CHAPTER V

SPECTROPHOTOMETRIC AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY OF IRBESARTAN

166

Section 5.0

DRUG PROFILE AND LITERATURE SURVEY

5.0.1 DRUG PROFILE

Irbesartan (IRB) is chemically known as 2-butyl-3-[p-(o-1 H-tetrazol-5-

ylphenyl)benzyl]-1,3-diazospiro[4,4]non-1-en-4-one [1].Its molecular formula is

C25H28N6O and molecular weight 428.54 g mol-1. Physically, IRB is white to off-

white powder. IRB has the following chemical structure:

HN N

N N NO

NCH3

IRB is practically insoluble in water, sparingly soluble in methanol, soluble in

dichloromethane, acetonitrile and chloroform.

IRB is a potent, long-acting, nonpeptide angiotensin II receptor antagonist [2]

having high selectivity for the AT1 subtype (angiotensin I). Irbesartan inhibits the

activity of angiotensin II (AII) via specific, selective noncompetitive antagonism of

the AII receptor subtype 1 (AT1) which mediates most of the known physiological

activities of AII [3]. It is potentially safe and more tolerable than other classes of

antihypertensive drugs. It is indicated for hypertension. Irbesartan reduces the

chances of cardiac failure, myocardial infarction, sudden death, and death from

progressive systolic failure. Irbesartan may also delay progression of diabetic

nephropathy and is also indicated for the reduction of renal disease progression in

patients with type 2 diabetes, hypertension, and microalbuminuria or proteinuria [4].

IRB is officially listed in Martindale: The Extra Pharmacopoeia [5] and USP [6].

167

5.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

IRBESARTAN

5.0.2.1 Visible spectrophotometry

There are only three reports dealing with the spectrophotometric methods for

irbesartan. Five methods based on the formation of ion-pair complex, extraction into

organic solvent and measurement of absorbance at the wavelength of maximum

absorption using five different ion-pair reagents viz, picric acid, bromocresol green,

bromothymol blue, cobaltthiocyanate and molybdenum thiocyanate have been

described by Abdellatef [7]. Based on a similar reaction but employing Erichrome

Black-T as ion-pair reagent in an acidic buffer of pH 3.5, an extractive

spectrophotometric method for the determination of IRB in 50-250 µg ml-1 range has

recently [8] been reported . A kinetic spectrophotometric method, based on the

reaction of carboxylic acid group of the IRB with mixture of KIO3 and KI in which

the yellow colored triiodide ion is formed, has been described by Ding et al., [9]. The

reaction was followed spectrophotometrically by measuring the rate of change of

absorbance at 352 nm. Both initial rate and fixed-time methods were used to compute

the unknown concentrations.

5.0.2.2 HPLC methods.

Perhaps the most widely used technique for the assay of IRB in both

pharmaceuticals and body fluids has been the HPLC. One of the first reports for IRB

in tablets uses CLC-ODS column with a mixture of H2O, acetonitrile and

triethylamine (50:50:0.15) as mobile phase at a flow rate of 1.0 ml min-1 with UV-

detection at 245 nm. The method was applicable over a concentration range of 49-146

µg ml-1 [10]. Using Diamonsil C18 column (250 mm× 4.6 mm, 5µm) and a mobile

phase consisting of acetonitrile-0.02% KH2PO4 (45:55) (pH adjusted to 2.6 with

H3PO4) and a flow rate of 1.0 ml min -1 with UV-detection at 245 nm, the drug in

sustained tablets [11] was determined in the concentration range, 12.4-185.4 µg ml-1.

Jiang Dongbo et al.[12] have determined main components and related substances in

IRB-sustained capsules by RP-HPLC. This was performed on a Kromasil C18 column

using acetonitrile-0.02 M KH2PO4 (48:52, pH 2.60 adjusted by H3PO4 solution) as the

168

mobile phase at a flow rate of 1.0 ml min-1 with UV-detection at 245 nm. The

detector response was linear in the 13.22-185.08 µg ml-1 concentration range.

Recently, Reddy et al [13] have used HPLC for the separation and

simultaneous determination of process-related substance of IRB in bulk drug. The

bulk drug was subjected to acid, base, hydrolytic, oxidative, thermal and photolytic

degradation. Considerable degradation was observed under acid, base, and oxidation

conditions. Separation was achieved on a symmetry shield RP 18 LC column using a

mobile phase consisting of a mixture of aqueous KH2PO4 and acetonitrile. A stability

indicating RP-HPLC method for IRB in pure and pharmaceutical dosage form was

developed and validated by Praveen Kumar and Sreeramulu [14].The

chromatographic conditions comprised of reversed phase C18 column (250×4.6 mm,

5µm) with a mobile phase consisting of a mixture of acetonitrile-0.03 M KH2PO4, pH

3.0) in the ratio 15:85 at a flow rate of 1 ml min-1 and UV-detection at 275 nm. The

calibration plot showed good linear relationship in the concentration range 2-12 µg

ml-1. The drug was found to undergo degradation under acidic, basic, photo and

thermal forced stress conditions. IRB in bulk drug and tablet dosage form was also

determined by an isocratic RP-HPLC method [15] employing a symmetry C8

(150×4.6 mm, 5µm) at ambient temperature. The mobile phase was a mixture of 0.01

M NaH2PO4 (pH 3.0) and acetonitrile (50:50) and the UV detection wavelength was

209 nm. Very recently [16], an RP-HPLC-PDA method with a C18column (150×4.6

mm, 5µm) and methanol: formic acid (0.02%) (70:30) as mobile phase at a flow rate

of 1 ml min-1 with UV-detection at 234 nm was reported for 10-50 µg ml-1 IRB.

Apart from methods when IRB was present alone in dosage forms [10-16],

several HPLC methods have been reported for the simultaneous determination of IRB

and hydrochlorothiozide in combined dosage forms [17-25].

5.0.2.3 Other methods.

Many other methods reported for IRB in pharmaceuticals include HPTLC

when present alone [26] and in combined dosage forms with hydrochlorothiozide [27,

28], UPLC [29], derivative UV-spectrophotometry [30-34] absorbance ratio uv-

spectrophotometry [35] and difference UV-spectrophotometry [36] all in combined

dosage forms [30-36], spectrofluorimetry [34] and voltammetry [37, 38]. Besides,

169

using LC-MS/TOF, Msn, and H/D exchange and LC-NMR the degradation products

of IRB have been identified and characterized [39].

5.0.2.4 Methods for body fluids.

Quite a good number of reports are found in the literature which are devoted

to body fluids. IRB in human blood plasma has been assayed by HPLC [40-48], LC-

MS [49-53] and capillary HPLC [18]. HPLC also finds application in the

determination of IRB in human serum [54] rat plasma [55], and human plasma and

urine [56]. IRB in rat plasma has also been determined by LC/MS/MS [57].

Voltammetry is one more technique that has been used for the determination of IRB

in body fluids such as human blood [37] and human blood serum [58].

From the foregoing paragraphs, it is clear that the reported visible

spectrophotometric methods [7-9] suffer from such disadvantages as rigid pH control

and extraction step [7, 8] and judicious control of all experimental variables [9]. More

number of HPLC methods are found for IRB when it is present in combined dosage

forms along with hydrochlorothizide [17-25] than when it is present alone in its

dosage forms [10-16]. The only stability-indicating HPLC method [14] available for

IRB is applicable over a narrow-linear range of concentration (2-12 µg ml-1).

In an attempt to overcome the limitations of the existing methods, the author

has developed five visible spectrophotometric methods and one HPLC method, the

last being the stability-indicating. The details concerning the method development

and validation of these new methods are presented in this Chapter.

170

Section 5.1

SIMPLE AND SELECTIVE SPECTROPHOTOMETRIC DETERMINATION

OF IRBESARTAN IN TABLETS USING TWO NITROPHENOLS AS

CHROMOGENIC AGENTS

5.1.0 INTRODUCTION The chemistry and analytical utility of charge transfer complexation reaction

in spectrophotometric assay of organic compounds of pharmaceutical importance has

been reviewed in Section 2.3.1.

From the literature survey presented in Section 5.0.2, it is clear that there is no

report dealing with the determination of IRB in pharmaceutical formulations, based

on its reaction with nitrophenols such as 2, 4, 6-trinitrophenol (picric acid; PA) or

2,4-dinitrophenol (DNP). The reagents under study, i.e., PA and 2,4-DNP have

numerous applications as analytical reagents and they have been used for the

spectrophotometric determination of many drugs in pharmaceutical formulations [59-

62]. In this Section (5.1), the author has used PA and DNP as chromogenic agents to

develop two spectrophotometric methods for the determination of IRB in tablet

formulation. Both methods are based on the charge transfer from the Lewis acid such

as PA and DNP to the amino group of IRB which works as Lewis base and formation

of yellow colored ion-pair complexes. The colored complexes formed show

absorption maximum at 420 and 425 nm for DNP (method A) and PA (Method B),

respectively. The details about the reaction chemistry, method development and

validation as well as applications of both methods are presented in this Section (5.1).

5.1.2 EXPERIMENTAL

5.1.2.1 Apparatus

The instrument used for absorbance measurements was the same as described

in Section 2.2.2.1.

5.1.2.2 Materials

All chemicals used were of analytical reagent grade. Pure IRB

(Pharmaceutical grade) sample was kindly provided by Jubiliant Life Sciences Ltd,

Nanjangud, Mysore, India, as a gift and used as received. Two brands of tablets,

171

namely, Irovel-150 and Irovel-300 (both from Sun Pharmaceuticals Ltd. India) were

obtained from the commercial sources.

2,4-Dinitrophenol (DNP, 0.1%): A 0.1% (w/v) solution of DNP (Loba Chemie Pvt.

Ltd., Mumbai, India) was prepared in dichloromethane and used for the assay in

method A.

Picric acid (PA, 0.1%): A 0.1% (w/v) solution of PA (Sisco Research Laboratories

Pvt. Ltd., Mumbai, India) was prepared in dichloromethane and used for the assay in

method B.

Standard IRB solution: A stock standard solution containing 300 g ml-1 IRB was

prepared by dissolving 30 mg of pure drug in dichloromethane and diluting to the

mark in a 100 ml calibrated flask with same solvent. The stock standard solution was

diluted appropriately with dichloromethane to get working concentrations of 150 and

100 µg ml-1 IRB for use in method A and method B, respectively.

5.1.2.3 Recommended procedures

Calibration curves

Method A (using DNP)

Different aliquots (0.2-3.5 ml) of a standard IRB (150 µg ml-1) solution were

accurately transferred into a series of 5 ml calibrated flasks using a micro burette and

the total volume was adjusted to 3.5ml by adding adequate quantity of

dichloromethane. One ml of 0.1% DNP solution was added to each flask and the

mixture was diluted to the volume with dichloromethane and mixed well. The

absorbance of each solution was measured at 420 nm against a reagent blank after 5

min.

Method B (Using PA)

Aliquots (0.2-3.5 ml) of a standard IRB (100 µg ml-1) solution were accurately

transferred into a series of 5 ml calibrated flasks, as described above. To each flask

was then added 1.0 ml of 0.1% PA and the content was diluted to volume with

dichloromethane and was mixed well. After 5 min, the absorbance was measured at

425nm against a reagent blank prepared simultaneously.

172

In both the cases, calibration curves were prepared and 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.

Procedure for tablets

The content of ten tablets each containing 150 or 300 mg of IRB was

weighed. An accurately weighed quantity equivalent to 30 mg of IRB was transferred

into a 100 ml calibrated flask and dissolved in 60 ml dichloromethane. The content

was shaken thoroughly for about 15-20 min, diluted to the mark with

dichloromethane, mixed well and filtered using a Whatman No. 42 filter paper. The

first 10 ml portion of the filtrate was discarded and a suitable aliquot of the filtrate

(300 µg ml-1 IRB) was diluted to get the working concentrations of 150 and 100 µg

ml-1 IRB for analysis by methods A and B, respectively, as described above.

Placebo blank analysis

A placebo blank of the composition: talc (45 mg), starch (35 mg), acacia (30

mg), methyl cellulose (35 mg), sodium citrate (40 mg), magnesium stearate (50 mg)

and sodium alginate (45 mg) was made and its solution was prepared by taking 50 mg

as described under “Procedure for tablets” and then subjected to analysis.

Procedure for synthetic mixture analysis

To the placebo blank of the composition described above, 30 mg of IRB was

added to 20 mg of placebo blank and homogenized, transferred to 100 ml calibrated

flask and the solution was prepared as described under “procedure for tablets” Then

the resulting solution was subjected to analysis using the procedure described above.

The analysis was done to study the interferences of excipients such as talc, starch,

acacia, methyl cellulose, sodium citrate, magnesium stearate and sodium alginate.

5.1.3. RESULTS AND DISCUSSION

5.1.3.1 Absorption spectra

The reaction of PA or DNP as Lewis acid with IRB as Lewis base results in

the formation of an intense yellow colored product. The absorption spectra of the

yellow colored products were recorded at 380 - 500 nm against the corresponding

blank solutions. The large difference between the absorbance of the blank and the

measured species was observed at 420 nm for IRB-DNP and the resulting yellow

173

colored CT complex showed maximum absorbance at 425 nm for IRB-PA (Fig.

5.1.1).

Figure 5.1.1 Absorption spectra of:

a. IRB-DNP C-T complex (60.0 µg ml-1 IRB), b. blank (method A), c. IRB-PA C-T complex (40.0 µg ml-1 IRB), d. blank (method B).

5.1.3.2 Reaction pathway Charge-transfer complex is a complex formed between an electron-donor and an

electron-acceptor, characterized by electronic transition(s) to an excited state in which

there is a partial transfer of electronic charge from the donor to the acceptor moiety.

As a result, the excitation energy of this resonance occurs very frequently in the

visible region of the electro-magnetic spectrum [63]. This produces the usually

intense colors characteristic of these complexes.

Therefore, IRB, a nitrogenous base, an n-donor, was made to react with DNP

and PA to produce a coloured charge transfer complexes in dichloromethane.

Ttrinitrophenol (Picric acid) and dinitrophenol react with electron donor

molecule to form charge transfer and proton transfer complexes [60, 61].It was used

for the determination of some amine derivatives through formation of intense yellow

coloured complex. Interestingly, application of picric acid for quantitative estimation

of orphendrine citrate and phentolamine mesylate injections is official in the USP

[64].

When an amine is combined with a polynitrophenol, one type of force field

produces an acid-base interaction, and the other, an electron donor-acceptor

interaction. The former interaction leads to the formation of true phenolate by proton-

transfer, and the latter, to a true molecular compound by charge-transfer [65]. Based

174

on this, the reaction pathway can be discussed in terms of transfer of electronic

charge from IRB, an electron-rich molecule (a Lewis-base donor), to the ring of DNP

or PA, an electron-deficient molecule (a Lewis-acid acceptor), and at the same time

the proton of the hydroxyl group of DNP or PA will transfer to the secondary amine

of IRB. The interaction between IRB (D), an n-donor and nitrophenols (A), π-

acceptors, is a charge transfer complexation reaction followed by the formation of

radical ions as shown in Scheme 5.1.1.

D•• + A → [D••→ A] → D•+ + A• −

[Donor + Acceptor → Complex → Radical ions]

The explanation for the produced color in both methods lies in the formation of

complexes between the pairs of molecules IRB-DNP and IRB-PA, and this complex

formation leads to the production of two new molecular orbitals and, consequently, to

a new electronic transition [66].

NHN

NN

NON

H3C OHR1

R2

R3

DNP or PA

For DNP: R1=R2= NO2 and R3=H For PA: R1=R2=R3= NO2

C-T Complex measured at 420 nm for DNP, 425nm for PA

NHN

NN

NON

H3COH

R1

R2

R3

NHN

NN

NON

H3C

OR1

R2

R3

Scheme 5.1.1. The possible reaction pathway for the formation of IRB-DNP/PA C-T

complex.

5.1.3.3 Method development

Optimum conditions were established by measuring the absorbance of C-T

complexes at 420 and 425 nm, for method A and method B, respectively, by varying

one and fixing other parameters.

175

Effect of reagent concentration

To establish optimum concentrations of the reagents for the sensitive and

rapid formation of the IRB charge transfer complexes, the drug (IRB) was allowed to

react with different volumes of the reagents (0.5 – 2.5 ml of 0.1% DNP and 0.5 - 3 ml

of 0.1% PA). In both the cases, maximum and minimum absorbance values were

obtained for sample and blank, respectively, only when 1 ml of the reagent was used.

Therefore, 1 ml of reagent in a total volume of 5 ml was used throughout the

investigation.

Choice of solvent to dissolve drug and reagents

To dissolve IRB, dichloromethane was preferred to chloroform, acetonitrile,

acetone, 1,4-dioxane, methanol and ethanol because as the complex formed in these

solvents either had very low absorbance values or precipitated upon dilution. Where

as in the case of reagents, highly intense coloured products were formed when

dichloromethane medium was maintained as solvent to dissolve DNP and PA.

Therefore, dichloromethane was chosen as solvents to dissolve IRB and reagents.

Effect of reaction time and stability of the C-T complexes

The optimum reaction time was determined by following the absorbance of

the developed color upon the addition of DNP or PA solution to the IRB solution at

room temperature. For both methods, the reaction was found to be complete and

quantitative when the reaction mixture was allowed to stand for 5 min, and any delay

in the absorbance measurements of the yellow ion pair complexes had no effect on

the reaction stoichiometry which was determined to be 1: 1 (IRB: reagent) for the

ranges studied. The C-T complexes of IRB with DNP and PA which were used for

quantitation of the drug were found to be stable up to 2 and 2.5 hrs, respectively.

Investigation of composition of C-T complexes

The composition of the C-T complexes with either DNP or PA was evaluated

by following the Job’s continuous variations method [67]. The experiments were

performed by preparing and mixing equimolar solutions of drug and reagent (method

A: 6.73 × 10-7 M IRB and DNP; method B: 5.54 × 10-5 M IRB and PA) by

maintaining the total volume at 2.5 ml. The plots of the molar ratio between drug and

reagent versus the absorbance values were prepared (Figure 5.1.2a and 2b), and the

176

results revealed that the formation of C-T complex between drug and reagent

followed a 1:1 reaction stoichiometry.

(a) (b)

Figure 5.1.2 Job’s plots obtained for C-T complex from equimolar solutions of (a) IRB & DNP and (b) IRB & PA.

5.1.3.4 Method validation

Linearity and sensitivity

Under the optimized experimental conditions for IRB determination, the

standard calibration curves for IRB with DNP and PA were constructed by plotting

absorbance versus concentration (Fig. 5.1.3). The linear regression equations were

obtained by the method of least squares and the Beer's law range, molar absorptivity,

Sandell’s sensitivity, correlation coefficient, standard deviation of intercept (Sa),

standard deviation of slop (Sb), limits of detection and quantification for both

methods are summarized in Table 5.1.1.

Method A Method B

Figure. 5.1.3 Calibration Curves.

0

0.1

0.2

0.3

0.4

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratioVIRB/(VIRB+VPA)

00.20.40.60.8

11.2

0 20 40 60 80

Abso

rban

ce

Concentration of IRB, , µg ml-1

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120

Abso

rban

ce

Concentration of IRB, µg ml-1

00.10.20.30.40.50.6

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratioVIRB/(VIRB+VDNP)

177

Table 5.1.1 Sensitivity and regression parameters Parameter Method A Method B max, nm 420 425 Color stability, hrs 2.0 2.5 Linear range, µg ml-1 6-105 4-70

Molar absorptivity (ε), l mol-1cm-1 3.8 × 103 6.4 × 103

Sandell sensitivity*, µg cm-2 0.1121 0.0670 Limit of detection (LOD), µg ml-1 0.49 0.53 Limit of quantification (LOQ), µg ml-1 1.49 1.62 Regression equation, Y**

Intercept (a) 0.0005 0.0120 Slope (b) 0.0088 0.0144 Standard deviation of a (Sa) 0.0008 0.0107 Standard deviation of b (Sb) 1.3 × 10-4 2.5 × 10-4 Regression coefficient (r) 0.9995 0.9993

a Limit of determination as the weight in µg ml-1 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.b Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1 Accuracy and precision

In order to evaluate the precision of the proposed methods, solutions

containing three different concentrations of IRB were prepared and analyzed in seven

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

day precision) and the results were summarized in Table 5.1.2. The low values of the

percentage relative standard deviation (RSD ≤ 1.78% for intra-day) and (RSD ≤

2.18% for inter-day) indicate the high precision of the proposed methods. Also, the

accuracy of the proposed methods was evaluated as percentage relative error (RE %)

and from the results shown in Table 5.1.2, it is clear that the accuracy is good (RE ≤

2.84%).

Selectivity

The selectivity of the proposed methods for the analysis of IRB was evaluated

by analysis of placebo blank solution as shown under Section 5.1.2.3 and the

resulting absorbance readings in both methods were same as reagent blank, inferring

no interference from the placebo. Non interference from placebo was further

confirmed by carrying out recovery study from synthetic mixture which gave percent

recoveries of 99.42 ± 2.25 and 98.56 ± 2.63 for method A and method B,

178

respectively. These results confirm the selectivity of the proposed methods in the

presence of the commonly employed excipients added to the formulations.

Robustness and ruggedness

The evaluation of the method robustness was done by making small

incremental changes in two experimental variables, reagent volume and reaction time,

and performing the analysis under the altered experimental conditions. The effect of

the changes on the absorbance reading of the resulted complexes in both methods was

studied and found to be negligible confirming the robustness of the proposed

methods. Method ruggedness was expressed as % RSD of the same procedure applied

by four analysts and also by a single analyst performing analysis on four different

cuvettes. The results presented in Table 5.1.3 showed that no statistical differences

between different analysts and instruments suggesting that the proposed methods

were rugged.

Applications to analysis of tablets

The proposed methods were applied to the determination of IRB in two

representative tablets Irovel-150 and Irovel-300. The results obtained are compiled in

Table 5.1.4 and were compared with those obtained by the official method [6] by

means of Student’s t- test for accuracy and F- tests for precision at 95% confidence

Table 5.1.2 Results of intra-day and inter-day accuracy and precision study

Method IRB

taken, µg ml-1

Intra-day accuracy and precision

(n=7)

Inter-day accuracy and precision

(n=5) IRB

found, µg ml-1

RE,% RSD,% IRB

found, µg ml-1

RE, % RSD,%

A 30.0 60.0 90.0

30.40 59.08 91.62

1.35 1.51 1.92

1.27 1.78 1.19

30.46 59.32 91.76

1.37 1.61 1.98

1.34 1.88 2.18

B 20.0 40.0 60.0

20.23 39.86 61.26

1.18 0.79 2.08

1.50 1.77 1.67

20.31 39.79 61.45

1.23 1.03 2.84

1.74 1.66 1.78

RE. Percent relative error, RSD. relative standard deviation. n = Number of measurements

179

level. The official method involved analysis by chromatography on a column (4.6 mm

X 25 cm) containing packing L1 with a mobile phase consisting of phosphate buffer

and acetonitrile (67:33), at a flow rate of 1.0 ml min -1 and the UV-detection being set

at 220 nm. As can be seen from the Table 5.1.4, the calculated t- and F- values at

95% confidence level did not exceed the tabulated values of 2.78 and 6.39,

respectively, indicating that there were no significant differences between the

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

Table 5.1.3 Results of robustness and ruggedness study expressed as intermediate precision ( %RSD)

Method IRB

taken, µg ml-1

Robustness Ruggedness

Parameters altered Inter-analysts (RSD, %),

(n=4)

Inter-cuvettes

(RSD, %), (n=4)

Volume of DNP/PA*

Reaction timeΨ

DNP 30.0 60.0 90.0

0.94 1.36 1.27

0.58 0.65 0.42

1.28 0.84 0.85

2.42 3.15 2.76

PA 20.0 40.0 60.0

0.66 0.74 1.03

0.36 0.85 0.64

0.96 0.78 0.54

1.98 2.38 1.62

*The volumes of DNP or PA added were 1±0.2. Ψ The reaction times were 5±1 min

*Mean value of five determinations.

Table 5.1.4 Results of analysis of IRB formulations by the proposed methods and statistical comparison of the results with the reference method

Brand name

Nominal amount#

Percent IRB found±SD* Reference

method Method A Method B

Irovel-150 150 99.25±0.92

98.58±1.03 t = 2.74 F = 1.03

98.76±1.02 t = 2.28 F = 1.22

Irovel-300 300 101.20±0.83

100.79±1.18 t = 1.49 F = 2.02

102.08±1.23 t = 2.35 F = 2.19

180

Recovery study

The accuracy and validity of the proposed methods were further confirmed by the standard addition procedure. Pre-analyzed

tablet powder (Irovel-150 and Irovel-300) was spiked with pure IRB at three different concentration levels (50, 100 and 150% of the

quantity present in the tablet powder) and the total was analyzed by the proposed method. The results of this study are presented in

Table 5.1.5 and indicate that the excipients present in the tablets did not interfere in the assay.

*Mean value of three determinations.

Table 5.1.5 Results of recovery study via standard-addition method

Tablet

studied

Method A Method B

IRB in tablet, µg ml-1

Pure IRB

added, µg ml-1

Total found, µg ml-1

Pure IRB recovered

(Percent±SD)

IRB in tablet, µg ml-1

Pure IRB

added, µg ml-1

Total found, µg ml-1

Pure IRB recovered

(Percent±SD)

Irovel -

150

29.58

29.58

29.58

15

30

45

45.08

60.13

74.27

102.17±1.25

101.12±1.98

99.04±1.14

19.76

19.76

19.76

10

20

30

29.52

40.57

50.49

99.51±2.56

102.2±1.82

101.3±1.46

Irovel-

300

30.22

30.22

30.22

15

30

45

45.79

59.41

74.52

102.68±1.87

100.78±2.13

98.84±0.98

20.40

20.40

20.40

10

20

30

30.82

40.73

50.01

101.51±2.25

101.64±1.82

99.18±1.86

181

Section 5.2 SIMPLE AND SENSITIVE EXTRACTION-FREE

SPECTROPHOTOMETRIC METHODS FOR THE ASSAY OF IRBESARTAN USING THREE SULPHONTHALEIN DYES

5.2.1 INTRODUCTION

The chemistry and analytical utility of ion-pair complexation reaction is

described in Section 4.2.1.

In the literature survey presented in Section 5.0.2, six extractive

spectrophotometric methods for the determination of IRB based on ion-pair complex

formation with picric acid, bromocresol green, bromothymol blue, cobaltthiocyanate,

molybdenum thiocyanate [7] Eriochrome Black-T [8] have been reported .These

methods require strict pH control, tedious and time consuming extraction step, and

are prone to inaccuracy due to incomplete extraction of the analyte. In this Section

(5.2), three simple extraction-free spectrophotometric methods using three

sulphonthalein dyes are described. The methods are based on formation of yellow

ion-pairs between irbesartan and three sulphonthalein dyes; bromophenol blue (BPB)

(method A), bromothymol blue (BTB) (method B) and bromocresol purple (BCP)

(method C) in dichloromethane medium followed by absorbance measurement at 415,

420 and 425 nm, respectively. The method development, validation and its

applications are presented in this Section (5.2).

5.2.2 EXPERIMENTAL

5.2.2.1 Instrument

The instrument is the same that was described in Section 2.2.2.1.

5.2.2.2 Reagents and materials

All reagents were of analytical reagent grade and HPLC grade organic solvents were

used throughout the investigation.

Bromophenol blue (0.05%), Bromothymol Blue (0.1%) and Bromocresol purple

(0.1%): The solutions of bromophenol blue (BPB, Merck India, Mumbai)

bromothymol blue (BTB, Loba Chemie, Mumbai, India), bromocresol purple (BCP,

Loba Chemie, Mumbai, India) were prepared in dichloromethane (Merck, Mumbai,

India, Sp. gr. 1.32) as described in Section 4.2.1.

182

Standard IRB solution:

A stock standard IRB solution (100 µg ml-1) was prepared by dissolving 10

mg of pure IRB in dichloromethane and diluting to the mark in a 100 ml calibrated

flask with dichloromethane. The working standard solution 40 µg ml-1 (for method A

and method B) and 30 µg ml-1 (for method C) was then prepared by suitable dilution

of the stock solution with dichloromethane.

The pharmaceutical preparations used in this study are the same mentioned in

previous section.

5.2.2.3 Assay procedures

Method A (using bromophenol blue)

Different aliquots (0.2-3.5 ml) of a standard IRB (40 µg ml-1) solution were

transferred into a series of 5 ml calibrated flasks using a micro burette and to each

flask was added 1 ml of 0.05% BPB solution. The mixture was diluted to the volume

with dichloromethane and mixed well. The absorbance of each solution was measured

at 415 nm against a reagent blank after 5 min.

Method B (using bromothymol blue)

Different aliquots (0.2-3.5 ml) of a standard IRB (40 µg ml-1) solution were

transferred into a series of 5 ml calibrated flasks, as described above. To each flask

was added 1 ml of 0.1% BTB solution and diluted to the volume with

dichloromethane and mixed well. The absorbance of each solution was measured at

420 nm against a reagent blank after 5 min.

Method C (using bromocresol purple)

Different aliquots (0.2-3.5 ml) of a standard IRB (30 µg ml-1) solution were

transferred into a series of 5 ml calibrated flasks, as described above. To each flask

was added 1 ml of 0.1% BCP solution and diluted to the volume with

dichloromethane and mixed well. The absorbance of each solution was measured at

425 nm against a reagent blank after 5 min.

Procedure for tablets

An amount of the powder equivalent to 10 mg of IRB was weighed into a 100

ml calibrated flask containing about 60 ml of dichloromethane. The flask was shaken

thoroughly for about 15-20 min; the content diluted to the mark with

183

dichloromethane, and filtered using a Whatman No. 42 filter paper. First 10 ml

portion of filtrate was discarded and subsequent portions were subjected to analysis

by the procedures described above after dilution to 40 and 30 µg ml-1 IRB with

dichloromethane.

Procedure for the analysis of placebo blank and synthetic mixture

A placebo blank was prepared as described in Section 5.1.2.3. A 20 mg of the

placebo blank was accurately weighed and its solution was prepared as described

under ‘procedure for tablets’, and then subjected to analysis by following the general

procedure.

A synthetic mixture was prepared by adding an accurately weighed 10 mg of

IRB to 10 mg of the placebo mentioned above. The extraction procedure described

for tablets was followed to prepare 100 µg ml-1 IRB solution. Then the resulting

solution after appropriate dilution was subjected to analysis using the procedures

described above.

5.2.3 RESULTS AND DISCUSSION

5.2.3.1 Absorption spectra

The absorption spectra of the ion-pair complexes, formed between IRB and

each of BPB, BTB and BCP were recorded at 360-540 nm against the blank solution

and are shown in Figure 5.2.1. The yellow ion-pair complexes showed maximum

absorbance at 415, 420 and 425 nm for IRB-BPB, IRB-BTB and IRB-BCP,

respectively. The measurements were thus made at these wavelengths.

00.10.20.30.40.50.60.7

360 380 400 420 440 460 480 500 520

Abso

rban

ce

Wavelength, nm

a

b

00.10.20.30.40.50.60.7

360 380 400 420 440 460 480 500 520

Abso

rban

ce

Wavelength, nm

c

d

184

Figure 5.2.1 Absorption spectra of:

a. IRB-BPB ion-pair complex (16.0 µg ml-1 IRB), b. blank (method A), c. IRB-BTB ion-pair complex (16.0 µg ml-1 IRB), d. blank (method B), e IRB-BCP ion-pair complex (12.0 µg ml-1 IRB) and f. blank (method C). 5.2.3.2 Reaction pathway

Chemically, the structure of IRB features its basic nature. This structure

suggests the possibility of utilizing an anionic dye as chromogenic reagent.

BPB, BTB and BCP are dyes of sulphonphthalein type and the color of such

dyes is due to the opening of lactoid ring and subsequent formation of quinoid group

[68]. IRB forms ion-pair complexes with acidic dyes such as BPB, BTB and BCP

since it contains basic secondary nitrogen atom, which can be protonated easily. After

protonation of the drug, the protonated IRB forms ion-pair complexes with these

anionic dyes. The possible reaction pathway proposed is illustrated in Scheme 5.2.1,

Scheme 5.2.2 and Scheme 5.2.3.

NHN

NN

N ON

H3C

IRB

(quinoid ring)

+H+

HO Br

BrOHBr

BrOSO2

BPB(lactoid ring)

HOBr

BrOBr

BrSO3H

HO Br

BrOBr

BrSO3

-

+HO Br

BrOBr

BrSO3

-

+ H+

NHN

N

N

N ON

H3C

H

HO Br

BrOBr

Br

SO3

1:1 IRB- BPB complex

Scheme 5.2.1. The possible reaction pathway for the formation of IRB-BPB ion- pair complex.

00.10.20.30.40.50.60.7

380 420 460 500 540

Abso

rban

ce

Wavelength,nm

e

f

185

C3H7HO

BrCH3 SO2

O

C3H7OH

Br

C3H7HO

BrCH3 SO3H

C3H7O

Br

C3H7HO

BrCH3

SO3-

C3H7O

Br+ H+

BTB(lactoid ring) (quinoid ring)

C3H7HO

BrH3C SO3

-

C3H7O

Br+ H+

NHN

N

N

NON

H3C

+

NHN

N

N

NON

H3C

HIRB

C3H7HO

BrCH3 SO3

C3H7O

Br

1:1 IRB-BTB complex

Scheme 5.2.2. The possible reaction pathway for the formation of IRB-BTB ion- pair complex.

BrHO

H3CSO2O

CH3OHBr

BrHO

H3CSO3H

CH3OBr

BrHO

H3CSO3

-

CH3OBr

+ H+

+

BrHO

H3C

CH3OBr

+ H+

BrHO

H3C

CH2OBr

BCP(lactoid ring) (quinoid ring)

1:1 IRB-BCP complex

NHN

N

N

N ON

H3C

NNN

NON

H3C

H

IRB

SO3- SO3

-

Scheme 5.2.3. The possible reaction pathway for the formation of IRB-BCP ion- pair complex.

5.2.3.3 Method development

Choice of solvent

In order to select the suitable solvent for the formation of ion-pair complex,

the reaction of IRB with BPB, BTB or BCP was studied in different solvents. Better

results were obtained when IRB was dissolved dichloromethane in all the three

methods than other solvents like chloroform, 1,2-dichloroethane, acetonitrile or

carbon tetrachloride. In the case of dyes, dichloromethane was preferred to

chloroform, acetone, acetonitrile, 2-propanol, 1,2-dichloroethane, 1,4-dioxane,

methanol and ethanol because as the complex formed in these solvents had very low

186

sample absorbance values or higher blank absorbance values. Therefore,

dichloromethane was chosen as solvent.

Effect of volume of dye, and reaction time, and stability of the ion-pair complex

The effect of the dye-concentration on the intensity of the color developed at

the selected wavelengths was studied by measuring the absorbances of solutions

containing different amounts of the reagents BPB, BTB and BCP and fixed

concentrations of IRB (16 g ml-1 IRB for Method A and B, and 12 g ml-1 IRB for

Method C). The results showed that maximum color intensity of the complex was

achieved with 1.0 ml of both BPB, BTB and BCG solutions (Fig. 5.2.2). The reaction

time or standing time after the addition of dye was also examined. A 5 min standing

time was sufficient for the complete formation of ion-pair complex. The absorbance

of the resulting ion-pair complex was found to be stable for at least 1.5 h in method

A, 2.5h in method B and 2.0 h in method C at room temperature (28±20C).

(a) (b)

(c)

Figure 5.2.2 Effect of dye on the formation of ion-pair complex: (a) Method A (16 µg ml-1 IRB, (b) Method B (16.0 µg ml-1 IRB), and (c)

Method C (12.0 µg ml-1 IRB)

00.10.20.30.40.50.6

0 0.5 1 1.5 2 2.5

Abso

rban

ce

Volume of 0.05 % BPB, ml

0

0.2

0.4

0.6

0.8

0 0.5 1 1.5 2 2.5

Abso

rban

ce

Volume of 0.1%BTB, ml

00.10.20.30.40.50.60.7

0 0.5 1 1.5 2 2.5

Abso

rban

ce

Volume of 0.1 % BCP, ml

187

Composition of the ion-pair complexes

Job's method of continuous variations of equimolar solutions was employed to

establish the composition of the ion-pair complex formed between IRB and

BPB/BTB/BCP. In this method, solutions of 8.65 × 10-5 M standard IRB and 8.65×

10-5 M dye (BPB/BTB) in Method A and Method B, and 5.39 × 10-5 M each of IRB

and BCP in Method C, were mixed in varying volume ratios in such a way that the

total volume of each mixture was kept the same at 5.0 ml. The absorbance of each

solution was plotted against the mole fraction of IRB (Figure. 5.2.3). The plot

reached a maximum value at a mole fraction of 0.5 which indicated the formation of

1:1 (IRB: dye) ion-pair complexes and confirms the presence of one basic nitrogen

containing group.

(a) (b)

(c)

Figure 5.2.3 Job’s plots for ion-pair complexes from equimolar solutions of (a) IRB & BPB , (b) IRB & BTB and (c) IRB & BCP.

0

0.1

0.2

0.3

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratioVIRB/(VIRB+VBPB)

0

0.1

0.2

0.3

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratioVIRB/(VIRB+VBTB)

0

0.1

0.2

0.3

0.4

0.5

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratioVIRB/(VIRB+VBCP)

188

5.2.3.4 Method validation

Linearity and sensitivity

Under optimum experimental conditions for IRB determination, the standard

calibration curves for IRB with BPB, BTB and BCP were constructed by plotting

absorbance versus concentration (Fig. 5.2.4). The regression parameters calculated

from the calibration graphs data, are presented in Table 5.2.1. Beer’s law was obeyed

over the concentration ranges given in Table 5.2.1 and the linearity of calibration

graphs was demonstrated by the high values of the correlation coefficient (r) and the

small values of the y-intercepts of the regression equations. The apparent molar

absorptivities of the resulting colored ion-pair complexes, Sandell sensitivities,

detection and quantification limits were calculated and shown in Table 5.2.1.

Method A Method B

Method C

Figure 5.2.4 Calibration Curves

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30

Abso

rban

ce

Concentration of IRB, µg ml-1

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

Abso

rban

ce

Concentration of IRB, , µg ml-1

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

Abso

rban

ce

Concentration of IRB, , µg ml-1

189

Table 5.2.1.Sensitivity and regression parameters

Parameter Method A Method B Method C

max, nm 415 420 425 Linear range, µg ml-1 1.6-28 1.6-28 1.2-21 Color stability, hrs 1.5 2.5 2 Molar absorptivity(ε), l mol-1 cm-1 1.3× 104 1.6× 104 2.0 × 104 Sandell sensitivitya, µg cm-2 0.0383 0.0268 0.0211

Limit of detection (LOD), g ml-1 0.13 0.32 0.36

Limit of quantification (LOQ), g ml-1 0.39 0.98 1.09 Regression equation, Yb

Intercept (a) 0.0049 0.0120 0.0009 Slope (b) 0.0332 0.0360 0.0477 Standard deviation of a (Sa) 0.00075 0.0107 0.00064 Standard deviation of b (Sb) 0.00045 0.00641 0.00051 Regression coefficient (r) 0.9996 0.9993 0.9977

a Limit of determination as the weight in µg ml-1of 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. Accuracy and precision

The precision of the proposed methods was calculated in terms of

intermediate precision (intra-day and inter-day). Three different concentration of IRB

(within the working limits) were analyzed in seven replicates during the same day

(intra-day precision) and five consecutive days (inter-day precision). The percentage

relative standard deviation (RSD %) values were ≤ 2.27% (intra-day) and ≤ 2.48%

(inter-day) indicating high precision of the proposed methods (Table 5.2.2). Also, the

accuracy of the proposed methods was evaluated as percentage relative error (RE %)

between the measured concentrations and taken concentrations for IRB (Bias %) and

from the results shown in Table 5.2.2, it is clear that the accuracy is satisfactory (RE

≤ 2.34%).

190

Table.5.2.2. Results of intra-day and inter-day accuracy and precision study

Method

IRB taken

µg ml-1

Intra-day accuracy and precision

(n=5)

Inter-day accuracy and precision

(n=5) IRB

found µg ml-1

RE,% RSD,% IRB

found µg ml-1

RE,% RSD,%

A 8.0 8.08 1.11 2.06 8.35 1.35 1.18

16.0 15.77 1.37 1.68 15.75 1.86 2.48 24.0 23.66 1.39 1.04 24.78 1.45 1.41

B

8.0 8.09 1.15 2.27 7.92 1.23 1.76 16.0 15.94 0.43 1.77 16.08 1.06 2.05

24.0 24.51 2.11 1.75 24.68 2.34 1.23

C 6.0 5.93 1.02 0.51 5.86 1.38 1.38

12.0 12.25 2.10 0.69 12.29 2.16 1.42 18.0 18.32 1.80 1.34 18.46 2.06 1.66

RE- Relative error and RSD- Relative standard deviation Robustness and ruggedness

The robustness of the proposed methods was evaluated by making small

incremental changes in two experimental variables, namely, volume of the dye and

the reaction time, and performing the analysis under the altered experimental

conditions. The effect of the changes on the absorbance reading of the resulted

complexes in both methods was studied and found to be negligible confirming the

robustness of the proposed methods. Ruggedness of the proposed methods was

expressed as % RSD of the same procedure applied by three analysts and also by a

single analyst performing analysis on three different cuvettes. The results presented in

Table 5.2.3 showed that no statistical differences between different analysts and

instruments suggesting that the proposed methods were rugged.

191

Table 5.2.3. Results of robustness and ruggedness study expressed as intermediate precision (%RSD).

a Dye (BPB, BTB and BCP) volumes used were 0.8, 1.0 and 1.2 ml. b Reaction time were 4.0, 5.0 and 6.0 min Selectivity

The placebo blank when subjected to analysis yielded absorbance readings

which were the same as reagent blank, inferring no interference from the placebo.

Non interference from placebo was further confirmed by carrying out recovery study

on synthetic mixture with percent recoveries of 99.34 ± 2.16, 98.67 ± 1.39and 98.72 ±

1.78 for Method A, Method B and Method C, respectively. These results confirm the

selectivity of the proposed methods in the presence of the commonly employed

excipients added to the formulations.

Applications to analysis of tablets

The proposed methods were applied to the determination of IRB in two

representative tablets Irovel-150 and Irovel-300. The results obtained are compiled in

Table 5.2.4 and were compared with those obtained by the official method [6] by

means of Student’s t- test for accuracy and F- test for precision at 95% confidence

level. As can be seen from the Table 5.2.4, the calculated t- and F- values at 95%

Method

IRB taken,

µg ml-1

Method robustness Method ruggedness Parameters altered

Dye, mla

RSD, % (n = 3)

Time, minb

RSD,% (n=3)

Inter-analysists’ RSD, % (n = 4)

Inter-cuvettes’ RSD, % (n = 3)

8.0 1.33 1.28 1.48 2.86

A 16.0 1.29 1.54 1.35 3.07

24.0 1.16 1.31 1.17 1.64

B 8.0 1.43 1.82 1.51 2.66

16.0 1.25 1.12 1.48 2.95 24.0 1.53 1.64 1.38 3.12

6.0 1.34 1.82 1.31 2.76

C 12.0 1.18 1.12 1.37 3.15 18.0 1.29 1.64 1.29 3.24

192

confidence level did not exceed the tabulated values of 2.78 and 6.39, respectively,

indicating that there were no significant differences between the proposed methods

and the official method with respect to accuracy and precision.

Table 5.2.4. Results of analysis of tablets by the proposed methods.

*mg/tablet in tablets 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. Recovery study

In order to further ascertain the accuracy of the proposed methods, pre-

analyzed tablet powder was spiked with pure drug at three different concentration

levels (50, 100 and 150% of the quantity present in the tablet powder) and the total

was measured by the proposed methods. The determination with each level was

repeated three times and the results of this study presented in Table 5.2.5 indicated

that the commonly excipients present in the formulations did not interfere in the

assay.

Tablet brand name

Label claim*

Founda (Percent of label claim ±SD)

Reference method

Proposed methods A B C

Irovel -

150

25

99.25±0.92

98.75±1.19 t =1.82 F=1.67

100.24±0.53 t =2.75 F =3.10

98.68±1.12 t =2.69 F =1.48

Irovel-300 100 101.20±0.83

100.48±0.92 t=2.51 F=1.22

100.73±0.78 t =1.92 F=1.13

101.49±1.31 t =1.20 F =2.49

193

Table 5.2.5. Results of recovery study via standard-addition method.

*Mean value of three determinations

Tablets

studied

Method A Method B Method C

IRB in

tablets,

µg ml-1

Pure

IRB

added,

µg ml-1

Total

found,

µg ml-1

Pure IRB

recovered*,

Percent±SD

IRB in

tablets,

µg ml-1

Pure

IRB

added,

µg ml-1

Total

found,

µg ml-1

Pure IRB

recovered*,

Percent±SD

IRB in

tablets

µg ml-1

Pure

IRB

added,

µg ml-1

Total

found,

µg ml-1

Pure IRB

recovered*,

Percent±SD

Irovel

150

7.90

7.90

7.90

4.0

8.0

12.0

11.86

15.96

19.90

99.45±2.24

100.48±1.47

99.92±0.81

8.01

8.01

8.01

4.0

8.0

12.0

12.06

16.00

20.30

100.92±1.68

99.65±1.11

102.27±2.54

5.92

5.92

5.92

3.0

6.0

9.0

9.04

12.19

15.14

101.46±0.98

103.28±1.92

101.64±1.90

Irovel

300

8.03

8.03

8.03

4.0

8.0

12.0

12.13

16.16

19.96

101.68±1.72

101.57±1.50

99.01±1.14

8.05

8.05

8.05

4.0

8.0

12.0

12.11

16.09

20.10

102.55±2.51

101.02±1.11

100.76±0.50

6.08

6.08

6.08

3.0

6.0

9.0

8.86

11.98

14.83

97.76±0.84

99.77±1.92

98.18±1.16

194

Section 5.3

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY OF

IRBESARTAN AND ITS STABILITY STUDY

5.3.1 INTRODUCTION

At present, HPLC is the most widely used of all of the analytical separation

techniques prior to detection by diverse detectors. This is due mainly to the extensive

versatility of the technique [69].

In the realm of pharmaceutical analysis, HPLC offers enhanced detection

sensitivity, improved accuracy, and reproducibility of drug analysis in the course of

drug research, development and quality control testing of marketed drug products.

Many wet analysis and classical test methods for existing drug products have also

been replaced by HPLC methods for more accurate measurements, better precision

and much faster analytical run time. This translates into lower cost per test in

Research and Development and Quality Control Laboratories [70].

HPLC methods for IRB reported earlier [10-16] look less sensitive going by

linear ranges of applicability.

By introducing certain modifications in respect of column and mobile phase

composition, the author has developed an HPLC method for the determination of IRB

alone which does not require an internal standard. The method is applicable over a

wide linear dynamic concentration range. 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,

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

the validation results in detail are presented in this Section (5.3).

5.3.2 EXPERIMENTAL

5.3.2.1 Materials

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

used throughout the investigation. Pure IRB and tablets used were same as described

in Section 5.1. HPLC grade acetonitrile, methanol were purchased from Merck India

.Pvt. Ltd, Mumbai, India.

195

5.3.2.2 Reagents and solutions

HCl (1 M), NaOH (1M), and H2O2 (5%) for degradation studies were prepared as

described in Section 3.3.2

Preparation of stock solution

Accurately weighed 100 mg of pure IRB was dissolved in and diluted to mark

in a 100 ml standard flask with methanol to get 1000 µg ml-1 IRB solution.

5.3.2.3 Mobile phase preparation

Five ml of orthophosphoric acid (Merck, Mumbai, India) was diluted to 1000

ml of water and the pH was adjusted to 3.5 using triethylamine (Merck, Mumbai,

India). A 350 ml portion of this resulting buffer was mixed with 650 ml of acetonitrile

(35:65 v/v), shaken well and filtered using 0.22 µm Nylon membrane filter.

5.3.2.4 Chromatographic conditions and equipments

HPLC analysis was performed with a Waters HPLC system equipped with

Alliances 2695 series low pressure quaternary gradient pump, a programmable

variable wavelength UV-visible detector and autosampler. Data were collected and

processed using Waters Empower 2 software. Chromatographic separation was

achieved on a 150 mm × 4.6 mm i.d., 5-µm particle Intersil ODS 3V column. The

mobile phase flow rate was 1.0 ml min-1; and UV-detection was performed at 230 nm.

The column temperature was maintained at 35 °C.

5.3.2.5 Stress study

All stress decomposition studies were performed at an initial drug

concentration of 200 µg ml-1 in mobile phase. Acid hydrolysis was performed in 1 M

HCl at 80 °C for 2 h. The study in alkaline condition was carried out in 1 M NaOH at

80 °C for 2 h. Oxidative studies were carried out at 80 °C in 5% hydrogen peroxide

for 2 h. For photolytic degradation studies, pure drug in solid state was exposed to 1.2

million flux hours in a photo stability chamber. Additionally, the drug powder was

exposed to dry heat at 105 °C for 3 h. Samples were withdrawn at appropriate time,

cooled and neutralized by adding base or acid and subjected to HPLC analysis after

suitable dilution.

196

5.3.3 General procedures

Procedure for preparation of calibration curve

Working standard solutions containing 12-300 µg ml-1 IRB were prepared by

serial dilutions of aliquots of the stock solution. Aliquots of 2.0 µL were injected (six

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

conditions. The average peak area versus the concentration of IRB 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.

Preparation of tablet extracts and assay procedure

Powder equivalent to 100 mg IRB was transferred into a 100 ml volumetric

flask containing 60 ml of the methanol. The mixture was sonicated for 20 min to

achieve complete dissolution of IRB, and the content was then diluted to volume with

the same solvent to yield a concentration of 1000 µg ml-1 IRB, and filtered through a

0.45 µm nylon membrane filter. The tablet extract was injected on to the HPLC

column after appropriate dilution.

5.3.3 RESULTS AND DISCUSSION

5.3.3.1 Method 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 (water,

methanol and acetonitrile) were evaluated in-order to obtain suitable composition of

the mobile phase. Choice of retention time, tailing factor, theoretical plates and run

time were the major tasks considered while developing the method. 150 mm × 4.6

mm i.d., 5-µm particle Intersil ODS 3V column was used for the elution, but the peak

eluted before 2.0 minutes with a tailing factor of 2. Use of ion pair reagents also did

not yield the expected peak. The following gradient conditions were experimented;

the cycle time was set at 5 min, 10 min, 15 min or 20 min while the flow rate was set

at either 300 µL min-1 or 600 µl min-1. Except for the 5-min cycle time, all gradients

197

began with 100% buffer for 0.5 min and maintained for 1 min at the end of each

cycle for equilibration. For a cycle time of 5-min , conditions started with 100%

buffer for 0.5 min, then proceeded with a linear gradient to 100% acetonitrile for

3min, then returned to initial conditions and maintained upto 5 min. The gradient

method was successful in yielding good peak shape of drug. The effect of different

elution gradients was assessed under either linear (described above), curve or step

gradient which was controlled by the Waters Empower-2 software. At 65:35 ratio of

the mobile phase in the linear gradient program, a perfect peak was eluted. Thus the

mobile phase ratio was fixed at 65:35 (buffer: solvent) in an isocratic mobile phase

flow rate. The typical chromatograms obtained for blank and pure IRB in final

optimized UPLC conditions are depicted in Figure 5.3.1.

(a)

(b)

Figure 5.3.1 Typical chromatograms obtained under optimized conditions for: (a) 200 µg ml-1 IRB and (b) blank.

IRB

ES

AR

TAN

- 5.

465

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

198

Stability studies

All forced degradation studies were analyzed at 200 µg ml-1 concentration

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

IRB was found to be more stable under photolytic (1.2 million flux hours), thermal

(105 0C for 3 hours) in solid state, stress conditions. The drug was found to be

sensitive to acid and oxidative stress conditions resulting in the decomposition to the

extent of 47.6 and 50 %, respectively. Less degradation occurred under alkaline stress

conditions with percent decomposition being only 8.3%. The chromatograms

obtained for IRB after subjecting to degradation are presented in Figure 5.3.2. Assay

study was carried out by the comparison with the peak area of IRB sample without

degradation. The results of this study are shown in Table 5.3.1below

Table 5.3.1.Results of degradation study Degradation condition % degradation

Acid hydrolysis (1M HCl , 80°C, 2 hours) 47.6 Base hydrolysis (M NaOH , 80°C, 2 hours) 8.3 Oxidation (5% H2O2 , 80°C, 2 hours) 50 Thermal (105°C, 3 hours) No degradation Photolytic (1.2 million lux hours) No degradation

(a)

IRB

ES

AR

TAN

- 5.

545

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

199

(b)

(c)

(d)

IRB

ES

AR

TAN

- 5.

546

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

IRB

ES

AR

TAN

- 5.

527

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

IRB

ES

AR

TAN

- 5.

486

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

200

(e)

Figure 5.3.2 Chromatograms obtained for IRB after subjecting to stress studies by: (a) acid degradation, (b) base degradation, (c) oxidative degradation, (d) thermal degradation and (e) photo degradation.

5.3.3.2 Method validation

Analytical parameters

Linearity was studied by preparing standard solutions of different concentrations

from 12 to 3000 µg ml-1, plotting a graph of mean peak area against concentration

and determining the linearity by least-square regression. The calibration plot was

linear over the concentration range 12-300 µg ml-1 (n= 7) (Fig 5.3.3).

y = 69050x + 82656, r² = 0.9998

where y is the mean peak area, x is the concentration of IRB 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 5.3.2. These results confirm

the linear relation between concentration of IRB and the peak areas as well as the

sensitivity of the method.

Accuracy and precision

The percent relative error which is an indicator of accuracy is ≤1.0% 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 <1%. The results obtained for the evaluation of accuracy and

precision of the method are compiled in Tables 5.3.3 and 5.3.4.

IRB

ES

AR

TAN

- 5.

465

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

201

Figure 5.3.3. Calibration curve

Table 5.3.2 Linearity and regression parameters with precision data Parameter Value

Linear range, µg ml-1 12 -300

Limits of quantification, (LOQ), µg ml-1 12.0 Limits of detection, (LOD), µg ml-1 3.90 Regression equation Slope (b) 69050.4 Intercept (a) 82656.8 Correlation coefficient (r) 0.9998

Table 5.3.3 Results of accuracy study (n=5)

RE. relative error

0

5000000

10000000

15000000

20000000

25000000

0 200 400Ar

ea

Concentration of IRB, µg ml-1

Concentration of IRB injected,

µg ml-1

Intra-day Inter-day Concentration of IRB found,

µg ml-1 RE,%

Concentration of IRB found,

µg ml-1 RE,%

150 150.80 0.64 151.03 0.69 200 199.48 0.49 198.24 0.88 250 249.16 0.38 252.12 0.85

202

Table 5.3.4 Results of precision study (n=5)

Robustness and Ruggedness

The robustness of the method was investigated by making small deliberate

changes in the chromatographic conditions. The chromatographic conditions varied

were flow rate (0.9, 1.0 and 1.1 ml) and temperature (33, 35 and 37 °C). There was no

significant change in the retention time (Rt) when the flow rate or temperature was

changed slightly. The values of RSD (Table 5.3.5) indicate that the method is robust.

The ruggedness of the method was assessed by comparison of the intra-day

and inter-day results for the assay of IRB performed by three analysts in the same

laboratory. The RSD for intra-day and inter-day assay of IRB did not exceed 2.8%

indicating the ruggedness of the method.

Solution stability

The drug solution and mobile phase were injected separately at time intervals

of 0, 12 and 24, and chromatograms were recorded. At the specified time interval,

RSD (%) for the peak area obtained from drug solution stability and mobile phase

stability were within 1%. This shows no significant change in the elution of the peak

and its system suitability criteria (tailing factor, theoretical plates). The results also

confirmed that the standard solution of drug and mobile phase were stable at least for

24 hours during the assay performance.

Concentration

injected, µg ml1

Intra-day precision Inter-day precision Mean

area±SD RSDa (%)

RSDb (%)

Mean area±SD

RSDa (%)

RSDb (%)

150 10455227±1434 0.014 0.28 10386754±1408 0.013 0.48 200 14172082±1893 0.013 0.69 14257351±1929 0.012 0.34 250 17397551±1802 0.010 0.15 17153284±1818 0.010 0.85

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

203

Table 5.3.5 Results of robustness study (IRB concentration, 200 µg ml-1, n=3)

Chromatographic Conditions Alteration

Peak area precision Retention time precision

Mean area ± SD RSD,% Mean Rt±SD RSD,

%

Mobile phase flow

rate (ml min-1)

0.9 13890223±29491 0.21 5.50±0.008 0.14 1.0 14180798±12540 0.20 5.50±0.004 0.07

1.1 13952604 ±22974 0.16 5.51±0.006 0.10

Column

temperature (° C)

33 13971582 ±21313 0.15 5.51±0.007 0.12 35 1429154 ±40579 0.28 5.49±0.01 0.18 37 14048512± 49010 0.34 5.5 ± 0.008 0.14

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.

Application of the method for the analysis of commercial tablets

The developed method was applied to the determination of IRB in two brands

of tablets containing IRB in two strengths (150 and 300 mg per tablet).Quantification

was performed using the regression equation. The same tablet powder used for assay

by the proposed method was used for assay by a official method [6] for comparison.

The results were compared statistically by applying the Student’s test for accuracy

and F-test for precision. As shown by the results compiled in Table 5.3.6, the

calculated t-test and F-values did not exceed the tabulated values of 2.77 and 6.39 for

four degrees of freedom at the 95% confidence level, suggesting that the proposed

method and the reference method do not differ significantly with respect to accuracy

and precision.

Recovery experiment

The accuracy and validity of the proposed HPLC method were further

ascertained by performing recovery experiments. Pre-analyzed tablet powder was

spiked with pure IRB at three different concentration levels and the total was found

by the proposed method. Each determination was repeated three times. The recovery

204

of pure drug added was quantitative (Table 5.3.7) and revealed that co-formulated

substances did not interfere in the determination.

Table 5.3.6 Results of determination of IRB in tablet and statistical comparison with the official method

Tablet brand name

Nominal amount, mg

IRB found* (%) ± SD t-value F- value Official method

Proposed method

Irovel Irovel

150.0 300.0

99.25±0.92 101.2±0.83

99.65±0.65 100. 6±0.86

2.28 2.33

2.00 1.07

* 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 5.3.7 Results of recovery study by standard addition method

*Mean value of three determinations

Tablet studied

IRB in tablet, µg ml-1

Pure IRB added, µg ml-1

Total found, µg ml-1

Pure IRB recovered* (% IRB ±SD)

Irovel-150 99.65 99.65 99.65

50.0 100.0 150.0

150.1 199.1 249.4

100.2±0.98 100.1±0.87 99.8±0.75

Irovel-300 100.6 100.6 100.6

50.0 100.0 150.0

150.9 200.5 249.9

100.5±0.78 99.7±0.57 98.7±0.49

205

Section 5.4

SUMMARY AND CONCLUSIONS -Assessment of the methods

Five visible spectrophotometric methods and one HPLC method, the last

being stability-indicating were developed and validated as per the required protocol

for the determination of irbesartan in bulk drug and tablet dosage form. The

spectrophotometric methods have been fairly accurate and precise with RE (%) and

RSD (%) values being < 2% in most cases. The methods are applicable over wide

linear dynamic ranges. They are characterized by simplicity and ease of performance

when compared to published methods [7-9] which involve optimization of several

variables such as nature of buffer, pH, aqueous-organic phase ratio, contact time,

extraction time, equilibration time, besides a tedious and time consuming extraction

step. In sharp contrast, the proposed methods involve simple mixing of the drug and

reagent solution in dichloromethane. The methods based on charge-transfer reaction

are comparable to the published methods [7, 8] in terms of sensitivity (ε= 103 l mol

cm-1). However, the ion-pair reactions are, roughly, an order of magnitude more

sensitive than the published methods. Of the developed methods by author, ion-pair

methods (= 104 l mol cm-1) more sensitive than the charge-transfer methods (= 103 l

mol cm-1) and both are equally accurate, precise, selective, robust and rugged. The

performance characteristics are summarized in Table 5.4.1.

206

Table 5.5.1 Comparison of performance characteristics of proposed methods with the existing methods

A. Spectrophotometry Sl no

Reagent/s used Methodology max (nm)

Linear range (µg ml-1) ( in l mol-1 cm-1)

LOQ (µg ml-1) Remarks

Ref

1 a)Picric acid Measurement of chloroform extractable ion-pair complex

416

20-60 (4.8×103)

NA

Tedious and time consuming extraction step, less sensitive, narrow linear range

7

b) bromocresol green 410 32-72 (5.1×103)

NA Strict pH control, extraction step, narrow linear range and less sensitive

c)bromothymol blue 415 32-72 (5.0×103)

NA

d)cobaltthiocyanate 625 200-800 (1.8×103)

NA

e)Molybdenum thiocyanate

475 8-80 (3.0×103)

NA

2

Eriochrome Black-T

Chloroform extractable ion-pair complex measured

50-250

NA

pH dependent, less sensitive, time consuming extraction step

8

3 a) DNP Measurement of charge transfer complex

420 6-105 (3.8×103)

1.49 Free from optimization of experimental variables, involve a single step reaction

Present work

b) PA 425 4-70 (6.4×103)

1.62

207

4 a)BPB Measurement of yellow colored ion-pair complex

425 1.6-28 (1.3×104)

0.39 Free from optimization of experimental variable, involves a single step reaction, sensitive, wide linear dynamic ranges.

Present work

b) BTB 420 1.6-28 (1.6×104)

0.98

c) BCP 415 1.2-21 (2.0×104)

1.09

208

B. HPLC methods

Sl. No.

Chromatographic condition Linear range (µg ml-1)

LOD (µg ml-1)

Remarks Ref

1 CLC-ODS column, H2O-CH3CN (CH3)2N (50:50:0.15) mobile phase at 1.0 ml min-1 with UV-detection at 245 nm.

49-146 NA Not stability indicating

10

2 Diamonsil C18 column, CH3CN,0.02 % KH2PO4, pH 2.6 (45:55) at a flow rate of 1.0 ml min -1 as mobile phase and UV-detection at 245 nm

12.4-185.4 NA Not stability indicating

11

3 Kromasil C18 column CH3CN-0.02 M KH2PO4, pH 2.60 (48:52), at a flow rate of 1.0 ml min-1 as mobile phase and UV-detection at 245 nm.

13.22-185.08

NA Not stability indicating

12

4 RP 18 LC column, KH2PO4- CH3CN - - - 13

5 Reversed phase C18 column, acetonitrile-0.03 M KH2PO4, pH 3.0,(15:85) at a flow rate of 1 ml min-1 as mobile phase and UV-detection at 275 nm.

2-12 NA Not stability indicating

14

6 C8 column, 0.01 M NaH2PO4 (pH 3.0) and acetonitrile (50:50) mobile phase of and the UV-detection at 209 nm.

NA - 15

7 RP-HPLC-PDA, C18column, methanol-0.02% formic acid (70:30) at a flow rate of 1 ml min-1 as mobile phase with UV-detection at 234 nm

10-50 NA Not stability indicating

16

8 Intersil ODS 3V column, acetic acid, pH 3.5-CH3CN (65:35) at flow rate of 1 ml min-1 as mobile phase and UV-detection at 230 nm.

12-300 3.9 Stability indicating, wide linear range

Present work

209

An important feature of the present HPLC method is the wide linear dynamic

range (12-300 µg ml-1) compared to many published methods [10-16] and is

comparable to them in terms of specificity. The method was found to be stability-

indicating with the drug undergoing extensive degradation under acidic and oxidative

stress conditions; mild degradation under alkaline stress-conditions.

While comparing the HPLC method with the spectrophotometric methods

developed by the author, HPLC is highly accurate, precise, selective, robust and

rugged compared to the spectrophotometric methods although the latter methods

would seem more sensitive in terms of the linear ranges and LOD, 0.1-0.5 µg ml-1 (

spectrophotometric methods) vs 3.9 µg ml-1 ( HPLC method).

210

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