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
6.2.1.0 INTRODUCTION
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.
6.2.1.2 Method validation
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.
Table 6.2.3 Results of intra-day and inter-day accuracy and precision study
INH taken, µg ml-1
Intra-day accuracy and precision
(n=7)
Inter-day accuracy and precision
(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.
Table 6.2.6 Results of recovery study via standard addition method
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.
6.3.2.0 EXPERIMENTAL
6.3.2.1 Apparatus
The instrument is the same that was described in Section 2.1.2.1.
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.
6.3.3.0 ASSAY PROCEDURES
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
solution prepared as described under ‘Procedure for tablets’ and then analyzed using the
procedures described above.
To 10 mg of the placebo blank, 5 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.
6.3.4.0 RESULTS AND DISCUSSION
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
6.3.3.4 Method validation
Linearity and sensitivity
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.
Table 6.3.1 Sensitivity and regression parameters
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
Accuracy and precision
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.
Robustness and ruggedness
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-
Table 6.3.2 Results of intra-day and inter-day accuracy and precision study
Method
INH taken
µg ml-1
Intra-day accuracy and precision (n=7)
Inter-day accuracy and precision
(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.
Application to tablets
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
INH in tablet, µg ml-1
Pure INH
added, µg ml-1
Total found, µg ml-1
Pure INH recovered*
Percent ± SD
INH in tablet, µg ml-1
Pure INH
added, µg ml-1
Total found, 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
6.4.1.0 INTRODUCTION
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.
6.4.2.0 EXPERIMENTAL
6.3.2.1 Apparatus
The instrument is the same that was described in Section 2.1.2.1.
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.
6.4.3.0 ASSAY PROCEDURES
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
solution prepared as described under ‘Procedure for tablets’ and then analyzed using the
procedures described above.
To 20 mg of the placebo blank, 10 mg of INH was added and homogenized,
transferred to 100 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.
6.4.4.0 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
6.4.4.2 Method validation
Linearity and sensitivity
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
Accuracy and precision
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.
Table 6.4.1 Sensitivity and regression parameters
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.
Table 6.4.2 Results of intra-day and inter-day accuracy and precision study
Methods* INH
taken µg ml-1
Intra-day accuracy and precision
(n = 7)
Inter-day accuracy and precision
(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.
Robustness and ruggedness
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
Application to tablets
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
Table 6.4.5 Results of recovery study via standard addition method
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
6.5.1.0 INTRODUCTION
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.
6.5.2.0 EXPERIMENTAL
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
6.5.3.0 ASSAY PROCEDURES
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.
6.5.4.0 RESULTS AND DISCUSSION
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
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
(a)
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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
54
1.5
17
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(b)
ISONIA
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1.4
83
1.5
83
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0.00
0.02
0.04
0.06
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0.28
0.30
0.32
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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
(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.
6.5.4.3 Method validation
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
Accuracy and precision
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.
Robustness and ruggedness
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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(b)
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
Application to tablets
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
Table 6.5.8 Results of recovery study via standard addition method
Tablet studied
INH in tablet, µg ml-1
Pure INH added, µg ml-1
Total found, 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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