Chapter5

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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 FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND VALS IN THEIR COMBINED DOSAGE FORM

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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 AMLO HCTZ VALS

Reported melting point (0C) 199-201 273-275 116-117

Observed melting point (0C) 200-202 274-275 117-118

Table 25: Melting points of drugs 5.1.2 UV spectra of AMLO, HCTZ and VALS UV- spectrum of AMLO (20g/mL), HCTZ (20 g/ mL) and VALS (20 g/ 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.

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

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

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

Table 26: Wavelength maxima for AMLO, HCTZ and VALS Drug AMLO HCTZ VALS Reported maxima 239nm, 238nm 225nm, 271nm, 317nm 249nm Recorded maxima 237.6nm,210.8nm 270.2nm,316.6nm 249.2nm, 206nm, 243.8nm

5.1.3 Determination of Infrared (IR) Spectra:

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Fig.8: Infrared spectrum of AMLO Table 27: Justification for IR Frequency (cm-1) Found for AMLO SPECIFICATION OF AMLO -NH2 stretching -C-Cl C-H bending C=O Ester -C-O Ether THEORITCAL WAVE NUMBER(CM-1) (85) 3500-3100 785-540 1465-1375 1750-1730 1300-1000 RECORDED WAVE 3372 NUMBER(CM-1) 752.102 1440.56 1765.99 1265.07

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Fig. 9: Infrared spectrum of HCTZ Table 28: Justification for IR Frequency (cm-1) Found for HCTZ SPECIFICATION FOR HCTZ (S=O) C-H stretch Mono substituted benzene -C-N - Amine Amide group THEO RITICAL WAVE NUMBER (CM-1) (85) 1050 3000-2850 900-690 1350-1000 1680-1630 RECORDED WAVE NUMBER (CM-1) 1078.98 2963.09 996.053 1206.26 1601.59

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Fig.10: Infrared spectrum of VALS Table 29: Justification for IR Frequency (cm-1) Found for HCTZ

SPECIFICATION OF FOR VALS C-H stretch ketone (C=O) stretch -NH2 stretching Carboxylic acid(-COOH) -N-H Stretch

THEORITICAL WAVE NUMBER (CM-1)(85) 3000-2850 1750-1730 3200-3500 3400-2400 1640-1550

RECORDED WAVE NUMBER (CM-1) 2834.85 1730.8 3238.86 2577.4 1523.49

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5.2

DEVELOPMENT

AND

VALIDATION

OF

UV-VIS

SPECTROPHOTOMETRIC ESIMATION OF AMLO,

METHOD FOR SIMULTANEOUS 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) (160mg) Brand name : exforge hct, name of manufacturer and Valsartan

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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 g/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 g/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 (max of AMLO), 249.2 nm (max of VALS) and 270.2 nm (max 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 g/ml for AMLO, 5, 10, 15, 20, 25 g/ml for HCTZ and 10, 20, 30, 40, 50 g/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.
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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 (20g/ml), HCTZ (20g/ml) and VALS (20g/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

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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 g/ml at 237.6nm as well as estimation of HCTZ, calibration curve (n=5) was plotted in the range of 5-25 g/ml at 270.2nm and For estimation of VALS, calibration

curve (n=6) was plotted in the range of 10-50 g/ml at 249.2 nm. Linearity curve shows linearity in the range of 2-20 g/ml for AMLO, 5-25 g/ml for HCTZ and 1050 g/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 g/ml for AMLO and 15, 20, 25, g/ml for HCTZ and for VALS 30,40,50 g/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 g/ml for AMLO AND 15, 20, 25, g/ml for HCTZ and for VALS 30, 40, 50 g/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. 4g/ml of AMLO, 10 g/ml for HCTZ and 20 g/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.
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LOD = 3.3 x /S LOQ = 10 x /S Where, = 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 5g/ml of AMLO, 12.5 g/ml and 160 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 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)
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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 g/ml for AMLO, 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.

Fig 12: Linearity Curve for AMLO at 237.6 nm

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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 237.6nm Conc. g/ml 2 4 5 10 15 20 Absorptivity MeanAbs. S.D* 0.0450.016 0.1220.018 0.1590.023 0.3460.011 0.5580.012 0.7070.015 320 % RSD 1.55 1.47 1.44 0.31 0.17 0.21 270.2nm MeanAbs S.D* 0.0130.0021 0.0240.0024 0.0420.0016 0.0560.0013 0.0640.0017 0.0780.0016 177.7 % RSD 1.61 1.60 1.80 1.12 1.42 0.95 249.2nm Mean Abs.S.D* 0.0210.0013 0.0650.0017 0.0880.0014 0.2010.0015 0.3280.0012 0.4220.0016 178.63 % RSD 1.61 1.53 1.13 0.74 0.30 0.37

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Fig. 12: Linearity Curve for HCTZ at 270.2 nm

Fig. 12: Calibration Curve for HCTZ at 270.2 nm

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

237.6nm Conc. g/ml MeanAbs. %RSD S.D

*

270.2nm Mean Abs. S.D*

249.2nm Mean Abs.

%RSD S.D
*

%RSD

5 10 15 20 25 Absorptivity

0.0090.001 0.0420.001 0.0760.001 0.1130.001 0.1660.006 45.88

1.33 1.57 1.71 1.32 1.61

0.2970.002 0.6180.002 0.9250.001 1.2450.003 1.5640.003 615.55

0.70 0.37 0.16 0.12 0.12

0.0320.001 0.0830.001 0.1370.001 0.1920.002 0.2650.002 88.086

1.75

1.56 1.16 0.78 0.37

Fig.13: Calibration Curve of VALS at 249.2 nm

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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 237.6nm Conc. g/ml Mean Abs.S.D* % RSD Mean Abs.S.D* % RSD Mean Abs.S.D* 0.3100.02 7 0.5830.02 5 0.8950.02 1 1.1670.03 2 % RSD 270.2nm 249.2nm

10 20 30 40 50 Absorptivity

0.3260.003

0.98 0.34 0.25 0.30 0.97

0.1460.002 0.2790.004 0.4330.002 0.5530.003

1.02 0.53 0.34 0.56

0.80 0.42 0.23 0.25

0.6340.0022 0.9780.002 1.2630.0038 1.580.0015 320.07

0.6820.002 0.30 1 141.02

1.4620.02 0.14 1 295.75

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2) Precision Intraday 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. Table 33: Intraday precision for AMLO CONC. g/ml 2 237.6nm 4 5 2 270.2nm 4 5 2 249.2nm 4 5

WAVELENGTH

MEAN CONC.S.D* 1.990.0281 0.0281 3.880.0168 5.050.0129 0.0168 1.870.0135 0.0129 4.170.0132 0.0135 0.0132 5.020.0152 2.130.0144 0.0152 4.120.0211 5.230.0221

% R.S.D 1.41 0.43 0.25 0.72 0.31 0.30 0.676 0.512 1.837

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Table 34: Interday precision for AMLO WAVELENGTH CONC. g/ml 2 237.6nm 4 5 2 270.2nm 4 5 2 249.2nm 4 5 Table 35: Intraday precision for HCTZ WAVELENGTH CONC. g/ml 15 20 25 15 270.2nm 20 25 15 249.2nm 20 25 MEAN CONC.S.D* 15.210.0084 21.010.0043 24.870.0044 14.950.0034 20.090.0032 24.670.0042 15.020.0062 21.170.0055 26.010.0048 % R.S.D 0.55 0.68 0.57 0.90 0.65 0.57 0.94 0.64 0.49 MEAN CONC.S.D* 2.020.00114 4.310.00154 4.220.00137 2.180.00305 4.070.00120 5.060.00260 2.180.00340 4.210.00231 5.350.00162 % R.S.D 1.10 0.45 0.27 1.17 0.56 0.26 1.05 0.50 0.31

237.6nm

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Table 36: Interday precision for HCTZ WAVELENGTH CONC. g/ml 15 237.6nm 20 25 15 270.2nm 20 25 15 249.2nm 20 25 MEAN Conc. S.D* 14.560.0018 20.170.0040 24.880.0050 14.760.0011 20.140.0016 25.180.0012 15.090.0026 20.310.0026 25.080.0025 % R.S.D 0.81 1.99 2.02 0.75 0.57 0.44 0.83 1.11 0.89

Table 37: Intraday precision for VALS

WAVELENGTH

CONC. g/ml 15

MEAN CONC.S.D* 15.210.0084 21.010.0043 24.870.0044 14.950.0034 20.090.0032 24.670.0042 15.020.0062 21.170.0055 26.010.0048

% R.S.D 0.55 0.68 0.57 0.90 0.65 0.57 0.94 0.64 0.49

237.6nm

20 25 15

270.2nm

20 25 15

249.2nm

20 25

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Table 38: Interday precision for VALS

WAVELENGTH

CONC. g/ml 15

MEAN CONC.S.D* 14.560.0018 20.170.0040 24.880.0050 14.760.0011 20.140.0016 25.180.0012 15.090.0026 20.310.0026 25.080.0025

% R.S.D 0.81 1.99 2.02 0.75 0.57 0.44 0.83 1.11 0.89

237.6nm

20 25 15 20

270.2nm

25 15 20

249.2nm 25

Table 39: Repeatibilty WAVE LENGTH DRUG AMLO 237.6nm HCTZ VALS AMLO 270.2nm HCTZ VALS AMLO 249.2nm HCTZ VALS 3) LOD and LOQ
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CONC. g/ml 4 10 20 4 10 20 4 10 20

MEAN CONC. g/ml 4.2 10.12 20.13 3.94 9.93 19.78 4.15 10.21 20.11

% R.S.D 0.27 0.32 1.21 0.53 0.46 1.64 0.51 0.28 1.59

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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 LOD LOQ AMLO 0.025 g/mL 0.078 g/mL HCTZ 0.013 g/mL 0.041 g/mL VALS 0.029 g/mL 0.089 g/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 Sample conc. g/ml Amt of std. added g/ml 4 5 6 8 10 12 16 20 24 Total Conc. g/ml 9 10 11 18 20 22 36 40 44 Amt % recovered Recovery g/ml

DRUG %level

80 AMLO 100 120 80 HCTZ 100 120 80 VALS 100 120

5 5 5 10 10 10 20 20 20

8.856 10.05 10.93 17.82 19.78 21.89 35.47 39.86 43.65

98.4 100.5 99.61 99.00 98.9 99.5 98.53 99.67 99.22

Table 42: Validation parameters for AMLO, HCTZ and VALS

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PARAMETERS max Linear Range g/ml Correlation coefficient R2 Repeatability% RSD Intraday precision % RSD Interday precision % RSD LOD g/ml LOQ g/ml % Recovery

AMLO 237.6nm 2-20 0.9997 0.705 0.523 0.727 0.025 0.078 99.19

HCTZ 270.2nm 5-25 0.999 0.154 0.781 1.021 0.013 0.041 99.13

VALS 249.2nm 10-50 0.999 1.413 0.539 0.489 0.029 0.089 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. Table 43: Analysis of Marketed Dosage Form

DRUG

LABLE CLAIM (mg)

% ASSAYSD*

AMLO HCTZ VALS

5 12.5 160

98.250.0781 98.820.0458 98.930.0404

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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.

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5.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND VALS IN THEIR COMBINED DOSAGE FORM
5.3.1.1 Instrumentation 1) HPTLC Camag Applicator Linomat 5: Semiautomatic application, Band application by spray on technique (2 - 500l) 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 l Applicator syringe (Hamilton, Bonaduz, Schweiz) Data Resolution: 100m/step 2) Spectrometer 3) 4) Model : SHIMADZU 2450 double beam spectrometer, version 2.21 Slit width : 1 nm Analytical Balance Model : keroy Manufacturer: keroy (Balance) pvt. Ltd. Capacity : 0.0001 to 100 g 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)
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Brand name: Exforge HCTZ, Name of manufacturer 5.3.1.3 5.3.1.4 API Amlodipine Besylate (AMLO) gifted sample from API Hydrochlorthiazide (HCTZ) gifted sample API Valsartan (VALS) gifted sample 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 : 25-26C Wavelength of detection : 242 nm Slit dimension : 3x 0.20 mm (micro) Band width : 4 mm Syringe capacity : 100 L 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 g/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 g/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 g/mL of VALS in methanol.

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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 105C 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 100l 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.

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5.3.2.8 1)

Method validation Preparation of Linearity curve 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 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%).
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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 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 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.
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Table 44: Observation and remarks of mobile phase optimization Sr. No. Trials Chloroform : methanol : toluene : glacial acid (6:2:1:0.1, v/v/v/v),Run length =80mm Acetone :chloroform :Ethyl acetate : methanol (3:3:3:0.5, v/v/v/v),Run length =80mm Ethyl Act: methanol:Amm.sol (7.5:2.5:0.5, v/v/v/v),Run length = 80mm Chloroform: Methanol : Amm.sol (7.5:2:5:0.5, v/v/v/v),Run length = 80mm Ethyl Acetate :Methanol : TEA 5 (7.5: 2.5:0.5, v/v/v/v), Run length =80mm ACN :Methanol: TEA 6 (7.5:2.5:0.5, v/v/v),Run length= 80mm Observation AMLO, HCTZ and VALS were close to solvent front,Improper resolution Improper resolution and HCTZ run slovent front, poor Rf values of VALS Very good separation but Diffused spot of VALS Remarks Not satisfactory

Not satisfactory

Good but Not satisfactory Not Satisfactory

Not Good resolution,

VALS spot was less diffused but poor Rf value of VALS

Not satisfactory

Closeness b/w HCTZ and VALS and diffused spot of VALS

Not satisfactory

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

Cyclohexane : Methanol:Ammonia 8 ( 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

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ACN :Methanol: TEA 9. (7.5:2.5:0.5, v/v/v),Run length= 80mm Ethyl Acetate :Methanol: Toluene: Ammonia (6:3:3:0.5, v/v/v/v),Run length= 80mm Ethyl Acetate :Methanol :Toluene :glacial acid (7.5:3.5:2.5:0.1, v/v/v/v), Run length= 80mm Chloroform :Methanol :Toluene :glacial acid (7.5:3:2.5:0.1, v/v/v/v),Run length: 80mm Ethyl acetate : Methanol: Toluene :Ammonia (7.5:3:2.5:1, v/v/v/v),Run length= 80mm Ethyl acetate :Methanol : Toluene :Ammonia (7.5:3:2:0.8, v/v/v/v),Run length =80mm Ethyl acetate :Methanol : Toluene :Ammonia (7.5:3:2:0.8, v/v/v/v), Run length =70mm

Closeness b/w HCTZ and VALS and diffused spot of VALS Good resolution but VALS having less tailing VALS spot was not diffused but poor Rf value of AMLO

Not satisfactory

10

Not good separation

11

Not satisfactory

12

AMLO spot was splitted

Not satisfactory Resolution was less b/w AMLO and HCTZ Very good separation

13

Good resolution but was not reproducible Good resolution and reproducible but VALS still having less tailing

14

15

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.

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Table 45: Calibration data of AMLO by HPTLC with UV detection Conc. (ng/spot) 100 200 300 400 500 600 Peak Area(n=6) Mean SD* 108918.0 1873.16710.2 2646.551.3 3182.858.84 3819.5136.63 4375.93379.0 %RSD 1.652 0.548 1.962 1.849 0.959 1.807 Rf 0.54 0.54 0.54 0.54 0.54 0.54

Sr. No.

1 2 3. 4. 5. 6.

5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0 100 200 300 400 500 600 700

y = 6.534x + 547.4 R = 0.994

Fig.14: Linearity curve for AMLO

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

Table 46: Calibration data of HCTZ by HPTLC with UV detection Conc. (ng/spot) 150 300 450 600 750 900 Peak Area Mean SD* 1470.426.43 2547.93320.66 3650.33345.23 4284.16733.74 5245.26737.84 5953.137.03 %RSD 1.79 0.81 1.23 0.78 0.72 0.52 0.64 0.64 0.63 0.63 0.65 0.65

Sr. No. 1 2 3 4 5. 6.

Rf

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7000 6000 5000 4000 3000 2000 1000 0 0 200 400 600 800 1000

y = 5.931x + 744.6 R = 0.992

Concentration (ng/spot)

Fig. 16: Linearity Curve for HCTZ

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

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Peak Area Sr. No. Conc (ng/spot) Mean SD* %RSD 1 2 3 4 5 6 1200 1600 2000 2400 2800 3200 108933.52 1873.1619.51 2646.593.04 3182.8134.8 3819.5175.6 4375.93144 1.65 0.54 1.96 1.84 0.95 1.80 0.23 0.23 0.23 0.23 0.27 0.27 Rf

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

16000 14000 12000 10000 8000 6000 4000 2000 0 0 500 1000 1500 2000 2500 3000 3500

y = 3.480x + 2356 R = 0.991

Fig.18: Linearity Curve for VALS

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

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

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)

2) Precision
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Table 48: Intraday precision AMLO, HCTZ AND VALS by HPTLC with UV Drug Concentration (ng/spot) 200 AMLO 500 600 450 HCTZ 600 750 1600 VALS 2000 2400 Peak Area Mean SD* 3201.631.50 3673.86718.43 4171.33325.79 3741.726.99 3624.918.43 365599.26 8076.2894.57 9343.43323.95 10297.73133.15 %RSD 1.98 1.65 0.99 0.67 1.20 1.12 0.23 1.83 1.40 RfSD* 0.540.015 0.560.013 0.540.011 0.660.012 0.640.014 0.640.010 0.230.021 0.250.22 0.240.024

Table 49: Interday precision AMLO, HCTZ AND VALS by HPTLC Peak Area Mean SD* 3201.664.16 3673.86760.64 4171.33341.56 3812.83325.8 4216.63350.91 5502.161.85 8076.2819.17 9343.433171.65 10297.73144.49 %RSD 2.00 1.65 0.99 0.67 1.20 1.12 0.23 1.83 1.40

Drug

Conc. (ng/spot) 200 500 600 450 600 750 1600 2000 2400

Rf SD* 0.540.015 0.550.011 0.540.013 0.660.010 0.640.012 0.640.011 0.260.02 0.270.011 0.270.015

AMLO

HCTZ

VALS

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Table 50: Repeatibilty study of AMLO, HCTZ AND VALS

Sr.no. 1 2. 3. Accuracy

Drug AMLO (500ng/spot) HCTZ(500ng/spot) VALS (2000ng/spot)

Peak AreaSD* 291733.30 3609.7825.65 7061.6199.95

%RSD 1.98 1.86 0.84

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 INITI AL CON C. ng/spo t AMLO 55 STD. Adde d ng/sp ot 44 55 66 78.57 HCTZ 98.21 98.21 117.8 1005.6 VALS 1257.1 1257.1 1508.56 TOTA L CONC . ng/spo t 99 110 121 177.47 196.42 216.81 2262.7 4 Accuracy Conc. Recovered Mean SD* 99.141.07 109.390.66 120.810.76 176.80.34 199.122.12 215.980.94 2357.417.09 1.08 0.60 0.62 0.19 1.08 0.43 0.13 Mean SD* 100.14 1.32 99.440.74 5 99.840.7 65 99.090.93 2 99.790.761 100.140.675 101.400.7 85 99.650.907 99.300.703 1.32 0.74 0.76 0.92 0.76 0.67 0.77 0.91 0.70 % RSD %Recover y %R SD

DRUG

2514.28 2551.7719.24 1.31 2765.7 2796.6416.25 0.51

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

Parameter LOD LOQ

AMLO (ng/spot) 2.95 8.94

HCTZ(ng/spot) 17.89 53.9

VALS(ng/spot) 70.90 214.85

Table 53: Robustness Study of AMLO, HCTZ and VALS Peak Area SD * Parameter AMLO Mobile Phase composition Ethyl Acetate (5%) Wavelength (2422nm) HCTZ VALS
AMLO HCTZ VALS

Sr.no .

%RSD

1.

1107.8 14.47

2119.938.8

13749.4 175.3

1.3

1.64

1.2

2.

1119.63 9.99

1743.617.2

1082 125.29

0.89

0.98

1.15

3.

Developmen t Distance 1073.43 10.6 (705mm)

1132.431.0

13430.1 133

0.93

1.02

0.99

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Specificity:

Fig.25: Peak purity spectra of AMLO

Fig.26: Peak purity spectra of HCTZ

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Fig.27: Peak purity spectra of VALS

Table: 54 specificty data of AMLO, HCTZ and VALS

Drugs AMLO HCTZ VALS

Co-relation r(s,m) 0.99906 0.99958 0.99966

Co-relation r (m,e) 0.9994 0.9976 0.999

Peak purity Pass pass pass

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

Sr.no 1 2

Parameters Linearity range (ng/spot) Regression equation

AMLO 100-600 y= 6.534x+547.48

HCTZ 150-900 y =5.931x+744.6

VALS 1200-3200 Y=3.48x+2356

Correlation coefficient (r2) Intercept Slope Precision Intraday % RSD (n = 3)

0.9945

0.9926

0.9918

4 5

6.534 547.48

5.931 744.6

3.48 2356

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

Interday % RSD (n = 3) Repeatability of measurements% RSD (n=6)

7 8 9

Limit of detection Limit of quantification Specificity

2.95 (ng/spot) 8.94(ng/spot) Pass

17.84 (ng/spot) 53.9 (ng/spot) Pass

70.90 (ng/spot) 214.85(ng/spot) 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.
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Fig.28: HPTLC CHROMATOGRAM OF STANDARD MIXTURE

Fig.29: HPTLC CHROMATOGRAM OF MARKETED SAMPLE

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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 HCTZ Drug Label claim(mg) Amt estimated(mg) 5.01 5.06 4.96 12.74 12.65 12.40 162.27 161.37 157.49 100.271.57 100.81.43 100.321.02 Assay results S.D*

AMLO

5 5 5

HCTZ

12.5 12.5 12.5

VALS

160 160 160

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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.

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5.4 DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND VALS IN THEIR COMBINED DOSAGE FORM
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 l 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

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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 API Amlodipine Besylate (AMLO) gifted sample from API Hydrochlorthiazide (HCTZ) gifted sample API Valsartan (VALS) gifted sample

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., 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. 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.

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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 g/ml of AMLO and 100 g/ml of HCTZ 5.4.2.5 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 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.

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Table 57: Observation and remarks of mobile phase optimization

Sr. no.

Mobile phase composition ACN :0.025 M potassium dihydrogen ortho phosphate (60:40v/v) PH 3.7 ACN :0.025 M potassium dihydrogen ortho phosphate (50:50v/v), PH 3.7

Inference Peak was not suitable for quantitative VALS RT greater than 10 min

Conclusion

M.P was not suitable

M.P was not suitable

ACN :0.025 M potassium dihydrogen ortho phosphate (57:43v/v) , TEA 0.1%, PH 3.7 ACN : 0.025 M potassium dihydrogen ortho phosphate (43:57v/v) TEA 0.2%, PH 3.7

Asymmetry greater than 1.5

M.P was not suitable

4.

Asymmetry was less as compare to 0.1% TEA

M.P was not suitable

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)

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

Fig.32: HPLC Chromatogram of HCTZ

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Fig.33: HPLC chromatogram of standard drug of AMLO

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

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Table 58: System suitability parameter by RP-HPLC method Sr. no Parameters AMLO HCTZ VALS

1 2. 3. 4. 5.

Capacity Factor Tailing factor Resolution factor Theoretical plates % RSD of Peak Area

0.3695 1.30 3.175 5378 0.48

0.6434 1.41 6357 1.85

3.4130 1.05 10.37 4886 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 g/mL). For estimation of HCTZ calibration curve (n=3) was plotted in the range of (5-45 g/mL). For estimation of range of VALS calibration curve (n=3) was plotted in the

(20-150 g/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, 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.

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Repeatability In the repeatability the peak area of sample solutions for AMLO, HCTZ, VALS (5, 15, 100 g/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 g/mL for AMLO, 4 g/ml for HCTZ and for VALS 64 g/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 g/ml concentration of AMLO ,12.5 g/ml HCTZ and 160 g/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.

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Table 59: Calibration data of AMLO by RP-HPLC with UV PDA detection Peak Area Sr. No. Conc. (g/ml) Mean SD* 1 2 3 4 5 6 2 5 10 15 20 25 144942 1936.48 327329.51376.5 50262 2063 796769.36361.674 11170131489.079 13725909579.747 %RSD 1.33 0.42 0.41 0.79 0.13 0.69

1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 5

y = 53407x + 24828 R = 0.994

10

15

20

25

30

Fig.35: Linearity curve for AMLO

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Table 60: Calibration data of HCTZ by RP-HPLC with UV PDA detection Peak Area Mean SD* 1 2 3 4 5 6 5 10 15 25 35 45 826897.34122.691 148119517761.38 197599515134.72 35574276076.3 503966041762.14 606955936571.47 %RSD 0.49 1.19 0.76 0.45 0.82 0.60

Sr. No.

Conc. (g/ml)

7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0 10 20 30 40 50

y = 13528x + 11458 R = 0.996

Fig.36: Linearity curve for HCTZ

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Table 61: Calibration data of VALS by RP-HPLC with PDA UV detection Peak Area Sr. No. Conc (g/ml) Mean SD* 1 2 3 4 5 6 20 40 60 80 120 150 161341316557.68 325834232479.94 617468382500.03 80224449798.269 1303326245680.19 1589395296053.33 %RSD 1.02 0.99 1.33 0.12 0.35 0.60

18000000 16000000 14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 0

y = 11282x - 83839 R = 0.997

50

100

150

200

Fig. 37: Linearity curve for VALS

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

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

Drug

Concentration (g/ml) 5

Peak Area Mean SD* 306648.72519.684 562226.71300.598 865629.75262.363 13668687087.164 219059445155.87 271234923962.09 772358530225.43 989746860556.46 13142484202472.7 %RSD 0.82 0.23 0.60 0.51 2.06 0.88 0.39 0.61 1.54

AMLO

10 15 10

HCTZ

15 20 80

VALS

100 120

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2) Precision Table 63: Intraday precision data of AMLO, HCTZ AND VALS by HPLC Peak Area Drug Concentration (g/ml) 5 AMLO 10 15 10 HCTZ 15 20 80 VALS 100 120 Mean SD* 309600.73256.28 502668.32644.688 773136.3253.4213 136914910733.71 211000710417.99 267864933853.87 777402543345.03 993643953407.12 1301815934631.55 %RSD 1.05 0.52 0.33 0.78 0.49 1.26 0.55 0.53 0.26

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Table 64: Repeatibilty study of AMLO, HCTZ and VALS

Drug

Conc. (g/ml)

Peak AreaSD*

%RSD

AMLO

29171968.42

0.71

HCTZ

15

18334406751.29

0.36

VALS

100

938193792487.03

0.98

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

DRUG

INITI AL CON C. g/ml

STD. Adde d g/m l

TOTA L CON C. After spikin g g /ml

ACCURACY

Conc. Recovered MeanSD * 3.5833 0.049 4.0146 0.046 4.4566 0.037 8.8766 056 9.9533 0.037 10.9033 0.08 117.806 0.101 129.466 2.085 140.3733 0.883

% RSD

%Recovery Mean SD*

% RSD

1.6 AMLO 2 2 2.4 4 HCTZ 5 5 6 51.2 VALS 64 64 76.8

3.6 4 4.4 9 10 11 115.2 128 140.8

1.13 1.42 0.69 0.82 0.49 0.95 0.18 1.61 0.62

99.57333 0.32 101.4233 0.75 101.4233 0.07 98.35333 0.01 98.35333 0.04 99.16667 0.76 102.01670. 074 101.0967 0.056 99.69 0.0637

1.33 0.75 0.76 0.95 1.47 0.76 0.74 1.56 0.63

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Table 66: LOD and LOQ Parameter AMLO HCTZ VALS

LOD

0.23 g/ml

0.48 g/ml

1.1

LOQ

0.71 g/ml

1.47 g/ml

3.3

Table 67- Robustness study of AMLO

Parameters

Change

Mean of Peak Area %RSD

%Assay

0.8 Flow Rate (0.2 ml/min) 1.2

2936241.74

98.25

2904940.52

100.0

3.65 PH ( 0.05) 3.75

382701.71.53

99.82

210569.21.74

100.2

230 Wavelength ( 2 nm) 234

12836530.29

100.42

12489260.51

101.04

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Table 68: Robustness study of HCTZ

Parameters

Change

Mean %RSD

%Assay

Flow Rate (0.2ml/min.)

0.8

19520661.17

98.44

1.2

681237.70.88

99.38

3.65 PH (0.05) 3.75

13658011.55

101.59

12805860.56

98.15

Wave Length ( 2nm)

230

14799090.95

99.77

234

15599061.79

98.51

Table 69 Robustness study of VALS Parameters Flow Rate (0.2 ml/min) Change 0.8 1.2 3.65 PH ( 0.05) 3.75 230 234 118075061.4 117413080.8 97272071.30 101.76 98.45 98.43 Mean %RSD 178625950.8 162600241.0 136887281.3 %Assay 98.60 98.79 98.48

Wave Length (2 nm)

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Table 70: Summary of Validation parameters of RP-HPLC

Sr.no

Parameters Linearity range (g/ml) Regression equation Correlation coefficient (r2) Precision Intraday % RSD (n = 3)

AMLO

HCTZ

VALS

1.

2-25

5-45

20-150

2.

y y=135283x+114584 =53047x+24828

y = 112822x83839

3.

0.9945

0.9967

0.9971

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

4.

Interday % RSD (n = 3) Repeatability of measurements % RSD

5.

Specificity LOD (g/ml) LOQ (g/ml)

Specific

Specific

Specific

6.

0.23

0.48

1.1

7.

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 1002.010.074

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SPECIFICITY

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Table 71: Analysis of Marketed Dosage Form Label claim(mg) 5 AMLO 5 5 12.5 HCTZ 12.5 12.5 160 VALS 160 160 Amt estimated(mg) 4.95 4.91 4.92 12.66 12.44 12.39 158.31 161.77 162.92 100.621.49 99.870.87 98.660.36 Assay results % recovery S.D*

Drugs

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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

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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

% Assay results Brand name Drugs U.V Exforge HCTZ AMLO 98.66 HPTLC 100.28 RP-HPLC 99.07

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98.45 100.3 98.82 HCTZ 98.40 98.20 98.33 VALS 98.75 99.20

101.37 98.79 101.97 101.23 99.26 98.59 99.15 101.61

98.36 98.55 100.8 99.52 99.13 101.10 101.82 98.94

Table 49 - Comparison of three Methods by ANOVA Test Brand name Drugs AMLO Exforge HCTZ HCTZ VALS Fcal 1.63 3.68 1.18 F crit 4.066 4.066 4.066

5.5.2 Conclusion
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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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