CHAPTER 6 TITRIMETRIC, SPECTROPHOTOMETRIC AND ...shodhganga.inflibnet.ac.in/bitstream/10603/36434/10/chapter 6.pdf · proposed for the assay of INH in dosage forms. Three redox titrimetric

CHAPTER 6

TITRIMETRIC, SPECTROPHOTOMETRIC

AND CHROMATOGRAPHIC ASSAY OF

ISONIAZID

244

SECTION 6.0

DRUG PROFILE AND LITERATURE SURVEY

6.0.1.0 DRUG PROFILE

Isoniazid (INH) is chemically known as pyridine-4-carboxylicacid hydrazide. Its

molecular formula is C6H7N3O, with a molecular weight of 137.14. The structural

formula is:

N

NO

H

NH2

Physically, INH is a white powder soluble in water; sparingly soluble in

chloroform, acetonitrile, insoluble in ether, benzene etc. Isoniazid was first synthesized in

1912 by Meyer and Mally [1].

It is a bacteriostatic drug and is now used together with other antituberculostatic

agents for the chemotherapy of tuberculosis. It also seems to be active for extra-

pulmonary illness such as meningitis and genitor-urinary infections [2].

INH is officially reported in British Pharmacopoeia (BP) [3] and United State

Pharmacopeia (USP) [4]. BP describes titration of the drug with potassium bromate in

presence of potassium bromide using methyl red indicator. USP describes HPLC method

using L1 column (4.6 mm× 25 cm) and a mobile phase consisting of methanol:water

(40:60) (pH adjusted to 2.5 with H2SO4) with a flow rate of 1.5 ml min-1 and UV-

detection at 254 nm.

245

6.0.2.0 LITERATURE SURVEY - ANALYTICAL FRAMEWORK

6.0.2.1Titrimetric methods

The literature survey reveals that only four titrimetric procedures have been

proposed for the assay of INH in dosage forms. Three redox titrimetric methods were

developed using bromamine-T [5], bromamine-B [6] and N-bromosuccinimide [7] as

titrants. A method developed by Das et al., [8], involves the titration of INH with sodium

methoxide in benzene-methanol medium using o-nitroaniline as indicator.

6.0.2.2 Spectrophotometric methods

Number of visible spectrophotometric methods based on different reaction

schemes are found in the literature for the quantification of INH on pure form and in its

dosage forms.

Kashyap et al., [9] developed a method based on condensation reaction between

the drug and ethyl vanillin in NaOH medium resulting in the formation colored

hydrazone complex having absorption maximum at 410 nm. Beer’s law was obeyed in

the range 2-16 µg ml-1. Based on similar reaction but using vanillin as chromogenic

agent, another method was developed by Oga [10] in HCl medium, the resulting product

was measured at 405 nm and quantification was achieved over a concentration range of

1-12 µg ml-1. INH is found to react with 1, 2-naphthoquinone-4-sulfonate (NQS) [11] in

the presence of buffer of pH 13 to form a pink-colored product which was measured at

495 nm. Beer’s law was obeyed in the range 0.5-30 µg ml-1. Abbas and Homoda [12]

developed another method based on the formation of isatin-isonicotinylhydrazone

product with isatin in aqueous solution at pH 1.4. The formed yellow colored product had

a strong absorption peak at 340 nm and the calibration graph was found rectilinear over a

concentration range of 0-32 µg ml-1. INH was found to react with 2-hydroxy-1,4-

napthoquinone (HNQ) at pH 3 in aqueous-methanolic solution yielding a yellow colored

product having absorption maximum at 365 nm [13]. The reaction between drug and 6-

methyl-2-pyridinecarboxaldehyde (MPA) yielded hydrazone derivative measurable at

328 nm [14] facilitating the assay in 2-16 µg ml-1 range.

246

Gowda et al., [15] developed a method based on oxidation of tiron by KIO4

followed by coupling with INH in alkaline medium resulting in the formation of a red

colored product having maximum absorbance at 505 nm. The method obeys Beer’s law

in the concentration of 1.5-18 µg ml-1. Same authors developed another method based on

same reaction where NaIO4 was used as oxidizing agent and absorbance of the coupled

product at 507 nm [16].

Two methods based on oxidative coupling reactions involving INH, tiron by KIO4

[15] and NaIO4 [16] have been reported in which the colored product was measured at

505 in the range of 1.5-18 µg ml-1.

Naidu et al., [17] developed a moderately sensitive method based on diazotization

of 4,4’-methylene-bis-m-nitroaniline followed by the coupling with INH in HCl medium

resulting in the formation of purple-colored chromogen having λmax at 495 nm. Beer’s

law was obeyed in the range 0.1-15 µg ml-1. Another method based on diazotization of

4,4’-sulphonyldianiline (dapsone) followed coupling with drug in NaOH medium [18].

The formed diazo-coupled product absorbs maximally at 460 nm. Divya et al., [19]

developed two methods based on reaction of INH with epichlorohydrine (EPI) and 4-

hydroxyphenacylchloride (HPC) in NaOH medium in which yellow colored chromogen

was measured at 405 and 402 nm for EPI and HPC methods respectively. INH was

treated with carbon disulfide in methanol medium followed by reaction with uranyl

acetate, the resulting yellow colored uranyl isonicotinyldisthiocarbazate complex was

measured at 410 nm [20]

In a method reported by Safavi et al., [21], INH has been determined using

copper(II) and neocuproine. The method is based on the reduction of copper(II) to

copper(I) ions by drug, which in presence of neocuproine produces green coloured Cu(I)-

neocuproine complex which was measured at 454 nm. Another method based on

reduction of Fe(III) by INH to Fe(II) followed by complexation with 3-(2-pyridyl)-5,6-

diphenyl-1,2,4-triazine (PDT) has also been reported. The complex showed absorption

maximum at 558 nm [22].The reaction of INH with Fe(III), in the presence of potassium

ferricyanide, under mild acidic conditions produced a blue color with maximum

247

absorption at 735 nm [23]. The first step in the colour development wasthe reduction of

iron(III) of to iron(II) which subsequently reacts with ferricyanide to form prussian blue

and formed the basis of assay.

In a related method, Cu(II) was reacted with INH and the unreduced Cu(II) was

made to react with 4-(2-Pyridylaxo)resorcinol to form a red colored complex and

measured at 510 nm [24]. The method obeyed Beer’s law in the concentration of 0.05-

4.50 µg ml-1. There are two repots based on the same sequence of reaction steps in which

the drug was reacted with known excess of NBS in acid medium and after the reaction

was judged complete, the residual NBS was determined by its reaction with excess iodide

and starch followed by the measurement of blue colored starch-iodine complex at 572 nm

[25, 26].

Based on charge-transfer reaction, Kamel [27] developed three methods using

chloranilic acid (CA), tetracyanoethylene (TCNE) and 2,3-dichloro-5,6-dicyano-1,4-

benzoquinone (DDQ) in acetonitrile medium and the colored complexes were measured

at 500, 480 and 580 nm, respectively. In the same article three more methods based on

extraction-free ion-pair complexation reaction in acetonitrile medium are presented. The

drug was reacted with thymol blue (TB), bromo phenol blue (BPB) and bromocresol

green (BCG) and the resulting yellow colored ion-pair complexes were at 390, 410 and

320 nm, respectively.

In a method reported by Amer et al., [28] the drug was treated with cadmium(II)

and rose bengal and the resulting ion-association ternary complex was extracted in

acetone-chloroform and measured at 555 nm.

Nevin [29] developed a derivative ratio spectrophotometric method for the

determination of INH in pharmaceutical dosage forms. The method involves the

measurement of absorbance of the drug solution in methanol at 250.7 nm. Graphical

absorbance ratio method and derivative spectrophotometric method was also developed

for the quantification of INH [30]. Simultaneous UV-spectrophotometric determination

of INH with pyridoxine was developed by Pawar et al., [31]. Another simultaneous

determination of INH with rifampicin is also found in the literature [32].

248

6.0.2.3 Chromatographic techniques

A good number of methods employing high performance liquid chromatography

(HPLC) [33-42], gas chromatography [43-45] have been developed for the

determination of INH in pure form and in tablet dosage either alone or in combination

with other drugs.

6.0.2.4 Other techniques

Many other methods reported for INH in pharmaceuticals include capillary

electrophoresis [46, 47], capillary zone electrophoresis [48-50], chemiluminescence

spectrophotometry [51-60], flow injection analysis [61-64], spectrofluorimetry [65,66],

cyclic voltammetry [67-73], voltammetry [74-76], polarography [77], differential pulse

polarography [78,79], amperometry [80,81] ion selective potentiometry [82-84].

From the literature survey presented in Section 6.0.2, it is observed that no

titrimetric method with HClO4 with potentiometric end point detection has ever been

reported for the assay of INH in pharmaceuticals, and even the reported visible

spectrophotometric methods suffer from one or other disadvantage such as poor

sensitivity, use of unstable reagents, use of expensive chemical as compiled in Table

6.6.1. This prompted the author to develop a potentiometric, four visible

spectrophotometric, one UV-spectrophotometric and one UPLC methods for INH. The

details are contained in five sections of this chapter.

249

SECTION 6.1

POTENTIOMETRIC DETERMINATION OF ISONIAZID IN

PHARMACEUTICAL FORMULATIONS

6.1.1.0 INTRODUCTION

Since INH posses three amino groups it acts as a weak base and this property has

not been exploited before for developing a titrimetric procedure in non-aqueous medium.

A validated potentiometric titration procedure is described without any sample pre-

treatment or prior extraction, in which the solution of drug in glacial acetic acid was

titrated potentiometrically with acetous perchloric acid using modified glass-saturated

calomel electrode system. The details relating to the development and validation of the

method for the assay of INH are presented in this Section 6.1.

6.1.2 EXPERIMENTAL

6.1.2.1 Apparatus

An Elico 120 digital pH meter provided with a combined glass-SCE electrode

system was used in the titration. The KCl of the salt bridge was replaced with saturated

solution of KCl in glacial acetic acid.

6.1.2.2 Materials

All chemicals used were of analytical reagent grade. All solutions were made in

glacial acetic acid (S. D. Fine Chem, Mumbai, India) unless mentioned otherwise. INH

pure drug was kindly provided by Cipla Ltd, Bangalore, India, India, as a gift and used as

received. Isokin-300 tablets (Pfizer) tablets were purchased from local market.

6.1.2.3 Reagents

Perchloric acid ( 0.02 M): The stock solution of (~0.1 M) perchloric acid (S. D. Fine

Chem, Mumbai, India) was diluted appropriately with glacial acetic acid (Merck,

Mumbai, India) to get a working concentration of 0.02 M and standardized with pure

potassium hydrogen phthalate (S. D. Fine Chem, Mumbai, India) [85].

Crystal violet indicator (0.1 %): Prepared by dissolving 50 mg of dye (S. D. Fine

Chem, Mumbai, India) in 50 ml of glacial acetic acid.

250

Standard drug solution

Stock standard solution containing 1.5 mg ml-1 INH was prepared in glacial acetic

acid.

6.1.2. 4 General procedure

An aliquot of the standard drug solution equivalent to 1.5-15.0 mg of INH was

measured accurately and transferred into a clean and dry 100 ml beaker and the solution

was diluted to 25 ml by adding glacial acetic acid. The combined glass-SCE (modified)

system was dipped in the solution. The contents were stirred magnetically and the titrant

(0.022M HClO4) was added from a microburette. Near the equivalence point, titrant was

added in 0.05 ml increments. After each addition of titrant, the solution was stirred

magnetically for 30 s and the steady potential was noted. The addition of titrant was

continued until there was no significant change in potential on further addition of titrant.

The equivalence point was determined by applying the graphical method. The amount of

the drug in the measured aliquot was calculated from

Amount (mg) = VMwR/n

where V = volume of perchloric acid required, ml; Mw = relative molecular mass of the

drug; and R = molarity of the perchloric acid and n = number of moles of perchloric acid

reacting with each mole of INH.

Analysis of tablets

Twenty tablets were weighed accurately and pulverised. A weighed quantity of

the tablet powdered equivalent to 150 mg INH was transferred into a clean and dry 100

ml volumetric flask. The flask was shaken for 20 min with 60 ml of acetic acid, the

volume was brought to 100 ml with the same solvent. After 5 min, the solution was

filtered through a Whatman No. 42 filter paper. First 10 ml of the aliquot was discarded.

A suitable aliquot was next subjected to analysis as described earlier.

Placebo blank and synthetic mixture analysis analyses

A placebo blank of the composition: talc (75 mg), starch (85 mg), acacia (65 mg),

methyl cellulose (80 mg), sodium citrate (75 mg), magnesium stearate (80 mg) and

sodium alginate (75 mg) was made and its solution was prepared as described under the

“Procedure for tablets” by taking about 100 mg; and then analysed using the procedures

described above.

251

To about 100 mg of the placebo blank of the composition described above, 75 mg

of pure INH was added, homogenized and transferred to 50 ml volumetric flask and the

solution was prepared as described under “Procedure for tablets”. The extract (1.5 mg

mL-1 in INH) was then subjected to analysis by the procedures described above, by taking

appropriate portions.

6.1.3 RESULTS AND DISCUSSION

The basis and chemistry involved in non-aqueous titrations are clearly presented

in Section 3.1.

Since, INH is having basic nitrogen atoms in its molecular structure, the enhanced

basicity of INH in acetic acid medium is due to non-levelling effect of acetic acid and the

determination of INH becomes much easier. The procedure involves the titration of INH

with perchloric acid with potentiometric end point detection. A steep rise in the potential

was observed at the equivalence point (Figure 6.1.1). A reaction stoichiometry of 1:2

(drug: titrant) was obtained which served as the basis for calculation. Using 0.022 M

perchloric acid, 1.5-15.0 mg of INH was conveniently determined. The relationship

between the drug amount and the titration end point was examined. The linearity between

two parameters is apparent from the correlation coefficient of 0.9985 obtained by the

method of least squares. From this it is implied that the reaction between INH and

perchloric acid proceeds stoichiometrically in the ratio 1:2 in the range studied. Based on

the above study the following reaction pathway is postulated.

Scheme 6.1.1 Possible reaction for the neutralization

252

Figure 6.1.1 Typical potentiometric titration curves for 7.5 mg of INH Vs 0.022 M

HClO4

6.1.3.1 Method validation

Intra-day and inter-day accuracy and precision

The precision of the methods was evaluated in terms of intermediate precision

(intra-day and inter-day). Three different amounts of INH within the range of study were

analysed in seven and five replicates during the same day (intra-day precision) and five

consecutive days (inter-day precision). For inter-day precision, each day analysis was

performed in triplicate and pooled-standard deviation was calculated. The RSD (%)

values of intra-day and inter-day studies for INH showed that the precision of the method

was good (Table 6.1.1). The accuracy of the method was determined by the percent mean

deviation from known concentration, and results are presented in Table 6.1.1.

Table 6.1.1 Results of intra-day and inter-day accuracy and precision study

INH taken

mg

Intra-day accuracy and precision

(n=7)

Inter-day accuracy and precision

(n=5) INH

found mg

%RE %RSD INH

found mg

%RE %RSD

3.0 2.92 2.22 2.03 2.91 2.83 3.14 6.0 6.10 2.34 1.46 5.89 1.88 2.62 9.0 9.15 2.58 2.59 8.77 2.59 1.93

RE- Relative error and RSD- Relative standard deviation

253

Ruggedness of the method

Method ruggedness was expressed as the RSD (%) of the same procedure applied

by four different analysts as well as using four different burettes. The inter-analysts RSD

(%) were within 1.78% whereas the inter-buretts RSD (%) for the same INH amounts

was less than about 1.38% suggesting that the developed method was rugged. The results

are shown in Table 6.1.2.

Table 6.1.2 Method ruggedness expressed as intermediate precision (RSD, %)

INH taken,

mg

Ruggedness

Inter-analysts (RSD, %),

(n=4)

Inter-instruments (RSD, %),

(n=4)

3.0 1.65 1.04

6.0

9.0

1.52

1.78

0.99

1.38

Selectivity

In the analysis of placebo blank, there was no measurable consumption of HClO4

for the blank titration was recorded, suggesting the non-interference by the inactive

ingredients added to prepare the placebo.

In method A, 5 ml of the resulting solution prepared by using synthetic mixture was

assayed titrimetrically (n = 5), and yielded a % recovery of 101.3 ± 0.62 INH.

Application

The described potentiometric procedure was applied to the determination of INH

in its pharmaceutical formulations (Isokin-300). The results obtained (Table 6.1.3) were

statistically compared with those obtained by titrating the drug with potassium bromate in

presence of potassium bromide using methyl red indicator [3]. The results obtained by the

proposed method agreed well with those of reference method and with the label claim.

The results were also compared statistically by Student’s t-test for accuracy and by

variance F-test for precision [86] with those of the reference method at 95 % confidence

level as summarized in Table 6.1.3. The results showed that the calculated t-and F-values

254

did not exceed the tabulated values inferring that proposed method is as accurate and

precise as the reference method.

Table 6.1.3 Results of assay in tablets and comparison with official method

*Average of five determinations. Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95% confidence level is 6.39.

Recovery study

To a fixed amount of drug in formulation (pre-analyzed): pure drug at three

different levels was added, and the total was found by the proposed method. Each test

was repeated three times. The results compiled in Table 6.1.4 show that recoveries were

in the range from 98.51 to 102.5% indicating that commonly added excipients to tablets

did not interfere in the determination

Brand name

Label claim,

mg/tablet

Found* (Percent of label claim ± SD) Reference

method Proposed method

Isokin-300 300 99.36±1.65 97.98± 0.86

t =2.61 F=3.68

Table 6.1.4 Results of recovery study via standard addition method

Tablet studied

INH in tablet,

mg

Pure INH

added, mg

Total found, mg

Pure INH recovered* Percent ±

SD

Isokin-300

4.43 3.0 7.32 98.51±1.45

4.43 4.5 9.01 100.9±1.02

4.43 6.0 10.58 102.5±0.98 *Mean value of three determinations

255

SECTION 6.2

DEVELOPMENT AND VALIDATION OF A UV-SPECTROPHOTOMETRIC

METHOD FOR THE DETERMINATION OF ISONIAZID AND ITS STABILITY

STUDIES


A smart profile and utilization of UV-spectrometry in different assay have been

presented in Section 3.4. From the literature survey presented Section 6.0.2.0 it is evident

that, INH in combination with a number of other drugs in tablet dosage form has been

assayed by UV- spectrophotometry [30, 31], ratio derivative spectrophotometry [28, 29].

In the literature, no stability-indicating UV-spectrophotometric methods have ever

been reported for the assay of INH. In the present Section 6.2, a simple, inexpensive,

accurate, reproducible, and stability-indicating UV- spectrophotometric method for INH

is described. The methods are based on the measurement of absorbance of INH solution

in 0.1 M H2SO4 at 266 nm. Besides, the method was used to study the degradation of the

drug under stress conditions as per the ICH guidelines [86].

6.2.2.0 EXPERIMENTAL

6.2.2.1 Apparatus

The instrument is the same that was described in Section 3.4.2.1.

6.2.2.2 Reagents

All chemicals used were of analytical reagent grade. Doubly-distilled water was

used to prepare solutions wherever required. Pure drug and tablets used were the same as

described in Section 6.1.2.

Hydrochloric acid (5 M), hydrogen peroxide (5 % v/v), sodium hydroxide solution

(5 M) were prepared as described under Section 4.2.2.2.

256

Standard drug solution

A stock standard solution of 200 µg ml-1 INH was prepared by dissolving 20 mg

of pure INH in 0.1 M H2SO4 and diluted to 100 ml with the same solvent in a calibrated

flask. A suitable aliquot of the above solution was further diluted to obtain 20 µg ml-1

INH and used.

6.2.3.0 ASSAY PROCEDURES

6.2.3.1 Preparation of calibration curve

Into a series of 10 ml calibration flasks, aliquots of standard drug solution (0.2 to

5 mL of 20 µg ml-1) equivalent to 0.4-10.0 µg ml-1 INH were accurately transferred and

the volume was made up to the mark with 0.1 M H2SO4. The absorbance of each solution

was then measured at 266 nm against 0.1 M H2SO4 as the blank.

A calibration curve was prepared by plotting the absorbance versus concentration

of drug. The concentration of the unknown was read from the respective calibration curve

or computed from the regression equation derived using the Beer’s law data.

6.2.3.2 Procedure for tablets

Ten Isokin tablets containing INH (300 mg/tablet) were weighed and pulverized.

The amount of tablet powder containing 20 mg INH was transferred into a 100 ml

volumetric flask. The content was shaken well with about 60 ml of 0.1 M H2SO4 for 20

min and the extract was diluted to the mark with the same solvent. It was filtered using

Whatman No 42 filter paper. First 10 ml portion of the filtrate was discarded and a

subsequent portion was diluted to get a working concentration of 20 µg ml-1 and

subjected to analysis following the general procedure described earlier.

6.2.3.3 Placebo blank and synthetic mixture analyses

Thirty mg of the placebo blank prepared in Section 6.1.3.3 was taken and its

solution prepared as described under ‘Procedure for tablets’ and then analyzed using the

procedures described above.

257

To 10 mg of the placebo blank, 10 mg of INH was added and homogenized,

transferred to 50 ml volumetric flask and the solution was prepared as described under

“Procedure for tablets”. A convenient aliquot was diluted and then subjected to analysis

by the procedures described above.

6.2.3.4 Forced degradation studies

One ml of 200 µg ml-1 INH was taken (in triplicate) in a 25 ml volumetric flask

and mixed with 5 ml of 5 M HCl (acid hydrolysis) or 5 M NaOH (alkaline hydrolysis) or

5% H2O2 (oxidative degradation) and boiled for 2 h at 80 °C in a hot water bath. The

solution was cooled to room temperature and diluted to the mark with 0.1 M H2SO4 after

neutralization with 5.0 mL of 5 M NaOH (for acid hydrolysis) and 5 ml of 5 M HCl (for

alkaline hydrolysis). In thermal degradation, solid drug was kept in Petri dish in oven at

100 °C for 24 h. After cooling to room temperature, 10 µg ml-1 INH solution in 0.1 M

H2SO4 was prepared and absorbance measured. For UV degradation study, the stock

solutions of the drug (10 µg ml-1) were exposed to UV radiation of wavelength 254 nm

and of 1.2 K flux intensity for 48 h in a UV chamber. The solutions after dilution with 0.1

M H2SO4 was assayed as described above.

6.2.4.0 RESULTS AND DISCUSSION

Spectral characteristics

The absorption spectrum of 10 µg ml-1 INH solution in 0.1 M H2SO4 recorded

between 200 and 400 nm showed an absorption maximum at 266 nm, and at this

wavelength 0.1 M H2SO4 had insignificant absorbance. Therefore, 266 nm was used as

analytical wavelength (λmax). Figure 6.2.1 represents the absorbance spectra of INH in 0.1

M H2SO4 along with blank.

258

Figure 4.2.1 Absorption spectrum of INH (10 µg ml-1) in 0.1 M H2SO4

6.2.4.1 Forced degradation studies

The INH was subjected to acid, neutral, base and hydrogen peroxide induced

degradation in solution state, and photo and thermal degradation in solid state. The study

was performed by measuring the absorbance of INH solution only after subjecting to

forced degradation. The results of this are presented in Table 6.2.1. The results revealed

that, the alkaline hydrolysis, photo and thermal degradation when compared with that of

pure INH solution. There was significant change in the absorbance due to oxidative

degradation in the presence of hydrogen peroxide. The absorption spectra (Figure. 6.2.2)

was recorded for this degraded INH and there was almost completely diminished signal

was observed. This confirms that INH is susceptible to oxidative degradation.

Table 6.2.1 Results of degradation study

Degradation condition % Degradation No degradation ( control) Zero Acid hydrolysis (5 M HCl , 80°C, 2 hours) 8.3 Base hydrolysis (5 M NaOH , 80°C, 2 hours) 38.2 Oxidation (5% H2O2 , 80°C, 2 hours) Undetectable Thermal (100°C, 24 hours) Zero Photolytic (1.2 K flux, 48 hours) Zero

259

(a)

(b)

(c)

260

(d)

(e)

Figure 6.2.2 Absorption spectra of 10 µg ml-1 INH after (a) acid hydrolysis, (b) base hydrolysis, (c) thermal degradation (d) photo degradation and (e) peroxide degradation.


Linearity and sensitivity

A linear correlation was found between absorbance at λmax and concentration of

INH (Figure 6.2.3). The slope (b), intercept (a) and correlation coefficient (r) for each

system were evaluated by using the method of least squares. Optical characteristics such

as Beer’s law limits, molar absorptivity and Sandell sensitivity values of the method was

calculated. The limits of detection (LOD) and quantitation (LOQ) were also calculated

according to ICH guidelines [87], and all these data are presented in Table 6.2.2.

261

0 2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0

Absorb

ance

Concentration of INH, µg ml-1

Figure 6.2.3 Calibration curve

Table 6.2.2 Sensitivity and regression parameters

Parameter Value

λmax, nm 266

Linear range, µg ml-1 1 – 10.0 Molar absorptivity(ε), l mol-1 cm-1 8.38 × 104

Sandell sensitivity*, µg cm-2 0.0401 Limit of detection (LOD), µg ml-1 0.39 Limit of quantification (LOQ), µg ml-1 1.18 Regression equation** Intercept (a) 0.0043 Slope (b) 0.0238 Sa 0.0998 Sb 0.0026 Regression coefficient (r) 0.9998 *Limit of determination as the weight in µg ml-1of solution, which corresponds to an absorbance of

A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, where Y is

the absorbance, X is concentration in µg/ml, a is intercept and b is slope

Accuracy and precision

Accuracy was evaluated as percentage relative error between the measured

concentrations and the concentrations taken for INH (Bias %). The results obtained are

compiled in Table 6.2.3 and they show that both accuracy and precision are good.

Precision of the method was calculated in terms of intermediate precision (intra-day and

inter-day). Three different concentrations of INH were analyzed in seven replicates

during the same day (intra-day precision) and for five consecutive days (inter-day

precision). RSD (%) values of the intra-day studies showed that the precision was good.

262

Robustness and ruggedness

Method robustness was tested by measuring the absorbance at 265, 266 and 267

nm whereas the method ruggedness was tested by comparing the RSD values of the

results obtained by four different analysts, and also with three different cuvettes by a

single analyst. The intermediate precision, expressed as percent RSD, which is a measure

of robustness and ruggedness was within the acceptable limits as shown in the Table

6.2.4.

Table 6.2.4 Results robustness and ruggedness expressed as intermediate precision(%RSD)

INH taken, µg ml-1

Robustness# (%RSD)

Ruggedness Inter-analysts

(%RSD), (n=4)

Inter-cuvettes (%RSD), (n=4)

3.0 6.0 9.0

1.93 1.52 0.98

0.64 0.52 0.76

3.04 2.86 2.14

#Wavelengths used were 365,366and 367 nm.


INH taken, µg ml-1


(n=7)


(n=5)

INH found, µg ml-1

%RE %RSD INH

found, µg ml-1

%RE %RSD

4.0 6.0 8.0

4.09 6.05 8.14

0.28 0.44 1.63

1.93 1.54 1.37

4.03 6.12 8.16

1.00 0.83 0.75

2.48 1.76 1.58

RE: Relative error and RSD: Relative standard deviation.

263

Table 6.2.4 Results of robustness and ruggedness expressed as intermediate precision(%RSD)

INH taken, µg ml-1

Robustness Ruggedness

λ, nm Inter-analysts (%RSD),

(n=4)

Inter-instruments

(%RSD), (n=4)

265 266 267

3.0 6.0 8.0

2.55 2.21 1.86

1.08 1.22 1.11

1.89 1.78 1.56

0.64 0.52 0.76

3.04 2.86 2.14

Selectivity

The proposed method was tested for selectivity by placebo blank and synthetic

mixture analyses. The placebo blank solution was subjected to analysis according to the

recommended procedure and found that there was no interference from the inactive

ingredients, indicating a high selectivity for determining INH in its tablets.

When the synthetic mixture solution was subjected to analyses at 3.0, 6.0 and 8.0

µg ml-1 INH concentration levels, the percent recoveries were 98.48, 97.36 and 98.94

respectively, with % RSD being less than 2.42% implying that the assay procedure is free

from matrix interference.

Application to tablets

In order to evaluate the analytical applicability of the proposed method to the

quantification of INH in commercial tablets, the results obtained by the proposed method

were compared to those of the reference method [3] by applying Student’s t-test for

accuracy and F-test for precision. The results (Table 6.2.5) showed that the Student’s t-

and F-values at 95 % confidence level did not exceed the tabulated values, which

confirmed that there is a good agreement between the results obtained by the proposed

method and the reference method with respect to accuracy and precision.

264

Recovery studies

The accuracy and validity of the proposed methods were further ascertained by

performing recovery studies. Pre-analysed tablet powder was spiked with pure INH at

three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was

found by the proposed methods. The added INH recovery percentage values ranged from

98.42–102.3 % with standard deviation of 0.95-1.83 (Table 6.2.6) indicating that the

recovery was good, and that the co-formulated substance did not interfere in the

determination.

Table 6.2.5 Results obtained by the analysis of tablets by the proposed method and statistical comparison of results with the reference method

Brand name

Label claim,

mg/tablet

Found* (Percent of label claim ± SD)

Reference method Proposed method

Isokin-300 300 99.36±1.65 98.57± 0.86

t =2.61 F=3.68

Mean value of 5 determinations. Tabulated t-value at the 95 % confidence level and for four degrees of freedom is 2.77.TabulatedF-value at the 95 % confidence level and for four degrees of freedom is 6.39.


Tablets studied

INH in tablet, µg ml-1

Pure INH

added, µg ml-1

Total found, µg ml-1

Pure INH recovered

(Percent±SD*)

Isokin-

300

2.95 2.95 2.95

1.5 3.0 4.5

4.38 6.08 7.39

98.42±1.83 102.3 ±1.24 99.19±0.95

*Mean value of three determinations.

265

SECTION 6.3

DETERMINATION OF ISONIAZID USING OXIDIZING PROPERTIES OF

POTASSIUM PERMANGANATE AND TWO DYES

6.3.1 INTRODUCTION

Potassium permanganate is a strong oxidizing agent [90] and it was first

introduced into titrimetric analysis by F. Margueritte for the titration of iron(II) [90]. The

salt is also known as permanganate of potash and in this salt, manganese is in the +7

oxidation state. The innate intense purple color solution of permanganate absorbs in the

vicinity of 550 nm. As a strong oxidant it does not generate toxic byproducts.

The Mn-containing products from redox reactions depend on the pH. In acid

solutions, permanganate is reduced to the faintly pink Mn2+ as represented by the

following equation:

MnO4- + 8H++ 5 e Mn2+ + 4H2O

The standard potential in acid solution, E, has been calculated to be 1.51 volts,

hence the permanganate ion in acid solution is a strong oxidising agent [90]. Sulphuric

acid is the most suitable acid, as it has no action upon permanganate in dilute solution.

With hydrochloric acid, there is the likelihood of the reaction:

2MnO4- + 10Cl- + 16H+ = 2Mn2+ + 5Cl2 + 8H2O

taking place, and some permanganate may be consumed in the formation of chlorine

.Potassium permanganate also finds some application in strongly alkaline solutions.

KMnO4 spontaneously reduced to green K2MnO4, wherein manganese is in +6 oxidation

state.

MnO4- + e MnO4

2-

The resulting green color manganese(VI) shows maximum absorbance at 610 nm.

266

In neutral solutions permanganate is only reduced by 3e- to give manganese dioxide

where Mn is in a +4 oxidation state. MnO2 is brown in color. The standard potential E is

0.59 volt [90]. The half-cell reaction is:

MnO4- + 2H2O + 3e MnO4

2-+ OH-

Exploiting the different colors of permanganate at different pH media, it has been

used for the assay of methyl thiouracil [91], chloramphenicol [92], amidopyrine [93],

valdecoxib [94], nicotine [95], tramodol HCl [96], cefuroxime [97], reloxifine[98],

pentaprazole [99], famotidine [100] and olanzapine [101]. The literature survey presented

in Section 6.0.2.0 reveals that no spectrophotometric methods for INH have been

reported using permanganate. The author developed two spectrophotometric methods for

the determination of INH in bulk drug and tablets employing KMnO4 as a oxidizing agent

and two dyes, methyl orange (MO) and indigo carmine (IC) as auxiliary reagents.

Methods involve the addition of a known excess of KMnO4 to INH followed by

estimation of residual KMnO4 by reacting with a fixed amount of either methyl orange

and measuring the absorbance at 520 nm (method A) or indigo carmine and measuring

the absorbance at 610 nm (method B). The details about the method development and

validation are presented in this Section 6.3.


6.3.2.1 Apparatus


6.3.2.2 Reagents

All the reagents were of analytical-reagent grade and distilled water was used

throughout the investigation. Pure INH and its tablets used were the same described in

Section 6.1.

Potassium permanganate (0.01 M and 600 µg ml-1): An approximately 0.01 M solution

was prepared by dissolving 395 mg of KMnO4 (Merck, Mumbai, India) in water and

diluting to 250 mL in a calibrated flask, and standardized using H.A Bright’s procedure

267

[102]. The standard solution was diluted appropriately with water to get 70 and 60 µg ml-1

for use in method A and method B, respectively.

Methyl orange (MO): A standard solution equivalent to 500 µg ml-1 methyl orange was

prepared by dissolving 58.8 mg of methyl orange (S. D. Fine Chem., Mumbai, India,

85% dye content) in water and diluting to 100 ml in a calibrated flask; and filtered using

glass wool. It was diluted to get a 50 µg ml-1 dye solution for use in method A.

Indigo carmine (IC): A standard solution containing 1000 µg ml-1 indigo carmine was

prepared by dissolving 107.6 mg of indigo carmine (Loba Chemie Pvt. Ltd., Mumbai,

India, 93% dye content) in water and diluting to the mark in a 100 ml calibrated flask.

The solution was diluted with water to get a working concentration of 200 µg ml-1 dye

solution for use in method B.

Sulphuric acid (5M): Concentrated acid (S.D. Fine Chem, Mumbai, India, sp. gr. 1.84)

was appropriately diluted with water to get the required concentration.

Standard INH solution: Ten mg of INH was dissolved in water and made up to 100 ml

with water and then diluted to 10 µg ml-1 INH concentration.


6.3.3.1 Method A (using methyl orange)

Different aliquots (0.25,0.5,1.0,----5.0 ml) of a standard INH (10 µg ml-1)

solution were accurately transferred into a series of 10 ml calibrated flasks and the total

volume was adjusted to 5.0 ml by adding adequate quantity of water. To each flask, 1 ml

of 5 M H2SO4 was added followed by 1.0 ml of 60 µg ml-1 KMnO4. The flasks were

stoppered, content mixed and allowed to stand for 5 min with occasional shaking. Then,

1.0 ml of methyl orange solution (50 µg ml-1) was added to each flask, and after 5 min,

the mixture was diluted to the volume with water and mixed well. The absorbance of

each solution was measured at 520 nm against a reagent blank.

6.3.3.2 Method B (using indigo carmine)

Varying aliquots (0.25,0.5,1.0,----5.0 ml) of a standard 10 µg ml-1 INH solution

were transferred into a series of 10 ml calibrated flasks using a micro burette and the total

268

volume was brought to 5 ml by adding water. To each flask 1 ml of 5 M H2SO4 and 1.0

ml of KMnO4 (70 µg ml-1) were added. The content was mixed well and the flasks were

kept aside for 5 min with intermittent shaking. Finally, 1.0 ml of indigo carmine solution

(200 µg ml-1) was added to each flask and the volume was adjusted to the mark with

water. The absorbance of each solution was measured at 610 nm against a reagent blank.

A standard graph was prepared by plotting absorbance against concentration and

the unknown concentration was read from the graph or computed from the regression

equation derived using Beer’s law data.

6.3.3.3 Procedure for tablets

Ten Isokin tablets containing INH (300 mg/tablet) were weighed and pulverized.

The amount of tablet powder containing 10 mg INH was transferred into a 100 ml

volumetric flask. The content was shaken well and diluted to the mark with water. It was

filtered using Whatman No 42 filter paper. First 10 ml portion of the filtrate was

discarded and a subsequent portion was diluted to get a working concentration of 10 µg

ml-1 and subjected to analysis following the general procedure described earlier in both

the methods.

6.3.3.3 Placebo blank and synthetic mixture analyses

Twenty mg of the placebo blank prepared in Section 6.1.3.3 was taken and its








The present work involves the oxidation of INH by KMnO4 followed by

determination of surplus KMnO4 after the reaction is ensured to be complete. The

unreacted KMnO4 is determined by reacting with a fixed amount of either methyl orange

and measuring the absorbance at 520 nm or indigo carmine and measuring the

269

absorbance at 610 nm. The methods make use of the bleaching action of KMnO4 on

either dye, where the discoloration is caused by oxidative destruction of the dye.

INH + Known excess of KMnO4 Reaction product of the drug + Unreacted KMnO4H+

Unreacted KMnO4

+ MO(Method A)

(Method B)

Unbleached color of MO measured at 520 nm

Unbleached color of IC measured at 610 nm

+IC

Possible reaction pathway for INH

Absorption spectra

The proposed methods are based on the determination of unreacted oxidant after

the reaction between the drug and KMnO4 is judged to be complete. The orange-red color

of the unreacted MO in acid medium absorbed maximally at 520 nm (method A) and blue

color of unreacted IC in acid medium peaked at 610 nm (method B). The absorption

spectra of both the methods are presented in Figure 6.3.1 (Absorption spectra).

Many dyes are irreversibly destroyed to colorless species by oxidizing agents in

acid medium [102] and this observation has been exploited for the indirect

spectrophotometric determination of some pharmaceutical compounds [103-109]. In the

proposed methods, the ability of KMnO4 to effect the oxidation of INH and irreversibly

destroy MO and IC to colorless products in acid medium has been capitalized. Both

methods are based on the oxidation of the drug by measured excess of KMnO4 and

subsequent determination of the latter by reacting with MO or IC, and measuring the

absorbance at 520 or 610 nm. In either method, the absorbance increased linearly with

increasing concentration of drug. INH when added in increasing concentrations to a fixed

concentration of KMnO4 consumes the latter and there will be a concomitant decrease in

the concentration of KMnO4. When a fixed concentration of either dye is added to

decreasing concentration of KMnO4 a concomitant increase in the concentration of dye is

obtained. This is observed as a proportional increase in the absorbance at the respective

wavelengths of maximum absorption with increasing concentration of INH.

270

Optimization of KMnO4 and dye concentrations

Preliminary experiments were performed to fix the upper limits of the MO and IC

that could produce a reasonably high absorbance, and these were found to be 5 µg ml−1

MO in method A and 20 µg ml−1 IC in method B. To fix the optimum concentration of

KMnO4, different concentrations of KMnO4 were reacted with a fixed concentration of

MO or IC (5 or 20 µg ml−1) in H2SO4 medium and the absorbance was measured at 520

or 610 nm. A constant and minimum absorbance resulted with 6.0 and 7.0 µg ml-1

KMnO4 for method A and method B, respectively. Different concentrations of INH were

reacted with 1ml each KMnO4 of 60 µg ml−1 in method A and 70 µg ml-1 in method B in

H2SO4 medium before determining the residual KMnO4. This facilitated the optimization

of the linear dynamic range over which each procedure could be applied for the assay of

INH.

Effect of reaction medium

The reaction between INH and KMnO4 was performed in different acid media

viz., sulphuric acid, hydrochloric acid and perchloric acid. Sulphuric acid was found to be

the ideal medium for the oxidation of INH by KMnO4 as well as the latter’s

determination employing either dye. The effect of acid concentration on the reaction

between INH and KMnO4 was studied by varying the concentration of H2SO4 keeping

the concentrations of KMnO4 and drug fixed. Higher the acid concentrations showed

lower sensitivity, hence 1 ml of 5 M H2SO4 in a total volume of 10 ml was found optimal.

Study of reaction time and stability

Under the described experimental conditions, for a quantitative reaction between

INH and KMnO4, contact time of 5 min was found necessary in both methods at room

temperature. After addition of dye, the reaction between KMnO4 and dye was

instantaneous and absorbance of the unreacted dye was stable at least 45 min in method

A and 60 min in method B.

271



The proposed methods were validated for linearity, selectivity, precision,

accuracy, robustness and ruggedness, and recovery. In both the methods a linear

correlation (Figure 6.3.2) was found between absorbance at λmax and concentration of

INH in the ranges given in Table 6.3.1. Regression analysis of the Beer’s law data using

the method of least squares was made to evaluate the slope (b), intercept (a) and

correlation coefficient (r) for each system and the values obtained from this investigations

are presented also presented in the same table. Sensitivity parameters such as apparent

molar absorptivity and Sandell sensitivity values and the limits of detection and

quantification are calculated as per the current ICH guidelines [87] which are compiled in

Table 6.3.1 that speaks of the excellent sensitivity of the proposed method.


Parameter Method A Method B

λmax, nm 520 610

Beer's law limits, µg ml-1 0.25 – 5.0 0.25 –4.0

Molar absorptivity, l mol-1 cm-1 2.5×105 3.0×105

Sandell sensitivity*, µg cm-2 0.0055 0.0045

Limit of detection, µg ml-1 0.08 0.06

Limit of quantification, µg ml-1 0.23 0.18

Regression equation, Y**

Intercept, (a) 0.0192 0.0102 Slope, (b) 0.1666 0.2167 Standard deviation of intercept (Sa) 0.2663 0.4720 Standard deviation of slope (Sb) 0.1088 0.1736 Regression coefficient (r) 0.9989 0.9990 a Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance ofA = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b

Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1

272

Method A Method B

Figure 6.3.2 Calibration curves


In order to study the precision and accuracy of the proposed methods, three

concentrations of pure INH within the linearity range were analyzed, each determination

being repeated seven times (intra-day precision) on the same day and one time each for

five days (inter-day precision). The percentage relative standard deviation (%RSD) was ≤

1.34 % (intra-day) and ≤ 2.98% (inter-day). In addition, the accuracy of the proposed

method was measured by calculating the percentage relative error (%RE), which varied

between 1.01% and 3%. The results of this study compiled in Table 6.3.2 indicate the

high accuracy and precision of the proposed methods.

273

Selectivity

Selectivity was evaluated by both placebo blank and synthetic mixture analyses.

The placebo blank, consisting the composition as mentioned under ‘Placebo blank

analysis’ was prepared and analyzed as described under the recommended procedures.

The resulting absorbance readings for the methods were same as the reagent blank,

inferring no interference from the placebo. The selectivity of the methods was further

confirmed by carrying out recovery study from synthetic mixture. The percent recoveries

of INH were 98.7±1.18 and 101.4±1.63 for method A and method B, respectively at 3.0

µg ml-1level in both methods. This confirms the selectivity of the proposed methods in

the presence of the commonly employed tablet excipients.


To evaluate the robustness of the methods, two important experimental variables,

viz., the amount of acid and reaction time, were slightly varied, and the capacity of the

methods was found to remain unaffected by small deliberate variations. The results of this

study are presented in Table 6.3.3 and indicate that the proposed methods are robust.

Method ruggedness is expressed as %RSD of the same procedure applied by three

analysts and using three different spectrophotometers by the same analyst. The inter-


Method

INH taken

µg ml-1

Intra-day accuracy and precision (n=7)


(n=5)

INH found µg ml-1

%RE %RSD INH

found µg ml-1

%RE %RSD

A

1.0 1.02 2.04 2.18 1.03 2.89 2.28

2.0 1.96 2.27 1.34 1.95 2.30 2.45 3.0 3.04 1.31 1.67 3.06 1.96 2.85

B

2.0 1.98 1.01 1.72 1.96 2.02 2.03 3.0 3.04 1.36 2.16 3.05 1.65 2.72

4.0 4.05 1.27 1.49 4.07 1.78 2.98 RE- Relative error and RSD- Relative standard deviation mg in titrimetry and µg ml-1 in spectrophotometry.

274

analysts’ and inter-instruments’ RSD values were ≤ 3.04 % indicating ruggedness of the

proposed methods. The results of this study are presented in Table 6.3.3.


The results presented in Table 6.3.4 showed that there was a close agreement

between the results obtained by the proposed methods and the label claim. The results

were also compared with those of the reference method [3] statistically by a Student's t-

test for accuracy and variance ratio F- test for precision at 95 % confidence level. The

calculated t- and F-values indicate that there is no significant difference between the

proposed methods and the reference method with respect to accuracy and precision.

Table 6.3.3 Results of robustness and ruggedness expressed as intermediate precision (% RSD)

Robustness Ruggedness

Method Nominal

concentration

Reaction times* (n=3)

Volume of acid$

Inter- analysts

(n=3)

Inter- instruments#

(n=3)

A 1.0

2.0 3.5

2.65 3.14 3.38

2.13 2.86 2.73

1.04 1.28 0.92

2.42 2.16 1.85

B 2.0

3.0 4.0

1.85 2.16 2.08

1.05 1.27 1.97

0.73 1.12 1.08

2.16 2.72 3.04

* Reaction time was 5±1 min, $

Volume of H2SO4, 1±0.1 ml

Table 6.3.4 Results of analysis of tablets by the proposed methods and statistical comparison with the official method

Tablet brand name

Label claim*

Founda (Percent of label claim ±SD)

Reference method

Proposed methods

Method A Method B

Isokin

300

99.36±1.65

98.84± 1.42 t =2.72 F =1.35

98.75± 1.38 t =2.82 F =1.42

a Mean value of five determinations, The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are 2.77 and 6.39, respectively.

275

Recovery studies

To further ascertain the accuracy of the proposed methods, a standard addition

technique was followed. A fixed amount of drug from pre-analyzed tablet powder was

taken and pure drug at three different levels (50, 100 and 150 % of that in tablet powder)

was added. The total was found by the proposed methods. The determination at each

level was repeated three times and the percent recovery of the added standard was

calculated. Results of this study presented in Table 6.3.5.

Table 6.3.5 Results of recovery study via standard addition method Method A Method B

Tablet studied


Pure INH

added, µg ml-1


Pure INH recovered*

Percent ± SD


Pure INH

added, µg ml-1


Pure INH recovered*

Percent ± SD

Isokin

1.48 0.75 2.18 97.85 ± 2.19 1.48 0.75 2.19 98.55 ± 1.81

1.48 1.50 3.02 101.5 ± 1.79 1.48 1.50 2.95 99.26± 2.58

1.48 2.25 3.82 102.5 ± 2.76 1.48 2.25 2.75 100.9 ± 2.24

276

SECTION 6.4

SPECTROPHOTOMETRIC DETERMINATION OF ISONIAZID IN

PHARMACEUTICALS USING IRON(III) CHLORIDE AND TWO CHELATING

AGENTS


Iron(III) chloride, also called ferric chloride, is an industrial scale commodity

chemical compound, with the formula FeCl3. Ferric chloride is a mild oxidising agent.

The standard reduction potential for the reduction of Fe3+ (aq) to Fe2+ (aq) under standard

acidic condition is 0.771 V.

Many pharmaceutical compounds have been determined using ferric chloride as

oxidizing agent. [111-117]. A scan of the literature survey presented in Section 6.0.2.0

and the above paragraph reveals that ferric chloride has not been applied to the

determination for INH. Utilizing the oxidizing property of ferric chloride, the author has

developed two visible spectrophotometric methods, which are convenient for the

determination of INH in pure form and in dosage forms. The procedures involved

oxidation of INH with iron(III)chloride followed by determination of the resuting iron

(II) by complexation with either 1, 10-phenanthroline (method A) or 2, 2΄-bipyridyl

(method B). The importance of such reaction in pharmaceutical analysis [118-119] has

long been recognized. The proposed methods are characterized by simplicity, sensitivity,

wide linear dynamic ranges, mild experimental conditions and above all cost-

effectiveness.


6.3.2.1 Apparatus


6.4.2.2 Reagents

All the reagents were of analytical-reagent grade and distilled water was used

throughout the investigation. Pure INH and its tablets used were the same described in

Section 6.1.

277

Ferric chloride (0.1 M): The aqueous solution of 0.1 M FeCl3. 6H2O (S.D. Fine Chem.,

Mumbai, India) was prepared by dissolving 2.8 g of the chemical in 100 ml of 0.1 M

HCl.

1,10-phenanthroline (phen) (0.01 M):The solution was prepared by dissolving 198 mg

of the chemical (Qualigens Fine Chemicals, Mumbai, India, assay 100%) in distilled

water and diluted to 100 ml with distilled water.

2,2’-Bipyridyl (bipy) (0.01 M):The solution was prepared by dissolving 156 mg of the

chemical (Qualigens Fine Chemicals, Mumbai, India, assay 100%) in distilled water and

diluted to volume in a100 ml calibrated flask.

Stock standard solution: A stock solution equivalent to 100 µg ml-1 of INH was

prepared by dissolving accurately weighed 10 mg of pure drug in water and diluting to

the mark in a 100 ml calibrated flask. This was diluted appropriately with water to get

working concentration 20 and 50 µg ml-1 INH for use in method A and method B,

respectively.


Method A (Using 1, 10-Phenanthroline)

Different aliquots (0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 ml) of the standard 20 µg ml-1

INH solution were accurately measured and transferred into a series of 10 ml calibrated

flasks and the total volume was adjusted to 4 ml by adding water. To each flask, 0.5 ml of

a ferric chloride (0.1 M) and 2 ml of 0.01 M 1,10-phenanthroline were added, and the

volume was brought to 10 ml with distilled water. The flasks were stoppered, the content

mixed well and the flasks were let stand for 10 min with occasional shaking. Then, the

absorbance of each solution was measured at 510 nm against the reagent blank.

Method B (Using 2, 2΄-Bipyridyl)

Varying aliquots (0.2, 0.5, 1.0, 2.0 and 3.0 ml) of the standard INH solution (50

µg ml-1) were accurately measured into a series of 10 ml calibrated flasks by means of a

micro-burette and the total volume was brought to 3 ml by adding water. To each flask

0.5 ml of 0.1 M ferric chloride and 2 ml of 0.01 M 2,2’-bipyridyl were added. The

content was mixed well and diluted to the mark with distilled water. The absorbance of

each solution was measured at 540 nm against reagent blank after 10 min.

278

A standard graph was prepared by plotting the increasing absorbance values

versus concentration of INH (µg ml-1). The concentration of the unknown was read from

the standard graph or computed from the respective regression equation derived using the

Beer’s law data.

Procedure for tablets

An amount of finely ground tablet powder equivalent to 10 mg of INH was

accurately weighed and transferred into a 100 ml calibrated flask, 60 ml of water was

added and the content shaken thoroughly for 15-20 min to extract the drug into the liquid

phase; the volume was finally diluted to the mark with water, mixed well and filtered

using a Whatman No. 42 filter paper. First 10 ml of the filtrate was discarded and a

suitable aliquot of the filtrate (100 µg ml-1 INH) was diluted stepwise with water to get 20

and 50 µg ml-1 concentrations for method A and method B, respectively.

Placebo blank and synthetic mixture analyses

Twenty mg of the placebo blank prepared in Section 6.1.2.4 was taken and its







6.4.4.0 RESULT AND DISCUSSION

INH was found to undergo oxidation with FeCl3 in neutral medium. The complex

formation of Fe2+ with 1, 10- phenanthroline and 2, 2΄-bipyridyl has long been

recognized. The proposed methods involved two steps:

The first step is the oxidation of INH with excess FeCl3 in neutral medium, and

the second step is the determination of the resulting Fe2+ by subsequent chelation with

either phen or bipy and measuring the absorbance at the respective wavelength (Figure

6.4.1). The probable reaction mechanism is shown in Scheme 6.4.1.The amount of Fe2+

formed was found to be proportional to the amount of INH serving as basis for its

279

quantification. INH, when added in increasing concentrations, consumes Fe3+, and

consequently there will be concomitant increase in Fe2+ concentration. This is observed as

a proportional increase in the absorbance of the colored species with increasing

concentration of INH and fixed concentration of reagent. The increasing absorbance

values at 510 nm in method A and at 540 nm in method B were plotted against the

concentration of INH to obtain the calibration graph.

400 450 500 550 600 650

0.0

0.1

0.2

0.3

0.4

0.5

0.6A

bsorb

ance

Wavelength, nm

a

b

c

d

Figure 6.4.1 Absorption spectra of:

a-Fe(II)-phen complex, b-blank (Method A) c-Fe(II)-bipy complex and d-blank (Method B)

280

Fe2+

+

N

N

N

N

Fe2+

Ferroin (orange-red colour)measured at 510 nm.

N

N

+

N

N

Fe2+

Method A

Method B

Fe(II)-2,2'-bipyridyl chelate (red colour)measured at 540 nm.2,2'-Bipyridyl

1,10-Phenanthroline

3

3

3

3

Excess of Fe3++ + Fe2+

Oxidation product of INHINH

Scheme 6.4.1 Possible reaction pathway

6.4.4.1 Optimization of the reaction conditions

Investigations were carried out to achieve maximum color development in the

quantitative determination of INH. When the Fe3+ concentration was increased, the

absorbance value of reagent blank was found to increase. Hence, by considering the

sensitivity of the reaction with a minimum blank absorbance, 0.5 ml of 0.1 M ferric

chloride in a total volume of 10 ml was found optimum in both methods. Several

experiments were carried out to study the effect of phen and bipy concentrations on the

color development. In order to determine the optimum concentration of phen, different

volumes (0.5-2.5 ml) of 0.01 M phen solution were used with a fixed concentration of

INH (6 µg ml-1) in a total volume of 10 mL and 1.5-2.5 ml of phen solution was found to

give constant absorbance readings. Hence, 2 ml of phen solution in a total volume of 10

ml is fixed. In method B, 2 ml of 0.01 M bipy solution in a total volume of 10 ml was

found to give the maximum absorbance value, hence, the same volume was used. The

standing times for full color development were found to be 10 min for both methods; and

the color was stable for 30 min thereafter in both methods.

281



A linear correlation was found between absorbance at λmax and concentration of

INH (Figure 6.4.2) in the ranges given in Table 6.4.1. Regression analysis of the Beer’s

law data using the method of least squares was made to evaluate the slope (b), intercept

(a) and correlation coefficient (r) for each system and the analytical results obtained from

this investigations are presented in Table 6.4.1. The optical characteristics such as Beer’s

law limits, molar absorptivity and Sandell sensitivity values of both the methods are also

given in Table 6.4.1. The high values of ε and low values of Sandell sensitivity and LOD

indicate the high sensitivity of the proposed methods.

Method A Method B

Figure 6.4.2 Calibration curves


The precision and accuracy of the proposed methods were studied by repeating

the experiment seven times within the day to determine the repeatability (intra-day

precision) and five times on different days to determine the intermediate precision (inter-

day precision). The assay was performed for three levels of analyte in each method. The

results of this study are summarized in Table 6.4.2. The percentage relative standard

deviation (%RSD) values were ≤ 1.92 % (intra-day) and ≤ 1.94 % (inter-day) indicating

good precision of the methods. Accuracy was evaluated as percentage relative error

282

(%RE) between the measured mean concentrations and taken concentrations of INH, and

it was ≤ 2.36 % demonstrating the high accuracy of the proposed methods.


Parameter Method A Method B

λmax, nm 510 540 Linear range, µg ml-1 0.4-8.0 1.0-15.0

Molar absorptivity(ε), l mol-1 cm-1 1.7 × 105 7.5 × 104

Sandell sensitivity*, µg cm-2 0.0079 0.0184

Limit of detection (LOD), µg ml-1 0.15 0.11

Limit of quantification (LOQ), µg ml-1 0.46 0.34

Regression equation, Y**

Intercept (a) 0.0061 0.0005 Slope (b) 0.1227 0.0572 Standard deviation of a (Sa) 0.0674 0.0079

Standard deviation of b (Sb) 0.0150 0.0011

Regression coefficient (r) 0.9997 0.9992 *Limit of determination as the weight in µg per ml of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1, a is intercept, b is slope.


Methods* INH

taken µg ml-1


(n = 7)


(n = 5) INH

found µg ml-1

%RE %RSD INH

found µg ml-1

%RE %RSD

A

2.00 2.03 1.60 1.92 2.04 2.36 1.94

4.00 3.95 1.02 0.90 3.94 1.35 0.82 6.00 6.04 0.75 1.29 6.06 1.06 1.32

B

7.50 7.45 0.74 0.62 7.46 0.83 0.66 10.0 9.89 1.10 1.02 9.85 1.48 1.04

12.5 12.31 1.50 0.45 12.28 1.80 0.42

RE. Relative error; RSD. Relative standard deviation.

Selectivity

A systematic study was performed to determine the effect of matrix on the

absorbance by analyzing the placebo blank. In the analysis of placebo blank solution the

absorbance in each case was equal to the absorbance of blank which revealed no

interference. To assess the role of the inactive ingredients on the assay of INH, the

283

general procedure was applied on the synthetic mixture extract by taking three different

concentrations of INH (2, 4 and 6 µg ml-1 in method A and 7.5, 10, and 12.5 µg ml-1 in

method B). The percentage recovery values were in the range 97.38 – 102.3% with RSD

< 3% indicating clearly the non-interference from the inactive ingredients in the assay of

INH.


The method robustness was tested by making small incremental changes in FeCl3

and phen/bipy concentrations (n = 3) and performing the experiments using 2, 4 and 6 µg

ml-1 (method A) and 7.5, 10, and 12.5 µg ml-1 INH (method B). The RSD with the

altered FeCl3 and Phen/Bipy concentrations were < 1.5%. In order to demonstrate the

ruggedness of the methods, a drug solution at different concentration levels were

analyzed by four different analysts, and also with three different cuvettes by a single

analyst. The inter-analysts RSD were < 1.07 %, whereas the inter-instrumental variation

expressed as RSD were < 1.5 %. These low values of intermediate precision demonstrate

the robustness and ruggedness of the proposed methods (Table 6.4.3).

Table 6.4.3 Results of robustness and ruggedness expressed as intermediate precision (%RSD)

Method

INH taken

(µg ml-1)

Method robustness Method ruggedness

Parameter altered Ferric

chloride* ml

%RSD (n = 3)

Reaction time**

%RSD (n=3)

Inter-analysts %RSD (n = 4)

Inter-instruments

%RSD (n = 3)

A

2.00 0.94 1.02 0.71 1.23 4.00 1.05 0.98 0.94 1.49 6.00 0.89 1.09 0.87 1.37

B

7.50 1.12 1.05 1.07 1.21

10.0 1.01 1.16 0.95 1.37 12.5 1.17 1.19 1.01 1.29

*Ferric chloride volumes used were 0.4, 0.5 and 0.6 ml **reaction time of 8, 10 and 12 min.

284


The proposed methods were applied to the quantification of INH in commercial

tablets. The tablets were assayed by the reference method [3]. The results obtained by the

proposed methods agree well with the claim and also are in agreement with those by the

reference method. Statistical analysis of the results did not detect any significant

difference between the performance of the proposed methods and reference method with

respect to accuracy and precision as revealed by the Student’s t-value and variance ratio

F-value. The results of assay are given in Table 6.4.4.

Recovery studies

To further assess the accuracy of the methods, recovery experiments were performed

by applying the standard-addition technique. The recovery was assessed by determining the

agreement between the measured standard concentration and added known concentration to

the sample. The test was done by spiking the pre-analyzed tablet INH with pure INH at three

different levels (50, 100 and 150 % of the content present in the preparation) and the total

was found by the proposed methods. Each test was repeated three times. In both the cases,

the recovery percentage values ranged between 98.20 and 102.0% with standard deviation in

the range 1.09-1.98%. Closeness of the results to 100% showed the fairly good accuracy of

the methods. The results are shown in Table 6.4.5.

Table 6.4.4 Results of analysis of tablets by the proposed methods and statistical comparison with the official method

Tablet brand name

Label claim*

Founda (Percent of label claim ±SD)

Reference method

Proposed methods

Method A Method B

Isokin

300

99.36±1.65

98.52± 1.42 t =2.73 F =1.35

98.39± 1.38 t =2.82 F =1.42

a Mean value of five determinations, The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are 2.77 and 6.39, respectively.

285


Method Tablet studied

INH in tablet µg ml-1

Pure INH added µg ml-1

Total found

µg ml-1

Pure INH * Percent ± SD

A

2.95 1.5 4.37 98.20 ± 1.28

Isokin-300 2.95 3.0 6.02 101.3 ±1.09

2.95 4.5 7.59 102.0 ± 1.71

B

4.92 2.5 7.45 100.5 ± 1.23

Isokin-300 4.92 5.0 9.81 98.85 ± 1.98 4.92 7.5 12.61 101.6 ± 1.37

* Mean value of three determinations

286

SECTION 6.5

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING RP-UPLC

METHOD FOR THE DETERMINATION OF ISONIAZID IN BULK DRUG AND

IN PHARMACEUTICAL DOSAGE FORM


The utility and importance of UPLC was discussed in Section 4.3.1. To the best

of the author’s knowledge, no UPLC method has been ever reported for the determination

of INH in pharmaceuticals, Thus, a need for a rapid, precise, accurate and validated

stability-indicating UPLC method for the determination of INH in bulk and tablets was

felt. This was accomplished with a Waters Acquity UPLC system and Acquity BEH

column (C18, 100 mm, 2.1 mm and 1.7 µm). The stability indicating power of the method

was established by comparing the chromatograms obtained under optimized conditions

before forced degradation with those after degradation via acidic, basic, hydrolytic,

oxidative, thermal and photolytic stress conditions. The optimization parameters and the

validation results in detail are presented in this Section 6.5.


6.5.2.1 Materials

All the reagents used were of analytical grade. Doubly distilled water was used

throughout the investigation. Pure INH used was the same as described in Section 6.1.

HPLC grade acetonitrile was purchased from Merck India. Pvt. Ltd., Mumbai, India,

Doubly distilled water was used throughout the investigation.

6.5.2.2 Reagents and solutions

Hydrochloric acid (1M), sodium hydroxide (1M) and hydrogen peroxide

(5%) were prepared as described in previous chapter.

Stock standard solution of INH

A stock standard solution of (1 mg ml-1) was prepared by dissolving an accurately

weighed 100 mg of pure drug in 100 ml volumetric flask using the mobile phase.

287

Chromatographic conditions and equipments

Analyses were carried out on a Waters Aquity UPLC with Tunable UV (TUV)

detector. The output signal was monitored and processed using Empower software. The

Chromatographic column used was Acquity UPLC BEH C-18 (100 × 2.1) mm and 1.7

µm particle size. Isocratic elution process was adopted throughout the analysis.

Mobile phase preparation

A 200 ml portion of water was mixed with 800 ml of acetonitrile, shaken well and

filtered using 0.22 µm Nylon membrane filter. This solution was also used as diluent in

all subsequent preparations of the sample.

Instrumental parameters

The isocratic flow rate of mobile phase was maintained at 0.25 ml min-1. The

column temperature was adjusted to 35 °C. The injection volume was 0.5 µl. Eluted

sample was monitored at 260 nm and the run time was 5.0 min. The retention time of the

sample was about 1.7 min.

Stress study

All stress decomposition studies were performed at an initial drug concentration

of 100 µg ml-1 in mobile phase and added 5 ml of 1 M HCl, 1 M NaOH or 5% H2O2

separately, and the flasks were heated for 2 h on a water bath maintained at 80 °C. Then

the solutions were cooled and neutralized by adding base or acid, the volume in each

flask was brought to the mark with mobile phase, and the appropriate volume (2.0 µl)

was injected for analysis. Solid state thermal degradation was carried out by exposing

pure drug to dry heat at 105 °C for 3 h. For photolytic degradation studies, pure drug in

solid state was exposed to 1.2 million lux hours in a photo stability chamber. The sample

after exposure to heat and light was used to prepare 100 µg ml-1 solutions in mobile phase

and the chromatographic procedure was followed.

288


Procedure for calibration curve

Working standard solutions containing 0.5-200 µg ml-1 INH were prepared by

serial dilutions of aliquots of the stock solution. Aliquots of 2 µl were injected (six

injections) and eluted with the mobile phase under the reported chromatographic

conditions. The average peak area versus the concentration of INH in µg ml-1 was

plotted. Alternatively, the regression equation was derived using mean peak area-

concentration data and the concentration of the unknown was computed from the

regression equation.

Procedure for tablets

Twenty tablets were accurately weighed and ground into a fine powder. Powder

equivalent to 20 mg INH was transferred into a 100 ml volumetric flask and 60 ml of the

mobile phase was added. The mixture was sonicated for 20 min to achieve complete

dissolution of INH, and the content was then diluted to volume with the mobile phase to

yield a concentration of 200 µg ml-1 INH, and filtered through a 0.22 µm nylon

membrane filter. The tablet extract was injected to the UPLC column after diluting to 100

µg ml-1.

Procedure for method validation

The validation parameters viz; accuracy and precision, LOD, LOQ, robustness

and ruggedness, solution stability and mobile phase stability were carried by following

the same procedure as described under Section 4.3.3.4.


6.5.4.1Method development

Different chromatographic conditions were experimented to achieve better

efficiency of the chromatographic system. Parameters such as mobile phase composition,

wavelength of detection, column, column temperature, pH of mobile phase and diluents

were optimized. Several proportions of buffer, and solvents were evaluated in-order to

289

obtain suitable composition of the mobile phase. Choice of retention time, tailing,

theoretical plates and run time were the major tasks while developing the method.

Alternative combinations of gradient and isocratic methods were also performed to obtain

a suitable peak. Finally, isocratic method was found suitable for the assay.

The separation was carried out with Agilent Zorbax, (50 × 2.1) mm, 5 µm

column. Better results were obtained when INH was eluted in acetonitrile, and water are

used. INH eluted late with higher peak shape and theoretical plates. The peak symmetry

was optimized with varying concentrations of buffer and acetonitrile. Best peak was

obtained with water-acetonitrile ratio (20:80 v/v). Flow rate of 0.5 ml min-1 was selected

with regard to the back pressure and analysis time as well. In order to achieve

symmetrical peak of INH, various stationary phases like C8 (different dimension), C18

(different brand), length (100 mm and 50 mm) and phenyl columns were studied. Agilent

Zorbax, (50 × 2.1) mm, 5 µm column was found to be have the ideal stationary phase for

the determination. The column oven temperature was studied at higher (40°C) and room

(30°C) temperatures and then found that 35°C is the optimum. Shimadzu Pharmaspec

1700 UV/Visible spectrophotometer was used for absorbance measurements. A 100 µg

ml-1 of INH solution in acetonitrile was scanned from 400 to 200 nm against acetonitrile

as blank and wavelength of the method was optimized to 260 nm. Overlay

chromatograms of blank and INH solutions are as shown in Figure 6.5.1.

AU

-0.004

-0.002

0.000

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0.006

0.008

0.010

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0.018

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Figure 6.5.1 Chromatograms of: a) blank, b) pure INH (100 µg ml-1).

6.5.4.2 Stability studies

All forced degradation studies were analyzed at 100 µg ml-1 concentration level.

The observation was made based on the peak area of the respective sample. INH was

found to be more stable under acid and alkaline, photolytic (1.2 million lux hours),

thermal (105 0C for 3 hours) in solid state, and hydrolytic (aqueous, 80 0C for 2 hours)

stress conditions. Less degradation occurred under oxidative stress conditions with

percent decomposition being only around 10 %. The chromatograms obtained for INH

after subjecting to degradation are presented in Figure 6.5.2. Assay study was carried out

by the comparison with the peak area of INH sample without degradation.

1.4

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1.4

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1.5

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(f)

Figure 6.5.2 Chromatograms obtained for INH (100 µg ml-1) after subjecting to stress studies by: (a) acid degradation, (b) base degradation, (c) hydrolytic degradation, (d) thermal degradation (e) photo degradation and (f) oxidative degradation.


The described method for the assay of INH was validated as per the current ICH

Guidelines. Parameters such as system suitability, specificity, precision, robustness,

ruggedness, linearity, accuracy, LOQ, LOD, solution stability, filter compatibility were

studied for the suitability of the method.

System suitability

System suitability is the measurement of performance verification of system,

method and column performance. Theoretical plates (should be more than 2000), tailing

factor (should be less than 1.5) and percentage relative standard deviation (should be less

than 2) for the area and retention time of replicate injections were verified on precision,

ruggedness (variation in column, analyst and day) and robustness (variation in

temperature, mobile phase and wavelength) of the validation. As seen from the data the

theoretical plates are found more than 2000, tailing factor is less than 1.4 and the

percentage relative standard deviation (%RSD) for area and retention time were less than

1.8.

293

Analytical parameters

A linear correlation was obtained between the peak area and the concentration in

the range of 0.5–200 µg ml-1 (Figure 6.5.3) INH from which the linear regression

equation was computed and found to be:

y = 34,989x + 7483 r² = 0.9999

where y is the mean peak area, x is the concentration of INH in µg ml-1 and r is

the correlation coefficient. The LOD and LOQ values, slope (m), y-intercept (a) and their

standard deviations are evaluated and presented in Table 4.4.3. These results confirm the

linear relation between concentration of INH and the peak areas as well as the sensitivity

of the method.

Table 6.5.1 Linearity and regression parameters

Parameter Value Linear range, µg ml-1 0.5-200 Limits of quantification, (LOQ), µg ml-1 0.5 Limits of detection, (LOD), µg ml-1 0.16 Regression equation Slope (b) 34989 Intercept (a) 7483 Correlation coefficient (r) 0.9999

Figure 6.5.3 Calibration curve

294


The percent relative error which is an indicator of accuracy is ≤ 0.89 and is

indicative of high accuracy. The calculated percent relative standard deviation (%RSD)

can be considered to be satisfactory. The peak area based and retention time based RSD

values were < 0.40%. The results obtained for the evaluation of accuracy and precision of

the method are compiled in Tables 6.5.2 and 6.5.3.


At the deliberate varied chromatographic conditions (flow rate, temperature, and

mobile phase composition), the analyte peak % RSD, tailing factor and theoretical plates

remained near to the actual values. The RSD values ranged from 0.1 to 2.0 % resumes the

robustness of the proposed method. In method ruggedness, different columns (same lot),

Table 6.5.2 Results of accuracy study (n=5)

Concentratio

n of INH injected, µg ml-1

Intra-day Inter-day

Concentration of INH found,

µg ml-1

RE,%

Concentration of INH found,

µg ml-1

RE,%

50.0 50.31 0.61 50.45 0.89 100.0 99.50 0.49 99.42 0.58 150.0 150.8 0.54 150.9 0.62

Table 6.5.3 Results of precision study (n=5)

INH

injected (µg ml-1)

Intra-day precision (n=7) Inter-day precision (n=5)

Mean area ±SD

%RSDa

Mean

retention time ±SD

%RSDb

Mean

area ±SD

%RSDa

Mean

retention time ±SD

%RSb

50 1785432± 3568

0.19 1.728 ± 0.009

0.52 1875495± 3984

0.21 1.732 ± 0.010

0.57

100

3466798±

7558

0.21

1.705 ±

0.01

0.58

3532847±

8926

0.26

1.717 ± 0.012

0.69

150 5213454± 8528

0.16 1.719 ± 0.016

0.92 5298258± 9853

0.18 1.708 ± 0.018

1.04

a Relative standard deviation based on peak area; b Relative standard deviation based on retention time.

295

on different days by different analysts were performed. The results were summarized in

Table 6.5.4 and 6.5.5.

Table 6.5.4 Results of method robustness study expressed as intermediate precision (%RSD)

Condition Modification Mean peak area

±S D* %RSD

Mean Rt±SD*

%RSD

Mean theoretical

plates ± SD*

%RSD

Mean tailing factor

± SD*

%RSD

Temperature 30±2 ºC 3547823±7083 0.19 1.737± 0.013

0.74 5624 ±112 1.99 1.08

±0.004 0.39

Mobile phase

composition

(acetonitrile: water) 80±10

3489364±9251 0.26 1.728

± 0.010

0.57 5759 ±119 2.06 1.05 ± 0.006

0.57

Flow rate 0.5±0.05

min 3658921±9635 0.27

1.708 ±

0.018 1.05 5449 ±99 1.81

1.07 ± 0.005

0.45

Wavelength 260±2 nm 3389257±10989 0.32 1.741

± 0.019

1.09 5841 ±128 2.19 1.05 ± 0.004

0.36

*Mean value of three determinations

Selectivity

Selectivity of the method was evaluated by injecting the mobile phase, placebo

blank, pure drug solution and tablet extract. No peaks were observed for mobile phase

and placebo blank and no extra peaks were observed for tablet extracts (Figure 6.5.4)

Table 6.5.5 Results of method ruggedness study expressed as intermediate precision (%RSD)

Variable Mean peak area

±SD* %RSD

Mean Rt ± SD*

%RSD Mean

theoretical plates ± SD

%RSD Mean tailing factor± SD*

%RSD

Analyte (n=3)

3532847± 8926

0.26 1.737± 0.013

0.74 5689±115 2.02 1.12±0.003 0.36

Column (n=3)

3489853±10876 0.31 1.705 ± 0.01

0.58 5728±124 2.11 1.07±0.004 0.46

*Mean value of three determinations

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Figure 6.5.4 Chromatograms obtained for (a) tablet extract (100 µg ml-1) and (b)

placebo blank

Stability of the solution and mobile phase stability

At the specified time interval, % assay of INH obtained from drug solution

stability and mobile phase stability were within 1%. For solution stability same sample

solution was injected at 0, 6, 12 and 24 hours and for mobile phase stability, separately

prepared INH sample injected at the same time interval as above. The results confirmed

that the INH solution of drug and mobile phase were stable at least for 24 hours during

the assay performance, which are represented in Table 6.5.6.

Table 6.5.6 Solution stability results

Initial 6 hours 12 hours 18 hours 24 hours

% Assay for solution stability

99.85 100.1 99.66 99.69 99.36

% Assay for mobile phase stability

99.75 100.0 100.1 99.83 99.75

297


A 100 µg ml-1 solution of tablets was prepared as per ‘Procedure for tablets’ and

injected in triplicate to the UPLC system. The mean peak area of the tablet extract for this

concentration was found to be equivalent to that of pure drug solution of the same

concentration and the results were compared with those of a reference method [3]. The

accuracy and precision of the proposed method was further evaluated by applying

Student’s t- test and variance ratio F- test, respectively. The t- and F- values at 95%

confidence level did not exceed the tabulated values and this further confirms that there is

no significant difference between the reference and proposed methods with respect to

accuracy and precision. Table 6.5.7 illustrates the results obtained from this study.

Recovery studies

A standard addition procedure was followed to further evaluate the accuracy of

the method. The solutions were prepared by spiking pre-analyzed tablet with pure drug at

three different levels and injected to chromatographic column. The recovery of the

known amount of added analyte was computed. The percentage recovery of INH from

pharmaceutical dosage forms ranged from 99.78 to 100.4%. The results presented in

Table 6.5.8 reveal good accuracy of the proposed method.

Table 6.5.7 Results of determination of INH in tablet and statistical comparison with the reference method

Formulation brand name

Nominal amount,

mg

% INH found* ± SD t-value

F- value Reference

method Proposed method

Isokin

300

99.36 ±1.65

100.2 ±0.65

2.47

6.48

* Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.78; Tabulated F-value at 95% confidence level is 6.39


Tablet studied


Pure INH added, µg ml-1


Pure INH recovered* (%INH±SD)

Isokin 50.10 50.10 50.10

25.0 50.0 75.0

75.32 99.87 125.6

100.3±1.35 99.78±0.98 100.4±1.21

298

SECTION 6.6

CONCLUSION ON CHAPTER 6 –Assessment of methods

A simple, rapid and accurate potentiometric method with best performance

parameters when compared to existing titrimetric method was developed for the assay of

INH (Section 6.1). The method could be successfully applied to the determination over

1.5-15 mg of INH which shows broad applicability of the method to the tablets.

Considering the results obtained, it is possible to affirm that the method is rapid,

selective, accurate and precise and hence suitable for the determination of INH in tablets.

The proposed method has the distinct advantages over the existing methods in terms of

simplicity of technique and ease of performance and does not need expensive and highly

sophisticate equipment or high-cost organic solvents. The titration can be carried out with

precision and accuracy comparable to those obtained with aqueous titrations. Hence, the

method can be used in routine analysis in pharmaceutical quality control laboratories.

Spectrophotometry, particularly in the visible region of the electromagnetic

spectrum, is one of the most widely used methods of analysis in clinical, environmental

and pharmaceutical analysis labs because many substances can be selectively converted

to a colored derivative. In addition, the instrumentation is readily available and generally

fairly easy to operate. However, most of the reported methods suffer from one or other

disadvantage such as poor sensitivity, Use of unstable oxidants, heating step, poor

selectivity, tedious time consuming liquid-liquid extraction step, strict pH control and

narrow linear range, etc., as indicated in Table 6.6.1. In this Chapter, the author has made

an attempt to develop and validate four visible spectrophotometric methods for the

determination of INH using different reagents and based on different reaction schemes.

Among the four proposed methods, two methods employing KMnO4 are the most

sensitive one as can be seen from the molar absorptivity values which range from 2.5 ×

105 to 3.0 × 104 l mol-1 cm-1. The methods have been demonstrated to be free from rigid

experimental conditions like heating or extraction.

299

Table 6.6.1 Comparison of performance characteristics of the proposed methods with the existing methods

Sl. No.

Reagent/s used Methodology λλλλmax (nm)

Linear range (µg ml-1)

(ε = l mol-1cm-1) Remarks Ref

1

Ethyl vanillin

Measurement of yellow colored hydrazone complex

410

2-16

(7.1 x 103)

Less sensitive 9

2

Vanillin

Measurement of yellow colored hydrazone complex

405

1-12

Moderately sensitive

10

3 1, 2-naphthoquinone-4-sulfonate Measurement of pink colred condensed product

495

0.5-3.0 (1.18 x 104)

Requires close pH control

11

4 Isatin Measurement of yellow colored hydrazone complex

340

0-32 (1.2 x 104)

Time consuming, requires close pH control 12

5. 2-hydroxy-1,4-napthoquinone Measurement of derivatized product

365

5-25

Requires close pH control, use of non-aqueous medium

13

6 6-methyl-2-pyridinecarboxaldehyde

Measurement of hydrazone derivative 328 2-16

Measurement at lower analytical wavelength

14 7 Tiron-KIO4 Measurement of red colored oxidative

coupled product 505 1.5-18

(1.77 x 104) Use of unstable oxidant 15

8. Tiron-NaIO4 Measurement of red colored oxidative coupled product

507 1.0-15 (1.84 x 104)

Use of unstable oxidant 16

9 4,4’-methylene-bis-m-nitroaniline

Measurement of purplecolored diazo-coupled complex

495 0.1-15 (5.63 x 104)

Requires costly reagent 17

10

4,4’-sulphonyldianiline

Measurement of purplecolored diazo-coupled complex

440 335

0.5-20 (5.72 x 104)

Requires cooling, 18

11

Epichlorohydrine hydroxyphenacylchloride

Measurement of purplecolored coupled complex

405

402

2-22 (0.51 x 104)

20-120

(0.10 x 104)

Longer reaction time and heating 19

12 Uranyl acetate Measurement of yellow colored uranyl isonicotinyldisthiocarbazate complex

410

-

Multistep reaction, time consuming 20

300

13 Neocuproine Measurement of redox complex 454 0.3-3.5

Requires close pH control 21

14 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine

Measurement of complex 558 0.62-6.15 Unstable oxidant 22

15 Potassium ferricyanide Measurement of prussian blue 735 0.04-8 (3.92 x 104)

Unstable oxidant 23

16 4-(2-Pyridylaxo)resorcinol Measurement of complex 510 0.05-4.5

Unstable oxidant 24

17 NBS Measurement of starch-iodine complex

572 0.1-3.4

Multistep reaction 25

18

Chloranilic acid Tetracyanoethylene 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Thymol blue Bromo phenol blue Bromocresol green

Measurement of charge-transfer complex Measurement of ion-associate complex

500 480 580 390 410 320

1.37-8.2

6.85-34.27

10.96-21.93

6.85-41.13

1.37-6.85

1.37-8.22

Use of organic solvent, requires heating 26

15

Rose bengal

Measurement of acetone-chloroform extractable ion-pair complex

555

2.8-5.6

Requires time consuming and tedious extraction step, use of organic solvent.

27

16

KMnO4-Methyl orange

KMnO4Indigo carmine

Measurement of unbleached color of the dye

520 610

0.25-5.0 (2.5 x 105)

0.24-4.0 (3.0 x 105)

Simple, highly sensitive, no heating, no extraction step

Present work

17

Iron(III)-1,10-phenanthroline Iron(III)-2, 2΄-bipyridyl

Red colored chromogen (ferroin) formed is measured

Red colored chromogen [Fe(II)-Bipy complex]formed is measured

510

540

0.4-8.0 (1.7 x 105)

1-15 (7.5 x 104)

Simple, sensitive, aqueous medium and extraction free

Present work

301

The UV- spectrophotometric method is simple and cost-effective in terms of the

media employed (H2SO4). Wide linear dynamic ranges (1-10 µg ml-1), high sensitivity

(ε=8.38×104) and low LOQ values (< 1.2µg ml-1) are the features of the proposed UV-

spectrophotometric methods. Moreover, the developed UV-spectrophotometric is first of

its kind to demonstrate the stability-indicating property of method.

UPLC, which is becoming popular because of its speed and cost-effectiveness,

was not earlier used for the assay of INH. Using this technique, the author has developed

a rapid, isocratic RP-UPLC method for the drug in its pure form and tablet. The results

obtained are highly satisfactory with respect to accuracy, precision, selectivity,

robustness and ruggedness. A short retention time of 1.7 min enables rapid determination

of the drug which is of paramount importance in routine analysis. The method was

demonstrated to be stability-indicating with the results of the degradation study revealing

that the drug is stable to all other conditions with the exception of oxidative degradation

with slight degradation.

Selectivity of all the proposed methods was examined by both placebo blank and

synthetic mixture analyses. The methods are free from common excipients found in drug

formulations.

Statistical comparison of the proposed methods with those of the reference

method was performed by applying the Student’s t-test for accuracy and F-test for

precision. All calculated values were found to be less than the tabulated t- and F- values.

The results show that there is no significant difference between the results of the

proposed methods and reference method. The accuracy and validity of the methods were

further ascertained by recovery studies via spike method. The methods have been

demonstrated to be fairly accurate and precise in addition to being sensitive and simple.

Hence, the proposed methods can usefully be employed in quality control laboratories.

302

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CHAPTER 6 TITRIMETRIC, SPECTROPHOTOMETRIC AND ...shodhganga.inflibnet.ac.in/bitstream/10603/36434/10/chapter 6.pdf · proposed for the assay of INH in dosage forms. Three redox titrimetric