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80
CHAPTER III
DIFFERENCE SPECTROPHOTOMETRIC TECHNIQUE
3.0 SPECTROPHOTOMETRIC ANALYSIS USING DIFFERENCE
ABSORBANCE/DIFFERENCE ABSORPTION RATIO METHODS.
3.1 INTRODUCTION (THEORY).
3.2 SECTIONA: SIMULTANEOUS SPECTROPHOTOMETRIC ANALYSIS
OF SOME BINARY MEXTURE OF DRUGS USING pHINDUCED
DIFFERENCE ABSORBANCE/DIFFERENCE ABSORBANCE RATIO
TECHNIQUES.
3.3 SECTIONB: SIMULTANEOUS SPECTROPHOTOMETRIC ANALYSIS
OF A TERNARY MIXTURE OF DRUGS USING pHINDUCED
DIFFERENCE ABSORBANCE/DIFFERENCE ABSORBANCE RATIO
TECHNIQUES.
3.4 SECTION C: DIFFERENCE SPECTROPHOTOMETRIC ANALYSIS
BASED ON REACTIONINDUCED SPECTRAL CHANGES.
81
CHAPTER III
3.0 SPECTROPHOTOMETRIC ANALYSIS USING DIFFERENCE
ABSORBANCE/DIFFERENCE ABSORPTION RATIO METHODS.
3.1 INTRODUCTION:
Difference spectrophotometry is applicable to acidic, basic or amphoteric
drug substances that undergo reproducible spectral changes due to pH changes or
the effect of reagents. It is a method of compensating spectral interferences of
interfering components. It involves the measurement of the absorbance difference
at a defined wavelength between two equimolar solutions, one of which a physical
or chemical property of the drug to be determined has been changed, provided the
absorbance of interfering components remain unaltered. This permits estimation of
components of multicomponent drug mixtures without prior separation.(13)
THEORY
Difference spectra of a compound can be produced by two different ways:
(i) By change pH of two equimolar solutions; a pHinduced difference
spectrophotometry.
(ii) By alteration of chemical composition of one of the two equimolar
solutions; Reactioninduced difference spectrophotometry.
pHinduced: Changes in pH have been exploited more than any other change of
chemical conditions, when the differential method is applied, specially for
compounds showing bathochromic or hyperchromic shift together with
hypochromic or hypsochromic effect. If such shifts occur, the presence of a pH
insensitive irrelevant absorption ‘Z’ may be cancelled by changing the solvent
from ‘a’ (eg. acidic solvent) to ‘b’ eg. basic solvent). Thus, A = (Aa + Z) (Ab+Z).
In fact spectral changes are induced by simple reversible ionization of
groups directly conjugated to a chromophore. Thus the difference absorbance
82
method is based on the absorbance measurement of the ionic from against the
molecular form of the drug. This permits the determination of active components
without any interference from other coexisting components, not sensitive to pH
change.
It is important that acid or base solutions be selected so that both are at least
two pH units removed from the pKa, on opposite sides of this value. At pH values
closer to the pKa, small pH changes may result in appreciable changes in the
difference spectrum, resulting serious errors. The difference spectrum taken in the
usual hydrochloric acid or sodium hydroxide solutions is known to result from
pure cationic and anionic species and may, therefore, be used with confidence
despite the complexity of the intermediate transformations.
Reaction induced: While most instances of difference spectrum involve pH
effects, spectral changes may also results from factors other than ionization. These
permit sample and reference solutions to be prepared at an identical pH, with
improved likelihood for cancellation of interferences. The most suitable reactions
are rapid, clean, complete and employ mild reagents that are transparent over the
spectral region of interest. If both the drugs and its reaction product have distinct
spectra of comparable intensity, then isoabsorptive point should be produced.
In application of the method, the hydrolysis product in alkaline or acidic
medium can be read against the intact drug in a suitable medium maintaining its
stability. A superior procedure is to adjust the reaction solution back to the
medium of the intact drug, after complete hydrolysis.
If the hydrolysis is temperature controlled, then order of addition of reagents
remain the same but, heating is omitted for the unhydrolysed solution. If the
hydrolysis took place at normal temperature, then order of addition of reagents is
altered for the unhydrolysed solution. These may results a pronounced
83
reproducible difference spectra between hydrolysed and unhydrolysed solutions of
equimolar concentrations.
Application of DifferenceAbsorbance
If, in the determination of a component ‘X’, spectral interference of other
component ‘Y’ is totally cancelled at the measuring wavelength ‘I’ of the
component ‘X’. Then concentration ‘C’ of ‘X’ is calculated by direct correlation
of absorbance difference values of the test solution to that of the standard
solution within the linearity range, as under:
A1 test / A1 standard = Ctest/Cstandard …. (E24)
Application of Difference Absorbance Ratio
If, in the determination of a component ‘X’ at measuring wavelength ‘I’, the
other component ‘Y’ contributes +ve or ve absorbancedifference, and if the
component ‘X’ does not interfere in determination of ‘Y’ at the wavelength ‘2’,
then application of the absorbance ratio equation (E9; Chapter II, 2.1) is extended
to differenceabsorbance ratio equation as follows:
CX =
2
12
11
AA
xAAAC … (E25)
where ‘α’ and ‘β’ relates to standard solutions of ‘X’ ‘Y’ respectively.
84
CHAPTER III
SECTIONA
3.2 SIMULTANEOUS SPECTROPHOTOMETRIC ANALYSIS OF SOME
BINARY MEXTURE OF DRUGS USING pHINDUCED DIFFERENCE
ABSORBANCE/DIFFERENCE ABSORBANCE RATIO TECHNIQUES
3.2.1 INTRODUCTION:
In this section simultaneous spectrophotometric(47) determination of the
following combinations of drugs, using pHinduced difference absorbance/
difference absorbance ratio techniques have been described categorically:
(A) Epirubicin/Epirubicin benzoateMitoxantrone (M2)
As detailed under Chapter II, Section A, 2.2.1 (B).
The present investigation deals with difference spectrophotometric
determination of EPB or EPBB as EPB, and direct spectrophotometric determination
of MTS, which are applicable for simultaneous determination of both the components
without prior separation.
(B) MercaptopurineEpirubicin/Fudarabine Phosphate (M3)
As described under ChapterII, Section A, 2.2.1 (C). The present investigation
deals with simultaneous difference spectrophotometric determination of both the
components.
(C) VinblastineVincristine (M5)
As described under Chapter II, Section A, 2.2.1 (E). The present investigation
deals with simultaneous determination of both the components using difference
absorbance and difference absorbance ratio techniques.
85
(D) PaceitaxelAltretamine (M8)
Combination of Paceitaxel (PCT) and Altretamine (ALT) are available in the
form of tablets and are used for the treatment of rheumatoid arthritis. PCT and ALT
can not be estimated by the official(810) alkalimetric titration, in their combination
due to mutual interference. Petrova et.al.(11) reported a direct spectrophotometric
method for estimation of ALT without any interference of PCT, however, ALT did
interfere in the estimation of PCT upto a certain extent. The difference
spectrophotometric procedures(1214) for estimation of PCT are not applicable in
combination because of mutual interference.(1517)
The present investigation deals with difference spectrophotometric procedure
for simultaneous determination of both the components without prior separation.
(E) Sulphamethoxy Pyridazine (M9)
Combined dosage form of sulphamethoxy pyridazine (SMPZ) and
Pyrimethamine (PYM) is official in U.S.P.(10), and is used for the prophylactic and
suppressive treatment of malaria. The assay procedure described in the U.S.P.
involves HPLC determination of each component. Parimoo(18) has reported an
absorption spectrophotometric procedure for determination of both the components,
which is less sensitive and require predetermination of several factors with different
pH media. SMPZ can also be determined by the official(8) titrimetric procedure.
In the present investigation, a difference spectrophotometric method have been
developed for rapid and accurate determination at SMPZ in formulation without prior
separation.
3.2.2 EXPERIMENTAL:
Apparatus
Spectrophotometers: (1) Beckman 24 UV/Visible spectrophotometer with recorder
and (2) Hitachi 15020 recording spectrophotometer with 1cm matched silica cells.
86
Operating Parameters: Scanning range: 380220 and 360200 nm; scan speed, 100
nm, min1; Ordinate; (+ 0.7) (0.3) and (+ 1.4)(0.6).
Procedure
A general procedure applicable to each combination is described below. The
corresponding experimental details regarding each combination are mentioned in
Table 3.13.3.
Standard stock solutions: ‘X’ mcg/ml in solvent (S1) of each component.
Standard determination: Three 2.0 ml aliquots of each of the standard stock
solution were separately diluted to 50 ml with solvent (S2) [Solution A], solvent (S3)
[Solution B] and solvent (S4) [Solution W]. (Table3.1), and difference absorption
spectra of the solutions were recorded, using one of the solution as blank as given in
the Table 3.2. The wavelength of maximum absorbancedifference (1 nm) of
(2nm), was selected for determination of the component in the formulation under the
measuring conditions (Table 3.2). Measured standard A values of the components
(Astd) at the wavelength of measurement. The appropriate solvent corrections were
carried out at the respective wavelength and the net A values of each substance were
calculated as the average of five determinations.
Sample determination: Appropriate amount of the sample equivalent to about (X1)
mg. of component (C1) was extracted with about 70 ml of solvent (S5), filtered if
necessary by washing the residue with small portions of the solvent and the combined
filtrate diluted to 100 ml with the solvent (S5). Three 2ml aliquots (for the
combination M2, the aliquot taken after diluting 25 ml to 100 ml with methanol) of
the solution were separately diluted to 50 ml with the solvent S2, S3 and S4 (Table 3.1)
and measured A of the resulting solutions as applicable to each combination, for the
standard determination (Table 3.2). Carried out the appropriate solvent corrections.
87
Calculation of results: For the combination M2, M3, M8 & M9, content of each
component was calculated by direct correlation of A values of the sample solution
to that of the standard solution at the respective wavelength as per the equation (E24).
For the combination M5, content of VIC was calculated as per the equation
(E24) at 246.5 nm, whereas VIB was calculated by application of the difference
absorbance ratio equation (E25). Where the subscripts ‘1’ and ‘2’ refer to the
wavelengths 267.5 nm and 246.5 nm respectively; A1 and A2 are the A values
of the sample solution at the respective wavelengths; Aβ1/Aβ2 is the ratio of A
values of the standard VIC solution at the respective wavelength with ve sign; Aα1
and C are the A value and concentration of the standard VIB solution respectively.
RESULTS AND DISCUSSIONS
pHinduced spectral characteristics: The specificity of the difference spectro
photometric assays of these formulations are due to pHinduced spectral changes
exhibited by the components, in acidic, basic and neutral medium. EPB, EPBB,
FLDP, VIB, VIC, PCT, ALT, SMPZ and PYM display bathochromic shift together
with hyperchromic effect from acidic to alkaline media, whereas MEP shows slight
hypsochromic shift from acidic to alkaline media and MTS, do not undergo such
changes. It has been observed that in acidic methanol, there is slight hyperchromic
shift in the absorption of MTS (max 358 nm) and consequently the absorbance of
EPB/EPBB solution in the solvent become regligible at 358 nm (Fig. 3.2), which
permitted determination of MTS at 358 nm, in acidic methanol without interference
of EPB/EPBB. It has also been observed that EPBB solution has lower A value at
322 nm (Fig. 3.1) than the solution having equivalent amount of pure EPB (i.e. 250
mcg/ml of EPB and 402.015 mcg/ml of EPBB). This is because slight variation in
spectral characteristics of both the base and ester form in acidic and aqueous media,
which necessitated the use of reference EPBB separately.
88
Choice of wavelengths: Choice of wavelengths are based on consideration of factors
as detailed under Chapter I (1.3.6). In the combinations M2, M3, M8 & M9, when ‘the
components were measured under the conditions described in the Table 3.2, they
exhibited maximum A value at the wavelength of measurement (Fig. 3.1, 3.3, 3.4,
3.6 & 3.7), whereas the other components in the combinations exhibited zero A
value when measured similarly at the same wavelength. In the combination (M9),
although, SMPZ exhibited isoabsorptive point of zeroA at 295 nm, which is near
the A maximum of PYM, at 293 nm (Fig. 3.7), but determination of PYM was not
possible, as very low fraction of PYM led to greater degree of error.
In the combination M5, VIB exhibited maximum A value at 267.5 nm and an
isoabsorptive point of zeroA at 246.5 nm, whereas VIC under similar condition
exhibited almost maximum A at 246.5 nm and negative A at 267.5 nm (Fig. 3.5).
Thus VIC was estimated at 246.5 nm without any interference of VIB, whereas VIB
was estimated at 267.5 nm, by application of the vector sum of the positive A due to
VIB and negative A due to VIC.
Selection of solvents: Selection of acidic and basic solvents of suitable strength were
based on studies as described under Chapter I (1.3.6). Plot of absorptivity versus
different measuring media (Fig. 3.83.12) reveal the range of different strength of
acids and bases within which the components exhibit constant A value. The ranges
are tabulated below:
89
Component Acidic range
(HCl) strength
Basic range
(NaOH) strength
(W: neutral solution in water)
1 2 3
EPB/EPBB 0.05 N 0.2 N W 0.15 N
FLDP 0.1 N 0.2 N W 0.1 N
MEP W 0.2 N 0.01 N 0.15 N
VIC W 0.1 N 0.01 N 0.1 N
PCT 0.01 N 0.2 N W 0.2 N
ALT W 0.2 N 0.01 N 0.2 N
SMPZ 0.2 N 0.5 N 0.01N 0.1 N
PYM W 0.5 N W 0.1 N
The strength of acids and bases chosen for analysis as given under Table 3.1
are within these ranges. It was also taken into consideration that the selected strength
was applicable to both the components and the components were sufficiently stable
(>90 min) in the media. For the combination M2, 0.01 N HCl was selected for
EPB/EPBB determination, because no appreciable difference in absorptivity value
was observed when measured in 0.01 N and 0.05 N HCl, however, MTS was found
more stable in 0.01 N HCl. MTS was also found more stable in acidic methanol than
in acidic aqueous solution. On the absis of this study, the solvents used in the analysis
were 0.01N HCl (pH~2); 0.02 N HCl (pH~1.7); 0.1 N HCl (pH~1.0); 0.2 N HCl
(pH~0.7); 0.02 N NaOH (pH~12.5); 0.1 N NaOH (pH~13); Water (pH~7).
Adherence to Beer’s Law: The components in the combinations exhibited linear,
relationship between A versus concentration within the following concentration
range:
90
Combination Component Beer’s law range (mcg/ml)
1 2 3
M2 EPB/EPBB
MTS
420
214
M3 MEP
EPB
FLDP
218
420
424
M5 VIB & VIC 420
M8 PCT & ALT 418
M9 SMPZ 416
Interference studies: Possible presence of interfering substances, depending on
the nature of formulation, were studied as described under Chapter 1 (1.3.11) and
were found to have no interference to the methods. Further to confirm any
intereference from formulation matrix graphs of log A versus were plotted, for
samples as well as authentic mixtures of the same ratio, as described under
Chapter I (1.3.6). The graphs were superimposable indicating that the methods
nullify any nonspecific irrelevant absorption due to formulation matrix which
may affect the accuracy of results. In the combination M3, presence of degradation
product will interfere.
Precision, Accuracy and Specificity: To prove the validity and applicability of
the proposed methods synthetic drug mixtures at different drug ratios and
commercial dosage forms were analysed by the developed methods. The
specificity and validity of the applicable equations were assessed by calculating
the recovery of the synthetic mixtures. The methods were also subjected to
recovery studies by adding known amount of the drugs to the preanalysed samples.
The analytical results (Table 2.62.8, 2.10, 3.4 & 3.5) represent that the mean %
91
RSD is less than 2 and mean per cent recoveries are between 98102%, indicating
that the methods are precise, accurate and reproducible.
CONCLUSION
The developed methods are fast, accurate, precise and can conveniently be
applied to control analysis of commercial formulations.
92
TABLE 3.1: Experimental Details of pHinduced Difference Spectrophotometric Method (3.2.2)
(Standard Stock Solutions)
Combination Concentration
(mcg/ml)(X)
Solvents
(S1) (S2)
(Solution A)
(S3)
(Solution B)
(S4)
(Solution W)
(M2) EPB/EPBBMTS 250
(402.015)*
DMF+MeOH
(1:3)**
0.01N HCl 0.1N HCl
in MeOH
Water
(M3) MEPEPB
MEPFLDP
250
250
MeOH
MeOH
0.1N HCl
0.1N HCl
0.1N NaOH
0.02 N NaOH
Water
Water
(M5) VIBVIC 250 MeOH 0.02 N HCl 0.02N NaOH Water
(M8) PCTALT 250 MeOH 0.1N HCl 0.1N NaOH Water
(M9) SMPZPYM 250 MeOH 0.2N HCl 0.02N NaOH Water
* : 402.015 mcg EPBB equivalent to 250 mcg EPB.
** : Dissolved in 25 ml of DMF, then diluted with MeOH.
93
TABLE 3.2: Experimental Details of pHinduced Difference Spectrophotometeric Method (3.2.2)
(Standard Determination)
Combination Determination
of component
Wavelength
(nm) (1)
A Measurement Wavelength
(nm) (2)
A Measurement
Soln. §
(Samp)
Vs. Soln. §
(Ref.)
Soln. §
(Samp)
Vs. Soln. §
(Ref.)
M2 EPB/EPBB
MTS
322
322
(W)
Vs.
(A)
358
(B)
Vs.
(C)*
M3 MEP
EPB
FLDP
267
267
267
(W)
(W)
(W)
Vs.
(B)**
322
320
(W)
(W)
Vs.
Vs.
(A)
(A)
M5 VIB
VIC
267
246.5
(W)
(B)
Vs.
(A)
267.5
267.5
(B)
(B)
Vs.
Vs.
(A)
(A)
M8 PCT
ALT
266
266
(W)
(W)
Vs.
Vs.
(A)
330
(B)
Vs.
(W)
M9 SMPZ 275 (B) Vs. (A)
§ : Solution (A), (B) & (W) as per the Table 3.1.
* : (C) = 1% DMF in 0.1 N methanolic HCl as solvent blank.
** : Alkaline solution of the respective combination used.
94
TABLE 3.3: Experimental Details of pHinduced Difference Spectrophotometeric Method (3.2.2).
(Sample Determination)
Combination Amount (X1) (mg) Component (C1) Solvent (S5) Fig. No.
M2 100 EPB DMF 3.1 & 3.2
M3 25 EPB / FLDP MeOH 3.3 & 3.4
M5 25 VIC MeOH 3.5
M8 25 ALT MeOH 3.6
M9 25 SMPZ MeOH 3.7
95
TABLE 3.4: Results of the Estimation of Paceitaxel and Altretamine Commerical Formulations by Difference
Spectrophotometric Method
Formulation Paceitaxel Altretamine
Claimed
(mg)
Found*
(mg)S.D.
Added
(mg)
Rec. (%) Claimed
(mg)
Found*
(mg)S.D.
Added
(mg)
Rec. (%)
T1 125 124.780.50 20 98.9 125 124.970.48 30 99.9
T2 100 98.82 0.46 25 99.3 250 249.9 0.80 25 99.7
T3 100 99.76 0.48 20 98.8 250 249.721.01 20 98.6
SM1 100 99.73 0.42 10 97.30 100 99.8 1.01 10 98.0
Mean 98.58 99.5
* : Average of five determinstions.
T1 T3: Tablets (mg/tablet)
SM1 : Laboratory preparation containing Paceitaxel and Altretamine
96
TABLE 3.5: Results of the Estimation of Sulphamethoxy pyridazine in Commercial Formulations Containing
Pyrimethamine by FirstDerivative (D1) and Difference (A) Spectrophotometric Methods.
Formulation Sulphamethoxy Pyridazine
Found* (% Claim S.D.)
Claimed (mg) A Method Official Method D1 Method
T1 500 94.04 0.74 94.56 0.36 94.94 0.83
T2 500 98.21 0.58 98.85 0.52 99.79 0.48
T3 500 103.19 0.57 103.76 0.31 104.46 0.70
tvalue (1.68) (2.05)
Fvalue (2.81) (3.63)
SM1 100 99.15 0.55 98.69 0.61
SM2 50 99.86 0.47 101.04 0.57
Mean 99.51 99.87
* : Average of five determinstions.
T1 T3 : Tablets (mg/tablet) contain 25 mg of PYM.
SM1SM2 : Standard Laboratory mixtures; Contain 10 mg of PYM.
97
CHAPTERIII
SECTIONA
1. G.D. Gupta and R.S.Gound, Ind. J. Pharm. Sci. 61 (1999) 229234.
2. D.N.Tipre & P.R.Vavia, Ind. Drugs. 37 (2000) 412416.
3. A.Markhan, G.L.Ploskar and K.L.Goa, Drugs, 60 (2000) 955974.
4. B.M.Gurupadayya, B.V.Vijaya, Y.N.Manohara, S.Hemalatha, Ind. Drugs.
45 (3) (2008) 19398.
5. A.Somers, M.Petrovic, H.Robays, M.Bogaert, Eur. J. Clin Pharmacol. 58
(2003) 707714.
6. A.V.Vedrana, T.Uladimir, L.Zdravko, Croat. Med. J. 46 (1) (2005) 7480.
7. E.Abahussain, L.K.Matowe, P.J.Nichols, Med. Princ. Pract. 14 (2005)
161164.
8. Pharmacopoeia of India 3rd Ed., Controller of Publications, Delhji (1985).
9. British Pharmacopoeia, University Press, Cambridge, 1980.
10. The United States Pharmacopoeia/National Formulary, United States
Pharmacopoeial Convention, Rock Ville, 1985.
11. Ya. Patrova, Izv. Durzh, Inst. Kontrol Lek, Sredstva 12 (1979) 3845; Anal.
Abst. 40(2) (1981) E38.
12. H.Abdine, M.A.H. Elasayed and M.E.Abdel Hamid, Ind. J. Pharm. Sci. 41
(3) 1979) 118120.
13. V.G.Belikov, E.N.Vergeichik, S.Mutsuevakh 33 (2) (1984) 4446; Throu.
Anal. Abst; 47(2) (1985) E31.
14. Z.Berakova, M.Bachrata, M.Blesova, and L.Knazko, Farm Obz. 49 (1980)
157167; Throu. Anal. Abstr. 39 (5) (1980) E40.
15. S.Bang, S.Sontakke, V.Thauwani, Ind. J. Pharm. 43 (2011) 275277.
98
16. S.D.Sontakke, C.S.Bajait, S.A.Pimpal Khute, K.M.Jaiswal, Int. J. Biomed.
Res. 2(2) (2011) 561564.
17. M.S.Uma Shankar, A.Aruna, V.V. Satish Madhav and M.K.Ranganathan,
Jour. of Pharm. Res. No. 2 Vol. 11 (2012) 6770.
18. P.Parimoo, Ind. J. Pharm. Sci. 49 (1987) (1) 2829.
99
CHAPTER III
(SECTIONB)
3.3 SIMULTANEOUS SPECTROPHOTOMETRIC ANALYSIS OF A
TERNARY MIXTURE OF DRUGS USING pHINDUCED DIFFERENCE
ABSORBANCE/DIFFERENCE ABSORBANCE RATIO TECHNIQUES.
3.3.1 INTRODUCTION:
In this section simultaneous spectrophotometric estimation of salicylamide.
Propyphenazone and Pyrithyldione, based on pHinduced difference
spectrophotometric technique has been described. The applicability of the
technique for binary mixtures, as described in the previous section, has been
extended to a ternary mixture.
Salicylamide (SAM) is official in I.P.(1) and, Propyphenazone (PPZ) and
Pyrithyldione (PDO) in E.P.(2). Combined dosage forms of SAMPPZ (Anafebrin)
and SAMPPZPDO containing Caffeine (Saridon) are available in the form of
tablets, and are commonly used for their analgesic and antipyretic action.
Literature review(36) reveals that so far on extraction followed by
spectrophotometric(7) and HPLC(813) methods for the combination containing PDO
and Caffine. Direct spectrophotometric and simultaneous spectrophotometry using
absorbance ratio techniques are not possible for the combinations because of
mutual interference and nonfulfillment of proper assay conditions.
In the present investigations, a difference spectrophotometric method has
been developed for simultaneous determination of SAM, PPZ and PDO in
presence of Caffeine, without prior separation.
3.3.2 EXPERIMENTAL:
Apparatus
Spectrophotometer: As described under Section A of this Chapter (3.2.2).
100
Procedure
Standard Stock Solutions: 250 mcg/ml of SAM, PPZ, PDO and Caffeine,
separately in methanol.
Standard determination: Three 2.0 ml aliquots of each of the standard stock
solution were separately diluted to 50 ml with Water, 0.1 N NaOH and 0.5 N HCl.
Recorded A spectra (Fig. 3.13 & 3.14) and simultaneously measured absorbance
at the wavelength of maximum difference absorbance of each of the standard
solutions, as follows:
The solution of SAM in alkali at 267.5 nm, relative to that of the solution in
water. The solution of PDO in alkali at 267.5 nm and 365 nm, relative to that of
the solution in water; the solution of PPZ in water at 271 nm relative to that of the
acidic solution. The appropriate solvent corrections were carried out at the
respective wavelengths and average A values of each substance were calculated
as the average of five determinations.
Sample determination: A quantity of the powdered tablets equivalent to about
30mg of SAM was accurately weighed and extracted with about 70 ml of
methanol, filtered by washing the residue with small portions of methanol and the
combined filtrate diluted to 100 ml with methanol. Three 2.0 ml aliquots of the
resulting solution were separately diluted to 50 ml with water, 0.1 N NaOH and
0.5 N HCl.
The absorbance of the resulting solution in water was measured at 271 nm
relative to that of the acidic solution and of the alkaline solution at 267.5 nm and
365 nm (in presence of PDO) relative to that of the solution in water. The
appropriate solvent corrections were carried out.
Calculation of results: For the SAMPPZ combination, contents of SAM and
PPZ were calculated by direct correlation of the A values of the sample solution
101
to that of the standard solution measured at 267.5 nm and 271 nm respectively, as
per the equation (E24).
For the SAMPPZPDO combination, contents of PPZ and PDO were
calculated by direct correlation of the A values of the sample solution to that of
the standard solution, measured at 271 nm and 365 nm respectively, whereas SAM
was calculated by application of the difference absorbance ratio equation (E25),
where the subscripts ‘1’ and ‘2’ refer to wavelengths 267.5 nm and 365 nm
respectively. C and A1 denote concentration and A value at 267.5 nm of the
standard SAM solution respectively; Aβ1/Aβ2 is the ratio of A values of
standard PDO solution at the respective wavelength with ve sign; A1 and A2
are the A values of the sample solution at the respective wavelengths.
RESULTS AND DISCUSSIONS
pH induced spectral characteristics: The selectivity of the difference
spectrophotometric estimation of SAM, PPZ and PDO are based on the use of
pHinduced spectral changes. SAM, PPZ and PDO display bathochromic shift
together with hyperchromic effect from acidic to alkaline media, whereas no such
spectral change is observed from acidic to neutral media with SAM and PDO and
from neutral to alkaline media with PPZ.(14) (Fig. 3.153.17).
Choice of wavelengths: Choices of wavelengths are based on consideration of
factors as detailed under Chapter 1 (1.3.6). The A spectra (Fig. 3.13) of the
alkaline solution of SAM relative to its identical solution in water shows
maximum A at 267.5 nm; PDO under identical condition exhibit maximum A
at 365 nm and negative A at 267.5 nm, whereas PPZ under the identical
condition shows zeroA value at 267.5 nm and 365 nm. Similarly the A spectra
(Fig. 3.14) of PPZ solution in water relative to the acidic solution exhibit
maximum A at 271 nm whereas SAM and PDO under similar condition shows
102
zeroA value at 271 nm. Thus PDO and PPZ were estimated at 365 nm and 271
nm respectively without any mutual interference whereas SAM was estimated at
267.5 nm, by application of the vector sum of the positive A due to SAM and
negative A due to PDO. In the combination SAMPPZ, both are estimated by
direct correlation of the standard and sample solutions without any mutual
interference.
Selection of solvents: Selection of acidic and basic solvents of suitable strength
are based on the studies as described under Chapter 1. (1.3.6). Plot of absorptivity
versus different measuring media (Fig. 3.153.17) reveal that at 267.5 nm, 271 nm
and 365 nm both SAM and PPZ have constant absorptivity values from 0.01 N to
0.2 N NaOH, whereas PDO exhibit similar behaviour from 0.07 N to 0.2 N NaOH.
Similarly SAM and PDO have constant absorptivity values from 0.01 N to 0.6 N
HCl, whereas PPZ exhibit similar behaviour from 0.45 N to 0.6 N HCl. Thus water
(pH~7), 0.1 N NaOH and 0.5 N HCl were chosen, which are within these
ranges.(1518)
Adherence to Beer’s Law: SAM and PPZ exhibit linear relationship between A
versus concentration within the range 420 mcg/ml whereas PDO in the range
214 mcg/ml.
Interference Studies: Possible presence of interfering substances, were studied as
described under Chapter 1 (1.3.11), and were found to have no interference to the
methods. Caffeine shows slight interference (about 2%) in the estimation of PPZ
(Fig. 3.14), whereas no such interference was observed in the determination of
SAM and PDO (Fig. 3.13).
Precision, Accuracy and Specificity: To prove the validity and applicability of
the proposed method, synthetic drug mixtures at different drug ratios and
commercial dosage forms were analysed. The specificity and validity of the
applicable equations were assessed by calculating the recovery of the synthetic
103
mixtures. The analytical results (Table 3.6) represent that mean % RSD is less than
2 and mean per cent recovery of the synthetic mixtures are between 98103%,
indicating that the proposed method is precise, accurate and reproducible, except
for the estimation of PPZ in presence of Caffeine.
CONCLUSION
The developed method is suitable for simultaneous determination of the
components without prior separation.
104
TABLE: 3.6: Results of the estimation of Propyphenazone, Salicylamide and Pyrithyldione in Formulations by Difference
(A) and DerivativeDifference (D1) Spectrophotometric Methods.
Formulation Propyphenazone Salicylamide Pyrithyldione
Claimed (mg) Found* (% Claim S.D.) Claimed
(mg)
Found* (%
Claim S.D.)
Claimed
(mg)
Found* (%
Claim S.D.) A Method D1 Method
T1 150 103.60.89 101.81.29 250 99.6 1.10 50 105.10.60
T2 150 101.40.76 98.4 1.23 250 97.7 0.83 50 104.8 0.56
T3 300 98.7 0.45 99.5 0.97 250 101.2 0.53
SM1 150 102.3 0.69 99.06 1.10 250 100.8 0.68 50 101.1 0.75
SM2 50 99.3 0.64 98.7 1.28 50 98.7 0.59
* : Average of five determinations.
T1 & T2 : Saridon Tablets; contain 46.75 mg Caffeine.
T3 : Anafebrin Tablets
SM1 : Synthetic mixture containing 46.75 mg Caffeine.
SM2 : Synthetic mixture without Pyrithyldione.
105
CHAPTERIII
SECTIONB
1. Pharmacopoeia of India, 3rd Ed. Controller of Publications, Delhi (1985).
2. Martindale, The Extra Pharmacopoeia, 28th Ed., The Pharmaceutical Press,
London.
3. B.H.M. Mruthiunja Swamy et.al., Ind. J. Pharm. Sci. 63(5) (2001) 433.
4. S.S.Zarapkar, N.P.Bhandari, Uphalkar, Ind. Drugs (2000) 295298.
5. Saranjit Singh et.al., Int. J. Pharm. 37 (2002) 245.
6. A.Bootz, et.al., Eur. J. Pharm. Bio. Pharm. 57 (2004) 369.
7. M.Peterkova, O.Matonsova and B.Kakaecesk Form 30(8) (1981) 270273;
Anal. Abst. 42(4) (1982) E44.
8. M.G.Mamalo, L.Vio and V.Maurich, J. Pharm. Biomed. Anal 3 (2) (1985)
157164.
9. J.K.Lalla, P.D.Hamrapukar & H.M.Mamania, Ind. Drugs 38 (2004) 87.
10. S.Manimaran, T.Subbaraju and B.Suresh, Ind. Drugs 49 (2003) 532.
11. N.J.Shah et.al., Ind. J. Pharm. Sci. 69 (2007).
12. D.R.Mehta, R.S.Mehta, K.K.Bhatt & M.B.Shankar, Ind. Drugs 42 (2005)39.
13. A.Biswa, Rita Mahanta, S.K.Bandyopadhyay and S.K.Bhattacharjee, Jour.
of Pharm. Res. Vol. 8, No. 2 (2009) 108111.
14. R.Revalthi, T.Ethiraj, L.Jhansi, Mareddy, V.Ganeshan, J. Pharm. Educ. Res.
Vol. 2, No. 2 (2011) 7177.
15. S.Sharma, Pharmazie 61 (2006) 495504.
16. A.M.Dyer, M.Hinchliffe, P.Watts, J.Castile, I.Jabbal Gill, R.Nankervis et.al,
Pharm. Res. 19 (2002) 9981008.
17. L.Illum, A.N.Fisher, I.Jabbal Gill, S.S.Davis, Int. J. Pharm. 222 (2001)
10919.
18. Hussain, T.Yang, A.A.Zaghloul, F.Ashan, Pharm. Res. 20 (2003) 15517.
106
CHAPTER III
SECTION C
3.4 DIFFERENCE SPECTROPHOTOMETRIC ANALYSIS BASED ON
REACTIONINDUCED SPECTRAL CHANGES
3.4.1 INTRODUCTION:
In this section an application of difference spectrophotometric technique
based on reaction induced spectral changes, for the determination of Frusemide in
presence of its degradation products, has been described.
Frusemide (FSM), and its dosage forms as tablets and injections are official
in I.P.(1), B.P.(2) and U.S.P.(3). The official direct spectrophotometric assay
procedure in alkaline medium is not selective for FSM in presence of its
degradation products, as 4Chloro5sulphamoylanthranilic acid (CSAA), the
major degradation product, interferes in the assay. Other reported methods are
visible spectrophotometric(46), n.m.r.(79), Xray diffraction(1013), Orthogonal
functions(14) and HPLC(15). The HPLC method has been reported(16) selective for
FSM in presence of the CSAA.
In the present investigation a difference spectrophotometric method has
been developed for the estimation of FSM in formulations, which overcomes the
nonspecificity of the direct spectrophotometric method in presence of degradation
products.
3.4.2 EXPERIMENTAL:
Apparatus
Spectrophotometers: As described under Section A of this Chapter (3.2.2).
Procedure
Standard stock solutions: 250 mcg/ml of FSM and CSAA, separately in
methanol.
107
Standard determination: Two 2.0ml aliquots of the FSM stock solution were
transferred separately into two 50 ml volumetric flasks (‘A’ & ‘B’). To the flask
‘A’, subsequently added 10 ml of 1 N HCl, 12 ml of 1 N NaOH and made upto
volume with 0.1 N NaOH (Solution S). To the flask ‘B’, added 10 ml of 1 N HCl,
heated on waterbath for 25 min, cooled, added 12 ml of 1 N NaOH and diluted to
volume with 0.1 N NaOH (Solution B). The absorbance of the ‘Solution S’ was
measured against the ‘Solution B’ as blank at 274.5 nm (Astd).
Sample determination: A suitable amount of sample was dissolved and diluted
with methanol to get a concentration of about 250 mcg/ml of FSM, filtered, if
necessary. The preparation of the solutions ‘S’ & ‘B’ and measurement of the
(Asamp.) were performed as described under the standard determination.
Calculation of results: The content of FSM was calculated by direct correlation
of the A values of the sample solutions (Asamp.) to that of the standard solutions
(Astd) at 274.5 nm.
RESULTS AND DISCUSSIONS
Reactioninduced spectral characteristics: Spectral interference of the
degradation products in the conventional spectrophotometric determination of
FSM, has been verified by spectral studies with the CSAA and hydrolysed FSM
(Solution B) solutions (Fig. 3.18). FSM on hydrolysis is converted into its
degradation products, furfuryl alcohol and CSAA. The hydrolysed solution
(Solution B) relative to the solution of intact drug (Solution S) exhibit
hypsochromic shift together with hypochromic effect. These results a pronounced
reproducible difference spectra between unhydrolysed and hydrolysed solutions of
equimolar concentrations.
Choice of wavelengths: When the ‘Solution S’ was measured against the
‘Solution B’, maximum A was observed at 235 nm and 274.5 nm (Fig. 3.19).
When the stock solution of CSAA was subjected to A measurement as per the
108
FSM solution, zeroA values were observed at these wavelengths. The results
were found more accurate and precise at 274.5 nm, thus the intact drug could be
determined selectivity without any interference of degradation products at
274.5nm.
Selection of solvents and heating parameters: FSM exhibit stable spectral
characteristics in alkaline medium (0.1 N NaOH) and readily undergo hydrolysis
in acidic medium on heating. Thus the blank solution was subjected to hydrolysis
by heating after addition of 1 N HCl and then brought to the alkaline medium by
neutralization with 1 N NaOH and subsequent addition of 0.1 N NaOH. It was
observed that the hydrolysis was temperature controlled. Thus order of addition of
reagents in the sample solution remained the same but heating was omitted.
For fixation of heating interval, the ‘Solution B’ was prepared after
hydrolysis at different time intervals and A values were measured. It was
observed that complete hydrolysis took place after 15 min. Thus 25 min was fixed
for preparation of ‘Solution B’ in order to ensure complete hydrolysis.
Adherence to Beer’s law: FSM exhibit linear relationship between A versus
concentration within the range 422 mcg/ml.
Selectivity studies: In order to illustrate the selectivity of the method, the
preanalysed samples were subjected to analysis after addition of CSAA and
hydrolysed frusemide solution. The results were found unaffected by the
degradation products. Common pharmaceutical aids do not interfere.
Precision and accuracy: To prove the validity and applicability of the developed
method, four commercial formulations were analysed by the method. The
analytical results (Table 3.7) represent that mean % RSD is less than 2. The
method was also subjected to recovery studies by adding known amount of
standard FSM to the preanalysed samples. Mean % recovery was 99.39. The
accuracy of the method was also tested by comparing its results with those
109
obtained by the official direct spectrophotometric procedure. The calculated
tvalues (1.86) and Fvalues (3.12) were found not exceeding the theoretical
tvalue (2.31 at P’ = 0.05) and Fvalue (6.39 at P’ = 0.05). Thus, the method is
accurate and reproducible.
CONCLUSION
The developed method is selective, accurate and precise for determination
of FSM in presence of degradation products and is useful for routine and control
analysis. The method require no preliminary separation or individual
determination of the degradation product. Irrelevant absorption due to formulation
matrix as well as decomposition products is totally nullified by this techniques.
Thus this method has an advantage over the official direct spectrophotometric
method.
110
TABLE 3.7: Results of the Analysis in Commercial Formulations by Difference (A) and Derivative (D2)
Spectrophotometric Methods.
Formulation Claimed (%) Found* (% Claim S.D.)
A Method Official Method D2 Method
T1 40 97.23 0.63 97.88 0.42 98.41 0.70
T2 15 99.54 0.55 100.23 0.37 99.36 0.80
I1 10 98.66 0.50 98.42 0.20 98.90 0.52
I2 10 97.40 0.58 98.16 0.40 98.68 0.64
Mean
Recovery (%)
99.39
99.50
tvalue (1.86) (1.73)
Fvalue (3.12) (3.92)
SM1 10 99.11 161.9 99.96
SM2 10 100.4 149.5 99.61
* : Average of five determinations. T1T2 : Tablets (mg/tab); I1I2 : Injections (mg/ml
SM1 : Synthetic mixture; Contains equivalent amount of CSAA.
SM2 : Synthetic mixture; Contains equivalent amount of hydrolysed Frusemide.
111
CHAPTERIII
SECTIONC REFERENCES
1. Pharmacopoeia of India, 3rd Ed. Controller of Publication, Delhi (1985).
2. British Pharmacopoeia, University Press, Cambridge (1980).
3. The United States Pharmacopoeia/National Formulary, United States
Pharmacopoeial Convention, Rock Ville 1985.
4. L.Heilmeyer, Spectrophotometry in Medicine, Adam Hilger Ltd., London
(1943).
5. M.A.H. Elsayed and C.O.Nwakanma, Pharmazie 34(4) (1979) 251252;
Throu; Anal. Abst. 37(5) (1979) E68.
6. H.Y.AboulEnein, A.A.AlBadr and M.S.E.D. Rasheed, Spectrosc. Lett.
12(4) (1979) 323331; Throu. Anal. Abst. 37(5) (1979) E69.
7. W.H.De Camp, J. Assoc. off. Anal. Chem. 67(5) (1984) 927933.
8. H.Abdine, A.H.Elsayed and Y.M.Elsayed, J.Assoc. Off. Anal. Chem. 61 (3)
(1978) 695701.
9. Munira Momin, A.F.Amin and K.Pundarikakshudu, Ind. J. Pharm. Sci. 66
(2004) 432.
10. U.K. Jain & V.K.Dixit, Ind. Drugs 41 (2004) 469.
11. U.P.Chaudhri, T.S.Patil, N.A.Shah, H.M. Dehghan, A.G.Nikalge, Jour. of
Pharm. Res. Vol. 7 No.2 (2008) 6869.
12. M.A.Hifiz et.al., Jordan J. Pharm. Sci.; 2 (2009) 55.
13. M.K.Chourasia, S.K. Jain, J. Pharm. Sci. 6 (2003) 3366.
14. Y.Pan, J.Li, H.Zhuo, In. J. Pharm. 24 (2002) 139147.
15. J. Patel, J.B.Dave, C.N. Patel and D.Patel, J. Chem. Pharma. Res. 2 (3)
(2010) 1014.
16. T.Siva Kumar, P.Venkatarsan, R.Manovalan, K.Valliappan, Ind. J. Pharm.
Sci. 69 (2007) 15457.