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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
4.1.2 UV spectra of AMLO, HCTZ and VALS
UV- spectrum of AMLO (20g/mL), HCTZ (20g/mL) and VALS (20g/mL) in
methanol was taken and it was compared with reported UV spectra as shown in
Table
Fig. 5: UV spectrum of AMLO (20g/mL) in methanol
Drug Reported melting point (0C)() Observed melting point (
0C)
AMLO 199-201 200-202
HCTZ 273-275 274-275
VALS 116-117 117-118
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Fig.6: UV spectrum of HCTZ (20g/mL) in methanol
Fig.7: UV spectrum of VALS (20g/mL) in methanol
Table 26: Reported max for AMLO, HCTZ and VALS
Drug Reported maxima Recorded maxima
AMLO 239nm, 238nm 237.6nm,210.8nm, 330nm
HCTZ 225nm, 271nm, 317nm 270.2nm,316.6nm
VALS 249nm 249.2nm, 206nm, 243.8nm
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4.1.3Determination of Infrared (IR) Spectra
Identification of drug sample was carried out by IR spectroscopy studies. A
mixture of drug sample and KBr (spectroscopic grade, drug: KBr ratio 1:20) was
prepared using mortar-pestle. This mixture was then analyzed in attenuated
reflectance FT-IR. The mixture was scanned from 4000-400 cm-1
and a spectrum
was recorded with the help of IR Spectrophotometer (JASCO Model: FT-IR 6100
type A).
Fig.8: Infrared spectrum of AMLO
Fig.8: Reported Infrared spectrum of AMLO
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Table 27: IR Frequency (Cm-1
) for AMLO
SPECIFICATION OFAMLO
THEORITCAL WAVENUMBER(Cm
-1)
(85)
RECORDED WAVENUMBER(Cm
-1)
-NH2 stretching 3500-3100 3372
-C-Cl 785-540 752.102
C-H bending 1465-1375 1440.56
C=O Ester 1750-1730 1765.99
-C-O Ether 1300-1000 1265.07
Fig. 9: Infrared spectrum of HCTZ
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Fig. 9: Reported Infrared spectrum of HCTZ
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Table 28: IR Frequency (Cm-1
) for HCTZ
Fig.10: Infrared spectrum of VALS
SPECIFICATION FOR
HCTZ
THEO RITICAL WAVE
NUMBER (Cm-1) (85)
RECORDED WAVE
NUMBER (Cm-1)
(S=O) 1050 1078.98
C-H stretch 3000-2850 2963.09
Mono substituted benzene 900-690 996.053
-C-N - Amine 1350-1000 1206.26
Amide group 1680-1630 1601.59
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Fig.10: Reported Infrared spectrum of VALS
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Table 29: IR Frequency (Cm-1
) for VALS
SPECIFICATION OF FORVALS
THEORITICAL WAVENUMBER (Cm
-1)(85)
RECORDED WAVE
NUMBER (Cm-1
)
C-H stretch 3000-2850 2834.85
ketone (C=O) stretch 1750-1730 1730.8
-NH2 stretching 3200-3500 3238.86
Carboxylic acid(-COOH) 3400-2400 2577.4
-N-H Stretch 1640-1550 1523.49
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4.2 DEVELOPMENT AND VALIDATION OF UV
SPECTROPHOTOMETRIC METHOD FOR
SIMULTANEOUS ESIMATION OF AMLO, HCTZ AND
VALS IN THEIR COMBINED TABLET DOSAGE
FORM
4.2.1 Instrumentation
UV-Visible Double-Beam spectrophotometer- UV-VIS is 2400, version-2.21 double beam spectrophotometer with spectral width of 2 nm, wavelength
accuracy of 0.5 nm, and a pair of 10 mm matched quartz cells (Shimadzu,
Columbia, MD)
Analytical Balance- Weighing capacity-10 to 220 mg, Citizen CX 220(Citizen Pvt. Ltd),
Sonicator- Capacity- 2 L, D-compact (Trans-O-Sonic, Mumbai),4.2.2Materials and Methods4.2.2.1 Reagents and Chemicals
AMLO, HCTZ, and VALS Standards were kindly gifted by Torrent
pharmaceutical Gujarat, India.All reagents and solvents were of analytical grade
(Central Drug House Pvt. LTD). Marketed tablet formulation Exforge HCT
(Novartis pharma stein AG, stein, Switzerland.) Containing Amlodipine besylate
(5mg), Hydrochlorothiazide (12.5 mg) and Valsartan (160mg) purchased from
USA market.
4.2.2.2Preparation of standard stock solution of AMLO, HCTZ and VALSAMLO (25 mg), HCZ (25mg) and VALS (25mg) were accurately weighed and
transferred to three separate 25 ml volumetric flasks and dissolved in methanol to
obtain stock solution of concentration 100 g/mL of each drug.
4.2.2.3 Preparation of calibration curve of AMLO, HCTZ and VALSAliquots of 2, 4, 5, 10, 15, 20 g/mL were prepared by using stock solution of
AMLO, aliquots of HCTZ of 5, 10, 15, 20, 25 g/mL by using stock solution of
HCTZ and aliquots of 10 ,20, 30, 40, 50 g/mL by using of VALS stock solution
for preparation of calibration curve.
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4.2.2.4Preparation of sample preparationAn amount equivalent to 160 mg of VALS containing (5 mg of AMLO and 12.5
mg HCTZ) was weighed and transfer to100ml calibrated volumetric flask
dissolved methanol. From above solution 5 ml aliquots was pipette out and
transfer to 10 ml calibrated volumetric flask to get final conc. of AMLO(5
g/mL), HCTZ(12.5 g/mL) and VALS(g/mL).
4.2.2.4 Selection of Analytical Wavelength and measurement
From stock solutions of AMLO, HTCZ and VALS, working standard solutions
were prepared by appropriate dilution of solvent to get final concentration of 20
g/mL each drug and were scanned in the spectrum mode from 200 to 400 nm.
From the overlain spectra of these drugs (fig.1), wavelengths 237.6 nm (max of
AMLO), 249.2 nm (max of VALS) and 270.2 nm (max of HCTZ) were selected for
analysis.
Fig. 11: overlay spectra of AMLO, HCTZ and VALS
4.2.2.5Method Validation4.2.2.5.1 Preparation of linearity curve
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Linearity was performed in conc. range 2-20 g/mL at 237.6nm for AMLO, 5-
25 g/mL at 270.2nm for HCTZ and 10-50 g/mL at 249.2 nm for VALS.
Solution preparation was done which as shown in section 4.2.2.3.
4.2.2.5.2 Precision
Intra-day precisionIntra-day precision was determined by measuring amplitudes of three different
concentrations 2, 4, 5 g/mL for AMLO and 15, 20, 25, g/mL for HCTZ and
30,40,50 g/mL for VALS individually for three times in a day.
Inter-day precisionInter-day precision was determined by measuring amplitudes of three different
concentrations 2, 4, 5 g/mL for AMLO, for HCTZ 15, 20, 25, g/mL and 30,
40, 50 g/mL for VALS individually for three days.
RepeatabilityRepeatability of this method was determined by measurement of six
determinations at 100% test concentrations 4g/mL, 10 g/mL, 20 g/mL of
AMLO, HCTZ and VALS respectively.
4.2.2.5.3 Limit of detection and limit of quantification (LOD/LOQ)
The LOD & LOQ were measured by using mathematical equations given below.
LOD = 3.3 x /S
LOQ = 10 x /S Where, = Standard deviation of the Intercept
S = slope of calibration curve
5 AccuracyTo study the accuracy, 7 tablets were weighed and powdered. The powder
equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of VALS)
were weighed and transferred to 100ml volumetric flask and dissolved in 70 ml of
methanol. Then solution was sonicated for 15 minutes and volume was made up
to the mark with methanol. The above solution was filtered with whatmann filter
paper (No. 41). Aliquot (5ml) was pipette out and transferred to 50ml volumetric
flask. Volume was made up to the mark with methanol to get a solution
containing 5g/mL of AMLO, 12.5 g/mL of HCTZ and 160 g/mL of VALS.
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Standard drug was added at three different concentration levels (80%, 100% and
120% of test sample concentration) to pre-analyzed sample and amplitudes of the
solution were measured at selected wavelengths for AMLO, HCTZ and VALS
drugs. Amplitudes were substituted into respective straight line equation to
calculate percentage recovery of the drugs.
6 Analysis of marketed formulationMarketed tablet formulation containing VALS 160 mg, amlodipine besylate
equivalent to AMLO 5mg and HCTZ 12.5 mg was analysed using this method.
From the triturate of 7 tablets, an amount equivalent to 160 mg of VALS, (5 mg
of AMLO and 12.5 mg HCTZ) was weighed and dissolved in 35 ml of methanoland solution was sonicated for 30 minutes and volume was made up to the mark
in a 100ml calibrated volumetric flask with methanol. The above solution was
filtered through whatmann filter paper. The filtrate was appropriately diluted with
the same solvent to obtain final concentration within Beer Lambert's range for
each drug.
The concentration of drugs was determined by using the Eqns 1, 2 and 3.
Eqn.1 A 1 = 320C AMLO + 45.88C HCTZ +320.07C VALS (1)
Eqn.2 A= 177.7C AMLO +615.55C HCTZ +141.02C VALS (2) and
Eqn.3 A 3 = 178.63C AMLO +88.086C HCTZ +295.75C VALS (3),
Where A1, A2 and A 3 are absorbance of the tablet sample solution at 237.6, 270.2
and 249.2 nm respectively. CAMLO is the concentration of AMLO, CHCTZ is the
concentration of the HCTZ, and C VALS is the concentration of the VALS.
6.2.2 Results and Discussion4.2.3.1 Method development
The values of absorbance were recorded at the selected wavelengths and the
absorptivity and molar absorptivity values for AMLO, HCTZ and VALS
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were determined .Molar absorptivity value for AMLO were 320, 177.7,
178.63 mol lit-1
, for HCTZ were 45.88, 615.55, 88.086 mol lit-1
and for
VALS were 320.07, 141.02, 295.75 mol lit-1
at 237.6 nm, 270.2 nm and
249.2 nm respectively. Found simultaneous equation by putting molar
absorptivity value
A1== 320C AMLO + 45.88C HCTZ +320.07CVALS...(1)
A2=177.7C AMLO +615.55C HCTZ +141.02CVALS.. (2)
A3=178.63C AMLO +88.086C HCTZ +295.75CVALS . (3)
Where A1, A2 and A 3 are absorbance of the sample solution at 237.6nm,
270.2 nm and 249.2 nm respectively. CAMLO is the concentration of AMLO,
CHCTZ is the concentration of the HCTZ, and C VALS is the concentration of
VALS
4.2.3.2 Validation parameters
7 Linearity-Linearity curve shows linearity in the range of 2-20 g/mL forAMLO, 5-25 g/mL for HCTZ and for VALS 10-50 g/mL. The correlation
coefficient (r2) was found to be 0.9997, 0.9990, and 0.9990 for AMLO,
HCTZ and VALS respectively. Calculate and record value of correlation co-
efficient (r), y-intercept, slope of regression line and residual sum of
squares. (Figure 6.12) and results (Table)
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Fig 12: Linearity Curve for AMLO at 237.6 nm
Fig 12: Calibration Curve for AMLO at 237.6 nm
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Table 30: Linearity of AMLO at 237.6 nm, 270.2 nm and 249.2 nm
Conc.
g/mL
237.6nm 270.2nm 249.2nm
MeanAbs.
S.D*
%
RSD
MeanAbs
S.D*
%
RSD
Mean
Abs.S.D*
%
RSD
2 0.0450.016 1.55 0.0130.0021 1.61 0.0210.0013 1.61
4 0.1220.018 1.47 0.0240.0024 1.60 0.0650.0017 1.53
5 0.1590.023 1.44 0.0420.0016 1.80 0.0880.0014 1.13
10 0.3460.011 0.31 0.0560.0013 1.12 0.2010.0015 0.74
15 0.5580.012 0.17 0.0640.0017 1.42 0.3280.0012 0.30
20 0.7070.015 0.21 0.0780.0016 0.95 0.4220.0016 0.37
Absorptivity 320 177.7 178.63
*(n=6)
Fig. 12: Linearity Curve for HCTZ at 270.2 nm
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Fig. 12: Calibration Curve for HCTZ at 270.2 nm
Table 31: Linearity of HCTZ at 237.6 nm, 249.2 nm and 270.2 nm
Conc.
g/mL
237.6nm 270.2nm 249.2nm
MeanAbs.
S.D* %RSD
Mean Abs.
S.D* %RSDMean Abs.
S.D* %RSD
5 0.0090.001 1.33 0.2970.002 0.70 0.0320.0011.75
10 0.0420.001 1.57 0.6180.002 0.37 0.0830.001 1.56
15 0.0760.001 1.71 0.9250.001 0.16 0.1370.001 1.16
20 0.1130.001 1.32 1.2450.003 0.12 0.1920.002 0.78
25 0.1660.006 1.61 1.5640.003 0.12 0.2650.002 0.37
Absorptivity 45.88 615.55 88.086
*(n=6)
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Fig.13: Linearity Curve of VALS at 249.2 nm
Fig.13: Calibration Curve of VALS at 249.2 nm
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Table 32: Linearity of VALS at 237.6 nm, 249.2nm at 270.2 nm
Conc.
g/mL
237.6nm 270.2nm 249.2nm
Mean
Abs.S.D*%
RSD
Mean
Abs.S.D*
%
RSD
Mean
Abs.S.D*
%
RSD
100.3260.003
0.980.1460.00
21.02
0.3100.02
70.80
20 0.6340.0022 0.34 0.2790.00
0.53 0.5830.02
0.42
30 0.9780.002 0.25 0.4330.00
0.34 0.8950.02
0.23
40 1.2630.0038 0.30 0.5530.00
0.56 1.1670.03
0.25
50 1.580.0015 0.97 0.6820.00
0.30 1.4620.02
0.14
Absorptivity 320.07 141.02 295.75
*(n=6)
8 PrecisionIntraday precision and Interday precision for AMLO, HCTZ and VALS was
done by analyzing three different concentrations (g/mL) within linearity ranges
and % RSD less than 2 given in Table Repeatiblity determined by six replicates
of sample were prepared at sample concentration by one analyst and analyzed on
same day. (Table 6.9)
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Table 33: Intraday precision for AMLO
WAVELENGTHCONC.
g/mLMEAN CONC.S.D* % R.S.D
237.6nm
2 1.990.0281 1.41
4 3.880.0168 0.43
5 5.050.0129 0.25
270.2nm
2 1.870.0135 0.72
4 4.170.0132 0.31
5 5.020.0152 0.30
249.2nm
22.130.0144
0.676
4 4.120.0211 0.512
5 5.230.0221 1.837
*(n=3) Table 34: Interday precision for AMLO
WAVELENGTHCONC.
g/mLMEAN CONC.S.D* % R.S.D
237.6nm
2 2.020.00114 1.10
4 4.310.00154 0.45
5 4.220.00137 0.27
270.2nm
2 2.180.00305 1.17
4 4.070.00120 0.56
5 5.060.00260 0.26
249.2nm
2 2.180.00340 1.05
4 4.210.00231 0.50
5 5.350.00162 0.31
*(n=3)
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Table 35: Intraday precision for HCTZ
WAVELENGTHCONC.
g/mLMEAN CONC.S.D* % R.S.D
237.6nm
15 15.210.0084 0.55
20 21.010.0043 0.68
25 24.870.0044 0.57
270.2nm
15 14.950.0034 0.90
20 20.090.0032 0.65
25 24.670.0042 0.57
249.2nm
15 15.020.0062 0.94
20 21.170.0055 0.64
25 26.010.0048 0.49
*(n=3)
Table 36: Interday precision for HCTZ
WAVELENGTHCONC.
g/mLMEANConc. S.D* % R.S.D
237.6nm
15 14.560.0018 0.81
20 20.170.0040 1.99
25 24.880.0050 2.02
270.2nm
15 14.760.0011 0.75
20 20.140.0016 0.57
25 25.180.0012 0.44
249.2nm
15 15.090.0026 0.83
20 20.310.0026 1.11
25 25.080.0025 0.89
*(n=3)
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Table 37: Intraday precision for VALS
*(n=3)
Table 38: Interday precision for VALS
*(n=3)
WAVELENGTH CONC. g/mL MEANCONC.S.D*%
R.S.D
237.6nm
15 15.210.0084 0.55
20 21.010.0043 0.68
25 24.870.0044 0.57
270.2nm
15 14.950.0034 0.90
20 20.090.0032 0.65
25 24.670.0042 0.57
249.2nm
15 15.020.0062 0.94
20 21.170.0055 0.64
25 26.010.0048 0.49
WAVELENGTH CONC.g/mL
MEANCONC.S.D* % R.S.D
237.6nm
15 14.560.0018 0.81
20 20.170.0040 1.99
25 24.880.0050 2.02
270.2nm
15 14.760.0011 0.75
20 20.140.0016 0.57
25 25.180.0012 0.44
249.2nm
15 15.090.0026 0.83
20 20.310.0026 1.11
25 25.080.0025 0.89
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Table 39: Repeatibilty
WAVE LENGTH DRUG
CONC.
g/mL
MEAN
CONC.g/mL
% R.S.D
237.6nm
AMLO 4 4.2 0.27
HCTZ 10 10.12 0.32
VALS 20 20.13 1.21
270.2nm
AMLO 4 3.94 0.53
HCTZ 10 9.93 0.46
VALS 20 19.78 1.64
249.2nm
AMLO 4 4.15 0.51
HCTZ 10 10.21 0.28
VALS 20 20.11 1.59
*(n=6)
9 LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was
repeated six times and measured by mathematical equation given in section
(5.2.2.5). LOD value were 0.025 g/mL, 0.013 g/mL, 0.029 g/mL and LOQ
value were 0.078 g/mL, 0.041 g/mL, 0.089 g/mL for AMLO, HCTZ and
VALS respectively.
10AccuracyStandard edition was done at three level 80%, 100% and 120% of a
concentration of sample in the linearity range and % recovery was found 98 to
100%. % recovery was calculated from regression equation of the calibration
curve as shown in Table 6.8
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Table 40: Recovery study of AMLO, HCTZ and VALS
DRUG %level
Sample
conc.g/ml
Amt of
std.
added
g/ml
Total
Conc.g/ml
Amt
recoveredg/ml
%
Recovery
AMLO
80 5 4 9 8.856 98.4
100 5 5 10 10.05 100.5
120 5 6 11 10.93 99.61
HCTZ
80 10 8 18 17.82 99.00
100 10 10 20 19.78 98.9
120 10 12 22 21.89 99.5
VALS
80 20 16 36 35.47 98.53
100 20 20 40 39.86 99.67
120 20 24 44 43.65 99.22
Table 41: Validation parameters for AMLO, HCTZ and VALS
PARAMETERS AMLO HCTZ VALS
max 237.6nm 270.2nm 249.2nm
Linear Range g/mL 2-20 5-25 10-50
Correlation coefficient R2
0.9997 0.999 0.999
Repeatability% RSD 0.705 0.154 1.413
Intraday precision % RSD 0.523 0.781 0.539
Interday precision % RSD 0.727 1.021 0.489
LOD g/mL 0.025 0.013 0.029
LOQ g/mL 0.078 0.041 0.089
% Recovery 99.19 99.13 99.14
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4.2.3.3 Analysis of Marketed formulation
The developed method was used to estimate AMLO, HCTZ AND VALS in
the tablet dosage form. Marketed formulation was procured for the
analysis by proposed method.
Table 43: Analysis of Marketed Dosage Form
*(n=3)
4.2.4 Conclusion
The Proposed UV-VIS spectrophotometric method was accurate, precise
and sensitive for the determination of AMLO, HCTZ and VALS in
combined dosage form. High recoveries show that the method is free from
the interference from the excipients used in the commercial pharmaceutical
preparations. Hence, it can be successful applied for the routine estimation
for AMLO, HCTZ and VALS in quality control laboratories. The result of
validation parameters are satisfactory level indicates the accuracy of
proposed method for estimation of AMLO, HCTZ and VALS.
DRUG LABLE CLAIM(mg) % ASSAYSD*
AMLO 5 98.250.0781
HCTZ 12.5 98.820.0458
VALS 160 98.930.0404
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4.3 DEVELOPMENT AND VALIDATION OF HPTLC METHOD
FOR SIMULTANEOUS ESIMATION OF AMLO, HCTZ
AND VALS IN THEIR COMBINED DOSAGE FORM
4.3.1Instrumentation11HPTLC-Applicator Linomat 5(Camag) - Semiautomatic application, band
application by spray on technique (2 - 500l), twin trough glass
chamber(Camag) -( 20 x 10 cm), TLC scanner 3 (Camag) -Scanning speed up
to 100mm/s, Spectral range 190 800nm, U.V cabinet with dual
wavelength U.V lamp (Camag) - Dual wavelength 254 / 366nm, Stationary
Phase - Pre- coated Silica gel on aluminum sheet G60 F254 , Applicator syringe
(Hamilton, Bonaduz, Schweiz) - 100 L, Data Resolution-100m/step
Analytical Balance -Citizen CX 220 (Citizen Pvt. Ltd), Weighing capacity: 10to 220 mg
Sonicator-D-compact (Trans-O-sonic), Capacity: 2L4.3.2Materials and Methods4.3.2.1 Reagents and chemicals
Methanol (AR Grade, S.D. Fine chemicals Ltd., Mumbai , India ,Ethyl
Acetate (AR Grade, S.D. Fine chemicals Ltd., Mumbai , India), Ammonia
(25%) ,Toluene AR Grade, API of AMLO, HCTZ, and VALS Standards
were kindly gifted by Torrent pharmceutcial Gujarat, India., Analytical
grade methanol (Central Drug House Pvt. LTD) was used. Marketed tablet
formulation Exforge hct (Novartis pharma stein AG, stein, Switzerland.)
Containing Amlodipine besylate (5mg), HCTZ (12.5mg) and Valsartan
(160mg)purchased from USA market.
4.3.2.2HPTLC conditions Mobile phase : Ethyl Acetate : Methanol : Toluene : Ammonia (7.5: 3:2: 0.8,
v/v/v/v)
Chamber saturation time : 25 min Distance run : 70 mm Ambient temperature : 25-26C Wavelength of detection : 242 nm
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Slit dimension : 3x 0.20 mm (micro) Band width : 4 mm
Syringe capacity : 100 L4.3.2.3 preparation of Mobile phase
A mixture of 7.5 ml of Ethyl Acetate, 3 ml of methanol, 2 ml of toluene and
0.8 ml of ammonia (25%) were mixed properly and it was used as a mobile
phase.
4.3.2.4 Preparation of Standard Stock Solution of AMLO25 mg AMLO was weighed accurately and dissolved in 25 ml methanol. 1
ml aliquots of the above solution were diluted to 10 ml with methanol to
produce 100 g/mL of AMLO in methanol.
4.3.2.5Preparation of Standard Stock Solution of HCTZ25 mg HCTZ was weighed accurately and dissolved in 25 ml methanol. 1
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 VALSSuitable aliquots of standard stock solution of AMLO, HCTZ and VALS are
mixed and diluted to volume with methanol to obtain different Ternary mixture
solutions containing AMLO, HCTZ and VALS in 1:1.5:8 ratios. Concentration
of solutions in the range 100-3200 ng/spot were applied to the plate for the
calibration curve of these drugs. Peak area of the spots was measured at 242 nm
in the absorbance mode with camag TLC scanner III.
PrecisionIntraday and interday precision
For intraday precision, the experiment was repeated three times in a day using
three different concentrations for AMLO (400, 500, 600 ng/spot), HCTZ
(450,600,750 ng/spot), and for VALS (1600, 2000, 2400 ng/spot)
For interday precision, the experiment was repeated on three different days
using different concentrations for AMLO (400, 500, 600 ng/spot), HCTZ
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(450,600,750 ng/spot), and for VALS (1600, 2000, 2400 ng/spot) .Precision
measured in terms of %RSD
Repeatibilty
Sample solution of AMLO, HCTZ (500 ng/spot) and for VALS 2000 (ng/spot)
was spotted 6 times on the same plate and peak area was recorded. Repeatability
was measured in terms of %RSD.
LOD AND LOQFrom the linearity curve equations, the standard deviations (SD) of the intercepts
(response) were calculated. Then LOD and LOQ were measured for all the three
drugs by using mathematical expressions.
AccuracyTo study the accuracy, 7 tablets were weighed and powdered. The powder
equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of
VALS) were weighed and transferred to 50ml volumetric flask and dissolved in
30 ml of methanol. Then solution was sonicated for 15 minutes and volume was
made up to the mark with methanol. The above solution was filtered with
whatmann filter paper (No. 41). Aliquot (5.5ml) was pipette out and transferred
to 10 ml volumetric flask. Volume was made up to the mark with methanol to
get a solution containing 55g/ml of AMLO, 98.21 g/mL of HCTZ and 1257.1
g/ml of VALS. Standard drug was added at three different concentration levels
(80%, 100% and 120% of test sample concentration) to pre-analyzed sample and
amplitudes of the solution were measured at selected wavelengths 242 nm for
AMLO, HCTZ and VALS drugs. Amplitudes were substituted into respective
straight line equation to calculate percentage recovery of the drugs.
Robustness:The following parameters were changed one by one and their effect was
observed on system suitability. (Table)
a) Mobile phase composition Ethyl Acetate ( 5%)b) Wave length (242 2nm)c) Development distance (70mm5mm) Specificity
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Specificity of an analytical method is ability to measure specifically the analyte
of interest without interferences from blank and placebo.The purity of the
chromatographic peaks was analyzed by scanning all the three separated peaks
in spectral scanning mode of the WinCATs 1.4.2.8121 software. The peak purity
for AMLO, HCTZ and VALS was tested by correlation of spectra acquired at
the peak start (s), peak maximum (m), and peak end (e) positions.
4.3.2.10 Analysis of marketed formulation
Total 14 tablets were weighed accurately and powdered. An amount
equivalent to one tablet (containing 5 mg of AMLO, 12.5 HCTZ and 160
mg of VALS) was taken. Transfer to 100 ml volumetric flask and added
50ml methanol sonicated for 15 minutes and made up volume up to mark
Solution was filtered by using Whatmann filter paper N o.41 .Above
solution containing 50 g/mL concentration of AMLO, 125 g/mL HCTZ
and 1600 g/mL concentration of VALS. From this solution, aliquots of 2
ml sol. Transfer to 10 ml volumetric flask and diluted up to mark with
methanol and apply 10 L of this solution was spotted on activated TLC
plate4.3.3 RESULTS AND DISCUSSION4.3.3.1 Method Development
Selection and optimization of a proper mobile phase is a challenging task
in HPTLC method development. Several factors affects the selection of
mobile phase such as polarity of the drugs, desired Rf values, practical
problems such as diffusion of spots, tailing, proper peak shape after
scanning. Mobile phase trails are given Table
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Table 44: Observation and remarks of mobile phase optimization
Sr.
No.Trials Observation Remarks
1
Chloroform: Methanol: Toluene:
glacial acid (6:2:1:0.1, v/v/v/v),Run
length =80mm
AMLO, HCTZ and
VALS were close to
solvent front
Not
satisfactory
2
Acetone: Chloroform: Ethyl acetate:
Methanol (3:3:3:0.5, v/v/v/v),Run
length =80mm
Improper resolution
and poor Rfvalues
of VALS
Not
satisfactory
3
Ethyl Act: Methanol: Amm.sol
(7.5:2.5:0.5, v/v/v/v),Run length =
80mm
Very good separation
but Diffused spot of
VALS
Good but
Not
satisfactory
4
Chloroform: Methanol : Amm.sol
(7.5:2:5:0.5, v/v/v/v),Run length =
80mm
Not Good resolutionNot
Satisfactory
5
Ethyl Acetate :Methanol: TEA
(7.5: 2.5:0.5, v/v/v/v), Run length
=80mm
VALS spot was less
diffused but poor Rf
value of VALS
Not
satisfactory
6
ACN: Methanol: TEA
(7.5:2.5:0.5, v/v/v),Run length= 80mm
Closeness b/w HCTZ
and VALS and
diffused spot of
VALS
Not
satisfactory
7
Ethyl Acetate :Methanol:1,4 dioxane
:Ammonia (7:3:1:0.5, v/v/v/v), Run
length =80mm
Less resolution b/w
AMLO and HCTZ
Not
satisfactory
8
Cyclohexane:Methanol:Ammonia
( 7.5:2.5:0.5, v/v/v/v),Run length
=80mm
Diffused spot of
AMLO and high Rf
value of VALS and
HCTZ
Not
satisfactory
9.
ACN: Methanol: TEA
(7.5:2.5:0.5, v/v/v),Run length= 80mm
Closeness b/w HCTZ
and VALS and
diffused spot of
VALS
Not
satisfactory
10
Ethyl Acetate: Methanol: Toluene:
Ammonia (6:3:3:0.5, v/v/v/v),Run
length= 80mm
Good resolution but
VALS having less
tailing
Not good
separation
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11
Ethyl Acetate: Methanol: Toluene
:glacial acid (7.5:3.5:2.5:0.1, v/v/v/v),
Run length= 80mm
VALS spot was not
diffused but poor Rf
value of AMLO
Not
satisfactory
12
Chloroform: Methanol: Toluene:
glacial acid (7.5:3:2.5:0.1, v/v/v/v),Run
length: 80mm
AMLO spot was
splitted
Not
satisfactory
13
Ethyl acetate: Methanol: Toluene:
Ammonia (7.5:3:2.5:1, v/v/v/v),Run
length= 80mm
Good resolution but
was not reproducible
Resolution
was less
b/w AMLO
and HCTZ
14
Ethyl acetate: Methanol: Toluene:
Ammonia (7.5:3:2:0.8, v/v/v/v),Run
length =80mm
Good resolution and
reproducible but
VALS still having
less tailing
Very good
separation
15
Ethyl acetate :Methanol: Toluene:
Ammonia (7.5:3:2:0.8, v/v/v/v), Run
length =70mm
Good resolution and
VALS was not
diffused
Satisfactory
Optimized
Mobile
4.3.3.2Validation parameters Linearity
Linearity curve shows linearity in the range of 100-600 ng/spot for AMLO, 150-
900 ng/spot for HCTZ and for VALS 1200-3200 ng/spot. The correlation
coefficient (r2) was found to be 0.9945, 0.9926 and 0.9918 for AMLO, HCTZ
and VALS respectively. Calculate and record value of correlation co-efficient
(r), y-intercept, slope of regression line and residual sum of squares. (Figure
6.12) and results (Table)
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Table 45: Linearity of AMLO by HPTLC with UV detection
*(n=6)
Fig.14: Calibration curve of AMLO
Fig.15: Calibration curve of AMLO from Win CATS software
Sr. No.Conc.
(ng/spot)
Peak Area
RfMean SD* %RSD
1 100 108918.0 1.652 0.54
2 200 1873.16710.2 0.548 0.54
3. 300 2646.551.3 1.962 0.54
4. 400 3182.858.84 1.849 0.54
5. 500 3819.5136.63 0.959 0.54
6. 600 4375.93379.0 1.807 0.54
y = 6.5173x + 550.08
R = 0.9945
0
1000
2000
3000
4000
5000
0 200 400 600 800
P
e
a
k
A
r
e
a
Conc. ng/spot
Series1
Linear (Series1)
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Table 46: Linearity of HCTZ by HPTLC with UV detection
*(n=6)
Fig. 16: Calibration Curve for HCTZ
Sr. No.Conc.
(ng/spot)
Peak AreaRf
Mean SD* %RSD
1 150 1470.426.43 1.79 0.64
2 300 2547.93320.66 0.81 0.64
3 450 3650.33345.23 1.23 0.63
4 600 4284.16733.74 0.78 0.63
5. 750 5245.26737.84 0.72 0.65
6. 900 5953.137.03 0.52 0.65
y = 5.9829x + 707.78
R = 0.9927
0
1000
2000
3000
40005000
6000
7000
0 500 1000
P
e
a
k
A
r
e
a
Conc. ng/spot
Series1
Linear (Series1)
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Fig.17: Calibration curve for HCTZ from Win CATS software
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Table 47: Linearity of VALS by HPTLC with UV detection
*(n=6)
Fig.18: Linearity Curve for VALS
Sr. No. Conc (ng/spot)
Peak Area
RfMean SD* %RSD
1 1200 108933.52 1.65 0.23
2 1600 1873.1619.51 0.54 0.23
3 2000 2646.593.04 1.96 0.23
4 2400 3182.8134.8 1.84 0.23
5 2800 3819.5175.6 0.95 0.27
6 3200 4375.93144 1.80 0.27
y = 1.6293x - 753.28
R = 0.9945
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 1000 2000 3000 4000
P
e
a
k
A
r
e
a
Conc. ng/spot
Series1
Linear (Series1)
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Fig.19: Linearity curve for VALS from Win CATS software
Fig.20: HPTLC Chromatogram of AMLO (Rf = 0.54)
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Fig.21: HPTLC Chromatogram of HCTZ (Rf= 0.64)
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Fig.22: HPTLC Chromatogram of VALS (Rf = 0.23)
Fig.23: HPTLC chromatogram of VALS (Rf= 0.23), AMLO (Rf= 0.54) and
HCTZ (Rf =0.64) in standard mixture.
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Fig.24: HPTLC chromatogram (3D view) for Mix linearity of AMLO
(Rf=0.54), HCTZ (Rf=0.64) AND VALS (Rf=0.23)
PrecisionIntraday precision and Interday precision for AMLO, HCTZ and VALS was
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
DrugConcentration
(ng/spot)
Peak AreaRfSD*
Mean SD* %RSD
AMLO
200 3201.631.50 1.98 0.540.015
500 3673.86718.431.65 0.560.013
600 4171.33325.79 0.99 0.540.011
HCTZ
450 3741.726.99 0.67 0.660.012
600 3624.918.43 1.20 0.640.014
750 365599.26 1.12 0.640.010
VALS
1600 8076.2894.57 0.23 0.230.021
2000 9343.43323.95 1.83 0.250.22
240010297.73133.15
1.400.240.024
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*(n=3)
Table 49: Interday precision AMLO, HCTZ AND VALS by HPTLC
*(n=3)
Table 50: Repeatability study of AMLO, HCTZ AND VALS
*(n=6)AccuracyStandard edition was done at three level 80%, 100% and 120% of a concentration
of sample in the linearity range and % recovery was found 98 to 100%. % recovery
was calculated from regression equation of the calibration curve as shown in Table
6.8
DrugConc.
(ng/spot)
Peak Area
Rf SD*Mean SD %RSD
AMLO
200 3201.664.16 2.00 0.540.015
500 3673.86760.64 1.65 0.550.011
600 4171.33341.56 0.99 0.540.013
HCTZ
450 3812.83325.8 0.67 0.660.010
600 4216.63350.91 1.20 0.640.012
750 5502.161.85 1.12 0.640.011
VALS
1600 8076.2819.17 0.23 0.260.02
2000 9343.433171.65 1.83 0.270.011
2400 10297.73144.49 1.40 0.270.015
Sr.no. Drug Peak AreaSD* %RSD
1. AMLO (500ng/spot) 291733.30 1.98
2. HCTZ(500ng/spot) 3609.7825.65 1.86
3. VALS (2000ng/spot) 7061.6199.95 0.84
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Table 51: Recovery study of AMLO, HCTZ and VALS
*(n=3)
LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was
repeated six times and measured by mathematical equation .LOD value were
2.95, 17.89, 70.90 and LOQ value 8.94, 53.9, 214.85 for AMLO, HCTZ and
VALS respectively.
Robustness
DRUG
INITIAL
CONC.
ng/spot
STD.
Added
ng/spot
TOTAL
CONC.
ng/spot
Accuracy
Conc. Recover
MeanSD*
%
RSD
%Recovery
Mean SD*
%
RSD
AMLO 55
44 99 99.141.073 1.08 100.14 1.32
55 110 109.390.66 0.60 99.440.745 0.74
66 121 120.810.760 0.62 99.840.76
0.76
HCTZ 98.21
78.57 177.47 176.80.346 0.19 99.090.932 0.92
98.21 196.42 199.122.124 1.08 99.790.761 0.76
117.8 216.81 215.980.945 0.43 100.140.675 0.67
VALS 1257.1
1005.6 2262.74 2357.417.091 0.13101.400.78
50.77
1257.1 2514.28 2551.7719.20 1.31 99.650.907 0.91
1508.56 2765.7 2796.6416.258 0.51 99.300.703 0.70
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Table 53: Robustness Study of AMLO, HCTZ and VALS
Sr.no. Parameter
Peak Area SD*
%RSD
AMLO HCTZ VALS AMLO HCTZ VALS
1.
Mobile Phase
composition
Ethyl Acetate
(5%)
1107.8
14.47
2119.9
38.8
13749.4
175.3
1.3 1.64 1.2
2.Wavelength
(2422nm)
1119.63
9.99
1743.6
17.2
1082
125.29
0.89 0.98 1.15
3.
Development
Distance
(705mm)
1073.43
10.6
1132.43
10.0
13430.1
133
0.93 1.02 0.99
SpecificitySpecificity is carried out by taken peak purity of standard and sample of each drug
and overlay standard and sample peak spectra to check specificity of each
individual drug peak .The peak purity for AMLO, HCTZ and VALS was tested by
correlation of spectra acquired at the peak start (s), peak maximum (m), and peak
end (e) positions which was found pass. And results are shown in table 54.
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Fig.25: Peak purity spectra of AMLO
Fig.26: Peak purity spectra of HCTZ
Fig.27: Peak purity spectra of VALS
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Table 54: Specificty data of AMLO, HCTZ and VALS
Table 55: Summary of Validation parameters by HPTLC with UV detection
Sr.no. Parameters AMLO HCTZ VALS
1 Linearity range
(ng/spot)100-600 150-900 1200-3200
2 Regression equation y =6.534x+547.48 y=5.931x+744.6 y=3.48x+2356
3 Correlation coefficient
(r2)
0.9945 0.9926 0.9918
4 Intercept 6.534 5.931 3.48
5 Slope 547.48 744.6 2356
6
Precision
Intraday % RSD (n = 3)
Interday % RSD (n = 3)
Repeatability of
measurements% RSD
(n=6)
0.99 to 2.0
0.48 to 0.98
1.98
0.67 to 1.2
0.43 to 1.92
1.86
0.23 to 1.83
0.25 to 1.26
0.84
7 Limit of detection 2.95 (ng/spot) 17.84 (ng/spot) 70.90 (ng/spot)
8 Limit of quantification 8.94(ng/spot) 53.9 (ng/spot) 214.85(ng/spot)
9 Specificity Pass Pass Pass
Drugs Co-relation r(s,m) Co-relation r (m,e) Peak purity
AMLO 0.99906 0.9994 Pass
HCTZ 0.99958 0.9976 Pass
VALS 0.99966 0.999 Pass
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4.3.3.2 Analysis of marketed formulation
The developed method was used to estimate AMLO, HCTZ and VALS in
combined dosage form. The percentage of AMLO, HCTZ and VALS was foundfrom the calibration curve.
Fig.28: HPTLC CHROMATOGRAM OF STANDARD MIXTURE
FIG.29: HPTLC CHROMATOGRAM OF MARKETED FORMULATION
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Fig.30: HPTLC chromatogram (3D view) for sample in Mix linearity of
AMLO, HCTZ AND VALS (Track 2, 3 for Sample Spot)
Table 56: Analysis of Marketed Formulation for Exforge HCT
Drug Label
claim(mg)
Amt
estimated(mg)
Assay results
S.D*
AMLO 5 5.01
100.321.025 5.06
5 4.96
HCTZ 12.5 12.74
100.81.4312.5 12.65
12.5 12.40
VALS 160 162.27
100.271.57160 161.37
160 157.49
*(n=3)
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4.3.4 Conclusion :By the virtue developed method, it can be concluded that high performance
thin layer chromatography method is reliable technique for the analysis of
commercial formulations of AMLO, HCTZ and VALS in tablet dosage
form. The developed method is simple, sensitive, and specific which
renders it suitable analysis of AMLO, HCTZ and VALS in combined
dosage form and this method is specific which show developed method is
free from the interference of excipients used in formulation.
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4.4 DEVELOPMENT AND VALIDATION OF RP-HPLC
METHOD FOR SIMULTANEOUS ESIMATION OF
AMLO, HCTZ AND VALS IN THEIR COMBINED
DOSAGE FORM
4.4.1 Instrumentation High performance liquid chromatography JASCO 200 Series, JASCO,
Inc. JAPAN, JASCO plus Pump, MX-2080-31 Mixer, Rheodyne model
7125 with 20 l fixed loop Injector , JASCO-UV- VIS 2075 PLUS Detector
with Borwin software version 1.50
PH Meter- 11 E/101E (Analytical labs scientific instrument Ltd., PH: 0 to14,Resolution: 0.01
PH, Accuracy: 0.01 P
H 14 digit
Analytical Balance- Citizen CX 220 (Citizen Pvt. Ltd), Weighing capacity:10 to 220 mg
Sonicator: D-compact,( Trans-o-sonic., Mumbai),Capacity: 2 Lit.4.4.2 Materials and methods4.4.2.1 Chemicals and Materials -Methanol (AR Grade, S.D. Fine chemicals Ltd.,
Mumbai, India) Acetonitrile (HPLC Grade, S.D. Fine chemicals Ltd.,
Mumbai , India) Water HPLC & Spectroscopy ( central drug house (p) Ltd.,
New Delhi), API of AMLO, HCTZ, and VALS Standards were kindly
gifted by Torrent pharmceutcial Gujarat, India., Analytical grade methanol
(Central Drug House Pvt. LTD) was used. Marketed tablet formulation
Exforge hct (Novartis pharma stein AG, stein, Switzerland.) Containing
Amlodipine besylate (5mg), Hctz( 12.5 mg) and Valsartan (160mg)
purchased from USA market.
4.4.2.2Chromatographic Conditions4.4.2.2.1Optimized Chromatographic Conditions Stationary phase: Kromasil Column KR-5C 18 (250 mm 4.6mm i.d., 5m) Mobile phase: Acetronitrile : potassium dihyrogen ortho phosphate buffer with
0.2% TEA(44 :56, v/v) , PH 3.7 adjusted with OPA
Wavelength: 232 nm Runtime: 15 Min.
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Flow rate: 1ml/min Diluent : ACN and Millipore Water(50:50, v/v)
Retention time for HCTZ : 3.78 Min Retention time for AMLO: 3.15 Min Retention time for VALS : 10.15 Min4.4.2.3Preparation of Mobile phaseMobile phase A:HPLC grade Acetonitrile was degassed with sonicator for 15 min.
Mobile phase B: 3402.25 mg of KH2PO4 (potassium dihydrogen ortho phosphate)
was dissolved in 500 triple dist. Water and add 1 ml HPLC grade triethylamine
(0.2%) and pH
3.7 adjusted with ortho phosphoric acid.
4.4.2.4Preparation of Standard Stock Solution of AMLO, HCTZ and VALS25 mg AMLO, HCTZ and VALS was weighed accurately and dissolved
each standard drug in separately in 25 ml methanol in different volumetric
flasks. 1 ml aliquots of the above solutions were diluted to 10 ml with
methanol in different volumetric flasks to produce 100 g/mL of AMLO
and 100 g/mL of HCTZ
4.4.2.5Preparation of ternary mixtures of AMLO, HCTZ and VALSSuitable aliquots of standard stock solution of AMLO, HCTZ and VALS
are mixed and diluted to volume with ACN and Millipore water (50:50) to
obtain different ternary mixture solutions containing AMLO, HCTZ and
VALS in different ratio Concentration of solution in the range 2 to 150
g/mL was prepared for the calibration curve of three drugs.
4.4.2.6Method validation Preparation of Linearity curve
For estimation of AMLO, calibration curve (n=3) was plotted in the range of (2-
25 g/mL). For estimation of HCTZ calibration curve (n=3) was plotted in the
range of (5-45 g/mL). For estimation of VALS calibration curve (n=3) was
plotted in the range of (20-150 g/mL).Calibration curve of peak area v/s
concentration was plotted for the drug.
PrecisionIntraday and interday precision
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For intraday precision, the experiment was repeated three times in a day using
three different concentrations for AMLO (5, g/ml 10, 15 g/ml), for HCTZ
(10, 15, 20 g/ml) and for VALS (80,100,120 g/mL). For Interday precision,
the experiment was repeated on three different days using three different
concentrations respectively AMLO, HCTZ and for VALS Precision find out in
terms of %RSD
RepeatabilityPeak area of sample solutions for AMLO, HCTZ, VALS (5, 15, 100 g/mL)
were taken by 6 times and find out the % RSD.
LOD AND LOQFrom the linearity curve equations, the standard deviations (SD) of the
intercepts (response) were calculated. Then LOD and LOQ were measured for
all the three drugs by using mathematical expressions given is section ()
AccuracyTo study the accuracy, 7 tablets were weighed and powdered. The powder
equivalent containing (5mg of AMLO, 12.5 mg for HCTZ and 160 mg of
VALS) were weighed and transferred to 50ml volumetric flask and dissolved in
30 ml of methanol. Then solution was sonicated for 15 minutes and volume was
made up to the mark with methanol. The above solution was filtered with
whatmann filter paper (No. 41). Aliquot (0.2ml) was pipette out and transferred
to 10 ml volumetric flask. Volume was made up to the mark with methanol to
get a solution containing 2g/mL of AMLO, 4 g/mL of HCTZ and 64 g/mL
of VALS. Standard drug was added at three different concentration levels (80%,
100% and 120% of test sample concentration) to pre-analyzed sample and
amplitudes of the solution were measured at selected wavelengths 232 nm for
AMLO, HCTZ and VALS drugs. Amplitudes were substituted into respective
straight line equation to calculate percentage recovery of the drugs.
RobustnessThe following parameters were changed one by one and their effect was
observed on system suitability.
a)Flow rate of mobile phase ( 0.2 ml) to 0.8 ml/min and 1.2 ml/min. (Table6.11)
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b)PH ( 0.05 absolute) to 3.65 and 3.75 (Table 6.12)c)Wave length (2nm) to 230 and 234
SpecifictySpecificity of an analytical method is ability to measure specifically the analyte
of interest without interferences from blank and placebo.
4.4.2.7Analysis of marketed formulationTotal 14 tablets were weighed accurately and powdered. An amount
equivalent to one tablet (containing 5 mg of AMLO, 12.5 mg of HCTZ and
160 mg of VALS) was taken and dissolved in 100 ml methanol in 100 ml
volumetric flask Solution was sonicated for 15 minutes. After solution was
filtered by using Whatmann filter paper No.41.From this solution, 5 ml of
sample solution was taken in 50 ml volumetric flask and diluted with
diluents ACN:Water (50:50) final solution containing 5 g/mL
concentration of AMLO, 12.5 g/mL HCTZ and 160 g/mL concentration
of VALS.
4.4.3 Results and Discussion4.4.3.1 Method development
Optimization of the chromatographic condition was studied by checking the
effect of chromatographic variables such as temperature, back pressure,
flow rate and solvent ratio. The resulting chromatograms were recorded andthe chromatographic parameters which give the best peak resolution were
selected for analysis
Table 57: Observation and remarks of mobile phase optimization
Sr. no. Mobile phase composition Inference Conclusion
1
ACN :0.025 M potassium
dihydrogen ortho phosphate
(60:40, v/v) PH
3.7
Peak was not
suitable for
quantitative
M.P was not
suitable
2
ACN :0.025 M potassium
dihydrogen ortho phosphate
(50:50, v/v), PH
3.7
VALS RT greater
than 10 min M.P was not
suitable
3
ACN :0.025 M potassium
dihydrogen ortho phosphate
(57:43, v/v) , TEA 0.1%, PH
3.7
Asymmetry
greater than 1.5
M.P was not
suitable
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4.
ACN : 0.025 M potassium
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 Capacity Factor 0.6434 0.3695 3.4130
2. Tailing factor 1.30 1.41 1.05
3. Resolution factor 3.175 - 10.37
4. Theoretical plates 5378 6357 4886
5. % RSD of Peak Area 0.48 1.85 0.52
4.4.3.2Validation parameters Linearity
Linearity curve shows linearity in the range of 100-600 g/mL for AMLO, 5-
45g/mL for HCTZ and for VALS 20-120 g/mL. The correlation coefficient (r2)
was found to be 0.9945, 0.9965, and 0.9971 for AMLO, HCTZ and VALS
respectively Calculate and record value of correlation co-efficient (r), y-intercept,
slope of regression line and residual sum of squares. (Figure 6.12) and results
(Table)
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Table 59: Linearity of AMLO by RP-HPLC with UV PDA
*(n=3)
Fig.35: Calibration curve for AMLO
Sr. No. Conc.
(g/mL)
Peak Area
Mean SD* %RSD
1 2 144942 1936.48 1.33
2 5 327329.51376.5 0.42
3 10 50262 2063 0.41
4 15 796769.36361.674 0.79
5 20 11170131489.079 0.13
6 25 13725909579.747 0.69
y = 53407x + 24828
R = 0.9945
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 10 20 30
P
e
a
k
A
r
e
a
Conc. g/ml
Series1
Linear (Series1)
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Table 60: Linearity of HCTZ by RP-HPLC with UV PDA detection
*(n=6)
Fig.36: Calibration curve of HCTZ
y = 135283x + 114584
R = 0.996
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
0 10 20 30 40 50
P
ea
k
a
r
e
a
Conc. g/ml
Series1
Linear (Series1)
Sr. No.
Conc.
(g/mL)
Peak Area
Mean SD* %RSD
1 5 826897.34122.691 0.49
2 10 148119517761.38 1.19
3 15 197599515134.72 0.76
4 25 35574276076.3 0.45
5 35 503966041762.14 0.82
6 45 606955936571.47 0.60
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Table 61: Linearity of VALS by RP-HPLC with PDA UV detection
*(n=6)
Fig.36: Calibration curve of VALS
Sr. No.Conc.
(g/mL)
Peak Area
Mean SD* %RSD
1 20 161341316557.68 1.02
2 40 325834232479.94 0.99
3 60 617468382500.03 1.33
4 80 80224449798.269 0.12
5 120 1303326245680.19 0.35
6 150 1589395296053.33 0.60
y = 112822x - 838391
R = 0.9971
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
0 50 100 150 200
P
e
a
k
A
r
ea
Conc. g/ml
Series1
Linear (Series1)
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Fig.31: HPLC Chromatogram of Blank (diluents) ACN: Water (50:50, V/V)
Fig.32: HPLC Chromatogram of HCTZ
Fig.33: HPLC chromatogram of AMLO
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Fig.26: HPLC chromatogram of standard mixture of AMLO, HCTZ and
VALS
Fig.38: Mixture Linearity Curve of AMLO, HCTZ AND VALS BY HPLC
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Precision:Intraday precision and Interday precision for AMLO, HCTZ and VALS was
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
*(n=3)
Table 63: Intraday precision data of AMLO, HCTZ AND VALS by HPLC
*(n=3)
DrugConcentration
(g/mL)
Peak Area
Mean SD*
%RSD
AMLO
5 306648.72519.684 0.82
10 562226.71300.598 0.23
15 865629.75262.363 0.60
HCTZ
10 13668687087.164 0.51
15 219059445155.87 2.06
20 271234923962.09 0.88
VALS
80 772358530225.43 0.39
100 989746860556.46 0.61
120 13142484202472.7 1.54
Drug Concentration
(g/mL)
Peak Area
Mean SD %RSD
AMLO
5 309600.73256.28 1.05
10 502668.32644.688 0.52
15 773136.3253.4213 0.33
HCTZ
10 136914910733.71 0.78
15 211000710417.99 0.49
20 267864933853.87 1.26
VALS
80 777402543345.03 0.55
100 993643953407.12 0.53
120 1301815934631.55 0.26
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Table 64: Repeatability study of AMLO, HCTZ and VALS
Drug Conc. (g/mL) Peak AreaSD* %RSD
AMLO 5 29171968.42 0.71
HCTZ 15 18334406751.29 0.36
VALS 100 938193792487.03 0.98
*(n=6)
LOD and LOQFrom determination calibration curve for AMLO, HCTZ and VALS was
repeated six times and measured by mathematical equation .LOD value were
0.23 g/mL, 0.48 g/mL, 1.1 g/mL and LOQ value 0.71 g/mL, 1.47 g/mL,
3.3 g/mL for AMLO, HCTZ and VALS respectively.
SpecificityPeak purity spectra were taken of AMLO, HCTZ and VALS by UV-PDA
detection.Peak purity Front and Tail value given Table
Fig.25: Peak purity spectra of AMLO by UV-PDA
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Fig.25: Peak purity spectra of HCTZ by UV-PDA
Fig.25: Peak purity spectra of VALS by UV-PDA
Table 66: Specificity data of AMLO, HCTZ and VALS
Drugs Peak purity Front Peak purity Tail Peak
purity
AMLO 996.65 998.45 Specific
HCTZ 999.93 999.70 Specific
VALS 997.15 998.94 Specific
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AccuracyStandard edition was done at three level 80%, 100% and 120% of a concentration
of sample in the linearity range and % recovery was found 98 to 100%. % recoverywas calculated from regression equation of the calibration curve as shown in Table
6.8
Table 67: % Recovery study of AMLO, HCTZ and VALS
DRUG
INITIAL
CONC.
g/mL
STD.
Added
g/mL
TOTAL
CONC.
After
spiking
g /mL
ACCURACY
Conc.Recovered
MeanSD*
%
RSD
%Recovery
Mean SD*
%
RSD
AMLO 2
1.6 3.63.5833
0.0491.13
99.57333
0.321.33
2 44.0146
0.0461.42
101.4233
0.750.75
2.4 4.4 4.45660.037
0.69 101.42330.07
0.76
HCTZ 5
4 98.8766
0560.82
98.35333
0.010.95
5 109.9533
0.0370.49
98.35333
0.041.47
6 1110.9033
0.080.95
99.16667
0.760.76
VALS 64
51.2 115.2 117.8060.101
0.18 102.01670.074
0.74
64 128129.466
2.0851.61
101.0967
0.0561.56
76.8 140.8140.3733
0.8830.62
99.69
0.06370.63
*(n=3)
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RobustnessTable 68- Robustness study of AMLO
Parameters ChangeMean of Peak Area
%RSD%Assay
Flow Rate (0.2 ml/min)0.8 2936241.74 98.25
1.2 2904940.52 100.0
PH
( 0.05)
3.65 382701.71.53 99.82
3.75 210569.21.74 100.2
Wavelength ( 2 nm)
230 12836530.29 100.42
234 12489260.51 101.04
Table 69: Robustness study of HCTZ
Parameters Change Mean of Peak
Area%RSD
%Assay
Flow Rate
(0.2ml/min.)
0.8 19520661.17 98.44
1.2 681237.70.88 99.38
PH
(0.05)
3.65 13658011.55 101.59
3.75 12805860.56 98.15
Wave Length
( 2nm)
230 14799090.95 99.77
234 15599061.79 98.51
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Table 70: Robustness study of VALS
Parameters Change Mean %RSD %Assay
Flow Rate
(0.2 ml/min)
0.8 178625950.8 98.60
1.2 162600241.0 98.79
PH
( 0.05)
3.65 136887281.3 98.48
3.75 118075061.4 101.76
Wave Length
(2 nm)
230 117413080.8 98.45
234 97272071.30 98.43
Table 71: Summary of Validation parameters of RP-HPLC
Sr.
noParameters AMLO HCTZ VALS
1.Linearity range
(g/mL)2-25 5-45 20-150
2.
Regression
equation
y =53047x+24828 y=135283x+11458
4
y = 112822x-
83839
3.Correlation
coefficient (r2)
0.9945 0.9967 0.9971
4.
Precision
Intraday % RSD
(n = 3)
Interday % RSD
(n = 3)
Repeatability of
measurements %
RSD (n=6)
0.23-1.82
0.03-1.05
0.5-1.4
0.5-2.0
0.49-1.26
0.24-0.36
0.39-1.54
0.26-0.55
0.77-1.01
5. Specificit
Specific Specific Specific
6. LOD (g/ml) 0.23 0.48 1.1
7. LOQ (g/ml) 0..71 0.1.47 3.3
8. % Recovery 99.571.33 to
101.420.75
98.350.19 to
99.16 0.76
99.69 0. 63
to
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Table 72: Analysis of Marketed Dosage Form
*(n=3)
Fig.39: HPLC Chromatogram of Marketed Dosage Form
DrugsLabel
claim(mg)
Amt
estimated(mg)
Assay results
% recovery S.D*
AMLO
5 4.95
98.660.365 4.91
5 4.92
HCTZ
12.5 12.66
99.870.8712.5 12.44
12.5 12.39
VALS
160 158.31
100.621.49160 161.77
160 162.92
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4.4.4 ConclusionBy the virtue developed method, it can be concluded that high performance
Liquid chromatography method is reliable technique for the analysis of
commercial formulations of AMLO, HCTZ and VALS in tablet dosageform. The developed method is simple, sensitive, and specific which
renders it suitable analysis of AMLO, HCTZ and VALS in combined
dosage form and this method is specific which show developed method is
free from the interference of excipients used in formulation
4.5 COMPARISON OF UV-VIS SPECTROPHOTOMETRIC,HPTLC AND RP-HPLC METHOD
4.5.1 Comparison of developed chromatographic method
Comparison of developed methods (UV-VIS spectrophotometric, HPTLC
and RP-HPLC was performed by applying Student-ANOVA-test (single
factor)
Table73: COMPARISON OF UV-VIS SPECTROPHOTOMETRIC, HPTLC
AND RP-HPLC METHODS FOR ESTIMATION OF AMLO, HCTZ and
VALS IN TABLET DOSAGE FORM
Brand name Drugs% Assay results
U.V HPTLC RP-HPLC
Exforge HCT
AMLO
98.66 100.28 99.07
98.45 101.37 98.36
100.3 98.79 98.55
HCTZ
98.82 101.97 100.8
98.40 101.23 99.52
98.20 99.26 99.13
VALS
98.33 98.59 101.10
98.75 99.15 101.82
99.20 101.61 98.94
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Table 74: Comparison of three Methods by ANOVA Test
Brand name Drugs Fcal F crit
Exforge HCT
AMLO 1.63 4.066
HCTZ 3.68 4.066
VALS 1.18 4.066
4.5.2 Conclusion
ANOVA result was performed by using Microsoft excel and graph pad
instate, version 3.05, 32 bit. So developed methods were compared
statistically by ANOVA test. The results show that there is no significant
statistical difference between the results obtained by above mentioned
methods. In the cases, Fcal is less than Fcrit.
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