International Journal of Pharmacy and Pharmaceutical Sciences

Vol 2, Issue 1, 2010

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

will be seen as color. The basic principle

of Colorimetric analysis is the
measurement of color of a solution,
whose intensity corresponds to its
concentration [8]. The colorimetric
methods of analysis are at present
undergoing considerable development
andmaybeappliedtoagreatnumberof
estimationswithadequateaccuracy.
EXPERIMENTAL
Chemicalsandreagents
Methanol(ARgrade)waspurchasedfrom
Qualigens Fine Chemicals (Mumbai,
India),NaOHwaspurchasedfromEMerk
(India)Ltd(Mumbai,India),HCl(Ranbaxy
Fine Chemicals Ltd, New Delhi, India),
Doubled distilled water was used
throughout the study. Nitrazepam was
received as a gift sample from M/s
Bennett Pharmaceuticals, Vadodara,
India. All other chemicals used were of
analyticalreagent(AR)grade.
Formation of colored complex and
determination of absorption maxima
(max)
Nitrazepam (40 mg) was accurately
weighed and dissolved in methanol (100
ml) to make a standard stock solution of
strength 400 g/ml. From this, six
standard subdilutions of concentrations
4, 8, 12 16, 20 and 24 g/ml were
prepared in 10 ml aliquot volumetric
flasks. To each volumetric flask, NaOH
solution(0.5M,0.2ml)andFeCl3(0.05M,
0.02 ml) were added, shaken uniformly
and kept for 30 minutes to allow
formation of colored complex. After 30
minutes, each aliquot was scanned over
the entire colorimetric range (420720
nm)employingSystronicsColorimeterand

the wavelength of maximum absorbance

(max)wasdetermined(Figure1).
Determinationofstabilityofcomplex
The stability testing of developed
complex is based on the principle of
chemical kinetics, and was assessed
underdifferentconditionsoftemperature
and light. For stability study, aliquots of
concentration2,4,8,12,16and24g/ml
werepreparedandcomplexwasdone,as
stated in previous section. The samples
were kept in transparent and amber
colored glass vials under low (80.5C),
intermediate
(room
temperature;
250.5C) and elevated (500.5C)
temperature conditions, in controlled
oven for varying period of time. The
samples were analyzed initially and then
periodically(0.25,0.5,1,2,4,6,8,10and
12hr)forabsorbancedetermination.The
degradation rate constant at various
temperaturesweredetermined.Thedata
was analyzed to infer the effect of time
as well as storage conditions on the
stability of the formed complex (Figure
24).
Preparation of standard calibration
curveandmethodvalidation
Standard aliquots of concentration 4, 8,
12,16,20and24g/mlwerescannedat
wavelength of maximum absorbance
(max), absorbance of solution was
determined and standard calibration
curve was plotted (Y=0.039X0.039;
R2=1), the regression analysis as well as
optical properties of which are depicted
in Table 1. Validation, accuracy and
presion of the developed analytical
method was done by performing
systematically designed studies, as
describedinfollowingsections.
Recoverystudies
For recovery studies four standard
laboratorysamplesofNitrazepamNR 1,

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)

For establishment of accuracy and

precision of the developed method,
standard
laboratory
samples
of
Nitrazepam having concentration of 2
g/mlprepared(N1).Thestrengthofthis
solution was gradually raised (applying
alligation medial method; Jain and
Sharma, 2000) to yield solutions of
strength 6, 10, 16 and 24 g/ml,
respectively, which were labeled as N2,
49

N3, N4 and N5. Finally, the cumulative

strength of these solutions was
determined by developed method and
percent Nitrazepam detected was
calculated (Table 3). Statistical analysis
wasdoneasstatedinprevioussection.
Estimation
of
Nitrazepam
commercialformulations

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

Twenty tablets were weighed and

powdered in a pestlemortar. The
quantity of the powder equivalent to
amount labeled for each tablet was
weighed, mixed with 5 ml of water in
conical flask and allowed to stand for 15
min. Then, HCl (0.5 ml, 0.5 M) and
methanol (quantity sufficient; q.s) were
added to produce 100 ml solution. This
solution was finally filtered to collect the
filtrate containing extracted Nitrazepam
(I.P1996, with slight modification).
Nitrazepam from different marketed
brandsviz:Nitravet5mg(AngloFrench);
Nitrosun10mg(SunPharma)andNipam
5 mg (LaPharma), were extracted and
appropriately diluted to determine the
absorbanceatmaxandthecorresponding
amount of Nitrazepam in formulations
(Table4).Statisticalanalysiswasdoneas
statedinprevioussection.

Table 1: Regression and Optical

CharacteristicsofNitrazepam
Parameters

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

that the complex was stable in low

temperature, and showed no change in
absorbance value throughout the study.
The solution was also found to be retain
its stability for resonable period of time,
whenstoredat252C(Figure2).But,it
shouldbenotedthatthecolorofcomplex
starts fading when exposed to higher
temperature. The order of stability of
developed complex was found to be
81C>252C>502C(Figure34).This
suggests that the exposure of these
colored solutions to high temperature
shouldbeavoidedduringtheanalysis.
The colorimetric method so developed
wasfoundtoobeyLambertBeerslawin
the concentration range of 424 g/ml,
withcoefficientofdeterminationcloserto
1 (Y=0.039X0.039, R2=0.999; Table 1).
To validate this method for its accuracy
and precision, systematically designed
studies were performed, wherein it was
found that the method may successfully
determine the concentration of unknown
samples closer to their theoretical
strength,asisevincedfromtheoutcomes
ofrecoverystudies(Table2).

AmountofNitrazepam
Detected(mg)
5.0580.1862
9.8120.1012
4.92750.1597

Nitrazepam
Recovery(%)
101.163.725
98.121.012
98.553.145

To establish the accuracy of method,

standard laboratory sample of known
strength(2g/ml)wasprepared,andthe
strength of this solution was raised
graduallyfrom2to22g/ml.Duringeach
addition, the corresponding increase in
concentration of solution was confirmed
by estimating the sample for drug
content. The method was found
satisfactory in detecting the cumulative
rise in strength of solutions between
95.75to105.05%(Table3).Similarly,the
recoverystudieswerealsoperformedon
commercial
formulations,
wherein,
Nitravet 5 mg (Anglo French); Nitrosun
10mg(SunPharma)andNipam5mg(La
Pharma) showed an average recovery of
101.163.725,
98.121.012
and
98.553.145; %SD, respectively (Table
4). In both the studies, the recovery was
foundcloserto100%,withsmallerSD.
Theoutcomerevealedthatthedeveloped
method is accurate, precise, rapid,
reproducible and economic for routine
detection of Nitrazepam in sample
solutions. During product development
research, as well believed to provide a
supplementary alternative for daytoday
51

analysis of Nitrazepam in academia

practical
based
on
Nitrazepam
formulations.
ACKNOWLEDGEMENTS
TheauthorsarethankfultoMr.Siddharth
Jain (Managing Director), M/s Bennett
Pharmaceuticals, Vadodara, Gujrat for
industrial input regarding the need to
develop such economical method for
Nitrazepam estimation. Also, the authors
are thankful the same firm for providing
thegiftsampleofNitrazepam.

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