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Chapter5 Experimental Work

Institute of Pharmacy, Nirma University Page 100

CHAPTER 4

EXPERIMENTAL WORK

CONTENTS

4.1 IDENTIFICATION OF DRUG

4.2 DEVELOPMENT AND VALIDATION OF UV SPECTROMETRIC

METHOD FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ

AND VALS IN THEIR COMBINED TABLET DOSAGE FORM

4.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR

SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND VALS IN

THEIR COMBINED TABLET DOSAGE FORM

4.4 DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND VALS IN

THEIR COMBINED TABLET DOSAGE FORM

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4.1 IDENTIFICATION OF AMLO, HCTZ and VALS

Drug samples were kindly gifted 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.

4.1.1 Determination of Melting Point

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

and results are as shown in table.

Table25. Melting points of drugs

4.1.2 UV spectra of AMLO, HCTZ and VALS

UV- spectrum of AMLO (20g/mL), HCTZ (20g/mL) and VALS (20g/mL) in

methanol was taken and it was compared with reported UV spectra as shown in

Table

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

Drug Reported melting point (0C)() Observed melting point (

0C)

AMLO 199-201 200-202

HCTZ 273-275 274-275

VALS 116-117 117-118

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

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

Table 26: Reported max for AMLO, HCTZ and VALS

Drug Reported maxima Recorded maxima

AMLO 239nm, 238nm 237.6nm,210.8nm, 330nm

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

VALS 249nm 249.2nm, 206nm, 243.8nm

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4.1.3Determination of Infrared (IR) Spectra

Identification of drug sample was carried out by IR spectroscopy studies. A

mixture of drug sample and KBr (spectroscopic grade, drug: KBr ratio 1:20) was

prepared using mortar-pestle. This mixture was then analyzed in attenuated

reflectance FT-IR. The mixture was scanned from 4000-400 cm-1

and a spectrum

was recorded with the help of IR Spectrophotometer (JASCO Model: FT-IR 6100

type A).

Fig.8: Infrared spectrum of AMLO

Fig.8: Reported Infrared spectrum of AMLO

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Table 27: IR Frequency (Cm-1

) for AMLO

SPECIFICATION OFAMLO

THEORITCAL WAVENUMBER(Cm

-1)

(85)

RECORDED WAVENUMBER(Cm

-1)

-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

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Fig. 9: Reported Infrared spectrum of HCTZ

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) for HCTZ

Fig.10: Infrared spectrum of VALS

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

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Fig.10: Reported Infrared spectrum of VALS

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) for VALS

SPECIFICATION OF FORVALS

THEORITICAL WAVENUMBER (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

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4.2 DEVELOPMENT AND VALIDATION OF UV

SPECTROPHOTOMETRIC METHOD FOR

SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND

VALS IN THEIR COMBINED TABLET DOSAGE

FORM

4.2.1 Instrumentation

UV-Visible Double-Beam spectrophotometer- UV-VIS is 2400, version-2.21 double beam spectrophotometer with spectral width of 2 nm, wavelength

accuracy of 0.5 nm, and a pair of 10 mm matched quartz cells (Shimadzu,

Columbia, MD)

Analytical Balance- Weighing capacity-10 to 220 mg, Citizen CX 220(Citizen Pvt. Ltd),

Sonicator- Capacity- 2 L, D-compact (Trans-O-Sonic, Mumbai),4.2.2Materials and Methods4.2.2.1 Reagents and Chemicals

AMLO, HCTZ, and VALS Standards were kindly gifted by Torrent

pharmaceutical Gujarat, India.All reagents and solvents were of analytical grade

(Central Drug House Pvt. LTD). Marketed tablet formulation Exforge HCT

(Novartis pharma stein AG, stein, Switzerland.) Containing Amlodipine besylate

(5mg), Hydrochlorothiazide (12.5 mg) and Valsartan (160mg) purchased from

USA market.

4.2.2.2Preparation of standard stock solution of AMLO, HCTZ and VALSAMLO (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 g/mL of each drug.

4.2.2.3 Preparation of calibration curve of AMLO, HCTZ and VALSAliquots of 2, 4, 5, 10, 15, 20 g/mL were prepared by using stock solution of

AMLO, aliquots of HCTZ of 5, 10, 15, 20, 25 g/mL by using stock solution of

HCTZ and aliquots of 10 ,20, 30, 40, 50 g/mL by using of VALS stock solution

for preparation of calibration curve.

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4.2.2.4Preparation of sample preparationAn amount equivalent to 160 mg of VALS containing (5 mg of AMLO and 12.5

mg HCTZ) was weighed and transfer to100ml calibrated volumetric flask

dissolved methanol. From above solution 5 ml aliquots was pipette out and

transfer to 10 ml calibrated volumetric flask to get final conc. of AMLO(5

g/mL), HCTZ(12.5 g/mL) and VALS(g/mL).

4.2.2.4 Selection of Analytical Wavelength and measurement

From stock solutions of AMLO, HTCZ and VALS, working standard solutions

were prepared by appropriate dilution of solvent to get final concentration of 20

g/mL each drug 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 (max of

AMLO), 249.2 nm (max of VALS) and 270.2 nm (max of HCTZ) were selected for

analysis.

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

4.2.2.5Method Validation4.2.2.5.1 Preparation of linearity curve

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Linearity was performed in conc. range 2-20 g/mL at 237.6nm for AMLO, 5-

25 g/mL at 270.2nm for HCTZ and 10-50 g/mL at 249.2 nm for VALS.

Solution preparation was done which as shown in section 4.2.2.3.

4.2.2.5.2 Precision

Intra-day precisionIntra-day precision was determined by measuring amplitudes of three different

concentrations 2, 4, 5 g/mL for AMLO and 15, 20, 25, g/mL for HCTZ and

30,40,50 g/mL for VALS individually for three times in a day.

Inter-day precisionInter-day precision was determined by measuring amplitudes of three different

concentrations 2, 4, 5 g/mL for AMLO, for HCTZ 15, 20, 25, g/mL and 30,

40, 50 g/mL for VALS individually for three days.

RepeatabilityRepeatability of this method was determined by measurement of six

determinations at 100% test concentrations 4g/mL, 10 g/mL, 20 g/mL of

AMLO, HCTZ and VALS respectively.

4.2.2.5.3 Limit of detection and limit of quantification (LOD/LOQ)

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

LOD = 3.3 x /S

LOQ = 10 x /S Where, = Standard deviation of the Intercept

S = slope of calibration curve

5 AccuracyTo study the accuracy, 7 tablets were weighed and powdered. The powder

equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of VALS)

were weighed and transferred to 100ml volumetric flask and dissolved in 70 ml of

methanol. Then 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 pipette out and transferred to 50ml volumetric

flask. Volume was made up to the mark with methanol to get a solution

containing 5g/mL of AMLO, 12.5 g/mL of HCTZ and 160 g/mL of VALS.

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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.

6 Analysis of marketed formulationMarketed tablet formulation containing VALS 160 mg, amlodipine 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 methanoland solution was sonicated for 30 minutes and volume was made up to the mark

in a 100ml calibrated volumetric flask with methanol. The above solution was

filtered 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. CAMLO is the concentration of AMLO, CHCTZ is the

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

6.2.2 Results and Discussion4.2.3.1 Method development

The values of absorbance were recorded at the selected wavelengths and the

absorptivity and molar absorptivity values for AMLO, HCTZ and VALS

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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. Found simultaneous equation by putting molar

absorptivity value

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

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

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

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

270.2 nm and 249.2 nm respectively. CAMLO is the concentration of AMLO,

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

VALS

4.2.3.2 Validation parameters

7 Linearity-Linearity curve shows linearity in the range of 2-20 g/mL forAMLO, 5-25 g/mL for HCTZ and for VALS 10-50 g/mL. The correlation

coefficient (r2) was found to be 0.9997, 0.9990, and 0.9990 for AMLO,

HCTZ and VALS respectively. Calculate and record value of correlation co-

efficient (r), y-intercept, slope of regression line and residual sum of

squares. (Figure 6.12) and results (Table)

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Fig 12: Linearity Curve for AMLO at 237.6 nm

Fig 12: Calibration Curve for AMLO at 237.6 nm

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Table 30: Linearity of AMLO at 237.6 nm, 270.2 nm and 249.2 nm

Conc.

g/mL

237.6nm 270.2nm 249.2nm

MeanAbs.

S.D*

%

RSD

MeanAbs

S.D*

%

RSD

Mean

Abs.S.D*

%

RSD

2 0.0450.016 1.55 0.0130.0021 1.61 0.0210.0013 1.61

4 0.1220.018 1.47 0.0240.0024 1.60 0.0650.0017 1.53

5 0.1590.023 1.44 0.0420.0016 1.80 0.0880.0014 1.13

10 0.3460.011 0.31 0.0560.0013 1.12 0.2010.0015 0.74

15 0.5580.012 0.17 0.0640.0017 1.42 0.3280.0012 0.30

20 0.7070.015 0.21 0.0780.0016 0.95 0.4220.0016 0.37

Absorptivity 320 177.7 178.63

*(n=6)

Fig. 12: Linearity Curve for HCTZ at 270.2 nm

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Fig. 12: Calibration Curve for HCTZ at 270.2 nm

Table 31: Linearity of HCTZ at 237.6 nm, 249.2 nm and 270.2 nm

Conc.

g/mL

237.6nm 270.2nm 249.2nm

MeanAbs.

S.D* %RSD

Mean Abs.

S.D* %RSDMean Abs.

S.D* %RSD

5 0.0090.001 1.33 0.2970.002 0.70 0.0320.0011.75

10 0.0420.001 1.57 0.6180.002 0.37 0.0830.001 1.56

15 0.0760.001 1.71 0.9250.001 0.16 0.1370.001 1.16

20 0.1130.001 1.32 1.2450.003 0.12 0.1920.002 0.78

25 0.1660.006 1.61 1.5640.003 0.12 0.2650.002 0.37

Absorptivity 45.88 615.55 88.086

*(n=6)

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Fig.13: Linearity Curve of VALS at 249.2 nm

Fig.13: Calibration Curve of VALS at 249.2 nm

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Table 32: Linearity of VALS at 237.6 nm, 249.2nm at 270.2 nm

Conc.

g/mL

237.6nm 270.2nm 249.2nm

Mean

Abs.S.D*%

RSD

Mean

Abs.S.D*

%

RSD

Mean

Abs.S.D*

%

RSD

100.3260.003

0.980.1460.00

21.02

0.3100.02

70.80

20 0.6340.0022 0.34 0.2790.00

0.53 0.5830.02

0.42

30 0.9780.002 0.25 0.4330.00

0.34 0.8950.02

0.23

40 1.2630.0038 0.30 0.5530.00

0.56 1.1670.03

0.25

50 1.580.0015 0.97 0.6820.00

0.30 1.4620.02

0.14

Absorptivity 320.07 141.02 295.75

*(n=6)

8 PrecisionIntraday precision and Interday precision for AMLO, HCTZ and VALS was

done by analyzing three different concentrations (g/mL) within linearity ranges

and % RSD less than 2 given in Table Repeatiblity determined by six replicates

of sample were prepared at sample concentration by one analyst and analyzed on

same day. (Table 6.9)

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Table 33: Intraday precision for AMLO

WAVELENGTHCONC.

g/mLMEAN 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

22.130.0144

0.676

4 4.120.0211 0.512

5 5.230.0221 1.837

*(n=3) Table 34: Interday precision for AMLO

WAVELENGTHCONC.


237.6nm

2 2.020.00114 1.10

4 4.310.00154 0.45

5 4.220.00137 0.27

270.2nm

2 2.180.00305 1.17

4 4.070.00120 0.56

5 5.060.00260 0.26

249.2nm

2 2.180.00340 1.05

4 4.210.00231 0.50

5 5.350.00162 0.31

*(n=3)

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Table 35: Intraday precision for HCTZ

WAVELENGTHCONC.


237.6nm

15 15.210.0084 0.55

20 21.010.0043 0.68

25 24.870.0044 0.57

270.2nm

15 14.950.0034 0.90

20 20.090.0032 0.65

25 24.670.0042 0.57

249.2nm

15 15.020.0062 0.94

20 21.170.0055 0.64

25 26.010.0048 0.49

*(n=3)

Table 36: Interday precision for HCTZ

WAVELENGTHCONC.

g/mLMEANConc. S.D* % R.S.D

237.6nm

15 14.560.0018 0.81

20 20.170.0040 1.99

25 24.880.0050 2.02

270.2nm

15 14.760.0011 0.75

20 20.140.0016 0.57

25 25.180.0012 0.44

249.2nm

15 15.090.0026 0.83

20 20.310.0026 1.11

25 25.080.0025 0.89

*(n=3)

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Table 37: Intraday precision for VALS

*(n=3)

Table 38: Interday precision for VALS

*(n=3)

WAVELENGTH CONC. g/mL MEANCONC.S.D*%

R.S.D

237.6nm

15 15.210.0084 0.55

20 21.010.0043 0.68

25 24.870.0044 0.57

270.2nm

15 14.950.0034 0.90

20 20.090.0032 0.65

25 24.670.0042 0.57

249.2nm

15 15.020.0062 0.94

20 21.170.0055 0.64

25 26.010.0048 0.49

WAVELENGTH CONC.g/mL

MEANCONC.S.D* % R.S.D

237.6nm

15 14.560.0018 0.81

20 20.170.0040 1.99

25 24.880.0050 2.02

270.2nm

15 14.760.0011 0.75

20 20.140.0016 0.57

25 25.180.0012 0.44

249.2nm

15 15.090.0026 0.83

20 20.310.0026 1.11

25 25.080.0025 0.89

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Table 39: Repeatibilty

WAVE LENGTH DRUG

CONC.

g/mL

MEAN

CONC.g/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

*(n=6)

9 LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was

repeated six times and measured by mathematical equation given in section

(5.2.2.5). LOD value were 0.025 g/mL, 0.013 g/mL, 0.029 g/mL and LOQ

value were 0.078 g/mL, 0.041 g/mL, 0.089 g/mL for AMLO, HCTZ and

VALS respectively.

10AccuracyStandard 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%. % recovery was calculated from regression equation of the calibration

curve as shown in Table 6.8

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Table 40: Recovery study of AMLO, HCTZ and VALS

DRUG %level

Sample

conc.g/ml

Amt of

std.

added

g/ml

Total

Conc.g/ml

Amt

recoveredg/ml

%

Recovery

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 41: Validation parameters for AMLO, HCTZ and VALS

PARAMETERS AMLO HCTZ VALS

max 237.6nm 270.2nm 249.2nm

Linear Range g/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 g/mL 0.025 0.013 0.029

LOQ g/mL 0.078 0.041 0.089

% Recovery 99.19 99.13 99.14

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4.2.3.3 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.

Table 43: Analysis of Marketed Dosage Form

*(n=3)

4.2.4 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.

DRUG LABLE CLAIM(mg) % ASSAYSD*

AMLO 5 98.250.0781

HCTZ 12.5 98.820.0458

VALS 160 98.930.0404

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4.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD

FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ

AND VALS IN THEIR COMBINED DOSAGE FORM

4.3.1Instrumentation11HPTLC-Applicator Linomat 5(Camag) - Semiautomatic application, band

application by spray on technique (2 - 500l), twin trough glass

chamber(Camag) -( 20 x 10 cm), TLC scanner 3 (Camag) -Scanning speed up

to 100mm/s, Spectral range 190 800nm, U.V cabinet with dual

wavelength U.V lamp (Camag) - Dual wavelength 254 / 366nm, Stationary

Phase - Pre- coated Silica gel on aluminum sheet G60 F254 , Applicator syringe

(Hamilton, Bonaduz, Schweiz) - 100 L, Data Resolution-100m/step

Analytical Balance -Citizen CX 220 (Citizen Pvt. Ltd), Weighing capacity: 10to 220 mg

Sonicator-D-compact (Trans-O-sonic), Capacity: 2L4.3.2Materials and Methods4.3.2.1 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, API of AMLO, HCTZ, and VALS Standards

were kindly gifted by Torrent pharmceutcial Gujarat, India., Analytical

grade methanol (Central Drug House Pvt. LTD) was used. Marketed tablet

formulation Exforge hct (Novartis pharma stein AG, stein, Switzerland.)

Containing Amlodipine besylate (5mg), HCTZ (12.5mg) and Valsartan

(160mg)purchased from USA market.

4.3.2.2HPTLC 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 : 25-26C Wavelength of detection : 242 nm

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Slit dimension : 3x 0.20 mm (micro) Band width : 4 mm

Syringe capacity : 100 L4.3.2.3 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.

4.3.2.4 Preparation of Standard Stock Solution of AMLO25 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 g/mL of AMLO in methanol.

4.3.2.5Preparation of Standard Stock Solution of HCTZ25 mg HCTZ was weighed accurately and dissolved in 25 ml methanol. 1


produce 100 g/mL of HCTZ in methanol.

4.3.2.6 Preparation of Standard Stock Solution of VALS

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


produce 100 g/mL of VALS in methanol.

4.3.2.7 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.

4.3.2.8 Selection of wavelength for detection

Standard solution 500ng/spot of AMLO, HCTZ and VALS on developed

plate were scanned and selected 254 nm wavelength at which each drug

show considerable absorbance but in marketed formulation AMLO

proportion is very less compare to VALS. So that it was not feasible to

quantify in sample so selected 242nm detection wavelength for increasing

area of AMLO to quantify assay of sample.

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Fig.13: Over Lay UV Spectra of AMLO, HCTZ and VALS by HPTLC for

Selection of Wavelength

4.3.2.9 Method validation

Preparation of Linearity curve of AMLO, HCTZ and VALSSuitable 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 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.

PrecisionIntraday 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

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(450,600,750 ng/spot), and for VALS (1600, 2000, 2400 ng/spot) .Precision

measured in terms of %RSD

Repeatibilty

Sample solution of AMLO, HCTZ (500 ng/spot) and for VALS 2000 (ng/spot)

was spotted 6 times on the same plate and peak area was recorded. Repeatability

was measured in terms of %RSD.

LOD AND LOQFrom 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.

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

equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of

VALS) were weighed and transferred to 50ml volumetric flask and dissolved in

30 ml of methanol. Then 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 (5.5ml) was pipette out and transferred

to 10 ml volumetric flask. Volume was made up to the mark with methanol to

get a solution containing 55g/ml of AMLO, 98.21 g/mL of HCTZ and 1257.1

g/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 242 nm for

AMLO, HCTZ and VALS drugs. Amplitudes were substituted into respective

straight line equation to calculate percentage recovery of the drugs.

Robustness:The following parameters were changed one by one and their effect was

observed on system suitability. (Table)

a) Mobile phase composition Ethyl Acetate ( 5%)b) Wave length (242 2nm)c) Development distance (70mm5mm) Specificity

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Specificity of an analytical method is ability to measure specifically the analyte

of interest without interferences from blank and placebo.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.

4.3.2.10 Analysis of marketed formulation

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 g/mL concentration of AMLO, 125 g/mL HCTZ

and 1600 g/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 L of this solution was spotted on activated TLC

plate4.3.3 RESULTS AND DISCUSSION4.3.3.1 Method Development

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. Mobile phase trails are given Table

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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

Not

satisfactory

2

Acetone: Chloroform: Ethyl acetate:

Methanol (3:3:3:0.5, v/v/v/v),Run

length =80mm

Improper resolution

and poor Rfvalues

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 =

80mm

Not Good resolutionNot

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

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11

Ethyl Acetate: Methanol: Toluene

:glacial 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

splitted

Not

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

4.3.3.2Validation parameters Linearity

Linearity curve shows linearity in the range of 100-600 ng/spot for AMLO, 150-

900 ng/spot for HCTZ and for VALS 1200-3200 ng/spot. The correlation

coefficient (r2) was found to be 0.9945, 0.9926 and 0.9918 for AMLO, HCTZ

and VALS respectively. Calculate and record value of correlation co-efficient

(r), y-intercept, slope of regression line and residual sum of squares. (Figure

6.12) and results (Table)

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Table 45: Linearity of AMLO by HPTLC with UV detection

*(n=6)

Fig.14: Calibration curve of AMLO

Fig.15: Calibration curve of AMLO from Win CATS software

Sr. No.Conc.

(ng/spot)

Peak Area

RfMean SD* %RSD

1 100 108918.0 1.652 0.54

2 200 1873.16710.2 0.548 0.54

3. 300 2646.551.3 1.962 0.54

4. 400 3182.858.84 1.849 0.54

5. 500 3819.5136.63 0.959 0.54

6. 600 4375.93379.0 1.807 0.54

y = 6.5173x + 550.08

R = 0.9945

0

1000

2000

3000

4000

5000

0 200 400 600 800

P

e

a

k

A

r

e

a

Conc. ng/spot

Series1

Linear (Series1)

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Table 46: Linearity of HCTZ by HPTLC with UV detection

*(n=6)

Fig. 16: Calibration Curve for HCTZ

Sr. No.Conc.

(ng/spot)

Peak AreaRf

Mean SD* %RSD

1 150 1470.426.43 1.79 0.64

2 300 2547.93320.66 0.81 0.64

3 450 3650.33345.23 1.23 0.63

4 600 4284.16733.74 0.78 0.63

5. 750 5245.26737.84 0.72 0.65

6. 900 5953.137.03 0.52 0.65

y = 5.9829x + 707.78

R = 0.9927

0

1000

2000

3000

40005000

6000

7000

0 500 1000

P

e

a

k

A

r

e

a

Conc. ng/spot

Series1

Linear (Series1)

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

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Table 47: Linearity of VALS by HPTLC with UV detection

*(n=6)

Fig.18: Linearity Curve for VALS

Sr. No. Conc (ng/spot)

Peak Area

RfMean SD* %RSD

1 1200 108933.52 1.65 0.23

2 1600 1873.1619.51 0.54 0.23

3 2000 2646.593.04 1.96 0.23

4 2400 3182.8134.8 1.84 0.23

5 2800 3819.5175.6 0.95 0.27

6 3200 4375.93144 1.80 0.27

y = 1.6293x - 753.28

R = 0.9945

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 1000 2000 3000 4000

P

e

a

k

A

r

e

a

Conc. ng/spot

Series1

Linear (Series1)

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

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

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

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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.

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

(Rf=0.54), HCTZ (Rf=0.64) AND VALS (Rf=0.23)

PrecisionIntraday precision and Interday precision for AMLO, HCTZ and VALS was


and % RSD less than 2 given in Table.Repeatiblity determined by six replicates

of sample were prepared at sample concentration by one analyst and analyzed on


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

DrugConcentration

(ng/spot)

Peak AreaRfSD*

Mean SD* %RSD

AMLO

200 3201.631.50 1.98 0.540.015

500 3673.86718.431.65 0.560.013

600 4171.33325.79 0.99 0.540.011

HCTZ

450 3741.726.99 0.67 0.660.012

600 3624.918.43 1.20 0.640.014

750 365599.26 1.12 0.640.010

VALS

1600 8076.2894.57 0.23 0.230.021

2000 9343.43323.95 1.83 0.250.22

240010297.73133.15

1.400.240.024

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*(n=3)

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

*(n=3)

Table 50: Repeatability study of AMLO, HCTZ AND VALS

*(n=6)AccuracyStandard 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%. % recovery

was calculated from regression equation of the calibration curve as shown in Table

6.8

DrugConc.

(ng/spot)

Peak Area

Rf SD*Mean SD %RSD

AMLO

200 3201.664.16 2.00 0.540.015

500 3673.86760.64 1.65 0.550.011

600 4171.33341.56 0.99 0.540.013

HCTZ

450 3812.83325.8 0.67 0.660.010

600 4216.63350.91 1.20 0.640.012

750 5502.161.85 1.12 0.640.011

VALS

1600 8076.2819.17 0.23 0.260.02

2000 9343.433171.65 1.83 0.270.011

2400 10297.73144.49 1.40 0.270.015

Sr.no. Drug Peak AreaSD* %RSD

1. AMLO (500ng/spot) 291733.30 1.98

2. HCTZ(500ng/spot) 3609.7825.65 1.86

3. VALS (2000ng/spot) 7061.6199.95 0.84

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Table 51: Recovery study of AMLO, HCTZ and VALS

*(n=3)

LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was

repeated six times and measured by mathematical equation .LOD value were

2.95, 17.89, 70.90 and LOQ value 8.94, 53.9, 214.85 for AMLO, HCTZ and

VALS respectively.

Robustness

DRUG

INITIAL

CONC.

ng/spot

STD.

Added

ng/spot

TOTAL

CONC.

ng/spot

Accuracy

Conc. Recover

MeanSD*

%

RSD

%Recovery

Mean SD*

%

RSD

AMLO 55

44 99 99.141.073 1.08 100.14 1.32

55 110 109.390.66 0.60 99.440.745 0.74

66 121 120.810.760 0.62 99.840.76

0.76

HCTZ 98.21

78.57 177.47 176.80.346 0.19 99.090.932 0.92

98.21 196.42 199.122.124 1.08 99.790.761 0.76

117.8 216.81 215.980.945 0.43 100.140.675 0.67

VALS 1257.1

1005.6 2262.74 2357.417.091 0.13101.400.78

50.77

1257.1 2514.28 2551.7719.20 1.31 99.650.907 0.91

1508.56 2765.7 2796.6416.258 0.51 99.300.703 0.70

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Table 53: Robustness Study of AMLO, HCTZ and VALS

Sr.no. Parameter

Peak Area SD*

%RSD

AMLO HCTZ VALS AMLO HCTZ VALS

1.

Mobile Phase

composition

Ethyl Acetate

(5%)

1107.8

14.47

2119.9

38.8

13749.4

175.3

1.3 1.64 1.2

2.Wavelength

(2422nm)

1119.63

9.99

1743.6

17.2

1082

125.29

0.89 0.98 1.15

3.

Development

Distance

(705mm)

1073.43

10.6

1132.43

10.0

13430.1

133

0.93 1.02 0.99

SpecificitySpecificity is carried out by taken peak purity of standard and sample of each drug

and overlay standard and sample peak spectra to check specificity of each

individual drug peak .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 which was found pass. And results are shown in table 54.

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Fig.25: Peak purity spectra of AMLO

Fig.26: Peak purity spectra of HCTZ

Fig.27: Peak purity spectra of VALS

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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

3 Correlation 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

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

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4.3.3.2 Analysis of marketed formulation

The developed method was used to estimate AMLO, HCTZ and VALS in

combined dosage form. The percentage of AMLO, HCTZ and VALS was foundfrom the calibration curve.

Fig.28: HPTLC CHROMATOGRAM OF STANDARD MIXTURE

FIG.29: HPTLC CHROMATOGRAM OF MARKETED FORMULATION

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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 HCT

Drug Label

claim(mg)

Amt

estimated(mg)

Assay results

S.D*

AMLO 5 5.01

100.321.025 5.06

5 4.96

HCTZ 12.5 12.74

100.81.4312.5 12.65

12.5 12.40

VALS 160 162.27

100.271.57160 161.37

160 157.49

*(n=3)

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4.3.4 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.

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4.4 DEVELOPMENT AND VALIDATION OF RP-HPLC

METHOD FOR SIMULTANEOUS ESIMATION OF

AMLO, HCTZ AND VALS IN THEIR COMBINED

DOSAGE FORM

4.4.1 Instrumentation High performance liquid chromatography JASCO 200 Series, JASCO,

Inc. JAPAN, JASCO plus Pump, MX-2080-31 Mixer, Rheodyne model

7125 with 20 l fixed loop Injector , JASCO-UV- VIS 2075 PLUS Detector

with Borwin software version 1.50

PH Meter- 11 E/101E (Analytical labs scientific instrument Ltd., PH: 0 to14,Resolution: 0.01

PH, Accuracy: 0.01 P

H 14 digit

Analytical Balance- Citizen CX 220 (Citizen Pvt. Ltd), Weighing capacity:10 to 220 mg

Sonicator: D-compact,( Trans-o-sonic., Mumbai),Capacity: 2 Lit.4.4.2 Materials and methods4.4.2.1 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), API of AMLO, HCTZ, and VALS Standards were kindly

gifted by Torrent pharmceutcial Gujarat, India., Analytical grade methanol

(Central Drug House Pvt. LTD) was used. Marketed tablet formulation

Exforge hct (Novartis pharma stein AG, stein, Switzerland.) Containing

Amlodipine besylate (5mg), Hctz( 12.5 mg) and Valsartan (160mg)

purchased from USA market.

4.4.2.2Chromatographic Conditions4.4.2.2.1Optimized Chromatographic Conditions Stationary phase: Kromasil Column KR-5C 18 (250 mm 4.6mm i.d., 5m) 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.

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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 Min4.4.2.3Preparation of Mobile phaseMobile 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.

4.4.2.4Preparation of Standard Stock Solution of AMLO, HCTZ and VALS25 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 g/mL of AMLO

and 100 g/mL of HCTZ

4.4.2.5Preparation of ternary mixtures of AMLO, HCTZ and VALSSuitable aliquots of standard stock solution of AMLO, HCTZ and VALS

are mixed 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

g/mL was prepared for the calibration curve of three drugs.

4.4.2.6Method validation Preparation of Linearity curve

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

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

range of (5-45 g/mL). For estimation of VALS calibration curve (n=3) was

plotted in the range of (20-150 g/mL).Calibration curve of peak area v/s

concentration was plotted for the drug.

PrecisionIntraday and interday precision

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

three different concentrations for AMLO (5, g/ml 10, 15 g/ml), for HCTZ

(10, 15, 20 g/ml) and for VALS (80,100,120 g/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

RepeatabilityPeak area of sample solutions for AMLO, HCTZ, VALS (5, 15, 100 g/mL)

were taken by 6 times and find out the % RSD.

LOD AND LOQFrom 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 given is section ()

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

equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of

VALS) were weighed and transferred to 50ml volumetric flask and dissolved in

30 ml of methanol. Then 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 (0.2ml) was pipette out and transferred

to 10 ml volumetric flask. Volume was made up to the mark with methanol to

get a solution containing 2g/mL of AMLO, 4 g/mL of HCTZ and 64 g/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 232 nm for

AMLO, HCTZ and VALS drugs. Amplitudes were substituted into respective

straight line equation to calculate percentage recovery of the drugs.

RobustnessThe following parameters were changed one by one and their effect was

observed on system suitability.

a)Flow rate of mobile phase ( 0.2 ml) to 0.8 ml/min and 1.2 ml/min. (Table6.11)

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b)PH ( 0.05 absolute) to 3.65 and 3.75 (Table 6.12)c)Wave length (2nm) to 230 and 234

SpecifictySpecificity of an analytical method is ability to measure specifically the analyte

of interest without interferences from blank and placebo.

4.4.2.7Analysis of marketed formulationTotal 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

diluents ACN:Water (50:50) final solution containing 5 g/mL

concentration of AMLO, 12.5 g/mL HCTZ and 160 g/mL concentration

of VALS.

4.4.3 Results and Discussion4.4.3.1 Method development

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 andthe chromatographic parameters which give the best peak resolution were

selected for analysis

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

(60:40, v/v) PH

3.7

Peak was not

suitable for

quantitative

M.P was not

suitable

2



(50:50, v/v), PH

3.7

VALS RT greater

than 10 min M.P was not

suitable

3



(57:43, v/v) , TEA 0.1%, PH

3.7

Asymmetry

greater than 1.5

M.P was not

suitable

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4.

ACN : 0.025 M potassium


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

3.7

Asymmetry was

less as compare to

0.1% TEA

M.P was not

suitable

5.

ACN : 0.050 M potassium


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

3.7

Peak was suitable

for quantitative

M.P was

suitable

(Optimized)

Table 58: System suitability parameter by RP-HPLC method

Sr. no Parameters AMLO HCTZ VALS

1 Capacity Factor 0.6434 0.3695 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

4.4.3.2Validation parameters Linearity

Linearity curve shows linearity in the range of 100-600 g/mL for AMLO, 5-

45g/mL for HCTZ and for VALS 20-120 g/mL. The correlation coefficient (r2)

was found to be 0.9945, 0.9965, and 0.9971 for AMLO, HCTZ and VALS

respectively Calculate and record value of correlation co-efficient (r), y-intercept,

slope of regression line and residual sum of squares. (Figure 6.12) and results

(Table)

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Table 59: Linearity of AMLO by RP-HPLC with UV PDA

*(n=3)

Fig.35: Calibration curve for AMLO

Sr. No. Conc.

(g/mL)

Peak Area

Mean SD* %RSD

1 2 144942 1936.48 1.33

2 5 327329.51376.5 0.42

3 10 50262 2063 0.41

4 15 796769.36361.674 0.79

5 20 11170131489.079 0.13

6 25 13725909579.747 0.69

y = 53407x + 24828

R = 0.9945

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 10 20 30

P

e

a

k

A

r

e

a

Conc. g/ml

Series1

Linear (Series1)

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

*(n=6)

Fig.36: Calibration curve of HCTZ

y = 135283x + 114584

R = 0.996

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

0 10 20 30 40 50

P

ea

k

a

r

e

a

Conc. g/ml

Series1

Linear (Series1)

Sr. No.

Conc.

(g/mL)

Peak Area

Mean SD* %RSD

1 5 826897.34122.691 0.49

2 10 148119517761.38 1.19

3 15 197599515134.72 0.76

4 25 35574276076.3 0.45

5 35 503966041762.14 0.82

6 45 606955936571.47 0.60

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

*(n=6)

Fig.36: Calibration curve of VALS

Sr. No.Conc.

(g/mL)

Peak Area

Mean SD* %RSD

1 20 161341316557.68 1.02

2 40 325834232479.94 0.99

3 60 617468382500.03 1.33

4 80 80224449798.269 0.12

5 120 1303326245680.19 0.35

6 150 1589395296053.33 0.60

y = 112822x - 838391

R = 0.9971

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

0 50 100 150 200

P

e

a

k

A

r

ea

Conc. g/ml

Series1

Linear (Series1)

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

Fig.32: HPLC Chromatogram of HCTZ

Fig.33: HPLC chromatogram of AMLO

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Fig.26: HPLC chromatogram of standard mixture of AMLO, HCTZ and

VALS

Fig.38: Mixture Linearity Curve of AMLO, HCTZ AND VALS BY HPLC

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Precision:Intraday precision and Interday precision for AMLO, HCTZ and VALS was


and % RSD less than 2 Table.Repeatiblity determined by six replicates of

sample were prepared at sample concentration by one analyst and analyzed on


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

*(n=3)

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

*(n=3)

DrugConcentration

(g/mL)

Peak Area

Mean SD*

%RSD

AMLO

5 306648.72519.684 0.82

10 562226.71300.598 0.23

15 865629.75262.363 0.60

HCTZ

10 13668687087.164 0.51

15 219059445155.87 2.06

20 271234923962.09 0.88

VALS

80 772358530225.43 0.39

100 989746860556.46 0.61

120 13142484202472.7 1.54

Drug Concentration

(g/mL)

Peak Area

Mean SD %RSD

AMLO

5 309600.73256.28 1.05

10 502668.32644.688 0.52

15 773136.3253.4213 0.33

HCTZ

10 136914910733.71 0.78

15 211000710417.99 0.49

20 267864933853.87 1.26

VALS

80 777402543345.03 0.55

100 993643953407.12 0.53

120 1301815934631.55 0.26

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Table 64: Repeatability study of AMLO, HCTZ and VALS

Drug Conc. (g/mL) Peak AreaSD* %RSD

AMLO 5 29171968.42 0.71

HCTZ 15 18334406751.29 0.36

VALS 100 938193792487.03 0.98

*(n=6)

LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was

repeated six times and measured by mathematical equation .LOD value were

0.23 g/mL, 0.48 g/mL, 1.1 g/mL and LOQ value 0.71 g/mL, 1.47 g/mL,

3.3 g/mL for AMLO, HCTZ and VALS respectively.

SpecificityPeak purity spectra were taken of AMLO, HCTZ and VALS by UV-PDA

detection.Peak purity Front and Tail value given Table

Fig.25: Peak purity spectra of AMLO by UV-PDA

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Fig.25: Peak purity spectra of HCTZ by UV-PDA

Fig.25: Peak purity spectra of VALS by UV-PDA

Table 66: Specificity data of AMLO, HCTZ and VALS

Drugs Peak purity Front Peak purity Tail Peak

purity

AMLO 996.65 998.45 Specific

HCTZ 999.93 999.70 Specific

VALS 997.15 998.94 Specific

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AccuracyStandard 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%. % recoverywas calculated from regression equation of the calibration curve as shown in Table

6.8

Table 67: % Recovery study of AMLO, HCTZ and VALS

DRUG

INITIAL

CONC.

g/mL

STD.

Added

g/mL

TOTAL

CONC.

After

spiking

g /mL

ACCURACY

Conc.Recovered

MeanSD*

%

RSD

%Recovery

Mean SD*

%

RSD

AMLO 2

1.6 3.63.5833

0.0491.13

99.57333

0.321.33

2 44.0146

0.0461.42

101.4233

0.750.75

2.4 4.4 4.45660.037

0.69 101.42330.07

0.76

HCTZ 5

4 98.8766

0560.82

98.35333

0.010.95

5 109.9533

0.0370.49

98.35333

0.041.47

6 1110.9033

0.080.95

99.16667

0.760.76

VALS 64

51.2 115.2 117.8060.101

0.18 102.01670.074

0.74

64 128129.466

2.0851.61

101.0967

0.0561.56

76.8 140.8140.3733

0.8830.62

99.69

0.06370.63

*(n=3)

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RobustnessTable 68- Robustness study of AMLO

Parameters ChangeMean of Peak Area

%RSD%Assay

Flow Rate (0.2 ml/min)0.8 2936241.74 98.25

1.2 2904940.52 100.0

PH

( 0.05)

3.65 382701.71.53 99.82

3.75 210569.21.74 100.2

Wavelength ( 2 nm)

230 12836530.29 100.42

234 12489260.51 101.04

Table 69: Robustness study of HCTZ

Parameters Change Mean of Peak

Area%RSD

%Assay

Flow Rate

(0.2ml/min.)

0.8 19520661.17 98.44

1.2 681237.70.88 99.38

PH

(0.05)

3.65 13658011.55 101.59

3.75 12805860.56 98.15

Wave Length

( 2nm)

230 14799090.95 99.77

234 15599061.79 98.51

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Table 70: Robustness study of VALS

Parameters Change Mean %RSD %Assay

Flow Rate

(0.2 ml/min)

0.8 178625950.8 98.60

1.2 162600241.0 98.79

PH

( 0.05)

3.65 136887281.3 98.48

3.75 118075061.4 101.76

Wave Length

(2 nm)

230 117413080.8 98.45

234 97272071.30 98.43

Table 71: Summary of Validation parameters of RP-HPLC

Sr.

noParameters AMLO HCTZ VALS

1.Linearity range

(g/mL)2-25 5-45 20-150

2.

Regression

equation

y =53047x+24828 y=135283x+11458

4

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 (n=6)

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. Specificit

Specific Specific Specific

6. LOD (g/ml) 0.23 0.48 1.1

7. LOQ (g/ml) 0..71 0.1.47 3.3

8. % Recovery 99.571.33 to

101.420.75

98.350.19 to

99.16 0.76

99.69 0. 63

to

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Table 72: Analysis of Marketed Dosage Form

*(n=3)

Fig.39: HPLC Chromatogram of Marketed Dosage Form

DrugsLabel

claim(mg)

Amt

estimated(mg)

Assay results

% recovery S.D*

AMLO

5 4.95

98.660.365 4.91

5 4.92

HCTZ

12.5 12.66

99.870.8712.5 12.44

12.5 12.39

VALS

160 158.31

100.621.49160 161.77

160 162.92

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4.4.4 ConclusionBy 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 dosageform. 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

4.5 COMPARISON OF UV-VIS SPECTROPHOTOMETRIC,HPTLC AND RP-HPLC METHOD

4.5.1 Comparison of developed chromatographic method

Comparison of developed methods (UV-VIS spectrophotometric, HPTLC

and RP-HPLC was performed by applying Student-ANOVA-test (single

factor)

Table73: 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 HCT

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

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Table 74: Comparison of three Methods by ANOVA Test

Brand name Drugs Fcal F crit

Exforge HCT

AMLO 1.63 4.066

HCTZ 3.68 4.066

VALS 1.18 4.066

4.5.2 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 Fcrit.

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