ONDANSETRON HYDROCHLORIDE-ANTIEMITIC …shodhganga.inflibnet.ac.in/bitstream/10603/8230/12/12_chapter 4.pdf · Chapter-IV Ondansetron HCl Part-A: HPLC Method 247 QUANTIFICATION OF

Chapter-IV: Ondansetron HCl Introduction

245

ONDANSETRON HYDROCHLORIDE-ANTIEMITIC DRUG

4.01 Drug Profile

Ondansetron (OND) chemically known as 4H-Carbazol-4-one,1,2,3,9-

tetrahydro-9-methyl-3-(2-methyl-1H-imidazol-1-yl) methyl-,monohydrochloride,(±)-

,dehydrate. It is used as a selective blocking agent of the serotonin 5-HT3 receptor

type and also used for prevention of nausea and vomiting associated with highly

emetogenic cancer chemotherapy, radiotherapy or anesthesia and surgery. It is

officially reported in the United States of Pharmacopoeia [271] and British

Pharmacopoeia [272].

Physical Properties:

Molecular formula : C18H19N3O·HCl·2H2O

Molecular weight : 365.86 grams

Appearance : White to off-white powder

Solubility : Water and normal saline.

Structure:

O

N

NH3C

N

H3C

http://www.rxlist.com/script/main/art.asp?articlekey=5468




Chapter-IV: Ondansetron HCl Introduction

246

Pharmaceutical Formulations:

Ondem (Alkem) : 4mg.8mg Tablets

2mg/ml -2ml, 4ml Injections

Zofer Sun (Solares) : 4mg, 8mg Tablets

2mg/ml -2ml, 4ml Injections

Zofran (Glaxo Smith Kline) : 4-mg 8-mg Tablets

Osetron (Dr.Reddy’s) : 4mg.8mg Tablets

4mg/ml, 8mg/ml Injections

Inactive Ingredients:

Each tablet also contains the inactive ingredients aspartame, gelatin, mannitol,

methylparaben sodium, propylparaben sodium, strawberry flavor, lactose,

microcrystalline cellulose, pregelatinized starch, hypromellose, magnesium stearate,

titanium dioxide, triacetin, and iron oxide yellow (8mg tablet only)

Literature Survey:

An extensive literature survey is carried out, and it is evident that some

analytical methods such as reverse phase HPLC, spectrophotometric and derivative

spectrophotometric methods are reported for the determination of ondansetron in pure

and formulations and mostly in biological fluids. Reverse phase HPLC [273-293] are

reported in the literature for the determination of ondansetron mostly in biological

fluids [284-293] and in the study of impurities [294].The reported methods are costly

and not applicable at higher concentrations. A few spectrophotometric [295-296] and

derivative spectrophotometric [297] methods are also found in the literature for the

determination of ondansetron HCl in pure and combination with paracetamol.






Chapter-IV Ondansetron HCl Part-A: HPLC Method

247

QUANTIFICATION OF ONDANSETRON HYDROCHLORIDE

BY REVERSE PHASE HPLC METHOD

4.02 Introduction

High-performance liquid chromatography (HPLC) is a chromatographic

technique that can separate a mixture of compounds and is used in biochemistry and

analytical chemistry to identify, quantify and purify the individual components of the

mixture. HPLC typically utilizes different types of stationary phases, a pump that moves

the mobile phase(s) and analyte through the column, and a detector that provides a

characteristic retention time for the analyte. A few HPLC methods [273-293] are reported

in the literature for the determination of ondansetron in different biological fluids and in

combination with other drugs but there is a little HPLC work is done on pure and

formulations. The reported methods are costly and not applicable at higher

concentrations. Hence the author is interested in developing a reverse phase HPLC

method to estimate the amount of ondansetron pure drug and is successfully applied for

pharmaceutical formulations. The experimental details, materials, development of

method, chromatographic conditions and validated parameters are given below.

4.03 Experimental

Instrumentation: Shimadzu LC- 20AT Prominance liquid chromatographic system

equipped with binary gradient pump, UV-VIS SPD 20A detector, Column Oven and

controlled by spinchrom software is used for the analysis.

Chromatographic conditions

Column : Intertisil ODS 3V (150mm, 4.6mm- ID;

5µm particle size)


248

Detector : UV-VIS SPD 20A Prominance

Wavelength (nm) :305nm

Flow Rate : 0.5ml/minute

Injection Volume : 10 μl

Temperature : 25oC

Run Time : 25 minutes

Retention Time : 9.029 min.

4.04 Method Development

(i ) Materials and Reagents

Gift samples of Ondansetron hydrochloride and working reference standard

are obtained from Aurobindo Pharma Ltd. The formulations are purchased locally.

All the reagents and chemicals used are of HPLC grade. Acetonitrile, methanol,

sodium acetate and acetic acid are purchased from Merck, India and HPLC grade

water is used to prepare the mobile phase. Stock solutions of ondansetron

hydrochloride and sample solutions are prepared in the mobile phase. Fresh working

solutions are prepared daily. All solutions are filtered through 0.45 µm membrane

filter and degassed using a sonicator.

(ii) Preparation of Solutions:

Preparation of buffer solution of pH=4.5 (0.02M, Merck; v/w): About 0.820 grams

of sodium acetate is accurately weighed, transferred into 500ml volumetric flask and


249

dissolved in HPLC grade water and made up to the mark by adjusting the pH = 4.5 by

adding a few drops of acetic acid, sonicated and filtered

Preparation of mobile phase(Buffer: Acetonitril ; 65:35 v/v): Mobile phase is

prepared by mixing accurately measured volumes of 195ml of buffer and 105 ml of

acetonitrile in a 500ml beaker, stirred well, sonicated and used for the analysis.

Preparation of Standard (1mg/ml;w/v): About 50mg of ondansetron hydrochloride

is accurately weighed , transferred into a well cleaned 50ml volumetric flask,

dissolved in mobile phase and made up to the mark. The solution is degassed by

sonication, filtered through 0.45μ filter and used as a standard solution. Working

standard solutions of different concentrations (20%, 40%, 60% 80%, 100% and 120%

are prepared by measuring and diluting required volumes of standard.

Preparation of test solution: Twenty tablets (4.0mg/Tablet and 8.0mg/Tablet) of

Osetron/Ondem are powdered and mixed thoroughly, an amount of the powder

equivalent to 50mg of the drug is accurately weighed and dissolved in methanol,

shaken well and filtered. The filtrate is evaporated to dryness carefully and the

residue is dissolved in mobile phase and made up to 50ml. The resulting solution is

degassed by sonication and filtered through 0.45μ filter and 5.0ml of this test solution

is further diluted to 50ml and used for the analysis.

(iii) Procedure:

The chromatographic conditions are fixed for the Shimatzu HPLC system .The

mobile phase is allowed to pump from the mobile phase reservoir into the column

Intertisil ODS 3V (150mm, 4.6mm-ID; 5µm particle size) at a flow rate of 0.5

ml/min., keeping the column in thermostat at a temperature of 25oC. The response of


250

the instrument is recorded at maximum wavelength 305nm. After complete elution of

the mobile phase through the column, it is found that the base line is almost parallel to

x-axis in the chromatogram. Now 10μl of the standard / test solution of ondansetron

hydrochloride is injected into the system, and the chromatogram is recorded (Fig.4.01,

Fig.4.02 P: 255).

(iv) Optimization of the proposed method:

The developed method is optimized and optimum conditions are established

by varying the parameters such as concentration of the standard, type of column,

temperature of the column, flow rate, injection volume, composition of the mobile

phase (polarity), pH of the buffer ect one at a time, keeping the others fixed and

observing the effect on the retention time, tailing factor and other system suitability

parameters. It is found that the best suitable conditions to obtain the chromatogram in

reasonable retention time, tailing factor less than 2.0, and number of theoretical plates

more than 2000 are given above and maintained throughout the analysis.

4.05 Method Validation

Proper validation of analytical methods is important for pharmaceutical

analysis when ensurance of the continuing efficacy and safety of each batch

manufactured relies solely on the determination of quality. The ability to control this

quality is dependent upon the ability of the analytical methods, as applied under well-

defined conditions and at an established level of sensitivity, to give a reliable

demonstration of all deviation from target criteria. The most widely applied validation

characteristics are given below


251

(i) System Suitability and System Precision: The system suitability parameters and

system precision are evaluated from the area of the chromatograms of six replicate

injections and found to be within the limits. The system suitability parameters are

presented in Table-4.01(a) and Table-4.01(b), P: 257.

(ii) Linearity of detector response: Linearity of detector response to the concentration

of the standard drug is established by plotting a graph between the concentrations

versus average area of two peaks. A series of six solutions of the drug are prepared in

the concentration range of about 20μg/ml to 120μg/ml corresponding to 20% to 120%

of target concentration. Each solution is injected into the system and recorded the

chromatogram under the test conditions. A graph is plotted to concentration in µg/ml

on X-axis versus response on Y-axis (Fig.4.03 (a), P: 256). The detector response is

found to be linear with a correlation coefficient of 0.9999. The results are summarized

in Table-4.02, P: 257.

(iii) Precision of test method: Commercial formulations of ondansetron hydrochloride of

4.0mg and 8.0mg are successfully analyzed by the developed method. The precision of

the test method is evaluated by assaying six samples of tablets of 4.0mg and 8.0mg. The

average percent of assay of formulations in tablets is found to be 100.12 (±0.347) and

100.10 (±0.290). The results are summarized in Table-4.03, P: 258.

(iv) Accuracy of the method: To determine the accuracy of the method, solutions of

different concentrations (75%, 100%, and 125% of the target concentration) of

ondansetron hydrochloride sample are prepared in triplicate for each spike level and

assayed as per standard method. The % recovery is found to be100.18 (±0.671),

100.08 (±0.284) and 99.95 (±0.521) at 75%, 100% and 125% spike levels

respectively. The results are summarized in Table-4.04, P: 258.


252

(v) Limit of Detection and Limit of Quantitation: LOD and LOQ are calculated

based on the standard deviation of the response and the slope of the calibration curve

The results of LOD and LOQ are summarized in Table-4.02, P: 257.

(vi)Linearity of test method: The percent of recovery of the drug is determined by

adding different known amounts of the standard drug to equal amount of test sample.

A linear plot is drawn to amount of ondansetron added to average amount of the drug

recovered. The results of the recovery experiments by the developed method are

summarized in Table-4.05, P: 259.

(vii) Ruggedness

(d) Intra Day & Inter Day variability: This study is conducted on day-1 and day-2

using same columns on the same HPLC system by assaying six separately prepared

ondansetron sample solutions under similar conditions. The system suitability

parameters are evaluated as per the standard method on both the days and found to be

within limits. The average % assay for the two systems is found to be 100.00 (±0.256)

and 100.08 (±0.219). The results are summarized in Table-4.06(a), P: 259.

Comparison of the results obtained on two days shows that the assay method is

rugged for day to day variability.

(b) System to System variability: System to system variability study is conducted on

two HPLC systems by using the same column by assaying six separately prepared

ondansetron sample solutions under similar conditions. The system suitability

parameters are evaluated as per the standard method on both the systems and found to

be within limits. The average % assay for the two systems is found to be 100.05

(±0.287) and 100.25 (±0.348). The results are summarized in Table-4.06(b), P: 260.


253

Comparison of the results obtained on two systems shows that the assay method is

rugged for system to system variability.

(c)Column to Column variability: This study is conducted using two columns on the

same HPLC system by assaying six separately prepared ondansetron sample solutions

under similar conditions. The system suitability parameters are evaluated as per the

standard method on both the systems and found to be within limits. The average %

assay for the two columns is found to be 100.10 (±0.446) and 100.23(±0.363). The

results are summarized in Table-4.06(c), P: 260. Comparison of the results obtained

on two columns shows that the assay method is rugged for column to column

variability.

(viii) Robustness

A study is conducted to determine the effect of variation in chromatographic

conditions such as flow rate, detection wavelength, temperature of the column,

composition of the mobile phase and pH of the buffer solution. Sample solution

prepared in triplicate as per test method is injected into the HPLC system and the

system suitability parameters are evaluated as per the test method and found to be

within limits. The variation in parameters and the results obtained are incorporated in

Table-4.07, P: 261.

4.06 Results and Discussion:

To estimate the amount of ondansetron hydrochloride in pure and

pharmaceutical formulations by reverse high performance liquid chromatography

under optimized chromatographic conditions, chromatograms are recorded for

standard and test solutions. The system suitability parameters and system precision


254

are evaluated and found within the limits. The results are given in Table-4.01(a),

P: 257. A plot is drawn between concentration and the instrument response, it is linear

with good correlation coefficient(r=0.9999) (Table-4.02, P: 257; Fig.4.03 (a), P:

256). Precision and accuracy of the developed method are determined and are

expressed in %RSD and % of recovery of the active pharmaceutical ingredient. Low

%RSD value 0.347 and 0.290 and high percent of recovery 100.18(±0.671),

100.08(±0.284), 99.95(±0.521), at 80%, 100% and 120% concentration of the

standard indicate that the method is highly precise and accurate (Table-4.03 and

Table-4.04, P: 258).

A study is conducted between two different days, two different systems, and

two columns to compare the results. The system suitability parameters evaluated as

per the test method on both the systems, columns and between two days are found to

be within limits. The average % assay and relative standard deviation are within the

limits (Table-4.06(a) - Table-4.06(c), P: 259-260). The change in system suitability

parameters are evaluated by studying the effect of change in composition of the

mobile phase, flow rate, pH

of the buffer solution, wavelength and column

temperature and found to be acceptable (Table-4.07, P: 261).

Pharmaceutical formulations are analyzed by the developed method by

estimating the amount of drug recovered by standard addition method. A graph is

drawn between the amount of drug added and the amount of drug recovered, the plot

is linear and regression equation is given by y=1.0173x+0.5907with r2 =

0.9999

(Table-4.05, P: 259; Fig.4.03 (b), P: 256). Precision and Accuracy of the test sample

is determined by assaying formulations of 4.0mg, 8.0mg per tablet and 2.0mg/ml.

High percent of mean recovery and low %RSD values (Table-4.07, P: 261)


255

indicate that the method should be successfully applied for the analysis of

formulations.

Fig 4.01 Chromatogram of Ondansetron hydrochloride (Standard)

Fig4.02 Chromatogram of Onansetron hydrochloride (Formulation)


256

Linearity of detector responce to the concentration of the Ondansetron HCl

y = 28007x - 8898.8

R2 = 0.9998

0.0E+00

8.0E+05

1.6E+06

2.4E+06

3.2E+06

4.0E+06

0 30 60 90 120

Concentration of Ondansetron HCl µg/ml

Det

ecto

r R

esp

on

se

Fig 4.03(a) Linearity of the detector response

Linearity of the test Method

y = 1.0173x - 0.5907

R2 = 0.9999

0.0

25.0

50.0

75.0

100.0

125.0

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

Weight of Drug added in µg/ml

Weig

ht

of

dru

g r

eco

vered

in

µg

/ml

Fig.4.03 (b) Linearity of the Test Method


257

Table-4.01(a): System Suitability

System suitability parameters Observed value Acceptance criteria

USP tailing factor 1.524 NMT2.0

Number of theoretical plates 2746 NLT2000

RSD(six replicate measurements) 0.483 NMT 2.0%

Table-4.01(b): System Precision

S.No.

Concentration

(μg/ml)

Average Area

Statistical

Parameters

Value of the

parameters

1 100 2771955 Mean 2776428

2 100 2769832

3 100 2799604 SD 13420

4 100 2760823

5 100 2783188 %RSD 0.483

6 100 2773168

Table-4.02: Linearity of detector response

S.No.

Concentration

μg/ml

Average

Area

Regression Parameter

1 20 557656 Slope 27661

2 40 1129312 Intercept 7282

3 60 1668967 Correlation Coefficient 0.9999

4 80 2215323 LOD µg/ml 1.558

5 100 2772179 LOQ µg/ml 5.194

6 120 3324935


258

Table-4.03: Precision of the test method

Percent of Recovery of labeled amount

Sample

No.

Labeled Amount: 4.0mg/Tablet Labeled Amount: 8.0 mg/Tablet

Amount

Found(mg/Tab)

%Recovery

Amount

Found(mg/Tab)

%Recovery

1 3.991 99.78 8.024 100.3

2 3.994 99.86 7.990 99.87

3 4.024 100.60 7.998 99.98

4 3.997 99.93 7.991 99.89

5 4.020 100.50 7.982 100.6

6 4.002 100.04 7.997 99.96

Mean 4.005 100.12 7.997 100.10

SD 0.014 0.347 0.014 0.290

%RSD 0.346 0.346 0.180 0.290

Table-4.04: Accuracy

Spike

level

Sample

ID

Amount

Added(μg/ml)

Amount

Found(μg/ml)

% of

Recovery

Statistical Analysis

80% 1 80.00 79.58 99.47 Mean 100.18

2 80.00 80.22 100.27 SD 0.671

3 80.00 80.64 100.80 %RSD 0.670

100% 1 100.00 99.87 99.87 Mean 100.08

2 100.00 99.96 99.96 SD 0.284

3 100.00 100.40 100.40 %RSD 0.283

120% 1 120.00 119.33 99.44 Mean 99.95

2 120.00 120.58 100.48 SDV 0.521

3 120.00 119.92 99.93 %RSD 0.521


259

Table-4.05: Linearity of test method

Spike

level (%)

Drug

added(μg/ml)

Drug

Recovered(μg/ml )

Regression parameters

20 20.00 19.68 Slope 1.0173

40 40.00 40.72

60 60.00 59.79 Intercept 0.5007

80 80.00 80.63

100 100.00 101.32 Correlation Coefficient 0.9999

120 120.00 121.57

Table -4.06(a) Intra Day and Inter Day Variation

Percent of assay of the sample mg

Sample No. Day-1 Day-2

1 99.69 100.31

2 100.14 99.93

3 99.80 99.94

4 100.31 99.95

5 99.85 100.4

6 100.23 99.92

Mean 100.00 100.08

SD 0.256 0.219

%RSD 0.256 0.219

Comparison of the precision with method precision

F=1.840,t=1.117 F=2.511,t=0.503

Comparison of precisions between two analysts

F=1.366 t=0.540


260

Table -4.06(b) System to System Variation


Sample No. Systemt-1 System-2

1 99.97 100.50

2 99.93 99.97

3 100.3 99.93

4 99.82 100.60

5 100.5 99.91

6 99.78 100.60

Mean 100.05 100.25

SD 0.287 0.348

%RSD 0.287 0.347


F=1.463,t=0.598 F=1.003,t=0.928


F=1.476 t=1.205

Table -4.06(c) Column to Column Variation


Sample No. Column-1 Column-2

1 100.40 99.87

2 99.50 99.93

3 99.80 100.60

4 100.30 99.95

5 100.70 100.70

6 99.87 100.30

Mean 100.10 100.23

SD 0.446 0.363

%RSD 0.445 0.363


F=1.649,t=0.137 F=1.097,t=0.708


F=1.510 t=0.504


261

Table 4.07: Study of Robustness

Variable Variation USP Plate Count USP Tailing %RSD

Standard -------- 2259 1.537 0.000

Flow Rate 0.4 ml/min. 2284 1.653 0.316

0.6 ml/min. 2316 1.496 0.464

Wavelength 300 nm 2146 1.427 0.581

310 nm 2431 1.126 0.735

Column

Temperature

23oC

2197 1.254 0.673

28oC

2634 1.257 0.331

%Mobile

Phase

70%+30% 2422 1.197 0.215

60%+40% 2397 1.261 0.316

Buffer pH 4.8 2317 1.319 0.554

4.2 2521 1.652 0.523

Acceptance Criteria USP Tailing Factor not more than 2.0, Theoretical Plates

not less than 2000

4.07 Conclusion: The developed method is simple, rapid, precise, accurate, linear,

and robust and rugged. The developed method can be applied to determine

ondansetron in pharmaceutical formulations with good percent of recoveries and

hence it provides an alternative method to use in any analytical testing laboratories to

determine the amount of ondansetron in pure and formulations.

Chapter - IV Ondansetron HCl Part-B: Visible Spectrophotometry

262

VISIBLE SPECTROPHOTOMETRIC DETERMINATION OF

ONDANSETRON

4.08 Introduction

Analytically useful functional groups present in ondansetron hydrochloride

have not been fully exploited for designing suitable visible spectrophotometric

methods and therefore still offer a scope to develop more number of new visible

spectrophotometric methods. The author has made some attempts in this direction and

succeeded in developing visible spectrophotometric methods.

4.09 Experimental

4.09(i) Instrumentation

UV-Visible Spectrophotometer: Double beam Elico SL159 model UV-Visible

spectrophotometer with 2nm resolution, 1cm length quartz coated optics; Wavelength

range190-1100nm is used for all the spectral measurements.

Digital pH Meter: Digital pH Meter with combined electrode, Temperature probe ±0.01

accuracy is used for adjusting pH of the buffer solution.

4.09(ii) Preparation of solutions

(a) Standard Solution of Ondansetron HCl

Stock solution of Ondansetron HCl (0.1%) is freshly prepared by transferring

accurately weighed 100mg of Ondansetron HCl into 100mL volumetric flask,

dissolved in triple distilled water, and made up to the mark. Then working standard

solutions 250μg/ml, 200μg/ml and 100μg/ml are prepared by transferring 25.0ml,


263

20.0ml and 10.0ml of the stock solution into three 100ml standard flasks and made up

to the mark respectively. 100μg/ml working standard solution is used in M1(a), M18,

M19; 200μg/ml working standard solution is used for the following methods M5(c),

M7. M16, M17; 250μg/ml working standard solution is used for the following

methods M1(b), M5(a), M15.

(b) Preparation of reagents

Analytical grade chemicals, reagents and double distilled water are used in the

preparation of solutions.

Method M1(a), M1(b): ARS solution (Fluka) 0.2%, 5.49 x 10-3

M, BTB solution (0.1%,

1.60 x 10-3

M); Method M5(a), M5(c): MBTH Solution (Fluka): 0.2%, 8.56 x 10-3

M), Ce

(IV) solution (Merck):1%, 9.35 x 10-3

M), NaIO4 solution (BDH): 0.2%, 9.35 x 10-3

M),

AcOH solution (Qualigens): 2.3 M) ; Method – M15 : p-CA solution (Sd-fine; 0.1%,

4.785x10-3

M); Method M16 :MO solution ( 0.2%, 6.11 x 10-3

M);Method M17 :PA

solution ( 0.1%, 4.36 x 10-3

M), Buffer pH – 9.8. Preparations of reagents for the above

methods are as same as described in Chapter-II

Method – M7

NBS solution (Loba; 0.01%, 5.618x10-4

M): Prepared by dissolving accurately

weighed 50mg of N-Bromo succinimide (NBS) in 500mL of distilled water and

standardized iodometrically.

CB solution (Chroma; 0.005%, 5.497x10-4

M): Prepared by dissolving 50mg of

Celestine blue in 1000mL of distilled water.


264

Hydrochloric acid (E.Merck; 5M): Prepared by diluting 217.5mL of concentrated

HCI to 500mL with distilled water and standardized

Method M18

4-AP Solution(BDH; 0.5%, 2.45 x 10-2

M): 500mg of 4-AP is accurately weighed and

dissolved in 100ml of MeOH containing ml of Con. HCl.

Method M19

INH Solution (Sd.Fine; 0.8%,5.83 x 10-3

M) : 800mg of INH is accurately weighed

and dissolved in 100ml of MeOH containing ml of conc. HCl.

4.09(iii) Procedures for new methods

The procedures for the developed methods and optimum conditions are

described as given below.

Method – M1(a) and M1(b)

Into a series of 125ml separating funnels containing aliquots of standard OND

solution (3.3-20.0μg/ml, 8.3-50.0μg/ml for methods M1(a) and M1(b) respectively),

6.0ml of 0.1M HCI solution and 1.0ml of 0.2% ARS dye solution M1(a), 2.0ml BTB

dye solution M1(b) are added successively. The total volume of aqueous phase in

each separating funnel is adjusted to 15ml with distilled water. To each separating

funnel 10ml of chloroform is added and the contents are shaken for 2min. The two

phases are allowed to separate and the absorbance of the separated chloroform layer

is measured at max (415nm for ARS; 420nm for BTB) against a similar reagent


265

blank. The amount of OND is deduced from the calibration curve (Fig.4.14 - 4.15, P:

280).

Method – M5(a) and M5(c)

Aliquots of standard OND solution 10.0 - 60.0g/ml (3.2 – 19.2g/ml for

method M5(c)) are transferred into a series of 25ml calibrated tubes. Then 0.5ml of

MBTH (1.0ml in case of method M5(c)) solution is added and kept aside for 5min.

After that 2.0ml (1.58 x 10-2

M) of ceric ammonium sulphate (1.0ml (9.35 x 10-3

M) of

NaIO4 solution, 1.0ml of acetic acid solution for method M5(c)) is added and kept

aside for 10min. The volume is made up to the mark with distilled water. The

absorbance is measured at 630nm for both the methods against a similar reagent

blank. The amount of OND is computed from its calibration graph. (Fig.4.16 and Fig

4.17, P: 281).

Method – M7

Different aliquots of standard OND solution (8.0-48.0g/ml) are transferred

into a series of 25ml calibrated tubes. Then 1.25ml (1M) of HCl and 4.0ml (2.81 x

10-3

M) of NBS solutions are added and the total volume is brought to 15ml with

distilled water. After 10minutes 4.0ml of CB solution is added, after 5minutes

absorbance is measured at 460nm against reagent blank. The decrease in absorbance

corresponding to consumed NBS and in turn the dye concentration is obtained by

subtracting the decrease in absorbance of the solution that of blank solution. The

amount of drug is computed from calibration curve (Fig.4.18, P: 281).


266

Method – M15

Into a series of 10ml calibrated tubes containing aliquots of standard OND

solution (12.5 – 75.0 g/ml), 2.0ml of chloranilic acid (4.785 x 10-3

M) is added and

kept aside for 30 min at lab temperature. The volume in each tube is made up to the

mark with chloroform. The absorbance of the colored species is measured at 540nm

against a reagent blank. The amount of the drug is calculated from Beer’s law plot

(Fig.4.19, P: 281).

Method M16

To each of 10ml calibrated tubes different aliquots (10.0-60.0 g/ml) of

standard OND solution, 2.0ml of water and 8 Volumes H2O2 are added successively

and then kept aside for 10min. After that 1.0ml of MO solution is added and the total

volume in each flask is brought to 10ml by the addition of distilled water. The

absorbance is measured at 530nm against a reagent blank prepared in a similar way.

The concentration of drug in a sample is computed from Beer-Lambert plot (Fig.4.20,

P: 282).

Method M17

Into a series of 50 ml separating funnels containing aliquots of drug (6.7 –

40.0 g/ml) solutions, 2 ml of pH 9.8 buffer and 2.0 ml of 0.1% picric acid solutions

are added successively. Total volume of aqueous phase in each separating funnel is

adjusted to 15 ml with distilled water. To each separating funnel 10 ml of chloroform

is added and the contents are shaken for 2 min. The two phases are allowed to

separate and the absorbance of the separated chloroform layer is measured at 415 nm


267

against a reagent blank prepared under similar conditions. The amount of drug is

deduced from the calibration graph (Figs.4.21, P: 282).

Method: M18

Delivered aliquots of standard OND solution (5.0 – 30.0g/ml) into a series of

10ml calibrated tubes, then 3.0ml (5.83 x 10-3

M) of 4-AP is added to each tube and

kept aside for 15min. Later the solution in each tube is made up to 10ml with

methanol. The absorbance is measured at 440nm against the reagent blank. The

amount of OND is computed from its calibration graph. (Fig.4.22, P: 282)

Method: M19

Into a series of 10ml calibrated tubes delivered aliquots of standard OND

solution (5.0 – 30.0g/ml), then 2.0ml (2.45 x 10-3

M) of INH solution is added to

each tube and heated for 10min at 60oC. Later the solution in each tube is made up to

10ml with methanol. The absorbance is measured at 470 nm against the reagent

blank. The amount of OND is computed from its calibration graph (Fig.4.23, P: 282).

4.09(ii) Optimization of methods

Optimum conditions for the maximum color development of the proposed

methods (M1(a), M1(b), M5(a), M5(c), M7, M15, M16, M17, M18 and M19) are

established by varying the parameters one at a time, keeping the others fixed and

observing the effect produced on the absorbance of the colored species. The optimum

conditions necessary for rapid and quantitative formation of the colored product with

maximum stability and sensitivity for the methods Method - M1(a), M1(b); Method


268

– M5(a), Method – M5(c) and Method – M15 are as same as described in Chapter-

II, P: 97-109.

Method – M7

The effect of reagent concentration (acidity, NBS and CB), waiting period in

each step with respect to maximum sensitivity, minimum blank, adherence to Beer’s

law, reproducibility and stability of final color are studied by means of control

experiments varying one parameter at a time (Table: 4.08(a), P: 274)

Method M16

In developing this method, the effect of various parameters likes strength and

volume of H2O2 time required for oxidation, volume and strength of reagents MO,

strength of the acid, solvent for final dilution in developing color of maximum

stability and intensity are studied (Table: 4.08(b), P: 275).

Method – M17

The optimum conditions in this method are fixed based on the study of the

effects of various parameters such as type of buffer, concentration of picric acid,

choice of organic solvent, ratio of organic phase to aqueous phase, shaking time,

temperature, intensity and stability of the colored species in organic phase. The author

performed controlled in pediments by measuring absorbance at max 415nm of a

series of solutions varying one and fixing the other parameter (Table: 4.08(c), P:

276).


269

Method: M18

Optimum conditions are established based on the study of the effects of various

parameters such as volume of (2.54 x 10-2

M) 4-AP solution, volume of solvents

solution used initially and subsequently for final dilution and the stability of colored

species after final dilution, measuring absorbance at 440nm. The optimum conditions

developed and actual conditions chosen for the procedure (Table: 4.08(d), P: 277).

Method: M19

The optimum conditions in this method are found based on the study of the

effects of various parameters such as volume of (5.83 x 10-3

M) INH solution, volume

of solvents solution used initially and subsequently for final dilution and the stability of

colored species after final dilution is established by measuring absorbance at 450nm.

The optimum conditions developed and actual conditions chosen for the procedure

(Table: 4.08(e), P: 277).

4.10 Validation of the proposed methods

The proposed methods are validated based on sensitivity, linearity precision,

accuracy, specificity. Sensitivity of all the methods is evaluated by determining the

limit of detection (LOD) and limit of quantitation (LOQ). The average percent of

recovery obtained which indicates good accuracy of the developed methods.

4.10(i) Linearity

Calibration graphs are plotted by taking concentration on x-axis and

absorbance on y- axis for the proposed methods and found to be passing linearly

through the origin. Linear least square regression analysis is carried out for getting the


270

slope, intercept and correlation coefficient, standard deviation on slope, standard

deviation on intercept, limit of detection and limit of quantification are evaluated

Table 4.10(a)-Table 4.10(c) , P: 287

4.10(ii) Precision

Repeatability of proposed methods is determined from the absorbance values

for six replicates of constant amount of ondansetron hydrochloride within the Beer’s

law limits. The percent of relative standard deviation and percent range of error

(0.05confidence limit) are calculated for the proposed methods and represented in

(Table4.11 (a)-Table 4.11(c), P: 288)

4.10(iii) Accuracy

In order to determine the accuracy of the proposed methods, three different

levels of drug concentrations are prepared from the stock solution and analyzed by

taking three replicate measurements. Accuracy is expressed as mean percentage

recovery and percentage relative standard deviation. (Table4.12 (a)-Table4.12 (j), P:

289-293).

4.10(iv) Limit of Detection and Limit of Quantitation

The limit of detection and quantitation are calculated based on the standard

deviation of the intercept (Sa), and slope of calibration curve, b, and presented in

Table 4.10(a)-Table4.10(c), P: 287.


271

4.11(i) Assay of pharmaceutical formulations

Thirty tablets of 4.0mg/tablet or 8.0mg /tablet) are powdered and mixed

thoroughly. The amount of the powder equivalent to 100mg is dissolved in methanol,

shaken well and filtered .The filtrate is made up to 100ml with triple distilled water.

Working sample solutions of 250μg/ml, 200μg/ml are prepared by diluting 25.0ml

and 20.0ml of this solution to 100ml. The present of recoveries are determined by

adding different known amounts of the standard drug to equal amount of test sample;

reagents are added and diluted to the same volume. The absorption values are

measured for every solution and the results are plotted on a graph with the dependent

variable (absorbance) on y-axis and the amount of the drug added on x-axis.

Extrapolation of the straight line thus obtained to the point where the x-axis is cut

provides a measure of drug in the test solution. The results of the recovery

experiments by the proposed methods are listed in Table 4.13(a)-Table4.13 (e), P:

294-298.

4.11 (ii) Analysis of formulations

Commercial formulations containing Ondansetron HCl are successfully

analyzed by the proposed methods. The values obtained by the proposed and

reference methods for the formulations were compared statistically with F-test and t-

test and found to be not different significantly. The results are summarized in Table

4.13(a)-Table4.13 (e), P: 294-298. Percent recoveries are determined by adding

standard drug to preanalysed formulations. The results of the recovery experiments by

the proposed methods are also listed in Table 4.13(a)-Table4.13 (e), P: 294-298.


272

4.12 Results and Discussions

4.12(i) Spectral Characteristics

In order to ascertain the optimum wavelength of maximum absorption (max) of

the colored species formed in the each of nine spectrophotometric methods, certain

amount of the drug is taken and developed color by following the prescribed

procedure for each method. The absorption spectrum is scanned on a

spectrophotometer in the region of wavelength 370 to 900nm against similar reagent

blank. The reagent blank absorption spectrum of each method is also recorded against

the solvent. The results are graphically represented in Fig. 4.04 – Fig.4.13, P: 278-

280. The absorption curves of the colored species in each method show

characteristics absorption maxima where as the blank in each method has low or no

absorption in this region.

4.12(ii) Optical Characteristics

In order to know whether the colored species formed in the above methods, adhere

to Beer’s law, the absorbances at maximum wavelength of a series of solutions containing

different amounts of the drug and specified amounts of reagents (as given in the

recommended procedures for each method) are recorded against the corresponding reagent

blanks. The Beer’s law plots of these systems are recorded against the corresponding

reagent blanks (Fig. 4.14 – Fig. 4.23, P: 280-282). Beer’s law limits, molar absorptivity,

Sandell’s sensitivity and optimum photometric range for each method are calculated and

are represented in Table 4.09 (a)-Table4.09(c), P: 286).


273

4.12(iii) Validation Parameters

Least square regression analysis is carried out for getting the slope, intercept and

correlation coefficient and presented in (Table4.10 (a)-Table4.10(c), P:287). Precision

of each method is determined by measuring the absorbance of six replicate concentrations

of ondansetron hydrochloride and expressed as %RSD. The low percent of relative

standard deviation values (Tables4.11 (a)-Table 4.11(c), P: 288) show that these

methods are precise. Accuracy of these methods is expressed in terms of percent of

recovery of the drug added. High percent of recovery of the drug at different

concentration levels indicates that the developed methods are accurate. The results are

given in the Table4.12 (a)-Table4.12 (j), P: 289-293. Commercial formulations of the

drug are successfully analyzed by these methods. The %RSD and %Recovery values

obtained by the proposed are compared with reference method by applying statistical

tests such as F-test and t-test. These methods are found to be not significantly different

Table4.13 (a) - Table4.13 (e), P: 294-298.

Chapter – IV Ondansetron HCl Part-B: Visible Spectrophotometry

274

Table 4.08(a)

Optimum conditions established in method M7for OND Parameter Optimum range Conditions in

procedure

Remarks

max (nm) 500-500 460

Acid concentration (over all)

0.5-0.35 M HCl

0.10-0.25M H2SO4

0.3-0.5M ACOH

1.25 M HCl

The studies on the variation of acid concentration indicated that constant

absorbance value is obtained in 0.5-0.35M HCI, 0.10 to 0.25M H2SO4 or 0.3-

0.5M ACOH at NBS concentration of 200g. As the difference in absorbance

between the sample and the blank is found to be higher for the HCI medium,

subsequent studies are carried out in 0.25M HCI.

Amount of NBS (100 g/ml) and

amount of CB 50g/ml

1.0-5.0ml of NBS

8.0-12.0ml of CB

4.0ml of NBS

4.0ml of CB

In order to ascertain the linear relationship between the concentration of added

NBS and the corresponding decrease in the absorbance of CB, experiments are

carried out in 0.25M HCI medium with varying amounts of NBS. As the

decrease in absorbance is found to be linear up to an amount of 250g (2.5ml of

100 g/ml) of NBS, subsequent studies are carried out with 500g (10.0ml of

50g/ml) of CB and 250 g of NBS in 0.25M HCI medium.

Time and temperature for oxidation

with NBS

5-20 min at lab

temp (28+50C

10min 10 min waiting period at laboratory temperature (285

0C) is adequate in

oxidation stage of OND. Variation of oxidation time beyond the upper and

lower limits results in low absorbance values.

Time for oxidation of dye and

stability period of final color 2-30min 5 min

A minimum of 2 min is required for maximum color difference which remained

stable for 30 min. The difference in absorbance varied slowly and steadily

beyond 60 min.

Stability period after final dilution Immediate - 50 min 5min The intensity of the colored species decrease with time after the stability period


275

Table 4.08(b)

Optimum conditions established in method M16 for OND Parameter Optimum range Conditions in

procedure

Remarks

max (nm) 500-570 530

Effect of volume of (6.11x10-3

M) MO

on color development

0.5-2.0ml

1.0ml Addition of MO at lower limits resulted low absorbance values. Increasing the

volume beyond upper limits had no effect.

Nature of oxidant on color

development

H2O2 H2O2 Of the various oxidants such as Cr(VI), Mn(VII), Fe(III) ,Ce(IV and H2O2 are

tried in combination with MO for oxidation reaction, the combination MO-

H2O2 for the method is found to be best suited with respect to sensitivity and

stability of colored species formed .

Effect of volume of H2O2 for

maximum color development

0.5-2.5ml

2.0ml

Less than 1.0Ml of H2O2 is found to decrease the absorbance of the test solution

Time required for color development

after the addition of oxidant

1-10 min

10min

1 min of time is necessary to attain maximum color development.

Solvent for final dilution Water

Water

Other solvents like methanol, ethanol and propan-2-ol are found not to enhance

the intensity or stability of the final colored product.

Stability period after final dilution 1-45min

30 min

After the stability period, the intensity of the colored product is found to

decrease with time.


276

Table 4.08(c)

Optimum conditions established in method M17 for OND Parameter

Optimum range

Conditions in

procedure

Remarks

max (nm) PA

400-490

415

Effect of buffer on color development 9.0 - 10.0 pH-9.8

Variations of the pH less than 6.0 and greater than11.0 resulted in low

absorbance values

Effect volume of (4.36x10-3

M) PA for

M17 (6.11x10-3

M)

1.0-3.0ml (M17)

2.0ml (M17)

2.0ml dye solution of is necessary for covering broad range of Beer’s law limits

Choice of organic solvent for

extraction of the colored complex. Chloroform Chloroform

The water immiscible solvents tested for the extraction of the colored complex

into organic phase which include (chlorobenzene, carbon tetrachloride, benzene,

n-butanol and chloroform). Chloroform is preferred for its selective extraction

of the colored drug-dye complex from the aqueous phase.

Effect of ratio of organic to aqueous

phase on extraction

1:1.5

1:1.5

The extraction of the colored species into organic layer is incomplete when the

ratio of organic to aqueous phase is more than the specified ratio in each case

Effect of shaking time on extraction

1-5 min

2 min

Constant absorbance values are obtained for shaking periods between 1-5 min.

Effect of temperature on the colored

species

Laboratory

temperature

(28+30C)

Laboratory

temperature

At low temperature (less than200C) the extraction of colored species is found to

be improper. At high temperature (greater than350C) the stability of the colored

species is found to be less.

Stability of the colored species in

organic solvent.

1-60 min

5 min


277

Table 4.08(d)

Optimum conditions established in method M18 for OND

Parameter

Optimum range

Conditions in

procedure

Remarks

max (nm)

400 - 470

440

Effect of volume (2.45 x 10-2

M) of

4-AP in MeOH and waiting time.

2 - 4ml, 15min 3.0ml ,15min

3ml of 4-AP and 15min waiting time are preferred for covering broad

range in Beer’s law limits.

Solvent for final dilution. Methanol Methanol

MeOH has been found to be suitable for final dilution to give better

absorbance values.

Stability period after final dilution.

Immediate -

40min

10min

After 40min the absorbance of colored species diminish slowly with time.

Table 4.08(e)

Optimum conditions established in method M19 for OND

Parameter Optimum range Conditions in

procedure

Remarks

max (nm) 400 - 500 470

Effect of volume (5.83 x 10-3

M) of

INH in MeOH and heating (time

and temp).

1 - 3 ml, 600C,

5 - 15 min.

2.0 ml, 600C,

10 min.

Addition of INH (2ml) and heating (600C, 10min) have been found to be

necessary to cover broad range in Beer’s law limits.

Solvent for final dilution. Methanol Methanol

MeOH has been found to be suitable for final dilution to give better

absorbance values.

Stability period after final dilution.

Immediate - 50

min

Immediate

After the stability period, the absorbance of colored species decreased

slowly.


278

Method M1(a):ARS+CHCl3

[OND]=3.64x10-5

M

[ARS]=3.66x10-4

M, [HCl]=4.0x10-2

M

0.000

0.200

0.400

0.600

375 400 425 450 475 500

Wavelength nm

Ab

so

rba

nc

e

Blank

Test

Fig. 4.04 Absorption Spectrum of OND

with ARS M1(a)

Method M1(b):BTB+CHCl3

[OND]=9.11x10-5

M

[BTB]=2.14x10-4

M, Buffer PH=3.5

0.000

0.200

0.400

0.600

0.800

350 400 450 500 550

Wavelength nm

Ab

so

rba

nc

e

Blank

Test

Aq.

Dye


with BTB M1(b)

Method M5(a):MBTH+Ce(IV)

[OND]=5.47x10-5

M

[MBTH]=1.71x10-4

M,

[Ce(IV)]=1.27x10-3

M

0.000

0.050

0.100

0.150

0.200

500 550 600 650 700 750

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with MBTH+Ce(IV) M5(a)

Method M5(c):MBTH+NaIO4+AcOH

[OND]=4.37x10-5

M,

[MBTH]=3.42x10-4

M

[NaIO4]=3.47x10-4

M

[AcOH]=1.28x10-1

M

0.100

0.200

0.300

0.400

0.500

500 550 600 650 700 750

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with MBTH+NaIO4+AcOH M5(c)


279

Method M7:NBS+CB+HCl

[OND]=4.37x10-5

M,

[NBS]=8.99x10-5

M

[CB]=8.80x10-4

M, [HCl]=5.0x10-2

M

0.000

0.050

0.100

0.150

0.200

375 425 475 525 575

Wavelength nm

Ab

so

rba

nc

e

BlankTestAq.Dye


with NBC+CB M7

Method M15:p-CA+CHCl3

[OND]=1.37x10-4

M

[p-CA]=9.57x10-4

M

0.000

0.250

0.500

0.750

450 500 550 600 650

Wavelenght nmA

bs

orb

an

ce

BlankTest


with p-CA M15

Method M16:H2O2+MO

[OND]=5.47x10-5

M,

[MO]=3.06x10-4

M,

0.000

0.100

0.200

0.300

450 500 550 600 650Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with H2O2+MO M16

Method M17:PA+CHCl3

[OND]=7.29x10-5

M

[PA]=5.82x10-4

M, [HCl]=4.0x10-2

M

0.000

0.200

0.400

0.600

350 400 450 500

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with PA+CHCl3 M17


280

Method M18 4-AP

[OND]=5.47x10-5

M,

[4-AP]=2.45x10-2

M

0.000

0.100

0.200

0.300

0.400

400 420 440 460 480 500

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with 4-AP M18

Method M19:INH

[OND]=5.47X10-5

M

[INH]=5.83x10-3

M

0.000

0.100

0.200

0.300

0.400

400 425 450 475 500 525 550

Wavelength nm

Ab

so

rba

nc

e

BlankTest


with INH M19

MethodM1(a):ARS+CHCl3

[OND]=9.11x10-6

M - 5.47x10-5

M

[ARS]=3.66x10-4

M, [HCl]=4.0X10-2

M

0.000

0.200

0.400

0.600

0.800

0 5 10 15 20

Weight of the Drug in µg/ml

Ab

so

rba

nc

e

Fig. 4.14Beer’s Law Plot of OND with

ARS M1(a)

Method M1(b):BTB+CHCl3

[OND]=2.88x10-5

M - 1.37x10-4

M

[BTB]=2.14x10-4

M, Buffer PH=3.5

0.000

0.250

0.500

0.750

1.000

0 20 40 60


Ab

so

rba

nc

e

Fig. 4.15 Beer’s Law Plot of OND with

BTB M1(b)


281

Method M5(a):MBTH+Ce(IV)

[OND]=2.73x10-5

M - 1.64x10-4

M

[MBTH]=1.71x10-4

M,

[Ce(IV)]=1.27x10-3

M

0.000

0.250

0.500

0.750

0 20 40 60 80


Ab

so

rba

nc

e


MBTH+Ce(IV) M5(a)

Method M5(c):MBTH+NaIO4+AcOH

OND]=8.75x10-6

M -5.25x10-5

M

[MBTH]=3.42x10-3

M,

[NaIO4]=3.74x10-4

M

[AcOH]=1.4x10-1

M

0.000

0.200

0.400

0.600

0 10 20 30Weight of the Drug in µg/ml

Ab

so

rba

nc

e


MBTH+NaIO4+AcOH M5(c)

Method M7:NBS+CB+HCl

[OND]=2.19x10-5

M -1.31x10-4

M

[NBS]=1.35x10-4

M,[CB]=1.32x10-4

M

[HCl]=5.0x10-2

M

0.000

0.100

0.200

0.300

0.400

0 20 40 60 80Weght of the Drug in µg/ml

Ab

so

rba

nc

e


NBS+CB M7

Method M15:p-CA+CHCl3

[OND]=3.42x10-5

M - 2.05x10-4

M

[p-CA]=9.57x10-4

M

0.000

0.300

0.600

0.900

0 20 40 60 80Weight of the Drug in µg/ml

Ab

so

rba

nc

e


p-CA+CHCl3 M15


282

Method M16:H2O2+MO

[OND]=2.73x10-5

M-1.64x10-4

M

[MO]=3.06x10-4

M

0.000

0.250

0.500

0.750

1.000

0 20 40 60


Ab

so

rba

nc

e

Fig. 4.20Beer’s Law Plot of OND with

H2O2+MO M16

Method M17:PA +CHCl3

[OND]=1.82x10-5

M - 1.09x10-4

M

[PA]=5.82x10-4

M, [HCl]=4.0x10-2

M

0.000

0.200

0.400

0.600

0.800

0 10 20 30 40 50

Weight of the Drug in µg/mlA

bs

orb

an

ce


PA+CHCl3 M17

Method M18:OND+4-AP

[OND]=1.37x10-5

M - 8.20x10-5

M

[4-AP]=2.45x10-3

M

0.000

0.100

0.200

0.300

0.400

0.500

0 10 20 30 40

Weigth of the drug in µg/ml

Ab

so

rba

nc

e


4-AP M18

Method M19: OND+INH

[OND]=1.37x10-5

M - 8.20x10-5

M

[INH]=5.83x10-3

M

0.000

0.200

0.400

0.600

0 10 20 30 40

Weight of the drug in µg/ml

Ab

so

rba

nc

e


INH M19


283

Method M1(a):ARS+CHCl30.00

30.00

60.00

90.00

0.40 0.80 1.20 1.60

LOG(Concentration in μg/ml)

% o

f T

ran

sm

ita

nc

e

Fig. 4.24 Ringbom Plot of OND with

ARS M1(a)

Method M1(b):BTB+CHCl30.00

20.00

40.00

60.00

80.00

0.75 1.00 1.25 1.50 1.75

LOG(Concentration in μg/ml)%

of

Tra

ns

mit

an

ce


BTB M1(b)

Method M5(a):MBTH+Ce(IV)0.00

30.00

60.00

90.00

0.75 1.00 1.25 1.50 1.75 2.00


%o

f T

ran

sm

ita

nc

e


MBTH+Ce(IV) M5(a)

Method M5(c):MBTH+NaIO4+AcOH0.00

30.00

60.00

90.00

0.00 0.50 1.00 1.50


% o

f T

ran

sm

ita

nc

e


MBTH+NaIO4+AcOH M5(c)


284

Method M7:NBS+CB+HCl25.00

50.00

75.00

100.00

0.75 1.00 1.25 1.50 1.75 2.00

LOG (Concentration in μg/ml)

%o

f T

ran

sm

ita

nc

e


NBS M7

Method M15:p-CA+CHCl30.00

20.00

40.00

60.00

80.00

1.00 1.25 1.50 1.75 2.00


%o

f T

ran

sm

ita

nc

e

Fig. 4.29 Ringbom Plot of OND with p-

CA M15

Method M16:H2O2+MO0.00

20.00

40.00

60.00

80.00

0.75 1.00 1.25 1.50 1.75 2.00


%o

f T

ran

sm

ita

nc

e


H2O2+MO M16

Method M17: PA+CHCl30.00

20.00

40.00

60.00

80.00

0.75 1.00 1.25 1.50 1.75


%o

f T

ran

sm

ita

nc

e

Fig. 4.31 Ringbom Plot of OND with PA

M17


285

Method M18: OND+4-AP30.00

45.00

60.00

75.00

90.00

0.50 1.00 1.50 2.00

LOG (Concentration in μg/ml)

% T

ran

sm

ita

nc

e

Fig. 4.32Ringbom Plot of OND with 4-AP M18

Method M19:OND+INH0.00

30.00

60.00

90.00

0.50 1.00 1.50 2.00


% T

ran

sm

ita

nc

e

Fig. 4.33 Ringbom Plot of OND with INH M19


286

Table-4.09(a): Optical Characteristics of the proposed methods for OND

Name of the Parameter M1(a) M1(b) M5(a) M5(c)

Maximum Wavelength λmax 415 nm 420 nm 630 nm 630 nm

Beer's Law Limits µg/ml 3.3-20.0 8.3-50 10.0-60.0 3.2-19.2

Optimum Photometric Range µg/ml 6.7-20.0 25.0-41.67 30.0-60.0 9.6-16.0

Sandell's Sensitivity µg/cm2 / 0.001 Abs 2.62E-02 6.46E-02 1.00E-01 3.48E-02

Molar Absorptivity lt/mole/cm 1.33E+04 5.76E+03 3.47E+03 1.10E+04

Table-4.09(b): Optical Characteristics of the proposed methods for OND

Name of the Parameter M7 M15 M16 M17

Maximum Wavelength λmax 460 nm 540 nm 530 nm 415 nm

Beer's Law Limits µg/ml 8.0-48.0 12.5-75.0 10.0-60.0 6.7-40.0

Optimum Photometric Range µg/ml 16.0-40.0 25.0-75.0 20.0-50.0 20.0-33.3

Sandell's Sensitivity µg/cm2 / 0.001 Abs 1.27E-01 8.17E-02 7.35E-02 4.94E-02

Molar Absorptivity lt/mole/cm 2.93E+03 4.30E+03 5.04E+03 7.34E+03

Table-4.09(c): Optical Characteristics of the proposed methods for OND

Name of the Parameter M18 M19

Maximum Wavelength λmax 440 470

Beer's Law Limits µg/ml 5.0-30.0 5.0-30.0

Optimum Photometric Range µg/ml 10.0-30.0 10.0-30.0

Sandell's Sensitivity µg/cm2 / 0.001 Abs 6.85E-02 6.02E-02

Molar Absorptivity lt/mole/cm 5.62E+03 6.09E+03


287

Table-4.10(a): Linear least square regression analysis

Name of the Parameter M1(a) M1(b) M5(a) M5(c)

Slope (b) 3.64E-02 1.57E-02 9.47E-03 3.01E-02

Intercept(a) -1.53E-03 -4.73E-03 1.07E-03 3.53E-03

Standard Deviation on Slope(Sb) 3.67E-04 8.77E-05 7.93E-05 3.85E-04

Standard Deviation on Intercept(Sa) 4.76E-03 2.85E-03 3.09E-03 4.80E-03

Correlation coefficient (r ) 0.9997 0.9999 0.9998 0.9995

Limit of Detection (LOD) µg/ml 0.392 0.542 0.977 0.479

Limit of Quantification (LOQ) µg/ml 1.307 1.808 3.259 1.597

Table-4.10(b): Linear least square regression analysis

Name of the Parameter M7 M15 M16 M17

Slope (b) 8.01E-03 1.17E-02 1.38E-02 2.01E-02

Intercept(a) -1.33E-04 7.67E-03 -2.20E-03 -3.87E-03

Standard Deviation on Slope(Sb) 5.46E-05 1.13E-04 1.17E-04 1.41E-04

Standard Deviation on Intercept(Sa) 1.70E-03 5.52E-03 4.57E-03 3.66E-03

Correlation coefficient (r ) 0.9998 0.9998 0.9998 0.9999

Limit of Detection (LOD) µg/ml 0.637 1.410 0.996 0.548

Limit of Quantification (LOQ) µg/ml 2.124 4.700 3.321 1.826

Table-4.10(c): Linear least square regression analysis

Name of the Parameter M18 M19

Slope (b) 1.54E-02 1.67E-02

Intercept(a) -2.13E-03 2.33E-03

Standard Deviation on Slope(Sb) 8.73E-05 1.26E-04

Standard Deviation on Intercept(Sa) 1.70E-03 2.46E-03

Correlation coefficient (r ) 0.9999 0.9998

Limit of Detection (LOD) µg/ml 0.332 0.442

Limit of Quantification (LOQ) µg/ml 1.107 1.475


288

Table 4.11(a) Precision of the test method

S.No Statistical Parameter Value M1(a) M1(b) M5(a) M5(c)

1 Concentration (μg/ml) 13.33 33.33 40.00 12.80

2 Mean( of six replicates) (μg/ml) 13.34 33.31 39.96 12.82

3 Standard Deviation (s) 0.131 0.165 0.224 0.132

4 %Relative Standard Deviation(%RSD) 0.978 0.496 0.559 1.031

5 0.05 level confidence limit µg/ml 0.215 0.272 0.368 0.217

Table 4.11(b) Precision of the test method

S.No Statistical Parameter Value M7 M15 M16 M17

1 Concentration (μg/ml) 32.00 50.00 40.00 26.67

2 Mean( of six replicates) (μg/ml) 31.96 50.05 39.97 26.69

3 Standard Deviation (s) 0.256 0.271 0.228 0.176

4 %Relative Standard Deviation(%RSD) 0.802 0.542 0.570 0.659

5 0.05 level confidence limit µg/ml 0.422 0.446 0.375 0.289

Table 4.11(c) Precision of the test method

S.No Statistical Parameter Value M18 M19

1 Concentration (μg/ml) 20.00 20.00

2 Mean( of six replicates) (μg/ml) 19.98 20.04

3 Standard Deviation (s) 0.107 0.109

4 %Relative Standard Deviation(%RSD) 0.536 0.544

5 0.05 level confidence limit µg/ml 0.176 0.180


289

Table-4.12(a): Accuracy of the proposed Method M1(a)

Sample

ID

Amount

Taken µg/ml

Amount

Found µg/ml

Percent of

Recovery

Statistical analysis

1 10.00 9.98 99.82 Mean 100.36

2 10.00 10.13 101.33 SD 0.839

3 10.00 9.99 99.92 %RSD 0.836

1 13.33 13.32 99.92 Mean 100.15

2 13.33 13.35 100.15 SD 0.225

3 13.33 13.38 100.38 %RSD 0.225

1 16.66 16.76 100.59 Mean 100.01

2 16.66 16.54 99.26 SD 0.675

3 16.66 16.69 100.17 %RSD 0.675

Table-4.12(b): Accuracy of the proposed methodM1(b)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 25.00 25.42 101.69 Mean 100.757

2 25.00 24.93 99.73 SD 0.983

3 25.00 25.21 100.85 %RSD 0.976

1 33.33 33.29 99.88 Mean 100.450

2 33.33 33.54 100.63 SD 0.505

3 33.33 33.61 100.84 %RSD 0.502

1 41.66 41.79 100.31 Mean 100.066

2 41.66 41.82 100.38 SD 0.479

3 41.66 41.46 99.51 %RSD 0.479


290

Table-4.12(c): Accuracy of the proposed methodM5(a)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 30.00 30.12 100.40 Mean 99.489

2 30.00 29.78 99.27 SD 0.823

3 30.00 29.64 98.80 %RSD 0.827

1 40.00 40.41 101.03 Mean 99.917

2 40.00 39.78 99.45 SD 0.964

3 40.00 39.71 99.28 %RSD 0.965

1 50.00 49.72 99.44 Mean 99.900

2 50.00 49.81 99.62 SD 0.647

3 50.00 50.32 100.64 %RSD 0.648

Table-4.12(d): Accuracy of the proposed methodM5(c)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 9.60 9.64 100.42 Mean 100.000

2 9.60 9.57 99.69 SD 0.376

3 9.60 9.59 99.90 %RSD 0.376

1 12.80 12.71 99.30 Mean 100.156

2 12.80 12.86 100.47 SD 0.753

3 12.80 12.89 100.70 %RSD 0.752

1 16.00 16.22 101.38 Mean 100.250

2 16.00 15.93 99.56 SD 0.982

3 16.00 15.97 99.81 %RSD 0.980


291

Table-4.12(e): Accuracy of the proposed Method M7

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 24.00 24.34 101.42 Mean 100.583

2 24.00 23.96 99.83 SD 0.795

3 24.00 24.12 100.50 %RSD 0.790

1 32.00 32.46 101.44 Mean 100.542

2 32.00 32.27 100.84 SD 1.079

3 32.00 31.79 99.34 %RSD 1.073

1 40.00 40.18 100.45 Mean 99.683

2 40.00 39.79 99.48 SD 0.687

3 40.00 39.65 99.13 %RSD 0.689

Table-4.12(f): Accuracy of the proposed Method M15

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 37.50 37.97 101.25 Mean 100.444

2 37.50 37.38 99.68 SD 0.788

3 37.50 37.65 100.40 %RSD 0.784

1 50.00 50.32 100.64 Mean 100.087

2 50.00 49.85 99.70 SD 0.492

3 50.00 49.96 99.92 %RSD 0.491

1 62.50 62.98 100.77 Mean 99.653

2 62.50 61.94 99.10 SD 0.965

3 62.50 61.93 99.09 %RSD 0.969


292

Table-4.12(g): Accuracy of the proposed methodM16

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 30.00 30.38 101.27 Mean 100.400

2 30.00 29.87 99.57 SD 0.850

3 30.00 30.11 100.37 %RSD 0.847

1 40.00 40.32 100.80 Mean 99.808

2 40.00 39.63 99.08 SD 0.891

3 40.00 39.82 99.55 %RSD 0.893

1 50.00 50.33 100.66 Mean 99.933

2 50.00 49.71 99.42 SD 0.647

3 50.00 49.86 99.72 %RSD 0.647

Table-4.12(h): Accuracy of the proposed methodM17

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 20.00 20.11 100.54 Mean 99.838

2 20.00 20.08 100.39 SD 1.085

3 20.00 19.72 98.59 %RSD 1.087

1 26.67 26.84 100.64 Mean 99.704

2 26.67 26.52 99.44 SD 0.833

3 26.67 26.41 99.04 %RSD 0.836

1 33.34 33.58 100.73 Mean 100.217

2 33.34 33.24 99.71 SD 0.510

3 33.34 33.41 100.22 %RSD 0.509


293

Table-4.12(i): Accuracy of the proposed methodM18

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 15.00 14.95 99.67 Mean 100.111

2 15.00 14.98 99.87 SD 0.605

3 15.00 15.12 100.80 %RSD 0.604

1 20.00 20.31 101.55 Mean 100.683

2 20.00 20.21 101.05 SD 1.097

3 20.00 19.89 99.45 %RSD 1.090

1 25.00 25.21 100.84 Mean 99.933

2 25.00 24.83 99.32 SD 0.801

3 25.00 24.91 99.64 %RSD 0.802

Table-4.12(j): Accuracy of the proposed methodM19

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 15.00 15.22 101.47 Mean 100.667

2 15.00 14.97 99.80 SD 0.835

3 15.00 15.11 100.73 %RSD 0.830

1 20.00 20.16 100.80 Mean 100.483

2 20.00 20.19 100.95 SD 0.683

3 20.00 19.94 99.70 %RSD 0.679

1 25.00 25.11 100.44 Mean 100.080

2 25.00 25.06 100.24 SD 0.461

3 25.00 24.89 99.56 %RSD 0.461


294

Table4.13 (a): Assay of Formulations of Ondansetron Hydrochloride

Reference Method [296]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery

Ref.Method Proposed methods**

M1a M1b M1a M1b

Osetron 4 Mean 3.988 3.985 %REC 99.42 99.704 99.633

SD ±0.015 ±0.022 %RSD ±0.61 ±0.379 ±0.549

F-test 1.389 2.815

t-test 0.974 0.640

Ondem 4 Mean 4.008 3.995 %REC 99.91 100.204 99.872

SD ±0.036 ±0.031 %RSD ±0.71 ±0.905 ±0.788

F-test 1.636 1.231

t-test 0.624 0.088

Osetron 8 Mean 7.982 7.975 %REC 100.05 99.773 99.690

SD ±0.101 ±0.090 %RSD ±0.66 ±1.263 ±1.129

F-test 3.642 2.904

t-test 0.391 0.583

Ondem 8 Mean 8.054 8.027 %REC 99.86 100.671 100.338

SD ±0.084 ±0.054 %RSD ±0.68 ±1.038 ±0.670

F-test 2.368 1.980

t-test 1.614 1.250

*Average of six determinations are considered, AVG=Average, SD=Standard deviation,

F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,

t=1.833.

**%REC=% of Recovery, %RSD=%of Relative standard deviation; Recovery studies,

4.0 and 8.0mg added to the preanalyzed formulations (Average of six determinations)


295

Table4.13 (b): Assay of Formulations of Ondansetron Hydrochloride


Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M5a M5c M5a M5c

Osetron 4 Mean 3.978 3.991 %REC 99.42 99.454 99.775

SD ±0.023 ±0.009 %RSD ±0.61 ±0.580 ±0.222

F-test 2.906 3.133

t-test 0.100 1.347

Ondem 4 Mean 3.999 4.002 %REC 99.91 99.970 100.050

SD ±0.020 ±0.023 %RSD ±0.71 ±0.508 ±0.574

F-test 2.513 1.655

t-test 0.169 0.376

Osetron 8 Mean 7.974 7.967 %REC 100.05 99.669 99.585

SD ±0.091 ±0.110 %RSD ±0.66 ±1.136 ±1.383

F-test 2.937 4.351

t-test 0.619 0.665

Ondem 8 Mean 8.027 8.033 %REC 99.86 100.331 100.415

SD ±0.046 ±0.107 %RSD ±0.68 ±0.576 ±1.328

F-test 1.726 3.860

t-test 1.322 0.924



t=1.833.




296

Table4.13(c): Assay of Formulations of Ondansetron Hydrochloride


Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M7 M15 M7 M15

Osetron 4 Mean 3.957 3.992 %REC 99.42 98.917 99.792

SD ±0.032 ±0.011 %RSD ±0.61 ±0.810 ±0.263

F-test 1.746 2.188

t-test 1.227 1.378

Ondem 4 Mean 4.000 4.009 %REC 99.91 100.000 100.229

SD ±0.020 ±0.033 %RSD ±0.71 ±0.504 ±0.836

F-test 2.506 1.394

t-test 0.253 0.712

Osetron 8 Mean 7.977 7.978 %REC 100.05 99.713 99.725

SD ±0.104 ±0.090 %RSD ±0.66 ±1.301 ±1.131

F-test 3.861 2.918

t-test 0.484 0.515

Ondem 8 Mean 8.042 8.050 %REC 99.86 100.519 100.629

SD ±0.082 ±0.101 %RSD ±0.68 ±1.022 ±1.255

F-test 2.290 3.459

t-test 1.331 1.332



t=1.833.




297

Table4.13 (d): Assay of Formulations of Ondansetron Hydrochloride


Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M16 M17 M16 M17

Osetron 4 Mean 3.989 3.988 %REC 99.42 99.725 99.704

SD ±0.008 ±0.011 %RSD ±0.61 ±0.212 ±0.280

F-test 1.122 2.212

t-test 1.164 1.043

Ondem 4 Mean 4.009 4.010 %REC 99.91 100.221 100.238

SD ±0.034 ±0.033 %RSD ±0.71 ±0.842 ±0.827

F-test 1.414 1.366

t-test 0.691 0.735

Osetron 8 Mean 7.982 7.978 %REC 100.05 99.771 99.729

SD ±0.078 ±0.098 %RSD ±0.66 ±0.983 ±1.231

F-test 2.204 3.456

t-test 0.475 0.476

Ondem 8 Mean 8.033 8.054 %REC 99.86 100.417 100.673

SD ±0.075 ±0.085 %RSD ±0.68 ±0.932 ±1.057

F-test 1.900 2.458

t-test 1.201 1.597



t=1.833.


4.0 and 8.0mg added to the preanalyzed formulations (Average of six determi


298

Table4.13 (e): Assay of Formulations of Ondansetron Hydrochloride


Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M18 M19 M18 M19

Osetron 4 Mean 3.990 3.981 %REC 99.42 99.758 99.533

SD ±0.014 ±0.028 %RSD ±0.61 ±0.363 ±0.703

F-test 2.356 1.333

t-test 1.174 0.300

Ondem 4 Mean 4.007 4.007 %REC 99.91 100.172 100.175

SD ±0.037 ±0.035 %RSD ±0.71 ±0.912 ±0.879

F-test 1.661 1.542

t-test 0.554 0.574

Osetron 8 Mean 7.984 7.986 %REC 100.05 99.794 99.823

SD ±0.100 ±0.079 %RSD ±0.66 ±1.248 ±0.993

F-test 3.558 2.251

t-test 0.358 0.364

Ondem 8 Mean 8.050 8.051 %REC 99.86 100.621 100.631

SD ±0.084 ±0.092 %RSD ±0.68 ±1.039 ±1.138

F-test 2.373 2.845

t-test 1.515 1.438



t=1.833.




299

4.13 Nature of the colored species

Ondansetron HCl possesses tertiary nitrogen atom and keto functional group

in substituted Carbazol ring and tertiary nitrogen atom substituted imidazol. These

groups are responsible for color development in the analysis. Tertiary nitrogen atom

of imidazol in OND is responsible for the development of ion-ion association

complex formation with acid dyes such as Alizarin Red-S (ARS), Bromothymol

blue(BTB) [M1(a) &M1(b),]; α-position of substituted imidazol is favorable for

electrophlic substitution reactions in oxidative coupling reactions [M5(a) and M5(c)].

The tertiary nitrogen in imidazol is also responsible for ion-ion association complex

with PA in basic buffer medium [M17]. Keto functional group is responsible for the

color development with 4-AP and INH reagents due to condensation reaction with the

elimination of water molecule [M18 and M19]

Method - M1(a) and M1(b)

When the drug is treated with hydrochloric acid, protonation takes place on

tertiary nitrogen atom of substituted imidazol ring. The protonated nitrogen having

positive charge and is associated with anion of the acid dyes ARS and BTB and

behaves as a single unit being held together by electrostatic force of attraction which

is extractable into chloroform from aqueous phase. The scheme of the colored product

is given below (Scheme4.01)

O

N

NH3CN

H3C

H

O

N

NH3C

NH3C

H

OndansetronOndansetron Cation

Protonation


300

O

N

N

H3C

NH3C

H

O O

-O3S OH

O

O

SO3-

OH

Anion of ARSColored Product

O

N

N

CH3

N CH3

H

Cation of the Drug

O

N

N

CH3

N CH3

H

C

BrMe2HC

CHMe2

O

SO3H3C

Br

Me2HC

HO

C

BrMe2HC

CHMe2

O

SO3H3C

Br

Me2HC

HO

O

N

N

H3C

NH3C

H

Anion of BTBColored ProductCation of the Drug

Scheme 4.01

Method – M5(a) and M5(c)

Under the reaction conditions, MBTH on oxidation in the presence of oxidants

such as Ce (IV), NaIO4 in acetic acid medium, loses two electrons and one proton

forming and electrophilic intermediate, which is the active coupling species that reacts

with the coupler (OND) by electrophillic attack on ortho position of the most

nucleophilic site on cyclic ring (substituted imidazol ring).

N

S

CH3

N-NH2

MBTH

N

S

CH3

N NH

Electrophilic intermediate

-2e , H

Ce(IV) / NaIO4+AcOH


301

N

S

CH3

N


NHN

S

CH3

N NH


O

N

N

H3CN

H3C

O

N

N

H3C

NH3C

+N

S

H3C

N

NH


N

S

CH3

NNH

Ondansetron

Colored Product

Scheme 4.02

Method – M7

The first step is the oxidation of drug in the presence of NBS. The unreacted

NBS is reacted with excess CB. The decrease in the intensity of color is

corresponding to unreacted CB. The scheme of reactions are presented in scheme 4.03

+ CB Oxidation products of dye + Unreacted CB (Coloured)

(Colourless through disruption of chromophores and auxochromes)

NBS

Step - I

OND + NBS Oxidation product (s) of OND + Unreacted NBS

Step - II

(Unreacted)

O

N

N

H3C

NH3C

N

O

O

Br+ O

N

N

H3C

NH3C

HN

O

O

+ HBrOxidation

Oxidised Product of the DrugNBS (excess)Ondansetron

NBS (unreacted)+

Succinamide


302

HO

HO

CONH2

N

EtEt

NOClHN

O

OO

O

CONH2

N

EtEt

NOCl-HBr +

NBS (unreacted)

OH

OH

CONH2

N

Et

Et

N

OCl

unreacted CB (colored dye)

Spectrophotometrically estimatedOxidised form of CB(coloer less) SuccinamideCB (colored dye)

Scheme 4.03

Method – M15

As drug possesses tertiary amine group, it functions as an electron donor and

participate in charge transfer interaction with p-CA which is known as electron

acceptor. This complex is formed by the lone pair of electrons donated by the

ondansetron as n-donor and the charge transfer reagent as an electron acceptor,

through which a partial ionic bond

O

O

OH

ClHO

Cl

O

N

N

H3C

NH3C

2+

p-CA

OH

Cl

HO

Cl

ON

N

H3C

N

H3C

HO

Cl

OH

Cl

O

O

O

O

Ondansetron

Colored Product

Scheme 4.04

Method – M16

Tertiary amines can be converted to amine by oxidation. Hydrogen peroxide is

often used. The positive ion of the oxidized form of the drug is associated with


303

negative ion of the methyl orange molecule to form ion-ion associated complex which

is extracted in to chloroform solvent.

O

N

NH3C

N

H3C

O

N

NH3C

N

H3C

OH

Ondansetron Oxidised form of the Drug

H2O2

O

N

N

H3C

NH3C

OH

N

N

SO3

N(CH3)2

+O

N

N

H3C

NH3C

OH

N

N

SO3

N(CH3)2Cation of the Drug Anion of the Methyl Orange Colored Product

Scheme 4.05

Method M17

The phenolic group in picric acid is more acidic, so in basic medium it

immediately loses the acidic proton, to produce a stable anionic species. The drug

OND possesses a tertiary nitrogen atom; it immediately accepts the proton. The

positive ion thus formed and negative ion of the dye forms a stable ion-ion associated

complex which is extractable into chloroform solvent. The scheme of the colored

product is represented in scheme 4.06

OH

NO2

NO2

O2N

O

NO2

NO2

O2N+ H

Basic Buffer

Stable Anion of Picric Acid


304

O

N

NH3CN

H3C

H

O

N

NH3C

NH3C

H

Ondansetron Protonated Ondansetron

Protonation

O

NO2

O2N

NO2

O

N

NCH3 N

CH3

H

O

NO2

O2N

NO2

O

N

NCH3 N

CH3

H

+

Colored Product

Scheme 4.06

Method: M18 and M19

It is well known that 4 – amino phenazone (4- AP) and isonicotinic hydrazide

(INH) give a colored hydrazone and Schiff’s base respectively with keto groups of the

drug (OND).The colored species formation may be represented as under scheme 4.07

and scheme 4.08.

CH3

CH3H2N

O

C6H5

O

N

N

H3C

NH3C

CH3H3C

C6H5

N

N

H3C

NH3C

N

+

-H2O

Condensation

Ondansetron 4-AP Colored Product

O

Scheme 4.07


305

C

HN

O

N

O

N

NH3C

N

H3C

H H

C

HN

O

N

NH3C

N

H3C

N-H2O

CondensationINH

Ondansetron Colored Product

Scheme 4.08

4.14 Conclusions: The proposed methods are simple, sensitive and reliable and can

be used for routine determination of OND in bulk and formulations. Statistical

analysis of the results shows that the proposed methods have good precision and

accuracy. Results of the analysis of formulations reveal that the proposed methods are

suitable for their analysis with virtually no interference of the usual additives present

in pharmaceutical formulations. The descending order of sensitivity (εmax) and

maximum wavelength (λmax) among the proposed methods are shown below.

The decreasing order of sensitivity (εmax) among the proposed methods is shown

below.

M17›M19›M1(b)›M18 ›M16 ›M15 ›M5(a) ›M7 ›M1(a) ›M5(c)

The descending order maximum wavelength (λmax) among the proposed methods is

shown below.

M5(a)›M5(c)› M15 ›M16 ›M19›M7›M18›M15›M1(a)›M17.

Chapter – IV Ondansetron HCl Part-C: Derivative Spectrophotometry

306

DERIVATIVE SPECTROPHOTOMETRIC DETERMINATION OF

ONDANSETRON HYDROCHLORIDE

4.15 Introduction

The influence of an impurity on the absorption spectrum of a substance can be

eliminated by considering derivative curves. For quantitative purpose, peak heights

are usually measured for the long-wave peak satellite of either the second or fourth

derivative curves. Single or multi-components can be analyzed in the presence of

broad, interfering background matrix absorption. Author developed four derivative

spectrophotometric methods based on the reactions such as ion-ion association

reaction with acidic dye ARS chloroform as extracting solvent (M1a),oxidative

coupling reactions with MBTH as oxidative coupler in the presence of an oxidizing

agent Ce(IV) (M5a), substitution reaction with NBS(M7) and n-electron transfer

reaction with p-CA (M15).

4.16(i) Experimental

UV-Visible Spectrophotometer: Elico SL159 model, 2nm high resolution, double

beam, 1cm length quartz coated optics; Wavelength range 390-900nm; High stability,

linearity, and precision instrument is used for all the spectral measurements.

4.16(ii) Preparation of solutions

Standard Solution of Ondansetron HCl: The stock solution of ondansetron HCl is

freshly prepared by transferring accurately weighed 100mg of ondansetron HCl into

100ml volumetric flask and dissolved in double distilled water, and then made up to

the mark. Then working standard solutions 250μg/ml( for methods M5(a), M7,M15),


307

and 100μg/ml ( for method M1(a)), are prepared by transferring 25.0ml and 10.0ml

of the stock solution into two 100ml standard flask and made up to the mark

respectively.

Preparation of reagents: The following reagents are prepared for the estimation of

the drug by second order derivative spectra. The procedure for the preparation is same

as described in Chapter-IV Part-B: Visible spectrophotometry.

ARS solution : Fluka, 0.2%, 5.49 x 10-3

M

HCl solution : Qualigens, 0.1M

MBTH Solution : Fluka, 0.2%, 8.56 x 10-3

M

Ce (IV) solution : MERCK, 1%, 9.35 x 10-3

M

NBS solution : Loba, 0.01%, 5.618x10-4

M

CB solution : Chroma, 0.005%, 5.497x10-4

M

Hydrochloric acid : E.Merck, 5M

p-CA solution : Sd-fine, 0.1%, 4.785x10-3

M

4.16(iii) Proposed Procedures

The following procedures are used to determine the amount of the

Ondansetron HCl by the second order spectrophotometry


308

Method – M1(a)

Into a series of 125ml separating funnels transferred aliquots of standard OND

solution (1.67 – 26.67μg/ml) 6.0ml of 0.1M HCI solution and 1.0ml of 0.2%ARSdye

solution are added successively. The total volume of aqueous phase is adjusted to

15ml with distilled water. To each separating funnel 10ml of chloroform is added and

the contents are shaken for 2min. The two phases are allowed to separate and the zero

order, first derivative and second derivative spectra are recorded using the prepared

solutions against reagent blank. The values of the second derivative absorbance (2D)

are obtained using five different concentrations by measuring each concentration

against solvent blank at the chosen wavelength. The amplitudes of 2D are plotted

against concentration to construct the calibration curve.

Method – M5(a)

Aliquots of standard OND solution (2.5 – 40.0g/ml) are transferred into a

series of 25ml calibrated tubes. Then 0.5ml (8.56 x 10-3

M) of MBTH solution is

added and kept aside for 5min. After that 2.0ml (1.58 x 10-2

M) of ceric ammonium

sulphate is added and kept aside for 10min. The volume is made up to the mark with

distilled water. The zero order, first derivative and second derivative spectra are

recorded using the prepared solutions against reagent blank. The values of the second

derivative absorbance (2D) are obtained using five different concentrations by

measuring each concentration against solvent blank at the chosen wavelength. The

values of 2D are plotted against corresponding concentrations to construct the

calibration curve.


309

Method – M7

Aliquots of standard OND solution (20.0 - -100g/ml) are transferred into a

series of 25ml calibrated tubes. Then 1.25ml (1M) of HCl and 4.0ml (2.81 x 10-3

M)

of NBS solutions are added and the total volume is brought to 15ml with distilled

water. After 10minutes4.0ml of CB solution is added, after 5minutes absorbance is

measured at 540nm against reagent blank. The blank (omitting drug) and dye

(omitting drug and oxidant) solutions are prepared in similar manner and their

absorbances are measured against distilled water. The decrease in absorbance

corresponding to consumed NBS and in turn the dye concentration is obtained by

subtracting the decrease in absorbance of the solution that of blank solution. The zero

order, first derivative and second derivative spectra are recorded using the prepared

solutions against reagent blank. The values of the second derivative absorbance (2D)

are obtained using five different concentrations by measuring each concentration

against solvent blank at the chosen wavelength. The values of 2D are plotted against

corresponding concentrations to construct the calibration curve.

Method – M15

Into a series of 10ml calibrated tubes containing aliquots of standard OND

solution (6.25-100g/ml), 2.0ml of chloranilic acid (4.785 x 10-3

M) is added and kept

aside for 30 min at lab temperature. The volume in each tube is made up to the mark

with chloroform. The zero order, first derivative and second derivative spectra are

recorded using the prepared solutions against reagent blank. The values of the second

derivative absorbance (2D) are obtained using five different concentrations by

measuring each concentration against solvent blank at the chosen wavelength. The


310

values of 2D are plotted against corresponding concentrations to construct the

calibration curve.

4.17 Method Development

The zero-order (0D) spectra of OND are showed in Fig.4.34 –Fig. 4.37, P:

312, the zero-crossing points are assigned from the 1D and

2D spectra of the proposed

methods are shown Fig.4.38- Fig.4.45, P: 313-314. The 1D zero-crossing point of

OND for the proposed methods are found at 425nm method M1(a), 640nm method

M5(a) , 455nm method M7 and 540 nm method M15 respectively (Fig.4.38- Fig.4.41,

P: 313); and 2D zero-crossing point of OND for the proposed methods are found 412

and 440nm method M1(a), 595nm, 695nm method M5(a); 425nm and 500 nm

method M7 and 485nm and 580 nm method M15 (Fig4.42-Fig4.45, P: 314)

respectively. The selection of wavelength is based on the fact that the absolute value

of the total derivative spectrum at the selected wavelength has the best linear response

to the analyte concentration. The 2D spectra showed better resolution and linearity of

the calibration curve of OND. Therefore second derivative amplitudes are chosen for

the determination of OND.

4.18 Method Validation

Linearity and Range

The calibration plots are linear in the range of 1.7 – 26.7μg/ml method M1(a);

2.5-40.0 μg/ml for method M5(a), 20.0-100.0 μg/ml method M7 and 6.25 – 50.0

μg/ml method M15 (Fig.4.46 – Fig 4.49, P: 315). Correlation coefficient for all the

methods is greater than 0.999. The mean values of correlation coefficient, slope and

intercept are shown in Table 4.14, P: 316.


311

Precision

The precision of each proposed methods is ascertained from the absorbance

values obtained by actual determination of five replicates of ondansetron sample

solution. The percent relative standard deviation and percent range of error (0.05

confidence limit) are calculated for the proposed methods (Table 4.15, P: 316).

Accuracy

To determine the accuracy of each proposed method, different amounts of

samples of ondansetron HCl within the Beer’s law limits are taken and analyzed by

the proposed method. The results (percent error) are recorded in Table 4.16(a)-

Table4.16 (d) P: 316-318.

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ of OND are calculated based on the standard deviation of

the intercept and s is the slope of the calibration curves (n=5).The data are presented

in Table 4.14, P: 316.


312

Zero Order Spectra of OND

with ARS,CHCl3

0.000

0.200

0.400

0.600

0.800

1.000

375 400 425 450 475 500

Wavelength (λ)nm

Ab

so

rpti

on

(A)

1.67 μg/ml3.33 μg/ml6.67μg/ml13.3 μg/ml26.6 μg/ml


with ARS methodM1(a)

Zero Order Spectra of OND with

MBTH,Ce(IV)

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

500 550 600 650 700 750

Wavelength (λ)nmA

bs

orb

an

ce

(A)

2.50 μg/ml5.00 μg/ml10.0 μg/ml20.0 μg/ml40.0 μg/ml


with MBTH method M5(a)


NBS,CB,HCl

0.000

0.200

0.400

0.600

0.800

375 425 475 525 575

Wavelength (λ) nm

Ab

so

rba

nc

e(A

)

20 μg/ml40 μg/ml60 μg/ml80 μg/ml100μg/ml


with NBS+CB method M7


p-CA,CHCl3

0.000

0.250

0.500

0.750

450 500 550 600 650

Wavelength(λ) nm

Ab

so

rba

nc

e(A

)

6.25 μg/ml12.5 μg/ml25.0 μg/ml50.0 μg/ml


with p-CA method M15


313

First Derivative Spectra of OND with

ARS, CHCl3

-3.E-02

-2.E-02

-2.E-02

-1.E-02

-5.E-03

0.E+00

5.E-03

1.E-02

2.E-02

2.E-02

3.E-02

375 400 425 450 475 500

Wavelength nm

ΔA

/Δλ


Fig. 4.38 First Derivative Spectra of

OND with ARS method M1(a)

First Derivative Spectra of OND with

MBTH ,Ce(IV)

-6.E-03

-4.E-03

-2.E-03

0.E+00

2.E-03

4.E-03

6.E-03

500 550 600 650 700 750

Wavelength nmΔ

A/Δ

λ



OND with MBTH method M5(a)

First Derivtive Spectra of OND with

NBS,CB,HCl

-1.E-02

-5.E-03

0.E+00

5.E-03

1.E-02

375 425 475 525 575

Wavelength nm

ΔA

/Δλ

20 μg/ml40 μg/ml60 μg/ml80 μg/ml100 μg/ml


OND with NBS+CB method M7

First Derivtiver Spectra of OND with

p-CA,CHCl3

-8.E-03

-5.E-03

-3.E-03

0.E+00

3.E-03

5.E-03

8.E-03

1.E-02

400 450 500 550 600 650

Wavelength nm

ΔA

/Δλ



OND with p-CA method M15


314

Second Derivative Spectra of OND

with ARS,CHCl3

-4.E-03

-3.E-03

-2.E-03

-1.E-03

0.E+00

1.E-03

2.E-03

400 425 450 475 500

Wavelength nm

Δ2A

/Δ2λ


Fig. 4.42Second Derivative Spectra of

OND with ARS method M1(a)


with MBTH,Ce(IV)

-5.E-04

-3.E-04

-2.E-04

0.E+00

2.E-04

500 550 600 650 700 750

Wavelength nmΔ

2A

/Δ2λ

2.5μg/ml5.0μg/ml10.0μg/ml20.0μg/ml40.0μg/mlSeries6

Fig. 4.43 Second Derivative Spectra of

OND with MBTH method M5(a)


with NBS,CB,HCl

-8.E-04

-5.E-04

-3.E-04

0.E+00

3.E-04

400 450 500 550

Wavelength nm

Δ2A

/Δ2λ

20 μg/ml40 μg/ml60 μg/ml80 μg/ml100 μg/ml


OND with NBS+CB method M7


with p-CA,CHCl3

-4.E-04

-3.E-04

-2.E-04

-1.E-04

0.E+00

1.E-04

2.E-04

450 500 550 600 650

Wavelength nm

Δ2A

/Δ2λ



OND with p-CA method M15


315

Plot of Second Derivative Amplitude

against Concentration of

OND(ARS+CHCl3)

0.0E+00

1.0E-03

2.0E-03

3.0E-03

4.0E-03

0 10 20 30Concentration in μg/ml

D2 A

mp

litu

de

Fig.4.46 Calibration curve of OND with

ARS method M1(a)

Plot of Second Derivtive Amplitude

agianst Concentration of

OND(MBTH+Ce(IV)

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

0 10 20 30 40 50Concentration in μg/ml

D2 A

mp

litu

de


MBTH method M5(a)


against Concentration of OND

(NBS+CB+HCl)

0.0E+00

2.0E-04

4.0E-04

6.0E-04

0 20 40 60 80 100 120

Concentration in μg/ml

D2 A

mp

litu

de


NBS+CB method M7


against Concentration of OND(p-

CA+CHCl3)

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

0 10 20 30 40 50 60

Concentration in μg/ml

D2 A

mp

litu

de


p-CA method M15


316

Table: 4.14: Linearity studies and regression parameters

S.No Name of the Parameter M1(a) M5(a) M7 M15

1 Linearity Limits µg/ml 1.7-26.7 2.5-40.0 20.0-100.0 6.25-50.0

2 Slope (b) 1.38E-04 8.46E-06 5.54E-06 7.50E-06

3 Intercept(a) -3.75E-06 1.46E-06 -1.20E-06 -1.58E-06

4 Correlation Coefficient ( r ) 1.0000 0.9999 0.9999 0.9999

5 Limit of Detection (LOD) µg/ml 0.069 0.244 0.323 0.598

6 Limit of Quantification (LOQ) µg/ml 0.229 0.813 1.076 1.993

Table 3.15 Precision of the proposed methods

S.No Name of the Parameter M1(a) M5(a) M7 M15

1 Amount Taken (µg/ml) 13.33 20.00 60.00 25.00

2 Mean (n=5)( µg/ml) 13.328 19.97 60.43 24.64

3 Standard Deviation (S) 0.026 0..53 0.633 0.173

4 %Relative Standard Deviation 0.194 0.260 1.048 0.695

5 0.05 Confidence level 0.314 0.142 0.215 0.352

5 %Recovery 99.62 99.85 100.72 99.76

Table-4.16(a): Accuracy of the method M1(a)

Concentration-1 Concentration-2 Concentration-3

Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery

10.00 10.06 100.63 13.33 13.35 100.15 16.66 16.68 100.11

10.00 9.97 99.72 13.33 13.32 99.92 16.66 16.14 96.86

10.00 9.98 99.82 13.33 13.36 100.23 16.66 16.63 99.80

10.00 10.07 100.73 13.33 13.31 99.85 16.66 16.69 100.17

10.00 9.98 99.82 13.33 13.30 99.77 16.66 16.67 100.05

Mean 10.012 100.145 13.328 99.985 16.562 99.397

SD 0.049 0.026 0.237

%RSD 0.486 0.194 1.431


317

Table-4.16(b): Accuracy of the method M5(a)



15.00 15.09 100.60 20.00 19.94 99.70 25.00 24.96 99.84

15.00 15.06 100.40 20.00 20.05 100.25 25.00 24.92 99.68

15.00 14.97 99.80 20.00 20.01 100.05 25.00 25.21 100.84

15.00 14.92 99.47 20.00 19.94 99.70 25.00 25.60 102.40

15.00 15.06 100.40 20.00 19.93 99.65 25.00 25.08 100.32

Mean 15.020 100.133 19.974 99.870 25.154 100.616

SD 0.072 0.053 0.274

%RSD 0.478 0.266 1.089

Table-4.16(c): Accuracy of the method M7



45.00 44.67 99.27 60.00 59.70 99.50 75.00 75.72 100.96

45.00 44.83 99.62 60.00 59.87 99.78 75.00 74.72 99.63

45.00 45.21 100.47 60.00 60.98 101.63 75.00 74.31 99.08

45.00 45.61 101.36 60.00 61.10 101.83 75.00 75.84 101.12

45.00 44.87 99.71 60.00 60.50 100.83 75.00 75.73 100.97

Mean 45.038 100.084 60.430 100.717 75.264 100.352

SD 0.375 0.633 0.701

%RSD 0.834 1.048 0.931


318

Table-4.16(d): Accuracy of the method M15



18.75 18.92 100.91 25.00 25.03 100.12 31.25 31.29 100.13

18.75 18.85 100.53 25.00 25.06 100.24 31.25 31.33 100.26

18.75 18.53 98.83 25.00 24.74 98.96 31.25 31.76 101.63

18.75 18.32 97.71 25.00 24.78 99.12 31.25 30.99 99.17

18.75 18.45 98.40 25.00 25.12 100.48 31.25 31.07 99.42

Mean 18.614 99.275 24.946 99.784 31.288 100.122

SD 0.260 0.173 0.300

%RSD 1.395 0.695 0.960

4.19 Results and Discussions

The plots drawn between second derivative amplitude against concentration of

the drug are linear. Correlation coefficient values (more than 0.999) show that there is

good correlation between the response and the concentration of the drug in the

developed method. Low percent of relative standard deviation values 0.194, 0.266,

1.048 and 0.695 for the developed methods indicate that these methods are precise.

High mean percent of recovery ranging 99.27% to 100.72% for the four methods in

three concentration levels reveals that the methods are highly accurate.

4.20 Conclusion

These methods are accurate, simple, rapid, precise, reliable, sensitive,

reproducible and economical. The developed methods could be readily adapted to

routine quality control analysis of ondansetron by ordinary laboratories

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