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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

Drug AMLO HCTZ VALS

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

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

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

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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 AMLO HCTZ VALS Reported maxima 239nm, 238nm 225nm, 271nm, 317nm 249nm Recorded maxima 237.6nm,210.8nm, 330nm 270.2nm,316.6nm 249.2nm, 206nm, 243.8nm

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4.1.3 Determination 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 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 NUMBER(Cm-1) 3372 752.102 1440.56 1765.99 1265.07

Fig. 9: Infrared spectrum of HCTZ

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Fig. 9: Reported Infrared spectrum of HCTZ

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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

Table 28: IR Frequency (Cm-1) for HCTZ Fig.10: Infrared spectrum of VALS

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Fig.10: Reported Infrared spectrum of VALS

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Table 29: IR Frequency (Cm-1) for VALS

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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4.2

DEVELOPMENT

AND

VALIDATION

OF

UV FOR

SPECTROPHOTOMETRIC

METHOD

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, version2.21 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), 4.2.2 Materials and Methods 4.2.2.1 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. 4.2.2.2 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 of each drug. 4.2.2.3 Preparation of calibration curve of AMLO, HCTZ and VALS Aliquots 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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4.2.2.4 Preparation of sample preparation An 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 4.2.2.5 Method Validation 4.2.2.5.1 Preparation of linearity curve

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Linearity was performed in conc. range 2-20 g/mL at 237.6nm for AMLO, 525 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 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 30,40,50 g/mL for VALS individually for three times in a day. Inter-day precision Inter-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. Repeatability Repeatability 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 Accuracy To 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 formulation Marketed 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 methanol and 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 Discussion 4.2.3.1 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 VALS were 320.07, 141.02, 295.75 mol lit
-1 -1

and for

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 +320.07C 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. 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 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. Calculate and record value of correlation coefficient (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 237.6nm Conc. g/mL 2 4 5 10 15 20 Absorptivity *(n=6) 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

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 237.6nm Conc. g/mL MeanAbs. S.D* 270.2nm Mean Abs. S.D* 249.2nm Mean Abs. S.D*

%RSD

%RSD

%RSD

5 10 15 20 25 Absorptivity *(n=6)

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

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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 237.6nm Conc. g/mL Mean Abs.S.D* % Mean RSD Abs.S.D* 0.1460.00 2 0.2790.00 4 0.4330.00 2 0.5530.00 3 % 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

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

0.6820.00 0.30 21 141.02

1.4620.02 0.14 1 295.75

*(n=6) 8 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 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 WAVELENGTH CONC. g/mL 2 237.6nm 4 5 2 270.2nm 4 5 2 249.2nm 4 5 *(n=3) WAVELENGTH MEAN CONC.S.D* 1.990.0281 3.880.0168 5.050.0129 1.870.0135 4.170.0132 5.020.0152 2.130.0144 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

Table 34: Interday precision for AMLO CONC. g/mL 2 237.6nm 4 5 2 270.2nm 4 5 2 249.2nm 4 5 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

*(n=3)

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Table 35: Intraday precision for HCTZ WAVELENGTH CONC. g /mL 15 237.6nm 20 25 15 270.2nm 20 25 15 249.2nm 20 25 *(n=3) 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 *(n=3) 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 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

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Table 37: Intraday precision for VALS

WAVELENGTH CONC. g/mL 15 237.6nm 20 25 15 270.2nm 20 25 15 249.2nm *(n=3) 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

Table 38: Interday precision for VALS WAVELENGTH CONC. g/mL 15 237.6nm 20 25 15 20 270.2nm 25 15 20 249.2nm 25 *(n=3) 25.080.0025 0.89 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 % R.S.D 0.81 1.99 2.02 0.75 0.57 0.44 0.83 1.11

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Table 39: Repeatibilty 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

WAVE LENGTH

DRUG

% R.S.D

AMLO 237.6nm HCTZ VALS AMLO 270.2nm HCTZ VALS AMLO 249.2nm HCTZ VALS *(n=6) 9 LOD and LOQ

0.27 0.32 1.21 0.53 0.46 1.64 0.51 0.28 1.59

From 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. 10 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%. % 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 Sample conc. g/ml 5 5 5 10 10 10 20 20 20 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 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

DRUG

%level

80 AMLO 100 120 80 100 120 80 VALS 100 120

HCTZ

Table 41: Validation parameters for AMLO, HCTZ and VALS 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

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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

DRUG

LABLE CLAIM (mg)

% ASSAYSD*

AMLO HCTZ

5 12.5

98.250.0781 98.820.0458

VALS *(n=3)

160

98.930.0404

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.

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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
4.3.1 Instrumentation 11 HPTLC-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: 10 to 220 mg Sonicator-D-compact (Trans-O-sonic), Capacity: 2L 4.3.2 Materials and Methods 4.3.2.1 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. 4.3.2.2 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
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Slit dimension : 3x 0.20 mm (micro) Band width : 4 mm Syringe capacity : 100 L 4.3.2.3 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 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. 4.3.2.5 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. 4.3.2.6 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. 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 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. 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

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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 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. Accuracy To 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 plate 4.3.3 RESULTS AND DISCUSSION

4.3.3.1 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. 1 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 (7.5: 2.5:0.5, v/v/v/v), Run length =80mm ACN: Methanol: TEA (7.5:2.5:0.5, v/v/v),Run length= 80mm Ethyl Acetate :Methanol:1,4 dioxane :Ammonia (7:3:1:0.5, v/v/v/v), Run length =80mm Cyclohexane:Methanol:Ammonia ( 7.5:2.5:0.5, v/v/v/v),Run length =80mm ACN: Methanol: TEA (7.5:2.5:0.5, v/v/v),Run length= 80mm Observation AMLO, HCTZ and VALS were close to solvent front Improper resolution and poor Rf values of VALS Very good separation but Diffused spot of VALS Not Good resolution VALS spot was less diffused but poor Rf value of VALS Closeness b/w HCTZ and VALS and diffused spot of VALS Less resolution b/w AMLO and HCTZ Diffused spot of AMLO and high Rf value of VALS and HCTZ Closeness b/w HCTZ and VALS and diffused spot of VALS Good resolution but VALS having less tailing Remarks Not satisfactory Not satisfactory Good but Not satisfactory Not Satisfactory Not satisfactory

Not satisfactory

Not satisfactory

Not satisfactory

9.

Not satisfactory

10

Ethyl Acetate: Methanol: Toluene: Ammonia (6:3:3:0.5, v/v/v/v),Run length= 80mm

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 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

VALS spot was not diffused but poor Rf value of AMLO

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 Good resolution and VALS was not diffused

14

15

Satisfactory Optimized Mobile Phase

4.3.3.2 Validation parameters Linearity Linearity curve shows linearity in the range of 100-600 ng/spot for AMLO, 150900 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 Conc. (ng/spot) 100 200 300 400 500 600 Peak Area 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. *(n=6)

5000 P e a k 4000 A r 3000 e 2000 a 1000 0 0 200 400 600 Conc. ng/spot 800 y = 6.5173x + 550.08 R = 0.9945 Series1 Linear (Series1)

Fig.14: Calibration curve of AMLO

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

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

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

Rf 0.64 0.64 0.63 0.63 0.65 0.65

Table 46: Linearity of HCTZ by HPTLC with UV detection *(n=6)

P e a k A r e a 7000 6000 5000 4000 3000 2000 1000 0 0 500 Conc. ng/spot y = 5.9829x + 707.78 R = 0.9927 Series1 Linear (Series1)

1000

Fig. 16: Calibration Curve for HCTZ

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

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Peak Area Sr. No. 1 2 3 4 5 6 *(n=6) Conc (ng/spot) Mean SD* 1200 1600 2000 2400 2800 3200 108933.52 1873.1619.51 2646.593.04 3182.8134.8 3819.5175.6 4375.93144 %RSD 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: Linearity of VALS by HPTLC with UV detection

P e a k A r e a

5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0 1000 2000 Conc. ng/spot 3000

y = 1.6293x - 753.28 R = 0.9945 Series1 Linear (Series1)

4000

Fig.18: Linearity Curve for VALS

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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) 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 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) Table 48: Intraday precision AMLO, HCTZ AND VALS by HPTLC with UV Drug Concentration (ng/spot) AMLO 200 500 600 450 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

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*(n=3) Conc. (ng/spot) 200 AMLO 500 600 450 HCTZ 600 750 1600 VALS 2000 2400 Peak Area Mean SD* %RSD 3201.664.16 2.00 3673.86760.64 4171.33341.56 3812.83325.8 4216.63350.91 5502.161.85 8076.2819.17 9343.433171.65 10297.73144.49 1.65 0.99 0.67 1.20 1.12 0.23 1.83 1.40

Drug

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

Table 49: Interday precision AMLO, HCTZ AND VALS by HPTLC *(n=3) Table 50: Repeatability study of AMLO, HCTZ AND VALS Sr.no. 1. 2. 3. *(n=6) 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%. % recovery was calculated from regression equation of the calibration curve as shown in Table 6.8 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

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Table 51: Recovery study of AMLO, HCTZ and VALS STD. Added ng/spot 44 AMLO 55 55 66 78.57 HCTZ 98.21 98.21 117.8 1005.6 VALS 1257.1 1257.1 1508.56 *(n=3) LOD and LOQ From 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 2514.28 2765.7 2551.7719.20 2796.6416.258 1.31 0.51 99.650.907 99.300.703 0.91 0.70 Accuracy TOTAL CONC. ng/spot 99 110 121 177.47 196.42 216.81 2262.74 99.141.073 109.390.66 120.810.760 176.80.346 199.122.124 215.980.945 2357.417.091 Conc. Recover MeanSD* % RSD 1.08 0.60 0.62 0.19 1.08 0.43 0.13 %Recovery Mean SD* 100.14 1.32 99.440.745 99.840.76 1115 99.090.932 99.790.761 100.140.675 101.400.78 5 % RSD 1.32 0.74 0.76 0.92 0.76 0.67 0.77

DRUG

INITIAL CONC. ng/spot

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Table 53: Robustness Study of AMLO, HCTZ and VALS Peak Area SD * Sr.no. Parameter AMLO Mobile Phase composition Ethyl Acetate (5%) Wavelength (2422nm) HCTZ 2119.9 38.8 1743.6 17.2 1132.43 10.0 VALS 13749.4 1.3 175.3 1.64 1.2
AMLO HCTZ VALS

%RSD

1.

1107.8 14.47

2.

1119.63 9.99

1082 125.29

0.89

0.98

1.15

3.

Development 1073.43 Distance 10.6 (705mm)

13430.1 0.93 133 1.02 0.99

Specificity Specificity 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 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

Table 55: Summary of Validation parameters by HPTLC with UV detection Sr.no. 1 2 3 4 5 Parameters Linearity range (ng/spot) Regression equation Correlation coefficient (r2) Intercept Slope Precision Intraday % RSD (n = 3) 6 Interday % RSD (n = 3) Repeatability of measurements% RSD (n=6) 7 Limit of detection 0.99 to 2.0 0.48 to 0.98 0.67 to 1.2 0.43 to 1.92 0.23 to 1.83 0.25 to 1.26 AMLO 100-600 y =6.534x+547.48 0.9945 6.534 547.48 HCTZ 150-900 y=5.931x+744.6 0.9926 5.931 744.6 VALS 1200-3200 y=3.48x+2356 0.9918 3.48 2356

0.84 1.98 1.86

2.95 (ng/spot)

17.84 (ng/spot)

70.90 (ng/spot)

Limit of quantification

8.94(ng/spot)

53.9 (ng/spot)

214.85(ng/spot)

Specificity

Pass

Pass

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 found from 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) 5 5 5 HCTZ 12.5 12.5 12.5 VALS 160 160 160 *(n=3)
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Amt estimated(mg) 5.01 5.06 4.96 12.74 12.65 12.40 162.27 161.37 157.49

Assay results S.D*

AMLO

100.321.02

100.81.43

100.271.57

Chapter5

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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 METHOD FOR

AND

VALIDATION

OF

RP-HPLC OF

SIMULTANEOUS

ESIMATION

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 to 14,Resolution: 0.01 PH, Accuracy: 0.01 PH 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 methods 4.4.2.1 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) purchased from USA market. 4.4.2.2 Chromatographic Conditions 4.4.2.2.1 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. and Valsartan (160mg)

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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 Min 4.4.2.3 Preparation of Mobile phase Mobile phase A:HPLC grade Acetonitrile was degassed with sonicator for 15 min. Mobile phase B: 3402.25 mg of KH2PO4 (potassium dihydrogen ortho phosphate) was dissolved in 500 triple dist. Water and add 1 ml HPLC grade triethylamine (0.2%) and pH 3.7 adjusted with ortho phosphoric acid. 4.4.2.4 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 4.4.2.5 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. 4.4.2.6 Method validation Preparation of Linearity curve For estimation of AMLO, calibration curve (n=3) was plotted in the range of (225 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 Precision Intraday and interday precision (20-150 g/mL).Calibration curve of peak area v/s

concentration was plotted for the drug.

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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 Repeatability Peak 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 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 given is section () Accuracy To 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. Robustness The 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. (Table 6.11)
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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 Specificty Specificity of an analytical method is ability to measure specifically the analyte of interest without interferences from blank and placebo. 4.4.2.7 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 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 Discussion 4.4.3.1 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 and the 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 ACN :0.025 M potassium dihydrogen ortho phosphate (60:40, v/v) PH 3.7 ACN :0.025 M potassium dihydrogen ortho phosphate (50:50, v/v), PH 3.7 ACN :0.025 M potassium dihydrogen ortho phosphate (57:43, v/v) , TEA 0.1%, 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

Asymmetry greater than 1.5

M.P was not suitable

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4.

ACN : 0.025 M potassium dihydrogen ortho phosphate (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 dihydrogen ortho phosphate (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 2. 3. 4. 5.

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

0.6434 1.30 3.175 5378 0.48

0.3695 1.41 6357 1.85

3.4130 1.05 10.37 4886 0.52

4.4.3.2 Validation parameters Linearity Linearity curve shows linearity in the range of 100-600 g/mL for AMLO, 545g/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 Peak Area Sr. No. 1 2 3 Conc. (g/mL) 2 5 10 15 20 25 Mean SD* 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

4 5 6 *(n=3)

1600000 P 1400000 e 1200000 a 1000000 k 800000 A r e a 600000 400000 200000 0 0 10 20 30 Conc. g/ml y = 53407x + 24828 R = 0.9945

Series1 Linear (Series1)

Fig.35: Calibration curve for AMLO

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Table 60: Linearity of HCTZ by RP-HPLC with UV PDA detection Conc. (g/mL) 5 10 15 25 35 45 Peak Area Mean SD* 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. 1 2 3 4 5 6 *(n=6)

7000000 P e 5000000 a k 4000000 a 3000000 r 2000000 e a 1000000 0 0 10 6000000

y = 135283x + 114584 R = 0.996

Series1 Linear (Series1)

20

30

40

50

Conc. g/ml

Fig.36: Calibration curve of HCTZ

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

Sr. No.

Conc. (g/mL)

Peak Area

Mean SD* 1 2 3 4 5 6 *(n=6)

18000000 16000000 P 14000000 e a 12000000 k 10000000 A r e a 8000000 6000000 4000000 2000000 0 0 50 100 Conc. g/ml 150 200 Series1

%RSD 1.02 0.99 1.33 0.12 0.35 0.60

20 40 60 80 120 150

161341316557.68 325834232479.94 617468382500.03 80224449798.269 1303326245680.19 1589395296053.33

y = 112822x - 838391 R = 0.9971

Linear (Series1)

Fig.36: Calibration curve of VALS

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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

done by analyzing three different concentrations (g/mL) within linearity ranges and % RSD less than 2 Table.Repeatiblity determined by six replicates of sample were prepared at sample concentration by one analyst and analyzed on same day. (Table 6.9) Table 62- Intraday precision data of AMLO, HCTZ AND VALS by HPLC Concentration (g/mL) 5 10 15 HCTZ 10 15 20 80 VALS *(n=3) Table 63: Intraday precision data of AMLO, HCTZ AND VALS by HPLC Drug Concentration (g/mL) 5 AMLO 10 15 10 HCTZ 15 20 80 VALS 100 120 *(n=3) Peak Area Mean SD* %RSD 309600.73256.28 502668.32644.688 773136.3253.4213 136914910733.71 211000710417.99 267864933853.87 777402543345.03 993643953407.12 1301815934631.55 1.05 0.52 0.33 0.78 0.49 1.26 0.55 0.53 0.26 100 120 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

Drug

AMLO

%RSD 0.82 0.23 0.60 0.51 2.06 0.88 0.39 0.61 1.54

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Table 64: Repeatability 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

*(n=6) LOD and LOQ From 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. Specificity Peak 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 AMLO HCTZ VALS Peak purity Front 996.65 999.93 997.15 Peak purity Tail 998.45 999.70 998.94 Peak purity Specific Specific Specific

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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%. % recovery was calculated from regression equation of the calibration curve as shown in Table 6.8 Table 67: % Recovery study of AMLO, HCTZ and VALS ACCURACY INITIAL CONC. g/mL STD. Added g/mL TOTAL CONC. After spiking g /mL

DRUG

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 *(n=3)

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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Robustness Table 68- Robustness study of AMLO Parameters Change 0.8 Flow Rate (0.2 ml/min) 1.2 3.65 P ( 0.05) 3.75 230 Wavelength ( 2 nm) 234 12489260.51 101.04 210569.21.74 12836530.29 100.2 100.42
H

Mean of Peak Area %RSD 2936241.74 2904940.52 382701.71.53

%Assay 98.25 100.0 99.82

Table 69: Robustness study of HCTZ Parameters Flow Rate (0.2ml/min.) Change 0.8 1.2 3.65 PH (0.05) 3.75 Wave Length ( 2nm) 230 234 12805860.56 14799090.95 15599061.79 98.15 99.77 98.51 Mean of Peak Area%RSD 19520661.17 681237.70.88 13658011.55 %Assay 98.44 99.38 101.59

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Table 70: Robustness study of VALS Parameters Flow Rate (0.2 ml/min) PH ( 0.05) 3.75 Wave Length (2 nm) 230 234 118075061.4 117413080.8 97272071.30 101.76 98.45 98.43 Change 0.8 1.2 3.65 Mean %RSD 178625950.8 162600241.0 136887281.3 %Assay 98.60 98.79 98.48

Table 71: Summary of Validation parameters of RP-HPLC Sr. no 1. 2. 3. AMLO 2-25 y =53047x+24828 0.9945 HCTZ 5-45 y=135283x+11458 4 0.9967 VALS 20-150 y = 112822x83839 0.9971

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

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 (n=6)

5. 6. 7. 8.

Specificit y LOD (g/ml) LOQ (g/ml) % Recovery

Specific 0.23 0..71 99.571.33 to 101.420.75

Specific 0.48 0.1.47 98.350.19 to 99.16 0.76

Specific 1.1 3.3 99.69 0. 63 to 1002.010.074

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Table 72: Analysis of Marketed Dosage Form Drugs Label claim(mg) 5 AMLO 5 5 12.5 HCTZ 12.5 12.5 160 VALS 160 160 *(n=3) 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*

Fig.39: HPLC Chromatogram of Marketed Dosage Form

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4.4.4

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

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 % Assay results U.V 98.66 AMLO 98.45 100.3 98.82 Exforge HCT HCTZ 98.40 98.20 98.33 VALS 98.75 99.20 HPTLC 100.28 101.37 98.79 101.97 101.23 99.26 98.59 99.15 101.61 RP-HPLC 99.07 98.36 98.55 100.8 99.52 99.13 101.10 101.82 98.94

Brand name

Drugs

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Table 74: Comparison of three Methods by ANOVA Test Brand name Drugs AMLO Exforge HCT HCTZ VALS Fcal 1.63 3.68 1.18 F crit 4.066 4.066 4.066

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

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