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).

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