Exp Project Final Last

Chapter5 Introduction

CHAPTER 5

EXPERIMENTAL WORK

CONTENTS

5.1 IDENTIFICATION OF DRUG

5.2 DEVELOPMENT AND VALIDATION OF SPECTROMETRIC

METHOD FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ

AND VALS IN THEIR COMBINED DOSAGE FORM

5.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD

FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND

VALS IN THEIR COMBINED DOSAGE FORM

5.4 DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD



Institute of Pharmacy, Nirma University Page 100


5.1 IDENTIFICATION OF DRUGS:

Drug samples were received from pharmaceutical companies; these samples were

subjected to identification of these drugs was carried out by melting point, IR

spectroscopy and U.V spectra studies.

5.1.1 Determination of Melting Point:

Melting point of AMLO, HCTZ and VALS were determined by capillary method and

obtained result is in table

Drug Reported melting point (0C)

Observed melting point (0C)

AMLO 199-201 200-202

HCTZ 273-275 274-275

VALS 116-117 117-118

Table 25: Melting points of drugs

5.1.2 UV spectra of AMLO, HCTZ and VALS

UV- spectrum of AMLO (20μg/mL), HCTZ (20 μg/ mL) and VALS (20 μg/ mL) in methanol was taken. AMLO was found show absorption maxima at 237.6nm; HCTZ was found to show absorption maxima at 270.2 nm and VALS was found to show absorption maxima at 249.2 nm.



Fig 5: UV spectrum of AMLO (20µg/mL) in methanol

Fig.6: UV spectrum of HCTZ (20μg/mL) in methanol

Fig.7: UV spectrum of VALS (20μg/mL) in methanol



Table 26: Wavelength maxima for AMLO, HCTZ and VALS

Drug Reported maxima Recorded maxima

AMLO 239nm, 238nm 237.6nm,210.8nm

HCTZ 225nm, 271nm, 317nm 270.2nm,316.6nm

VALS 249nm249.2nm, 206nm,

243.8nm

5.1.3 Determination of Infrared (IR) Spectra:

Fig.8: Infrared spectrum of AMLO

Table 27: Justification for IR Frequency (cm-1) Found for AMLO

SPECIFICATION OF

AMLO

THEORITCAL WAVE

NUMBER(CM-1) (85)

RECORDED

WAVE -NH2 stretching 3500-3100 3372

-C-Cl 785-540 752.102

C-H bending 1465-1375 1440.56

C=O Ester 1750-1730 1765.99



-C-O Ether 1300-1000 1265.07

Fig. 9: Infrared spectrum of HCTZ

Table 28: Justification for IR Frequency (cm-1) Found for HCTZ

SPECIFICATION FOR HCTZ

THEO RITICAL WAVE NUMBER (CM-1) (85)

RECORDED WAVE NUMBER (CM-1)

(S=O) 1050 1078.98

C-H stretch 3000-2850 2963.09

Mono substituted benzene 900-690 996.053

-C-N - Amine 1350-1000 1206.26

Amide group 1680-1630 1601.59



Fig.10: Infrared spectrum of VALS

Table 29: Justification for IR Frequency (cm-1) Found for HCTZ

SPECIFICATION OF FOR VALS

THEORITICAL WAVE NUMBER (CM-1)(85)

RECORDED WAVE NUMBER (CM-1)

C-H stretch 3000-2850 2834.85

ketone (C=O) stretch 1750-1730 1730.8

-NH2 stretching 3200-3500 3238.86

Carboxylic acid(-COOH)

3400-2400 2577.4

-N-H Stretch 1640-1550 1523.49



5.2 DEVELOPMENT AND VALIDATION OF UV-VIS

SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS

ESIMATION OF AMLO, HCTZ AND VALS IN THEIR

COMBINED DOSAGE FORM

5.2.1 INSTRUMENTATION

5.2.1.1 UV-Visible Double-Beam spectrophotometer:

Matched quartz cell (1cm), Model: UV-2450 Pc series, Manufacturer: Shimadzu Inc.

Japan, Wavelength range: 200.00 to 400.00 nm

5.2.1.2 Analytical Balance:

Model: KEROY, Manufacturer: Keroy (balance) pvt. Ltd. Varanasi, India. Weighing

capacity: 100gm

5.2.1.3 Sonicator:

Model: TRANS-O-SONIC; D-compect., Capacity: 2 Lit.

5.2.2 MATERIAL AND METHODS

5.2.2.1 Reagents and Chemicals

API Amlodipine Besylate (AMLO) gifted sample from

API Hydrochlorthiazide (HCTZ) gifted sample

API Valsartan (VALS) gifted sample

Methanol (AR Grade, S.D.Fine Chemicals Ltd., Bombay, INDIA)

Tablets: containing Amlodipine besylate (5mg) ,Hctz( 12.5 mg) and Valsartan

(160mg) Brand name : exforge hct, name of manufacturer



5.2.2.2 Preparation of standard stock solution of AMLO, HCTZ and VALS

AMLO (25 mg), HCZ (25mg) and VALS (25mg) were accurately weighed and

transferred to three separate 25 ml volumetric flasks and dissolved in methanol to

obtain stock solution of concentration 100 µg/ml each. .Aliquots of 2, 4, 5, 10, 15, 20

ml were prepared by using this stock solution of AMLO, aliquots of HCTZ of 5, 10,

15, 20, 25 ml by using stock solution of HCTZ and aliquots of 10 ,20, 30, 40, 50 ml

by using of VALS stock solution ,for preparation of calibration curve .

5.2.2.3 Preparation of ternary mixture of AMLO, HCTZ and VALS

Suitable aliquots of standard stock solution of AMLO, HCTZ and VALS were mixed

and diluted to volume with methanol to obtain different ternary mixture solutions in

concentration range of 2-50 were prepared for the calibration curve for AMLO, HCTZ

and VALS

5.2.2.4 Selection of Analytical Wavelength and measurement

From these stock solutions, working standard solutions were prepared by appropriate

dilution of solvent to get final concentration of 20 µg/ml each and were scanned in the

spectrum mode from 200 to 400 nm. From the overlain spectra of these drugs (fig.1),

wavelengths 237.6 nm (λmax of AMLO), 249.2 nm (λmax of VALS) and 270.2 nm (λmax

of HCTZ) were selected for analysis.

By appropriate dilution of standard drug solution with methanol six working

concentration containing 2, 4, 5, 10, 15, 20 µg/ml for AMLO, 5, 10, 15, 20, 25 µg/ml

for HCTZ and 10, 20, 30, 40, 50 µg/ml for VALS were prepared separately prepared

and scanned in the range of 200-400 nm .The values of absorbance were recorded at

the selected wavelengths and the absorptivity and molar absorptivity values for

AMLO, HCTZ and VALS were determined .Molar absorptivity value for AMLO

were 320, 177.7, 178.63 mol lit -1, for HCTZ were 45.88, 615.55, 88.086 mol lit -1

and for VALS were 320.07, 141.02, 295.75 mol lit -1 at 237.6 nm, 270.2 nm and 249.2

nm respectively. Molecular weight of AMLO, HCTZ and for VALS is respectively.



A1== 320C AMLO + 45.88C HCTZ +320.07C VALS……….……………..(1)

A2=177.7C AMLO +615.55C HCTZ +141.02C VALS……………………..(2)

A3=178.63C AMLO +88.086C HCTZ +295.75C VALS ………………….(3)

Where A1, A2 and A 3 are absorbance of the sample solution at 237.6nm, 270.2 nm

and 249.2 nm respectively.C AMLO is the concentration of AMLO, CHCTZ is the

concentration of the HCTZ, and C VALS is the concentration of the VALS.

Fig. 11: overlay spectra of AMLO, HCTZ and VALS

Overlay UV-spectrum of AMLO (20μg/ml), HCTZ (20μg/ml) and VALS (20μg/ml)

in methanol was taken. AMLO was found to show absorption maxima at 237.6 nm,

HCTZ at 270.2 nm and VALS was found to show absorption maxima at 249.2nm



5.2.2.5 Method Validation

1. Preparation of linearity curve

For estimation of AMLO, calibration curve (n=6) was plotted in the range of 2-20

µg/ml at 237.6nm as well as estimation of HCTZ, calibration curve (n=5) was plotted

in the range of 5-25 µg/ml at 270.2nm and For estimation of VALS, calibration

curve (n=6) was plotted in the range of 10-50 µg/ml at 249.2 nm. Linearity curve

shows linearity in the range of 2-20 µg/ml for AMLO, 5-25 µg/ml for HCTZ and 10-

50 µg/ml for VALS

2. Precision

The precision of analytical method is the degree of agreement among individual

results when the method is applied to multiple sampling of homogenous samples. It

provides an indication of random error in results and was expressed as coefficient of

variance (CV).

2.1 Intra-day precision: Intra-day precision was determined by measuring

amplitudes of three different concentrations 2, 4, 5 µg/ml for AMLO and 15, 20, 25,

µg/ml for HCTZ and for VALS 30,40,50 µg/ml individually for three times in a day.

2.2 Inter-day precision: Inter-day precision was determined by measuring

amplitudes of three different concentrations 2, 4, 5 µg/ml for AMLO AND 15, 20, 25,

µg/ml for HCTZ and for VALS 30, 40, 50 µg/ml individually for three days.

2.3 Repeatability: It is a measure of precision under the same operating conditions over a short interval of time. It is sometimes referred to as intra-assay precision. To study the repeatability, six determinations at 100% test concentrations (i.e. 4µg/ml of AMLO, 10 µg/ml for HCTZ and 20 µg/ml of VALS) were carried out.

3. Limit of detection and limit of quantification (LOD/LOQ)

For this determination Calibration curve for both the drugs was repeated six times The

LOD & LOQ were measured by using mathematical equations given below.

LOD = 3.3 x σ/S



LOQ = 10 x σ/S

Where, σ = Standard deviation of the Intercept

S = slope of calibration curve

4. Accuracy

To study the accuracy, 7 tablets were weighed and powdered. The powder equivalent

to 5mg of AMLO, 12.5 mg for HCTZ and 160 mg of VALS were weighed and

transferred to 100ml volumetric flask containing 70 ml of methanol. The solution was

sonicated for 15 minutes and volume was made up to the mark with methanol. The

above solution was filtered with whatmann filter paper (No. 41). Aliquot (5ml) was

pipetted out and transferred to 50ml volumetric flask. Volume was made up to the

mark with methanol to get a solution containing 5µg/ml of AMLO, 12.5 µg/ml and

160 µg/ml of VALS. Standard drug was added at three different concentration levels

(80%, 100% and 120% of test sample concentration) to pre-analyzed sample and

amplitudes of the solution were measured at selected wavelengths for AMLO, HCTZ

and VALS drugs. Amplitudes were substituted into respective straight line equation to

calculate percentage recovery of the drugs.

5. Analysis OF Tablet Dosage form

Marketed tablet formulation containing VALS 160 mg, AMLO besylate equivalent to

AMLO 5mg and HCTZ 12.5 mg was analysed using this method. From the triturate of

7 tablets, an amount equivalent to 160 mg of VALS, (5 mg of AMLO and 12.5 mg

HCTZ) was weighed and dissolved in 35 ml of methanol and sonicated for 30

minutes. After 30 min. Sonication the solution was filtered in a 100ml calibrated

volumetric flask through whatmann filter paper. The filtrate was appropriately diluted

with the same solvent to obtain final concentration within Beer Lambert's range for

each drug.

The concentration of drugs was determined by using the Eqns 1, 2 and 3.

Eqn.1 A 1 = 320C AMLO + 45.88C HCTZ +320.07C VALS (1)

Eqn.2 A = 177.7C AMLO +615.55C HCTZ +141.02C VALS (2) and



Eqn.3 A 3 = 178.63C AMLO +88.086C HCTZ +295.75C VALS (3),

Where A1, A2 and A 3 are absorbance of the tablet sample solution at 237.6, 270.2 and 249.2 nm respectively.C AMLO is the concentration of AMLO, CHCTZ is the concentration of the HCTZ, and C VALS is the concentration of the VALS.

5.2.3 Results and Discussion

5.2.3.1 Method Validation

1) Linearity

Linearity curve shows linearity in the range of 2-20 µg/ml for AMLO, 5-25 µg/ml for

HCTZ and for VALS 10-50 µg/ml. The correlation coefficient (r2) was found to be

0.9997, 0.9990, and 0.9990 for AMLO, HCTZ and VALS respectively.

Fig 12: Linearity Curve for AMLO at 237.6 nm



Fig 12: Calibration Curve for AMLO at 237.6 nm

Table 30: calibration curve data of AMLO at 237.6 nm, 270.2 nm and 249.2 nm

Conc.

μg/ml

237.6nm 270.2nm 249.2nm

MeanAbs. ± S.D*

%

RSD

MeanAbs ±S.D*

%

RSD

Mean Abs.±S.D*

%

RSD

20.045±0.01

61.55

0.013±0.0021

1.61 0.021±0.0013 1.61

40.122±0.01

81.47

0.024±0.0024

1.60 0.065±0.0017 1.53

50.159±0.02

31.44

0.042±0.0016

1.80 0.088±0.0014 1.13

100.346±0.01

10.31

0.056±0.0013

1.12 0.201±0.0015 0.74

150.558±0.01

20.17

0.064±0.0017

1.42 0.328±0.0012 0.30



200.707±0.01

50.21

0.078±0.0016

0.95 0.422±0.0016 0.37

Absorptivity 320 177.7 178.63

Fig. 12: Linearity Curve for HCTZ at 270.2 nm



Fig. 12: Calibration Curve for HCTZ at 270.2 nm

Table 31: Calibration curve data of HCTZ at 237.6 nm, 249.2 nm and 270.2 nm

Conc.

μg/ml

237.6nm 270.2nm 249.2nm

MeanAbs.

±S.D*

%RSD

Mean Abs. ±S.D* %RS

D

Mean Abs.

±S.D*

%RSD

50.009±0.00

11.33 0.297±0.002 0.70

0.032±0.001

1.75

100.042±0.00

11.57 0.618±0.002 0.37

0.083±0.001

1.56

150.076±0.00

11.71 0.925±0.001 0.16

0.137±0.001

1.16

200.113±0.00

11.32 1.245±0.003 0.12

0.192±0.002

0.78

25 0.166±0.00 1.61 1.564±0.003 0.12 0.265±0.00 0.37



6 2

Absorptivity

45.88 615.55 88.086

Fig.13: Calibration Curve of VALS at 249.2 nm

Fig.13: Calibration Curve of VALS at 249.2 nm



Table 32: Calibration data of VALS at 237.6 nm, 249.2nm at 270.2 nm

Conc.

μg/ml

237.6nm 270.2nm 249.2nm

Mean Abs.±S.D*

% RSD

Mean Abs.±S.D*

% RSD

Mean Abs.±S.D*

% RSD

10 0.326±0.003 0.98 0.146±0.002 1.02 0.310±0.027

0.80

20 0.634±0.0022

0.34 0.279±0.004 0.53 0.583±0.025

0.42

30 0.978±0.002 0.25 0.433±0.002 0.34 0.895±0.021

0.23

40 1.263±0.0038

0.30 0.553±0.003 0.56 1.167±0.032

0.25

50 1.58±0.0015 0.97 0.682±0.0021

0.30 1.462±0.021

0.14

Absorptivity 320.07 141.02 295.75

2) Precision

Intraday precision and Interday precision for AMLO, HCTZ and VALS was done by

analyzing three different concentrations (μg/ml) within linearity ranges and % RSD

less than 2.

Table 33: Intraday precision for AMLO

WAVELENGTHCONC. μg/ml

MEAN CONC.±S.D* % R.S.D

237.6nm

2 1.990±.0281 1.41

4 3.880±.0168 0.43

5 5.050±.0129 0.25



270.2nm

2 1.870±.0135 0.72

4 4.170±.0132 0.31

5 5.020±.0152 0.30

249.2nm

2 2.13±0.0144 0.676

4 4.12±0.0211 0.512

5 5.23±0.0221 1.837

Table 34: Interday precision for AMLO



237.6nm

2 2.02±0.00114 1.10

4 4.31±0.00154 0.45

5 4.22±0.00137 0.27

270.2nm

2 2.18±0.00305 1.17

4 4.07±0.00120 0.56

5 5.06±0.00260 0.26

249.2nm

2 2.18±0.00340 1.05

4 4.21±0.00231 0.50



5 5.35±0.00162 0.31

Table 35: Intraday precision for HCTZ

WAVELENGTH CONC. μg/ml


237.6nm

15 15.21±0.0084 0.55

20 21.01±0.0043 0.68

25 24.87±0.0044 0.57

270.2nm

15 14.950.0034 0.90

20 20.09±0.0032 0.65

25 24.67±0.0042 0.57

249.2nm

15 15.02±0.0062 0.94

20 21.17±0.0055 0.64

25 26.01±0.0048 0.49



Table 36: Interday precision for HCTZ

WAVELENGTH

CONC.μg/ml

MEAN Conc. ± S.D* % R.S.D

237.6nm

15 14.56±0.0018 0.81

20 20.17±0.0040 1.99

25 24.88±0.0050 2.02

270.2nm

15 14.76±0.0011 0.75

20 20.14±0.0016 0.57

25 25.18±0.0012 0.44

249.2nm

15 15.09±0.0026 0.83

20 20.31±0.0026 1.11

25 25.08±0.0025 0.89

Table 37: Intraday precision for VALS

WAVELENGTH CONC. μg/ml MEAN CONC.±S.D*

% R.S.D

237.6nm

15 15.21±0.0084 0.55

20 21.01±0.0043 0.68

25 24.87±0.0044 0.57

270.2nm

15 14.95±0.0034 0.90

20 20.09±0.0032 0.65

25 24.67±0.0042 0.57

249.2nm

15 15.02±0.0062 0.94

20 21.17±0.0055 0.64

25 26.01±0.0048 0.49

Table 38: Interday precision for VALS





237.6nm

15 14.56±0.0018 0.81

20 20.17±0.0040 1.99

25 24.88±0.0050 2.02

270.2nm

15 14.76±0.0011 0.75

20 20.14±0.0016 0.57

25 25.18±0.0012 0.44

249.2nm

15 15.09±0.0026 0.83

20 20.31±0.0026 1.11

25 25.08±0.0025 0.89

Table 39: Repeatibilty

WAVE LENGTH DRUG CONC.μg/ml

MEAN CONC. μg/ml

% R.S.D

237.6nm

AMLO 4 4.2 0.27

HCTZ 10 10.12 0.32

VALS 20 20.13 1.21

270.2nm

AMLO 4 3.94 0.53

HCTZ 10 9.93 0.46

VALS 20 19.78 1.64

249.2nm

AMLO 4 4.15 0.51

HCTZ 10 10.21 0.28

VALS 20 20.11 1.59

3) LOD and LOQ

From determination calibration curve for AMLO, HCTZ and VALS was repeated six

times and LOD and LOQ value were measured by mathematical equation.



Table 40: LOD and LOQ value for AMLO, HCTZ and VALS

Limit of detection AMLO HCTZ VALS

LOD 0.025 μg/mL 0.013 μg/mL 0.029 μg/mL

LOQ 0.078 μg/mL 0.041 μg/mL 0.089 μg/mL

4) Accuracy Standard edition was done at three level 80%, 100% and 120% of a concentration of sample in the linearity range and % recovery was found 98 to 100%.

Table 41: Recovery study of AMLO, HCTZ and VALS

DRUG%level

Sample conc. μg/ml

Amt of std.

added μg/ml

Total Conc.

μg/ml

Amt recovered μg/ml

% Recover

y

AMLO

80 5 4 9 8.856 98.4

100 5 5 10 10.05 100.5

120 5 6 11 10.93 99.61

HCTZ

80 10 8 18 17.82 99.00

100 10 10 20 19.78 98.9

120 10 12 22 21.89 99.5

VALS

80 20 16 36 35.47 98.53

100 20 20 40 39.86 99.67

120 20 24 44 43.65 99.22

Table 42: Validation parameters for AMLO, HCTZ and VALS

PARAMETERS AMLO HCTZ VALS

λmax 237.6nm 270.2nm 249.2nm



Linear Range μg/ml 2-20 5-25 10-50

Correlation coefficient R2 0.9997 0.999 0.999

Repeatability% RSD 0.705 0.154 1.413

Intraday precision % RSD 0.523 0.781 0.539

Interday precision % RSD 0.727 1.021 0.489

LOD μg/ml 0.025 0.013 0.029

LOQ μg/ml 0.078 0.041 0.089

% Recovery 99.19 99.13 99.14

5.2.3.2 Analysis of Marketed formulation

The developed method was used to estimate AMLO, HCTZ AND VALS in the tablet dosage form. Marketed formulation was procured for the analysis by proposed method.

DRUG LABLE CLAIM (mg) % ASSAY±SD*

AMLO 5 98.25±0.0781

HCTZ 12.5 98.82±0.0458

VALS 160 98.93±0.0404

Table 43: Analysis of Marketed Dosage Form

5.2.4 Conclusion

The proposed UV-VIS spectrophotometric method was accurate, precise and sensitive

for the determination of AMLO, HCTZ and VALS in combined dosage form. High



recoveries show that the method is free from the interference from the excipients used

in the commercial pharmaceutical preparations. Hence, it can be successful applied for

the routine estimation for AMLO, HCTZ and VALS in quality control laboratories.

The result of validation parameters are satisfactory level indicates the accuracy of

proposed method for estimation of AMLO, HCTZ and VALS.

5.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD





5.3.1.1 Instrumentation

1) HPTLC

Camag Applicator Linomat 5: Semiautomatic application, Band application by

spray on technique (2 - 500µl)

Camag twin trough glass chamber: ( 20 x 10 cm)

Camag TLC scanner 3 : Scanning speed up to 100mm/s, Spectral range 190 –

800nm

Camag U.V cabinet with dual wavelength U.V lamp:

Dual wavelength 254 / 366nm

Stationary Phase: Pre- coated Silica gel on aluminum sheet G60 F254

Camag 100 µl Applicator syringe (Hamilton, Bonaduz, Schweiz)

Data Resolution: 100µm/step

2) Spectrometer

Model : SHIMADZU 2450 double beam spectrometer, version 2.21 Slit width : 1 nm 3) Analytical Balance Model : keroy® Manufacturer: keroy (Balance) pvt. Ltd. Capacity : 0.0001 to 100 g 4) Sonicator Model : Trans-O-sonic, D-compact Capacity : 2L

5.3.1.2 Material and methods

5.3.1.2.1 Reagents and chemicals

Methanol (AR Grade, S.D. Fine chemicals Ltd., Mumbai , India

Ethyl Acetate (AR Grade, S.D. Fine chemicals Ltd., Mumbai , India)

Ammonia (25%)

Toluene AR Grade

Tablet containing AMLO(5 mg), HCTZ (12.5 mg) and VALS(160 mg)

Brand name: Exforge HCTZ, Name of manufacturer






5.3.1.3 HPTLC conditions

Mobile phase : Ethyl Acetate : Methanol : Toluene : Ammonia (7.5: 3:2: 0.8,

v/v/v/v)

Chamber saturation time : 25 min

Distance run : 70 mm

Ambient temperature : 25-26°C

Wavelength of detection : 242 nm

Slit dimension : 3x 0.20 mm (micro)

Band width : 4 mm

Syringe capacity : 100 μL

5.3.1.4 preparation of Mobile phase

A mixture of 7.5 ml of Ethyl Acetate, 3 ml of methanol, 2 ml of toluene and 0.8 ml of

ammonia (25%) were mixed properly and it was used as a mobile phase.

5.3.1.5 Preparation of Standard Stock Solution of AMLO

25 mg AMLO was weighed accurately and dissolved in 25 ml methanol. 1 ml

aliquots of the above solution were diluted to 10 ml with methanol to produce 100

μg/mL of AMLO in methanol.

5.3.1.6 Preparation of Standard Stock Solution of HCTZ

25 mg HCTZ was weighed accurately and dissolved in 25 ml methanol. 1 ml aliquots

of the above solution were diluted to 10 ml with methanol to produce 100 μg/mL of

HCTZ in methanol.

5.3.2.6 Preparation of Standard Stock Solution of VALS

25 mg VALS was weighed accurately and dissolved in 25 ml methanol. 1 ml

aliquots of the above solution were diluted to 10 ml with methanol to produce 100

μg/mL of VALS in methanol.

5.3.2.6 Preparation of Ternary mixtures of AMLO, HCTZ and VALS



Suitable aliquots of standard stock solution of AMLO, HCTZ and VALS are mixed

and diluted to volume with methanol to obtain different Ternary mixture solutions

containing AMLO, HCTZ and VALS in the range 100 - 3200 ng/spot were applied

to the plate for the calibration curve of three drugs.

5.3.2.7 HPTLC Analysis

1) Activation of Silica gel plate

Analysis was performed on 20 cm x 2o cm TLC silica gel 60 F 254 plates (EM science,

Gibbstown, NJ, USA, an affiliate of Merck KGaA, Darmstadt, Germany). Layers

were cleaned by predevelopment to the top with methanol and dried in a hot air oven

at 105°C for 20 minutes.

2) Spotting

Standard zones were applied to the layer as bands by Means of a camag (Wilmington,

NC, USA) Linomat V semi-automated spray-on applicator equipped with a 100 100-

μl syringe and operated with setting band length 4 mm, application rate 15 nl/s,

distance from the bottom of the plate 80 mm.

3) Development

Spotted plates were developed in saturated HPTLC twin-through chamber (20 cm x

20 cm) up to 70 mm solvent front. HPTLC twin- through chamber is presaturated with

mobile phase for 25 minutes with help of filter paper and after development; plates

were removed immediately in oven at 60 °C for 5 minutes.

4) Scanning of plates

The developed plates were scanned with help camag TLC scanner III with a deuterium

and tungsten source. Slit dimension was 3 x 0.20 mm, and a scanning rate of 10 mm

S-1 using WINCATS-3 software.



5.3.2.8 Method validation 1) Preparation of Linearity curve of AMLO, HCTZ and VALS


and diluted to volume with methanol to obtain different Ternary mixture solutions

containing AMLO, HCTZ and VALS in 1:1.5:8 ratios. Concentration of solutions in

the range 100 - 3200 ng/spot were applied to the plate for the calibration curve of

these drugs. Peak area of the spots was measured at 242 nm in the absorbance mode

with camag TLC scanner III.

2) Precision

Intraday and interday precision

For intraday precision, the experiment was repeated three times in a day using three

different concentrations for AMLO (400, 500, 600 ng/spot), HCTZ (450,600,750

ng/spot), and for VALS (1600, 2000, 2400 ng/spot)

For interday precision, the experiment was repeated on three different days using

different concentrations for AMLO (400, 500, 600 ng/spot), HCTZ (450,600,750

ng/spot), and for VALS (1600, 2000, 2400 ng/spot) .Precision measured in terms of

%RSD

Repeatibilty

In the repeatability the standard solution of AMLO, HCTZ (500 ng/spot) and for

VALS 2000 (ng/spot) was spotted 7 times on the same plate and peak area was

recorded. Repeatability was measured in terms of %RSD.

3) LOD AND LOQ

From the linearity curve equations, the standard deviations (SD) of the intercepts

(response) were calculated. Then LOD and LOQ were measured for all the three drugs

by using mathematical expressions.

4) Accuracy

Accuracy of the method was determined by recovery study by standard addition

method at three different levels (80%, 100% and 120%).

5) Specificity

The purity of the chromatographic peaks was analyzed by scanning all the three

separated peaks in spectral scanning mode of the WinCATs 1.4.2.8121 software.



The peak purity for AMLO, HCTZ and VALS was tested by correlation of

spectra acquired at the peak start (s), peak maximum (m), and peak end (e)

positions.

6) Flow Constant

The flow constant or velocity constant (K) is a measure of the migration rate of

the solvent front. It is an important parameter for the HPTLC and can be used to

calculate development times with different separation distances, provided that the

absorbent, solvent system, chamber type and temperature remain constant. The flow is

given by the following equation:

K = ZF/t

K= Flow constant (mm2/s)

ZF= distance between the solvent front and the solvent level (mm)

t = Development time (s)

5.3.2.9 Analysis of marketed dosage form Total 14 tablets were weighed accurately and powdered. An amount equivalent to one

tablet (containing 5 mg of AMLO, 12.5 HCTZ and 160 mg of VALS) was

taken.Transfer to 100 ml volumetric flask and added 50ml methanol sonicated for 15

minutes and made up volume up to mark Solution was filtered by using Whatmann

filter paper N o.41 .Above solution containing 50 μg/ml concentration of AMLO, 125

μg/ml HCTZ and 1600 μg/ml concentration of VALS. From this solution, aliquots of

2 ml sol. Transfer to 10 ml volumetric flask and diluted up to mark with methanol and

apply 10 μl of this solution was spotted on activated TLC plate

5.3.3 RESULT AND DISCUSSION

Selection and optimization of solvent and mobile phase:

Selection and optimization of a proper mobile phase is a challenging task in HPTLC

method development. Several factors affects the selection of mobile phase such as

polarity of the drugs, desired Rf values, practical problems such as diffusion of spots,

tailing, proper peak shape after scanning.

Table 44: Observation and remarks of mobile phase optimization



Sr. No.

Trials Observation Remarks

1

Chloroform : methanol : toluene : glacial acid (6:2:1:0.1,

v/v/v/v),Run length =80mm

AMLO, HCTZ and VALS were close to

solvent front,Improper resolution

Not satisfactory

2

Acetone :chloroform :Ethyl acetate : methanol (3:3:3:0.5, v/v/v/v),Run length =80mm

Improper resolution and HCTZ run slovent front, poor Rf values of VALS

Not satisfactory

3

Ethyl Act: methanol:Amm.sol (7.5:2.5:0.5, v/v/v/v),Run length

= 80mm

Very good separation but Diffused spot of

VALS

Good but Not

satisfactory

4

Chloroform: Methanol : Amm.sol (7.5:2:5:0.5,

v/v/v/v),Run length = 80mmNot Good resolution,

Not Satisfactory

5

Ethyl Acetate :Methanol : TEA

(7.5: 2.5:0.5, v/v/v/v), Run length =80mm

VALS spot was less diffused but poor Rf

value of VALS

Not satisfactory

6

ACN :Methanol: TEA

(7.5:2.5:0.5, v/v/v),Run length= 80mm

Closeness b/w HCTZ and VALS and diffused

spot of VALS

Not satisfactory

7

Ethyl Acetate :Methanol:1,4 dioxane :Ammonia (7:3:1:0.5, v/v/v/v), Run length =80mm

Less resolution b/w AMLO and HCTZ

Not satisfactory

8

Cyclohexane : Methanol:Ammonia

( 7.5:2.5:0.5, v/v/v/v),Run length =80mm

Diffused spot of AMLO and high Rf value of

VALS and HCTZ

Not satisfactory

9.

ACN :Methanol: TEA

(7.5:2.5:0.5, v/v/v),Run length= 80mm

Closeness b/w HCTZ and VALS and diffused

spot of VALS

Not satisfactory



10

Ethyl Acetate :Methanol: Toluene: Ammonia (6:3:3:0.5,

v/v/v/v),Run length= 80mm

Good resolution but VALS having less

tailing

Not good separation

11

Ethyl Acetate :Methanol :Toluene :g

lacial acid (7.5:3.5:2.5:0.1, v/v/v/v), Run length= 80mm

VALS spot was not diffused but poor Rf

value of AMLO

Not satisfactory

12

Chloroform :Methanol :Toluene :glacial acid (7.5:3:2.5:0.1, v/v/v/v),Run length: 80mm

AMLO spot was splittedNot

satisfactory

13

Ethyl acetate : Methanol: Toluene :Ammonia

(7.5:3:2.5:1, v/v/v/v),Run length= 80mm

Good resolution but was not reproducible

Resolution was less b/w AMLO and

HCTZ

14

Ethyl acetate :Methanol : Toluene :Ammonia

(7.5:3:2:0.8, v/v/v/v),Run length =80mm

Good resolution and reproducible but VALS still having less tailing

Very good separation

15

Ethyl acetate :Methanol : Toluene :Ammonia

(7.5:3:2:0.8, v/v/v/v), Run length =70mm

Good resolution and VALS was not diffused

Satisfactory Optimized

Mobile Phase

5.3.3.1 Validation parameters

1. Linearity

The correlation coefficient (r2) was found to be 0.9945, 0.9926 and 0.9918 for AMLO, HCTZ and VALS respectively.

Table 45: Calibration data of AMLO by HPTLC with UV detection

Sr. No. Conc. Peak Area(n=6)



(ng/spot) RfMean ± SD* %RSD

1 100 1089±18.0 1.652 0.54

2 200 1873.167±10.2 0.548 0.54

3. 300 2646.5±51.3 1.962 0.54

4. 400 3182.8±58.84 1.849 0.54

5. 500 3819.51±36.63 0.959 0.54

6. 600 4375.933±79.0 1.807 0.54

Fig.14: Linearity curve for AMLO


0 100 200 300 400 500 600 7000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

f(x) = 6.53428571428572 x + 547.483333333334R² = 0.994450427362076


Fig.15: Linearity curve for AMLO from Win CATS software

Table 46: Calibration data of HCTZ by HPTLC with UV detection

Sr. No.Conc.

(ng/spot)

Peak AreaRf

Mean ± SD* %RSD

1 150 1470.4±26.43 1.79 0.64

2 300 2547.933±20.66 0.81 0.64

3 450 3650.333±45.23 1.23 0.63

4 600 4284.167±33.74 0.78 0.63

5. 750 5245.267±37.84 0.72 0.65

6. 900 5953.1±37.03 0.52 0.65



Fig. 16: Linearity Curve for HCTZ

Fig.17: Linearity curve for HCTZ from Win CATS software

Sr. No. Conc (ng/spot)

Peak Area

RfMean ±

SD*%RS

D

1 1200 1089±33.52 1.65 0.2


100 200 300 400 500 600 700 800 900 10000

1000

2000

3000

4000

5000

6000

7000

f(x) = 5.93129904761905 x + 744.598R² = 0.99259489926599

Concentration (ng/spot)


3

2 16001873.16±19.5

10.54

0.23

3 2000 2646.5±93.04 1.960.23

4 2400 3182.8±134.8 1.840.23

5 2800 3819.51±75.6 0.950.27

6 3200 4375.93±144 1.800.27

Table 47: Calibration data of VALS by HPTLC with UV detection

1000 1500 2000 2500 3000 35000

2000

4000

6000

8000

10000

12000

14000

16000

f(x) = 3.48017035714286 x + 2356.00638095238R² = 0.991831074182329

Fig.18: Linearity Curve for VALS



Fig.19: Linearity curve for VALS from Win CATS software

Fig.20: HPTLC Chromatogram of AMLO (Rf = 0.54)



Fig.21: HPTLC Chromatogram of HCTZ (Rf = 0.64)

Fig.22: HPTLC Chromatogram of VALS (Rf = 0.23)



Fig.23: HPTLC chromatogram of VALS (Rf = 0.23), AMLO (Rf = 0.54) and HCTZ (Rf =0.64) in standard mixture.



Fig.24: HPTLC chromatogram (3D view) for Mix linearity of AMLO (Rf=0.54),

HCTZ (Rf=0.64) AND VALS (Rf=0.23)

2) Precision

DrugConc.

(ng/spot)

Peak AreaRf ±SD*

Mean ± SD* %RSD

AMLO200 3201.6±64.16 2.00 0.54±0.015

500 3673.867±60.64 1.65 0.55±0.011



600 4171.333±41.56 0.99 0.54±0.013

HCTZ

450 3812.833±25.8 0.67 0.66±0.010

600 4216.633±50.91 1.20 0.64±0.012

750 5502.1±61.85 1.12 0.64±0.011

VALS

1600 8076.28±19.17 0.23 0.26±0.02

2000 9343.433±171.65 1.83 0.27±0.011

2400 10297.73±144.49 1.40 0.27±0.015

Table 48: Intraday precision AMLO, HCTZ AND VALS by HPTLC with UV

DrugConcentratio

n (ng/spot)

Peak AreaRf±SD*Mean ± SD* %RSD

AMLO

3201.6±31.50 1.98 0.54±0.015

3673.867±18.43 1.65 0.56±0.013600 4171.333±25.79 0.99 0.54±0.011

HCTZ

450 3741.7±26.99 0.67 0.66±0.012

600 3624.9±18.43 1.20 0.64±0.014

750 3655±99.26 1.12 0.64±0.010

VALS

1600 8076.28±94.57 0.23 0.23±0.021

9343.433±23.95 1.83 0.25±0.22

2400 10297.73±133.15 1.40 0.24±0.024

Table 49: Interday precision AMLO, HCTZ AND VALS by HPTLC

Table 50: Repeatibilty study of AMLO, HCTZ AND VALS

Sr.no. Drug Peak Area±SD* %RSD



1 AMLO (500ng/spot) 2917±33.30 1.98

2. HCTZ(500ng/spot) 3609.78±25.65 1.86

3. VALS (2000ng/spot) 7061.61±99.95 0.84

Accuracy

Recovery study was determined by standard addition method. Standard addition was

done at three levels, 80%, 100% and 120% of sample concentration in linearity range.

Table 51: Recovery study of AMLO, HCTZ and VALS

DRUG

INITIAL

CONC.

ng/

STD. Adde

d

ng/spot

TOTAL

CONC.

ng/

Accuracy

Conc. Recovered

Mean± SD*

±%RSD

%Recovery

Mean

±%RS

D

AMLO

55

44 99 99.14±1.07 1.08 100.14 1.32

55 110 109.39±0.66 0.60 99.44±0.74 0.74

66 121 120.81±0.76 0.62 99.84± 0.76

HCTZ 98.21

78.57 177.47 176.8±0.34 0.19 99.09±0.93 0.92

98.21 196.42 199.12±2.12 1.08 99.79±0.761 0.76

117.8 216.81 215.98±0.94 0.43 100.14±0.675 0.67

VALS1257.

1

1005.62262.7

42357.4±17.09 0.13

101.40±0.785

0.77

1257.1 2514.282551.77±19.2

41.31 99.65±0.907 0.91

1508.56 2765.72796.64±16.2

50.51 99.30±0.703 0.70

Table 52: LOD and LOQ values of AMLO, HCTZ and VALS

Parameter AMLO (ng/spot) HCTZ(ng/spot) VALS(ng/spot)

LOD 2.95 17.89 70.90

LOQ 8.94 53.9 214.85



Table 53: Robustness Study of AMLO, HCTZ and VALS

Sr.no.

ParameterPeak Area ±SD * %RSD

AMLO HCTZ VALS AMLO HCTZ VALS

1.

Mobile Phase

composition Ethyl

Acetate (±5%)

1107.8 ±14.47

2119.9±38.813749.4±175.3

1.3 1.64 1.2

2.Wavelength (242±2nm)

1119.63 ±9.99

1743.6±17.21082±125.29

0.89 0.98 1.15

3.

Development Distance

(70±5mm)

1073.43±10.6

1132.43±1.013430.1

±1330.93 1.02 0.99

Specificity:

Fig.25: Peak purity spectra of AMLO



Fig.26: Peak purity spectra of HCTZ

Fig.27: Peak purity spectra of VALS

Drugs Co-relation r(s,m) Co-relation r (m,e) Peak purity

AMLO 0.99906 0.9994 Pass

HCTZ 0.99958 0.9976 pass



VALS 0.99966 0.999 pass

Table: 54 specificty data of AMLO, HCTZ and VALS



Table 55: Summary of Validation parameters by HPTLC with UV detection

Sr.no

Parameters AMLO HCTZ VALS

1 Linearity range (ng/spot)

100-600 150-900 1200-3200

2 Regression equation y = 6.534x+547.48

y =5.931x+744.6

Y=3.48x+2356

3Correlation coefficient

(r2)0.9945 0.9926 0.9918

4 Intercept 6.534 5.931 3.48

5 Slope 547.48 744.6 2356

6

Precision

Intraday % RSD (n = 3)

Interday % RSD (n = 3)

Repeatability of measurements% RSD

(n=6)

0.99 to 2.0

0.48 to 0.98

1.98

0.67 to 1.2

0.43 to 1.92

1.86

0.23 to 1.83

0.25 to 1.26

0.84

7 Limit of detection 2.95 (ng/spot) 17.84 (ng/spot) 70.90 (ng/spot)

8 Limit of quantification 8.94(ng/spot) 53.9 (ng/spot) 214.85(ng/spot)

9 Specificity Pass Pass pass

5.3.3.2 Analysis of marketed sample

The developed method was used to estimate AMLO, HCTZ and VALS in combined dosage form. The percentage of AMLO, HCTZ and VALS was found from the calibration curve.



Fig.28: HPTLC CHROMATOGRAM OF STANDARD MIXTURE

Fig.29: HPTLC CHROMATOGRAM OF MARKETED SAMPLE



Fig.30: HPTLC chromatogram (3D view) for sample in Mix linearity of AMLO, HCTZ AND VALS (Track 2, 3 for Sample Spot)

Table 56: Analysis of Marketed Formulation for Exforge HCTZ

Drug Label claim(mg) Amt estimated(mg)

Assay results ±S.D*

AMLO 5 5.01

100.32±1.025 5.06

5 4.96

HCTZ 12.5 12.74

100.8±1.4312.5 12.65

12.5 12.40

VALS 160 162.27

100.27±1.57160 161.37

160 157.49



5.3.4 Conclusion :

By the virtue developed method, it can be concluded that high performance thin layer chromatography method is reliable technique for the analysis of commercial formulations of AMLO, HCTZ and VALS in tablet dosage form. The developed method is simple , sensitive, and specific which renders it suitable analysis of AMLO, HCTZ and VALS in combined dosage form and this method is specific which show developed method is free from the interference of excipients used in formulation.



5.4 DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD



5.4.1 Instrumentation

5.4.1.1 High performance liquid chromatography

Model: JASCO 200 Series

Manufacturer: JASCO, Inc. JAPAN

Pump: JASCO PU-2080 plus

Mixer : JASCO MX-2080-31

Injector: Rheodyne model 7125 with 20 µl fixed loop

Detector: JASCO-UV-2075 PLUS

Software: Borwin software version 1.50 was used

5.4.1.2 PH Meter

Model : 11 E/101E

Manufacturer: Analabs scientific instrument Ltd.

PH : 0 to 14

Resoultion : ± 0.01 PH

Accuracy : ± 0.01 PH± 14 digit

5.4.1.3 Analytical Balance:

Model: KEROY

Manufacturer: Keroy (balance) pvt. Ltd. Varanasi, India.

Weighing capacity: 100gm

5.4.1.4 Sonicator:

Model: TRANS-O-SONIC; D-compect.

Capacity: 2 Lit.

5.4.2 Materials and methods



5.4.2.1 Chemicals and Materials

Methanol (AR Grade, S.D. Fine chemicals Ltd., Mumbai , India

Acetonitrile (HPLC Grade, S.D. Fine chemicals Ltd., Mumbai , India)

Water HPLC & Spectroscopy ( central drug house (p) Ltd., New Delhi

Tablet containing AMLO(5 mg), HCTZ (12.5 mg) and VALS(160 mg)

Brand name: Exforge HCTZ, Name of manufacturer




5.4.2.2 Chromatographic Conditions

5.4.2.2.1 Optimized Chromatographic Conditions

Stationary phase: Kromasil Column KR-5C 18 (250 mm × 4.6mm i.d., 5μm)

Mobile phase: Acetronitrile : potassium dihyrogen ortho phosphate buffer with )

0.2% TEA(44 :56, v/v) , PH 3.7 adjusted with OPA

Wavelength: 232 nm

Runtime: 15 Min.

Flow rate: 1ml/min

Diluent : ACN and Millipore Water(50:50, v/v)

Retention time for HCTZ : 3.78 Min

Retention time for AMLO: 3.15 Min

Retention time for VALS : 10.15 Min

Optimization of the chromatographic condition was studied by checking the effect of

chromatographic variables such as temperature, back pressure, flow rate and solvent

ratio. The resulting chromatograms were recorded and the chromatographic

parameters which give the best peak resolution were selected for analysis.

5.4.2.3 Preparation of Mobile phase

Mobile phase A: HPLC grade Acetonitrile was degassed with sonicator for 15 min.



Mobile phase B: 3402.25 mg of KH2PO4 (potassium dihydrogen ortho phosphate )

was dissolved in 500 triple dist. Water and add 1 ml HPLC grade triethylamine

(0.2%) and pH 3.7 adjusted with ortho phosphoric acid.

5.4.2.4 Preparation of Standard Stock Solution of AMLO, HCTZ and VALS

25 mg AMLO, HCTZ and VALS was weighed accurately and dissolved each standard

drug in separately in 25 ml methanol in different volumetric flasks. 1 ml aliquots of

the above solutions were diluted to 10 ml with methanol in different volumetric flasks

to produce 100 μg/ml of AMLO and 100 μg/ml of HCTZ

5.4.2.5 Preparation of ternary mixtures of AMLO, HCTZ and VALS


and diluted to volume with ACN and Millipore water (50:50) to obtain different

ternary mixture solutions containing AMLO, HCTZ and VALS in different ratio

Concentration of solution in the range 2 to 150 µg/ml was prepared for the calibration

curve of three drugs.



Table 57: Observation and remarks of mobile phase optimization


Sr. no. Mobile phase composition Inference Conclusion

1

ACN :0.025 M potassium dihydrogen ortho phosphate

(60:40v/v) PH 3.7

Peak was not suitable for quantitative

M.P was not suitable

2

ACN :0.025 M potassium dihydrogen ortho phosphate

(50:50v/v), PH 3.7

VALS RT greater than 10 min M.P was not

suitable

3ACN :0.025 M potassium

dihydrogen ortho phosphate (57:43v/v) , TEA 0.1%, PH 3.7

Asymmetry greater than 1.5


4.

ACN : 0.025 M potassium dihydrogen ortho phosphate

(43:57v/v) TEA 0.2%, PH 3.7

Asymmetry was less as compare to

0.1% TEA


5.

ACN : 0.050 M potassium dihydrogen ortho phosphate

(44:56v/v) , TEA 0.2%, PH 3.7

Peak was suitable for quantitative

M.P was suitable

(Optimized)


Fig.31: HPLC Chromatogram of Blank (diluents) ACN: Water (50:50, V/V)

Fig.32: HPLC Chromatogram of HCTZ



Fig.33: HPLC chromatogram of standard drug of AMLO

Fig.34: HPLC Chromatogram of standard mixture of AMLO, HCTZ and VALS



Table 58: System suitability parameter by RP-HPLC method

Sr. no Parameters AMLO HCTZ VALS

1 Capacity Factor 0.3695 0.6434 3.4130

2. Tailing factor 1.30 1.41 1.05

3. Resolution factor 3.175 - 10.37

4. Theoretical plates 5378 6357 4886

5. % RSD of Peak Area 0.48 1.85 0.52

5.4.2.6 Method validation

1. Preparation of Linearity curve

For estimation of AMLO, calibration curve (n=3) was plotted in the range of (2-25

µg/mL). For estimation of HCTZ calibration curve (n=3) was plotted in the range of

(5-45 µg/mL). For estimation of VALS calibration curve (n=3) was plotted in the

range of (20-150 µg/mL).Calibration curve of peak area v/s concentration was

plotted for the drug.

2. Precision

Intraday and Interday precision

For intraday precision, the experiment was repeated three times in a day using three

different concentrations for AMLO (5, µg/ml 10, 15 µg/ml), for HCTZ (10, 15, 20

µg/ml) and for VALS (80,100,120 µg/mL)

For Interday precision, the experiment was repeated on three different days using

three different concentrations respectively AMLO, HCTZ and for VALS Precision

find out in terms of %RSD.

Repeatability



In the repeatability the peak area of sample solutions for AMLO, HCTZ, VALS (5,

15, 100 µg/ml) were taken by 6 times and find out the % RSD.

1) LOD AND LOQ

From the linearity curve equations, the standard deviations (SD) of the intercepts

(response) were calculated. Then LOD and LOQ were measured for all the three drugs

by using mathematical expressions.

2) Accuracy Sample concentration was taken 2 µg/mL for AMLO, 4 µg/ml for HCTZ and for VALS 64 µg/ml. After that accuracy of the method was determined by standard addition method at three different levels (80%, 100% and 120%).

5.4.2.7 Analysis of marketed dosage form Total 14 tablets were weighed accurately and powdered.An amount equivalent to one tablet (containing 5 mg of AMLO, 12.5 mg of HCTZ and 160 mg of VALS) was taken and dissolved in 100 ml methanol in 100 ml volumetric flask Solution was sonicated for 15 minutes. After solution was filtered by using Whatmann filter paper No.41.From this solution, 5 ml of sample solution was taken in 50 ml volumetric flask and diluted with diluent ACN:Water (50:50) final solution containing 5 μg/ml concentration of AMLO ,12.5 µg/ml HCTZ and 160 μg/ml concentration of VALS.

5.4.2.8 Results and Discussion

5.4.3.1 Validation parameters

1) Linearity

The correlation coefficient (r2) was found to be 0.9945, 0.9965, and 0.9971 for AMLO, HCTZ and VALS respectively.



Table 59: Calibration data of AMLO by RP-HPLC with UV PDA detection

Sr. No. Conc. (µg/ml)

Peak Area

Mean ± SD* %RSD

1 2 144942± 1936.48 1.33

2 5 327329.5±1376.5 0.42

3 10 50262 ± 2063 0.41

4 15 796769.3±6361.674 0.79

5 20 1117013±1489.079 0.13

6 25 1372590±9579.747 0.69

Fig.35: Linearity curve for AMLO

Table 60: Calibration data of HCTZ by RP-HPLC with UV PDA detection


0 5 10 15 20 25 300

200000

400000

600000

800000

1000000

1200000

1400000

1600000

f(x) = 53406.5268230277 x + 24827.5391044776R² = 0.994479089552042


Sr. No. Conc. (µg/ml)Peak Area

Mean ± SD* %RSD

1 5 826897.3±4122.691 0.49

2 10 1481195±17761.38 1.19

3 15 1975995±15134.72 0.76

4 25 3557427±6076.3 0.45

5 35 5039660±41762.14 0.82

6 45 6069559±36571.47 0.60

Fig.36: Linearity curve for HCTZ

Sr. No.Conc

(µg/ml)

Peak Area


0 5 10 15 20 25 30 35 40 45 500

1000000

2000000

3000000

4000000

5000000

6000000

7000000

f(x) = 135283.193473684 x + 114583.696842105R² = 0.99600692082747


3 60 6174683±82500.03 1.33

4 80 8022444±9798.269 0.12

5 120 13033262±45680.19 0.35

6 150 15893952±96053.33 0.60

Table 61: Calibration data of VALS by RP-HPLC with PDA UV detection

Fig. 37: Linearity curve for VALS


0 20 40 60 80 100 120 140 1600

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

f(x) = 112822.220137931 x − 838391.244137931R² = 0.997113016063757


DrugConcentration

(µg/ml)

Peak Area

Mean ± SD* %RSD

AMLO

5 306648.7±2519.684 0.82

10 562226.7±1300.598 0.23

15 865629.7±5262.363 0.60

HCTZ

10 1366868±7087.164 0.51

15 2190594±45155.87 2.06

20 2712349±23962.09 0.88

VALS

80 7723585±30225.43 0.39

100 9897468±60556.46 0.61

120 13142484±202472.7 1.54

Fig. 38 MIXTURE LINEARTY CURVE OF AMLO, HCTZ AND VALS BY HPLC

Table 62- Intraday precision data of AMLO, HCTZ AND VALS by HPLC



2) Precision

Table 63: Intraday precision data of AMLO, HCTZ AND VALS by HPLC

DrugConcentration

(µg/ml)

Peak Area

Mean ± SD* %RSD

AMLO

5 309600.7±3256.28 1.05

10 502668.3±2644.688 0.52

15 773136.3±253.4213 0.33

HCTZ

10 1369149±10733.71 0.78

15 2110007±10417.99 0.49

20 2678649±33853.87 1.26

VALS

80 7774025±43345.03 0.55

100 9936439±53407.12 0.53

120 13018159±34631.55 0.26



Table 64: Repeatibilty study of AMLO, HCTZ and VALS

Drug Conc. (µg/ml) Peak Area±SD* %RSD

AMLO 5 2917±1968.42 0.71

HCTZ 15 1833440±6751.29 0.36

VALS 100 9381937±92487.03 0.98



Table 65: % Recovery study of AMLO, HCTZ and VALS

DRUG

INITIAL

CONC.

µg/ml

STD. Added

µg/ml

TOTAL

CONC.

After spikin

g µg /ml

ACCURACY

Conc. Recovere

d

Mean±SD*

%

RSD

%Recovery

Mean ±SD*

%

RSD

AMLO

2

1.6 3.63.5833±0.049

1.1399.57333

±0.321.33

2 44.0146±0.046

1.42101.4233

±0.750.75

2.4 4.44.4566±0.037

0.69101.4233

±0.070.76

HCTZ 5

4 98.8766±056

0.8298.35333

±0.010.95

5 109.9533±0.037

0.4998.35333

±0.041.47

6 1110.9033±0.08

0.9599.16667

±0.760.76

VALS 64

51.2 115.2117.806±0.101

0.18102.0167±0.

0740.74

64 128129.466±2.085

1.61101.0967±0.056

1.56

76.8 140.8140.3733±0.883

0.6299.69

±0.06370.63



Table 66: LOD and LOQ

Parameter AMLO HCTZ VALS

LOD 0.23 µg/ml 0.48 µg/ml 1.1

LOQ 0.71 µg/ml 1.47 µg/ml 3.3

Table 67- Robustness study of AMLO

Parameters ChangeMean of Peak Area ±

%RSD%Assay

Flow Rate (±0.2 ml/min)

0.8 293624±1.74 98.25

1.2 290494±0.52 100.0

PH (± 0.05)

3.65 382701.7±1.53 99.82

3.75 210569.2±1.74 100.2

Wavelength (± 2 nm)

230 1283653±0.29 100.42

234 1248926±0.51 101.04



Table 68: Robustness study of HCTZ

Parameters Change Mean ±%RSD %Assay

Flow Rate

(±0.2ml/min.)

0.8 1952066±1.17 98.44

1.2 681237.7±0.88 99.38

PH (±0.05)

3.65 1365801±1.55 101.59

3.75 1280586±0.56 98.15

Wave Length

( ±2nm)

230 1479909±0.95 99.77

234 1559906±1.79 98.51

Table 69 Robustness study of VALS

Parameters Change Mean ±%RSD %Assay

Flow Rate

(±0.2 ml/min)

0.8 17862595±0.8 98.60

1.2 16260024±1.0 98.79

PH (± 0.05)

3.65 13688728±1.3 98.48

3.75 11807506±1.4 101.76

Wave Length

(±2 nm)

230 11741308±0.8 98.45

234 9727207±1.30 98.43



Table 70: Summary of Validation parameters of RP-HPLC

Sr.no

Parameters

AMLO HCTZ VALS

1.Linearity range

(µg/ml)2-25 5-45 20-150

2.Regression equation

y =53047x+2482

8

y=135283x+114584

y = 112822x-83839

3.Correlation

coefficient (r2) 0.9945 0.9967 0.9971

4.

Precision

Intraday % RSD (n = 3)

Interday % RSD (n = 3)

Repeatability of measurements %

RSD

0.23-1.82

0.03-1.05

0.5-1.4

0.5-2.0

0.49-1.26

0.24-0.36

0.39-1.54

0.26-0.55

0.77-1.01

5. Specificity Specific Specific Specific

6.LOD

(µg/ml)0.23 0.48 1.1

7.LOQ

(µg/ml)0..71 0.1.47 3.3

8. % Recovery 99.57±1.33 to 101.42±0.75

98.35±0.19 to 99.16± 0.76

99.69 ± 0. 63 to

1002.01±0.07



4

SPECIFICITY



Table 71: Analysis of Marketed Dosage Form

DrugsLabel

claim(mg)Amt

estimated(mg)

Assay results

% recovery ±S.D*

AMLO

5 4.95

98.66±0.365 4.91

5 4.92

HCTZ

12.5 12.66

99.87±0.8712.5 12.44

12.5 12.39

VALS

160 158.31

100.62±1.49160 161.77

160 162.92



Fig.39: HPLC Chromatogram of Marketed Dosage Form

5.4.3 Conclusion By the virtue developed method, it can be concluded that high performance Liquid chromatography method is reliable technique for the analysis of commercial formulations of AMLO, HCTZ and VALS in tablet dosage form. The developed method is simple, sensitive, and specific which renders it suitable analysis of AMLO, HCTZ and VALS in combined dosage form and this method is specific which show developed method is free from the interference of excipients used in formulation



5.5 COMPARISON OF UV-VIS SPECTROPHOTOMETRIC, HPTLC

AND RP-HPLC METHOD

Table72: COMPARISON OF UV-VIS SPECTROPHOTOMETRIC, HPTLC AND RP-HPLC METHODS FOR ESTIMATION OF AMLO, HCTZ and VALS IN TABLET DOSAGE FORM

Brand name Drugs

% Assay results

U.V HPTLC RP-HPLC

Exforge HCTZ

AMLO

98.66 100.28 99.07

98.45 101.37 98.36

100.3 98.79 98.55

HCTZ

98.82 101.97 100.8

98.40 101.23 99.52

98.20 99.26 99.13

VALS 98.33 98.59 101.10

98.75 99.15 101.82

99.20 101.61 98.94



Table 49 - Comparison of three Methods by ANOVA Test

Brand name Drugs Fcal F crit

Exforge HCTZ

AMLO 1.63 4.066

HCTZ 3.68 4.066

VALS 1.18 4.066

5.5.2 Conclusion

ANOVA result was performed by using Microsoft excel and graph pad instate, version 3.05, 32 bit. So developed methods were compared statistically by ANOVA test. The results show that there is no significant statistical difference between the results obtained by above mentioned methods. In the cases, Fcal is less than Critical.












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