysis
(g/ml)
NR1
6.05480.2865
100.804.628
NR2
10
9.88370.3295
98.8053.242
NR3
12
11.8940.4946
99.1214.114
NR4
24
23.3661.1887
97.3552.984
Code
%Recovery
valuesarerepresentedasMeanSD,(n=3);NR1,NR2,NR3,NR4andNR5signifyauthentic
laboratorysamples.
50
Table3:Establishmentofaccuracyandprecisionofthemethod
Nitrazepam
Composition
(g/ml)
2
N1+2
N2+4
N3+6
N4+8
Code
N1
N2
N3
N4
N5
Cumulative
strengthofmixture
(g/ml)
2
4
8
14
22
Observedcumulative
Strength(g/ml)
%Detection
1.830.69
3.690.85
7.650.58
15.011.1
21.70.99
95.75
96.12
97.75
105.05
98.5
valuesarerepresentedasMeanSD,(n=3);N1,N2,N3,N4andN5signifyauthenticlaboratory
sampleswithpredefinedoverages.
Table4:EstimationofNitrazepaminVariousMarketedTablets(n=3)
Brand
Name
Nitravet
Nitrosun
Nipam
Company
AngloFrench
SunPharma
LaPharma
Labelled
Claim/Tablet(mg)
5
10
5
AmountofNitrazepam
Detected(mg)
5.0580.1862
9.8120.1012
4.92750.1597
Nitrazepam
Recovery(%)
101.163.725
98.121.012
98.553.145
acute
and
chronic
effects
of
benzodiazepines. Psychol Med, 1987, 17,
6017.
5.
6.
7.
8.
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