CHAPTER 4 CHEMOMETRICS ASSISTED QUANTITATIVE...

62

CHAPTER 4

CHEMOMETRICS ASSISTED QUANTITATIVE ANALYSIS

4.1 EXPERIMENTAL PROCEDURE

4.1.1 Instrument Employed

Perkin Elmer – Lamda 25 UV Visible Spectrophotometer with UVWinlab software, 1cm matched quartz cells and ± 0.01nm wavelengthaccuracy.

4.1.2 Chemicals Used

Sodium Hydroxide (AR), Ranchem, Mumbai

Distilled Water, MilliQ, Millipore.

4.1.3 Reference Standards

Pharmaceutical grade Amlodipine besylate, Hydrochlorothiazide,Irbesartan hydrochloride, Losartan potassium, Manidipine hydrochloride,Telmisartan (Madras Pharmaceuticals, Chennai) Ramipril, Trandolapril,Valsartan hydrochloride and Verapamil (Hetero Drugs Limited, Hyderabad)were used as working standards after confirming their purity.

4.1.4 Pharmaceutical Preparations

Different tablets containing the drugs under study were procuredfrom the local market and subjected to analysis by the proposed procedures.

4.1.5 Preparation of Stock Solutions

Into 100 ml volumetric flasks an accurately weighed amount of anyof the studied drugs was dissolved in about 50 ml of 0.1 M sodium hydroxideand diluted to volume with distilled water. The resulting solution was dilutedquantitatively with distilled water to obtain the appropriate dilutions for eachdrug according to its linear calibration range.

63

4.1.6 Sample Preparation

Twenty tablets were weighed and finely powdered. An accuratelyweighed amount of the powder equivalent to the average weight of one tabletwas transferred to 100 ml volumetric flask and diluted to about 50 ml with0.1M sodium hydroxide. The mixture was shaken for 10 min, sonicated foranother 10 min, diluted to 100ml with distilled water and filtered. Theobtained clear solution was used as stock sample solution. The stock solutionwas diluted quantitatively with distilled water to obtain suitable workingsample solutions for the UV measurements.

4.1.7 Standard Solutions for calibration set

In order to obtain the calibration matrices for applying multivariatetechniques, 25 solutions were prepared by mixing known amount of drugsunder study in different varying proportions. The ranges were previouslyverified to obey Beer’s law for each of the studied drugs. The mixtures wereprepared by accurately transferring aliquots of working solutions in to a seriesof 10 ml volumetric flask and the volume was completed using distilled water.

4.1.8 Preparation of Validation set

A prediction set of ten samples was randomly prepared to check theapplicability of the method. Validation set consisting of 10 samples were preparedfrom the working solutions in the same manner as that of calibration set.

4.1.9 Data Processing

The absorption spectra of all the mixtures were recorded in the UVrange and data Processing was carried out using Intel Pentium III processorcomputer equipped with essential softwares such as MATLAB R2007b(Mathworks Limited) and PLS-Toolbox ver5.0 (Eigen Vector TechnologiesLimited).

64

4.2 RESULTS AND DISCUSSION

4.2.1 Binary mixture 1 – Irbesartan (IRB) and Hydrochlorothiazide(HCZ)

4.2.1.1 Spectral Characteristics

The absorption spectra of IRB and HCZ were recorded over therange of 200 – 350nm in fundamental mode as shown in Figure 4.1. The firstderivative and second derivative spectra of both the drugs are represented inFigure 4.2 and 4.3 respectively. A clear overlap between the curves is evident

for all the spectra. The wavelength interval ( ) was optimized as 10nm and

15 nm for first and second order spectra respectively, which gave a goodsignal to noise ratio. A deep examination of all the spectra was done but nosignal proportional to the components was singled out. Multivariatecalibration methods appeared to be ideal for quantifying the drugs using fullspectral information.

Figure 4.1 Fundamental Absorption Spectra of Irbesartan,Hydrochlorothiazide and mixture

65

Figure 4.2 First Derivative Spectra of Irbesartan, Hydrochlorothiazideand mixture

Figure 4.3 Second Derivative Spectra of Irbesartan, Hydrochlorothiazideand mixture

66

4.2.1.2 Building the calibration models

Stock solutions of IRB (1 mg/ml) and HCZ (1 mg/ml) were used toset up the calibration set samples. Stock solutions were appropriately dilutedwith water separately to give working solutions of IRB and HCZ of 10µg/mlconcentration respectively. A calibration set of 25 different laboratorymixtures of IRB and HCZ were prepared with the concentration range of 1-5µg/ml for IRB and 0.5-2.5 µg/ml for HCZ (Table 4.1). The concentrationrange was established based on the fact that the absorbance value of theprepared mixture does not exceed 1.5 in the spectral range.

The absorption spectra of the mixtures were recorded in the rangeof 200-350 nm. The data points for each spectrum were collected at 1nmintervals using UV Winlab software and transferred to MS Excel, Matlab andPLS Toolbox for subsequent data analysis.

4.2.1.3 Optimized conditions for Partial Least Square (PLS)

Wavelength range : 200-350 nm

Data Points : 151 nm

Preprocessing : Autoscale, Mean Centre, orthogonal signalcorrection

Cross Validation : Leave one out

Latent Variables : 12

4.2.1.4 Validation using Prediction set

To investigate the prediction ability of the resulting PLS model, thecalibrated PLS model was used for quantization of the analytes in a separateprediction set mixtures that mostly did not have contribution in the modelbuilding steps.

The estimated amount of IRB and HCZ in calibration andprediction sets using the optimized PLS model are given in Tables 4.2 – 4.5.

67

Table 4.1 Concentrations of Calibration and Prediction sets expressed

in µg/ml

MixtureNumber

Calibration Set (µg/ml) Prediction Set (µg/ml)IRB HCZ IRB HCZ

M1 1 0.5 1 2M2 1 1 2 2M3 1 1.5 3 0.5M4 1 2 4 2.5M5 2 0.5 5 1.5M6 2 1 1.5 2M7 2 1.5 3 2.5M8 2 2 3 2M9 3 2.5 4 0.5M10 3 2 1.5 1.5M11 3 1.5 - -M12 3 1 - -M13 4 0.5 - -M14 4 1 - -M15 4 1.5 - -M16 4 2 - -M17 5 2.5 - -M18 5 1.5 - -M19 5 0.5 - -M20 5 1 - -M21 1.5 1 - -M22 2.5 2 - -M23 3.5 1 - -M24 4.5 1 - -M25 3.5 2 - -

68

Table 4.2 Estimated Concentrations of Irbesartan in the calibration set

under optimized PLS model

AmountPresent(µg/ml)

FundamentalMode

First DerivativeMode

Second DerivativeMode

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)% recovery

1 0.9884 98.8406 0.9899 98.9984 0.9931 99.31971 1.0026 100.2613 1.0058 100.5875 1.0124 101.24191 0.9991 99.9199 0.9720 97.2031 0.9860 98.60631 0.9988 99.8829 1.0306 103.0629 1.0378 103.78222 1.9979 99.8956 2.0118 100.5908 1.9711 98.55662 2.0041 100.2081 2.0145 100.7259 2.0060 100.30232 2.0133 100.6695 1.9625 98.1289 1.9609 98.04702 1.9960 99.8008 2.0401 102.0051 2.0442 102.21283 2.9993 99.9795 2.9387 97.9589 2.9162 97.20743 2.9897 99.6588 3.0271 100.9050 3.0651 102.17023 2.9976 99.9205 3.0116 100.3867 3.0100 100.33383 3.0024 100.0807 3.0139 100.4654 3.0218 100.72834 3.9950 99.8774 3.9632 99.0808 3.9832 99.58114 4.0310 100.7765 4.0022 100.0553 4.0357 100.89314 3.9994 99.9857 3.9887 99.7179 3.9574 98.93664 3.9981 99.9546 4.0257 100.6429 4.0193 100.48345 5.0093 100.1868 5.0182 100.3657 5.0191 100.38205 4.9732 99.4648 4.9500 99.0008 4.9300 98.60125 5.0096 100.1922 4.9936 99.8723 5.0049 100.09815 4.9976 99.9530 5.0484 100.9696 5.0199 100.3993

1.5 1.4971 99.8077 1.5025 100.1689 1.4720 98.13862.5 2.5058 100.2325 2.4931 99.7275 2.5046 100.18723.5 3.5072 100.2074 3.4982 99.9500 3.5101 100.29034.5 4.4851 99.6698 4.5104 100.2331 4.5152 100.33863.5 3.5013 100.0375 3.4861 99.6055 3.5029 100.0842

Mean 99.9786 Mean 100.0164 Mean 100.0369

69

Table 4.3 Estimated concentrations of Hydrochlorothiazide in the

calibration set under optimized PLS model


Fundamental Mode First Derivativemode

Second Derivativemode

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4971 99.4230 0.5278 105.5746 0.9931 99.31971 0.9994 99.9404 0.9617 96.1741 1.0124 101.2419

1.5 1.4997 99.9816 1.5193 101.2884 0.9860 98.60632 1.9978 99.8944 1.9941 99.7095 1.0378 103.7822

0.5 0.5020 100.4031 0.4996 99.9362 1.9711 98.55661 1.0081 100.8163 0.9589 95.8927 2.0060 100.3023

1.5 1.4946 99.6403 1.5691 104.6076 1.9609 98.04702 2.0009 100.0483 1.9994 99.9744 2.0442 102.2128

2.5 2.5058 100.2326 2.5119 100.4762 2.9162 97.20742 1.9915 99.5776 1.9628 98.1418 3.0651 102.1702

1.5 1.5126 100.8430 1.5065 100.4352 3.0100 100.33381 0.9942 99.4227 1.0096 100.9673 3.0218 100.7283

0.5 0.4890 97.8101 0.5279 105.5879 3.9832 99.58111 1.0194 101.9490 1.0080 100.8019 4.0357 100.8931

1.5 1.4927 99.5189 1.4000 93.3398 3.9574 98.93662 1.9986 99.9345 2.0384 101.9228 4.0193 100.4834

2.5 2.4977 99.9095 2.5118 100.4722 5.0191 100.38201.5 1.4948 99.6539 1.4885 99.2386 4.9300 98.60120.5 0.5066 101.3203 0.4838 96.7791 5.0049 100.09811 1.0011 100.1179 1.0277 102.7770 5.0199 100.39931 0.9989 99.8915 1.0009 100.0934 1.4720 98.13862 1.9962 99.8116 1.9991 99.9561 2.5046 100.18721 1.0036 100.3616 0.9797 97.9728 3.5101 100.29031 0.9885 98.8588 1.0415 104.1512 4.5152 100.33862 2.0081 100.4086 1.9708 98.5423 3.5029 100.0842

Mean 99.9908 Mean 100.1925 Mean 100.4786

70

Table 4.4 Estimated Concentrations of Irbesartan in the Prediction set

under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 0.9549 95.4982 0.9979 99.7932 1.0363 103.63482 2.0249 101.2457 1.9844 99.2202 2.0240 101.20303 2.8215 94.0533 2.7151 90.5043 2.7073 90.24604 3.8315 95.7886 3.9111 97.7786 3.9147 97.86955 4.7621 95.2436 4.9452 98.9052 4.9037 98.0751

1.5 1.4553 97.0217 1.4706 98.0451 1.5503 103.35613 3.0659 102.1995 2.9427 98.0929 2.9668 98.89373 2.9687 98.9594 2.8973 96.5794 2.9637 98.79034 3.8462 96.1561 3.8440 96.1001 3.8598 96.4972

1.5 1.4938 99.5917 1.4451 96.3431 1.4451 104.0495Mean 97.5758 Mean 97.1362 Mean 99.2616

Table 4.5 Estimated Concentrations of Hydrochlorothiazide in the

prediction set under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.1518 107.5916 2.1116 105.5831 2.1978 109.89462 2.1355 106.7775 2.1391 106.9583 1.8963 94.8157

0.5 0.4335 86.7072 0.5344 106.8966 0.5247 104.95962.5 2.3693 94.7750 2.4575 98.3010 2.5785 103.14001.5 1.6693 111.2906 1.4562 97.0852 1.5244 101.63142 2.0407 102.0356 1.9377 96.8883 1.9788 98.8424

2.5 2.5198 100.7955 2.5250 101.0008 2.5656 102.62412 1.9441 97.2059 1.8790 93.9521 1.9383 96.9152

0.5 0.4822 96.4572 0.4854 97.0992 0.5213 104.26251.5 1.5138 100.9243 1.4303 95.3534 1.5018 100.1233

Mean 100.4560 Mean 99.9118 Mean 101.7309

71

4.2.1.5 Statistical Parameters

Statistical Parameters such as mean % recovery, standard deviation(SD), Root Mean Square Error Calibration (RMSEC) and Root Mean SquareError Prediction (RMSEP) were calculated for each calibration and predictionset. The mean % recovery was close to 100 in all the systems. Though thestandard deviation values were found to increase with increase in spectralorder, the values of RMSEC and RMSEP were found to be nearly same asshown in Table 4.6.

Table 4.6 Statistical Parameters calculated from application of PLS

model to the spectral data of calibration and prediction set

samples

Partial Least SquaresParameters D0 D1 D2

Cal

ibra

tion

Set

IRB HCZ IRB HCZ IRB HCZMean(%) 99.9786 99.9908 100.0164 100.1925 100.0369 100.4786

SD 0.3718 0.7891 1.2254 2.9702 1.4515 5.5507R2 0.9999 0.9999 0.9996 0.9970 0.9994 0.9922

RMSEC 0.0103 0.0071 0.0266 0.0330 0.0066 0.0533

Pred

ictio

nSe

t

Mean(%) 97.5758 100.4560 97.1362 99.9118 99.2616 101.7309

SD 2.7589 7.1410 2.6390 4.8958 4.1528 4.3159R2 0.9965 0.9813 0.9963 0.9878 0.9952 0.9874

RMSEP 0.1358 0.0986 0.1163 0.0807 0.0363 0.0814

4.2.1.6 Optimized conditions for Principal Component Regression(PCR)


Data points : 151

Preprocessing : Mean Centre, Smoothing, Orthogonal Signalcorrection


Number of PCs : 13

72

Estimated concentrations of IRB and HCZ using the optimized

PCR model in both calibration and prediction sets are represented in Tables

4.7 – 4.10. The percentage recovery for both the drugs was found to lie in the

range of 97-103%.

4.2.1.7 Statistical Parameters

Statistical Parameters such as mean % recovery, SD, RMSEC and

RMSEP were calculated for each calibration and prediction set. The mean %

recovery was close to 100 in all the systems. RMSEC and RMSEP were found to

decrease for both drugs with increase in derivatization. Lesser values of RMSEC

and RMSEP show that the estimated concentrations are close to the reported

values. The correlation coefficient (R2) of both IRB and HCZ were found to

increase with increase in spectral order. Comparison of mean recovery values for

both the drugs in all three spectral modes is depicted in Figure 4.4.

73

Table 4.7 Estimated Concentrations of Irbesartan in the calibration setusing optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 1.0156 101.5641 0.9849 98.4900 1.0150 101.50731 1.0026 100.2628 1.0245 102.4592 1.0075 100.75301 1.0004 100.0435 0.9636 96.3669 0.9647 96.47941 0.9797 97.9713 1.0478 104.7825 1.0630 106.30962 2.0006 100.0318 1.9753 98.7681 1.9798 98.99012 1.9961 99.8081 2.0112 100.5640 2.0150 100.75152 2.0043 100.2161 1.9718 98.5949 1.9619 98.09602 2.0513 102.5659 2.0530 102.6504 2.0242 101.21413 2.9564 98.5497 2.9365 97.8855 2.9365 97.88373 2.9598 98.6614 3.0237 100.7923 3.0338 101.12993 3.0108 100.3601 2.9986 99.9563 3.0155 100.51763 3.0399 101.3312 3.0395 101.3181 2.9942 99.80664 3.9658 99.1468 3.9562 98.9073 3.9884 99.71194 4.0235 100.5892 4.0138 100.3452 4.0275 100.68974 4.0028 100.0714 3.9869 99.6730 3.9591 98.97844 3.9899 99.7494 4.0176 100.4411 4.0178 100.44705 5.0491 100.9821 5.0148 100.2971 5.0094 100.18975 4.9581 99.1638 4.9532 99.0642 4.9646 99.29215 5.0035 100.0714 4.9890 99.7818 4.9969 99.93815 5.0336 100.6727 5.0467 100.9357 5.0243 100.4873

1.5 1.5048 100.3222 1.5007 100.0531 1.4585 97.23392.5 2.4897 99.5900 2.4756 99.0260 2.5155 100.62293.5 3.4307 98.0227 3.4881 99.6623 3.5068 100.19694.5 4.4756 99.4585 4.5292 100.6498 4.5102 100.22683.5 3.5542 101.5502 3.4964 99.8975 3.5086 100.2481

Mean 100.0303 Mean 100.0545 Mean 100.0681

74

Table 4.8 Estimated concentrations of Hydrochlorothiazide in the

calibration set using optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4897 97.9420 0.6572 131.4599 0.5011 100.22201 1.0006 100.0648 0.9266 92.6690 0.8990 89.9090

1.5 1.4962 99.7533 1.5722 104.8136 1.5011 100.07332 1.9934 99.6732 1.9780 98.9022 1.8972 94.8601

0.5 0.5362 107.2441 0.5589 111.7860 0.5197 103.94601 0.9232 92.3208 1.0702 107.0228 1.1077 110.7700

1.5 1.4654 97.6972 1.4974 99.8303 1.5054 100.36532 2.0021 100.1071 2.0581 102.9081 2.0164 100.8243

2.5 2.5334 101.3360 2.482 99.2800 2.5594 102.37672 2.1032 105.1636 1.9794 98.9715 1.9695 98.4750

1.5 1.4775 98.5000 1.4860 99.0666 1.4764 98.42731 0.9918 99.1873 0.9318 93.1879 0.9870 98.7090

0.5 0.5194 103.8806 0.5140 102.8134 0.4323 86.46011 0.9945 99.4507 1.0894 108.9479 0.9731 97.3110

1.5 1.5307 102.0496 1.5012 100.0800 1.4812 98.74662 2.1040 105.2018 2.0638 103.1911 2.0751 103.7550

2.5 2.4416 97.6659 2.5127 100.5115 2.4900 99.60281.5 1.4919 99.4619 1.5015 100.1010 1.5377 102.51550.5 0.5166 103.3260 0.5357 107.1528 0.5282 105.64971 0.9945 99.4501 0.9381 93.8181 1.0640 106.40601 1.0187 101.8781 0.9820 98.2001 1.1035 110.35002 1.8790 93.9541 2.0648 103.2448 1.8315 91.57761 1.1345 113.4559 1.0411 104.1111 0.9484 94.84861 1.0028 100.2861 0.9804 98.0460 0.9893 98.93452 1.9480 97.4035 1.8864 94.3201 1.9650 98.2504

Mean 100.6581 Mean 102.1774 Mean 99.7346

75

Table 4.9 Estimated concentrations of Irbesartan in the prediction set

using optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 0.9283 92.8339 1.0423 104.2352 0.9766 97.66282 2.0548 102.7426 2.0317 101.5861 1.9843 99.21753 2.8258 94.1939 2.7086 90.2896 2.8206 94.02004 3.8996 97.4911 3.9202 98.0067 3.9207 98.01835 4.7768 95.5368 4.8835 97.6703 4.9533 99.0660

1.5 1.3534 90.2331 1.4492 96.6189 1.5315 102.10353 3.0144 100.4812 2.9836 99.4546 2.8783 95.94533 2.9313 97.71278 2.9396 97.9889 2.9346 97.82144 3.7871 94.6780 3.8583 96.4592 3.8383 95.9583

1.5 1.3796 91.9788 1.4640 97.6055 1.5218 101.4544Mean 95.7882 Mean 97.9915 Mean 98.1268

Table 4.10 Estimated concentrations of Hydrochlorothiazide in theprediction set using optimized PCR model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.1580 107.9005 2.1284 106.4206 2.1035 105.17772 2.1660 108.3003 1.8817 94.0855 2.0235 101.1751

0.5 0.4810 96.2093 0.4793 95.86172 0.5028 100.56722.5 2.6351 105.4053 2.4218 96.8740 2.4998 99.99231.5 1.4837 98.9158 1.4840 98.9375 1.5850 105.67222 2.0685 103.4268 1.8828 94.1405 1.9677 98.3871

2.5 2.7257 109.0300 2.5991 103.9667 2.4988 99.95492 1.8449 92.2457 1.9553 97.7694 1.9341 96.7097

0.5 0.5315 106.3023 0.5160 103.2079 0.5296 105.92771.5 1.4824 98.8285 1.4547 96.9819 1.4425 96.1681

Mean 102.6564 Mean 98.8246 Mean 100.9732

76

Figure 4.4 Plots of Calibration and Predictions sets of Irbesartan and

Hydrochlorothiazide in both PLS and PCR models

y = 0.999x + 0.000R² = 0.999

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

IRB-PLS-CAL-D0

y = 0.999x + 0.001R² = 0.999

0

1

2

3

4

5

6

0 1 2 3 4 5Amount Present

IRB-PLS-CAL-D1

y = 0.999x + 0.001R² = 0.999

0

1

2

3

4

5

6

0 1 2 3 4 5

Amount Present

IRB-PLS-CAL-D2

y = 0.946x + 0.073R² = 0.996

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

IRB-PLS-PRE-D0

y = 0.975x - 0.014R² = 0.996

0

1

2

3

4

5

6


IRB-PLS-PRE-D1

y = 0.948x + 0.094R² = 0.995

0

1

2

3

4

5

6


IRB-PLS-PRE-D2

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0

y = 0.997x + 0.004R² = 0.997

0

0.5

1

1.5

2

2.5

3

0 1 2 3Amount Present

HCZ-PLS-CAL-D1

y = 0.992x + 0.010R² = 0.992

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-CAL-D2

77

Figure 4.4 (Continued)

y = 1.012x + 0.004R² = 0.981

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.996x + 0.011R² = 0.987

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-PRE-D1

y = 1.012x + 0.001R² = 0.987

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-PRE-D2

y = 0.999x + 0.001R² = 0.999

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Am

ount

pre

dict

ed

Amount Present

IRB-PCR-CAL-D0

y = 0.999x + 0.001R² = 0.999

0

1

2

3

4

5

6


IRB-PCR-CAL-D1

y = 0.999x + 0.001R² = 0.999

0

1

2

3

4

5

6

0 1 2 3 4 5 6Amount Present

IRB-PCR-CAL-D2

y = 0.968x - 0.015R² = 0.995

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

IRB-PCR-PRE-D0

y = 0.961x + 0.036R² = 0.995

0

1

2

3

4

5

6


IRB-PCR-PRE-D1

y = 0.968x + 0.025R² = 0.997

0

1

2

3

4

5

6


IRB-PCR-PRE-D2

78

Figure 4.4 (Continued)

y = 0.99x + 0.017R² = 0.992

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.978x + 0.042R² = 0.990

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D1

y = 0.988x + 0.010R² = 0.989

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D2

y = 1.076x - 0.072R² = 0.983

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 0.997x - 0.015R² = 0.986

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-PRE-D1

y = 0.992x + 0.022R² = 0.994

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-PRE-D2

79

Table 4.11 Statistical parameters calculated from application of PCR

model to the spectral data of calibration and prediction set

samples

Principle Component Regression

Parameters D0 D1 D2

Cal

ibra

tion

Set

IRB HCZ IRB HCZ IRB HCZ

Mean

(%)100.0303 100.6581 100.0545 102.1774 100.0681 99.7346

SD 1.1033 4.2494 1.6707 7.7713 1.8079 5.6476

R2 0.9995 0.9921 0.9995 0.9907 0.9995 0.9893

RMSEC 0.0310 0.0539 0.0308 0.0600 0.0057 0.0629

Pred

ictio

n

Set

Mean

(%)95.7882 102.6564 97.9915 98.8246 98.1268 100.9732

SD 3.8719 5.7736 3.6185 4.2799 2.4856 3.5662

R2 0.9958 0.9830 0.9956 0.9858 0.9977 0.9941

RMSEP 0.1358 0.1234 0.1163 0.0807 0.2968 0.0529

4.2.1.8 Application of PLS and PCR models in PharmaceuticalPreparations

Suitably diluted samples of formulations were analysed for drug

content using optimized PLS and PCR models. The results are tabulated in

Table 4.12. Estimated amount and percentage of recovery are mean values of

six estimations. The percentage recovery was found to vary in the range of

99.17 – 100.96 for IRB and 98.72 – 101.28 for HCZ. The results show good

reproducibility of the proposed methods.

80

Table 4.12 Assay results from application of optimized PLS and PCR

models on pharmaceutical preparations

Method Tablet Method

IRB HCZAmountFound*

(mg/tab) ±SD

%recovery

AmountFound*

(mg/tab) ±SD

%recovery

PLS

T1D0

150.33 ± 0.05±0.05

100.220 12.58 ± 0.04 100.640

D1 150.23 ± 0.05 100.153 12.61 ± 0.05 100.880D2 149.77 ± 0.06 99.846 12.66 ± 0.03 101.280

T2D0 150.42 ± 0.06 100.280 12.57 ± 0.03 100.560D1 149.76 ± 0.03 99.840 12.59 ± 0.05 100.720D2 150.43 ± 0.03 100.286 12.47 ± 0.04 99.760

PCR

T1D0 148.76 ± 0.10 99.173 12.46 ± 0.10 99.680D1 151.21 ± 0.06 100.806 12.38 ± 0.20 99.040D2 150.51 ± 0.03 100.340 12.55 ± 0.08 100.400

T2D0 150.78 ± 0.21 100.520 12.36 ± 0.11 98.880D1 151.44 ± 0.12 100.960 12.34 ± 0.05 98.720D2 149.31 ± 0.09 99.540 12.57 ± 0.02 100.560

*Average of six estimations

T1 – IROVEL H – Sun Pharmaceuticals, J&K, India.

Irbesartan – 150 mg and Hydrochlorothiazide – 12.5 mg

T2 – XARB H – Hetero Labs, HP, India


81

Figure 4.5 Comparison of Mean Recovery of PLS and PCR models for

Fundamental (D0), First Derivative (D1) and Second

Derivative (D2) Spectra

96

98

100

102

104

D0 D1 D2

% R

ecov

ery

Irbesartan - PLS

Calibration set Prediction set

94

96

98

100

102

104

D0 D1 D2

% R

ecov

ery

Irbesartan - PCR


96

98

100

102

104

D0 D1 D2

% R

ecov

ery

Hydrochlorothiazide - PLS


96

98

100

102

104

D0 D1 D2

% R

ecov

ery

Hydrochlorothiazide - PCR


82

4.2.2 Binary Mixture 2 – Valsartan (VAL) and Hydrochlorothiazide(HCZ)


The absorption spectra of VAL and HCZ were recorded over therange of 200 – 350nm in fundamental mode as shown in Figure 4.6. The firstderivative and second derivative spectra of both the drugs are represented inFigure 4.7 and 4.8 respectively.

4.2.2.2 Building the calibration models

Stock solutions of VAL (1 mg/ml) and HCZ (1 mg/ml) wereappropriately diluted with water separately to give working solutions of VALand HCZ of 10µg/ml concentration respectively. A calibration set of 25different laboratory mixtures of VAL and HCZ were prepared with theconcentration range of 0.5-3 µg/ml for VAL and HCZ respectively (Table4.13). The absorption spectra of the mixtures were recorded in the range of200-350 nm. The data points were collected at 1nm intervals.

Figure 4.6 Fundamental Absorption Spectra of Valsartan,Hydrochlorothiazide and mixture

83

Figure 4.7 First Derivative Spectra of Valsartan, Hydrochlorothiazideand mixture

Figure 4.8 Second Derivative Spectra of Valsartan, Hydrochlorothiazideand mixture

84

4.2.2.3 Optimized conditions for Partial Least Square (PLS)


Data Points : 151 nm

Preprocessing : Autoscale, Mean Centre, orthogonal signalcorrection



4.2.2.4 Validation using Prediction set

The calibrated PLS model was used for quantization of the analytes

in a separate prediction set that varies from the calibration set. The estimated

amount of VAL and HCZ in calibration and prediction sets using the

optimized PLS model are given in Tables 4.14 – 4.17. Plots between amount

present and amount estimated for both the drugs in three different modes

under optimized PLS and PCR conditions are represented in Figure 4.9.

Statistical Parameters represented in Table 4.18 reveals that first

derivative method was found to be appropriate for calibration set as the R2

values were high 0.9999 for VAL and HCZ and the SD, RMSEC values were

low (2.42 and 0.0098 for VAL and 1.43 and 0.0051 for HCZ respectively)

compared to other two methods. Second derivative method was found to be

better than other methods in prediction set as SD and RMSEP values are low

(3.02 and 0.051 for VAL and 3.29 and 0.061 for HCZ respectively).

85

Table 4.13 Concentrations of Calibration and Prediction sets expressedin µg/ml

MixtureNumber

Calibration Set (µg/ml) Prediction Set (µg/ml)

VAL HCZ VAL HCZ

M1 0.5 0.5 0.5 2M2 0.5 1 1 2M3 0.5 1.5 1.5 2.5M4 0.5 2 2 2.5M5 1 0.5 1 0.5M6 1 1 1 1M7 1 1.5 0.5 2.5M8 1 2 1 2.5M9 1.5 0.5 1.5 2.5

M10 1.5 1 3 3M11 1.5 1.5 - -M12 1.5 2 - -M13 2 0.5 - -M14 2 1 - -M15 2 1.5 - -M16 2 2 - -M17 2.5 0.5 - -M18 2.5 1 - -M19 2.5 1.5 - -M20 2.5 2 - -M21 3 0.5 - -M22 3 1 - -M23 3 1.5 - -M24 3 2 - -M25 3 2.5 - -

86

Table 4.14 Estimated Concentrations of Valsartan in the calibration setunder optimized PLS model


FundamentalMode



Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4944 98.8935 0.4908 98.1640 0.4853 97.07360.5 0.4904 98.0823 0.4969 99.3985 0.5086 101.73790.5 0.5081 101.6343 0.5025 100.5176 0.4998 99.96600.5 0.4914 98.2944 0.5067 101.3521 0.4707 94.14431 1.0058 100.5848 1.0101 101.0149 1.0086 100.86371 0.9924 99.2422 0.9890 98.9005 1.0063 100.63701 0.9938 99.3885 1.0065 100.6549 0.9805 98.05321 1.0044 100.4454 0.9978 99.7866 1.0299 102.9978

1.5 1.5334 102.2269 1.4904 99.3658 1.5278 101.85461.5 1.4759 98.3983 1.4997 99.9819 1.4854 99.026681.5 1.5087 100.5830 1.5002 100.0144 1.4990 99.93571.5 1.5004 100.0328 1.5165 101.1014 1.5228 101.52262 1.9813 99.0653 2.0059 100.2954 1.9740 98.70492 2.0158 100.7915 1.9947 99.7352 1.9992 99.96332 2.0011 100.0593 1.9951 99.7566 1.9997 99.98772 2.0112 100.5601 1.9976 99.8833 2.0298 101.4903

2.5 2.4948 99.7945 2.5114 100.4586 2.4858 99.43262.5 2.5440 101.7635 2.4951 99.8079 2.4912 99.65172.5 2.5073 100.2931 2.5038 100.1550 2.5070 100.28222.5 2.4846 99.3859 2.4865 99.4615 2.5065 100.26143 2.9712 99.0432 3.0071 100.2398 2.9798 99.32993 3.0146 100.4893 3.0118 100.3944 3.0659 102.19843 2.9659 98.8633 2.9700 99.0007 2.9703 99.01083 3.0090 100.3023 3.0107 100.3584 3.0075 100.25253 2.9988 99.9612 3.0020 100.0670 2.9573 98.5796

Mean 99.9272 Mean 99.9947 Mean 99.8783

87

Table 4.15 Estimated concentrations of Hydrochlorothiazide in thecalibration set under optimized PLS model


Fundamental Mode First Derivativemode


Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5319 106.3869 0.5044 100.8849 0.5073 101.46151 1.0395 103.9526 0.9981 99.8187 1.0069 100.6907

1.5 1.5333 102.2245 1.5012 100.0821 1.4684 97.89442 1.9917 99.5876 1.9997 99.9864 2.0112 100.5647

0.5 0.4156 83.1218 0.4977 99.5553 0.4987 99.75051 1.0381 103.8130 0.9996 99.9621 1.0094 100.9409

1.5 1.5397 102.6531 1.5006 100.0428 1.5085 100.56892 1.9315 96.5785 1.9976 99.8834 2.0049 100.2451

0.5 0.5168 103.3771 0.5083 101.6780 0.4616 92.32911 1.0347 103.4793 0.9901 99.0116 1.0057 100.5723

1.5 1.4762 98.4198 1.4912 99.4188 1.4917 99.44742 1.9423 97.1176 2.0028 100.1423 2.0049 100.2476

0.5 0.4957 99.1403 0.4970 99.4010 0.5174 103.49291 0.9621 96.2134 1.0103 101.0307 0.9883 98.8333

1.5 1.5440 102.9342 1.5021 100.1402 1.5135 100.90132 1.9764 98.8237 1.9959 99.7968 1.9989 99.9493

0.5 0.5164 103.2932 0.4957 99.1567 0.5080 101.60291 0.9604 96.0408 1.0098 100.9875 1.0125 101.2574

1.5 1.4568 97.1214 1.5040 100.2697 1.4908 99.39022 2.0432 102.1643 2.0003 100.0178 2.0018 100.0914

0.5 0.5250 105.0161 0.4993 99.8686 0.4830 96.60161 1.0399 103.9975 0.9907 99.0708 1.0072 100.7251

1.5 1.4016 93.4424 1.4990 99.9343 1.5243 101.62592 2.0840 104.2002 2.0007 100.0385 1.9987 99.9387

2.5 2.5019 100.0789 2.5028 100.1129 2.4754 99.0174Mean 100.1271 Mean 100.0117 Mean 99.9256

88

Table 4.16 Estimated concentrations of Valsartan in the Prediction setunder optimized PLS model

Amount

Present

(µg/ml)

D0 D1 D2

Amount

found

(µg/ml)

%

recovery

Amount

found

(µg/ml)

%

recovery

Amount

found

(µg/ml)

%

recovery

0.5 0.5209 104.1805 0.4900 98.0082 0.5276 105.5375

1 1.0934 109.3481 1.0951 109.5110 1.0578 105.7874

1.5 1.5183 101.2262 1.5645 104.3005 1.5487 103.2479

2 1.9487 97.4367 1.8923 94.6197 1.9676 98.3807

1 1.0058 100.5848 1.0162 101.6257 1.0587 105.8734

1 0.9924 99.2422 1.0666 106.6666 1.0143 101.4371

0.5 0.4665 93.3167 0.5232 104.6413 0.4805 96.1064

1 1.0111 101.1135 1.0034 100.3414 1.0299 102.9978

1.5 1.5854 105.6998 1.6182 107.8816 1.5064 100.4290

3 3.1231 104.1047 3.0190 100.6354 3.0666 102.2225

Mean 101.6253 Mean 102.8231 Mean 102.2020

89

Table 4.17 Estimated Concentrations of Hydrochlorothiazide in thePrediction set under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.1009 105.0477 2.1825 109.1256 2.0630 103.1536

2 2.1747 108.7387 2.0440 102.2022 2.0677 103.3855

2.5 2.4673 98.6939 2.5148 100.5935 2.4827 99.3119

2.5 2.4197 96.7911 2.4509 98.0362 2.4836 99.3459

0.5 0.5048 100.9630 0.5264 105.2818 0.5203 104.0642

1 0.9849 98.4987 1.0198 101.9822 1.0661 106.6170

2.5 2.5563 102.2542 2.5008 100.0342 2.5085 100.3413

2.5 2.5746 102.9843 2.5266 101.0644 2.5049 100.1961

2.5 2.5395 101.5817 2.4832 99.3305 2.5450 101.8018

3 2.8950 96.5019 2.8946 96.4867 3.0682 102.2765

Mean 101.2055 Mean 101.4137 Mean 102.0494

90

Table 4.18 Statistical Parameters calculated from application of PLSmodel to the spectral data of calibration and prediction setsamples

Partial Least Squares

Parameters D0 D1 D2

Cal

ibra

tion

Set

VAL HCZ VAL HCZ VAL HCZ

Mean

(%)99.9272 100.1271 99.9947 100.0117 99.8783 99.9256

SD 1.0738 4.9150 0.7156 0.6233 1.8140 2.0685

R2 0.9996 0.9942 0.9999 0.9999 0.9993 0.9994

RMSEC 0.0172 0.0458 0.0098 0.0051 0.0232 0.0148

Pred

ictio

n

Set

Mean

(%)101.6253 101.2055 102.8231 101.4137 102.2020 102.0494

SD 4.5052 3.8183 4.6360 3.6224 3.2433 2.3380

R2 0.9958 0.9880 0.9930 0.9917 0.9981 0.9980

RMSEP 0.0189 0.0835 0.0666 0.0714 0.0411 0.0455

4.2.2.5 Optimized conditions for Principal Component Regression (PCR)


Data points : 151

Preprocessing : Mean Centre, Smoothing, Orthogonal

Signal correction


Number of PCs : 13

91

Table 4.19 Estimated Concentrations of Valsartan in the Calibrationset under optimized PCR model


Fundamental Mode First DerivativeMode


Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4959 99.1940 0.5005 100.1091 0.4941 98.83230.5 0.4842 96.8540 0.5125 102.5143 0.5165 103.30010.5 0.5095 101.9174 0.5097 101.9467 0.4851 97.02900.5 0.5065 101.3004 0.5075 101.5139 0.4961 99.23031 1.0128 101.2805 1.0067 100.6755 0.9943 99.43441 1.0388 103.8891 0.9598 95.9802 1.0049 100.49801 0.9353 93.5343 0.9524 95.2492 0.9093 90.93811 1.0078 100.7845 1.0614 106.1428 1.0152 101.5259

1.5 1.5384 102.5611 1.5295 101.9673 1.5250 101.66841.5 1.4749 98.3291 1.4828 98.8571 1.5256 101.70801.5 1.5150 101.0031 1.5668 104.4574 1.5365 102.43881.5 1.5377 102.5152 1.4724 98.1644 1.5051 100.34032 1.9679 98.3966 1.9662 98.3100 1.9891 99.45852 2.0031 100.1571 1.9850 99.2519 2.0099 100.49622 1.9924 99.6241 1.9868 99.3428 2.0033 100.16802 1.9953 99.7699 1.9885 99.4278 2.0402 102.0130

2.5 2.4877 99.5112 2.4768 99.0740 2.4734 98.93742.5 2.5113 100.4547 2.5535 102.1416 2.5097 100.39082.5 2.4899 99.5968 2.4665 98.6618 2.5076 100.30402.5 2.4740 98.9631 2.4846 99.3845 2.5407 101.62813 2.9930 99.7671 3.0222 100.7427 2.9500 98.33603 3.0514 101.7162 3.0405 101.3520 3.0481 101.60633 2.9369 97.8981 2.9325 97.7527 2.9485 98.28513 3.0375 101.2505 3.0610 102.0353 3.0061 100.20443 3.0015 100.0528 2.9727 99.0902 2.9645 98.8181

Mean 100.0128 Mean 100.1658 Mean 99.9036

92

Table 4.20 Estimated Concentrations of Hydrochlorothiazide in theCalibration set under optimized PCR model


FundamentalMode



Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5104 102.0867 0.5091 101.8212 0.4606 92.12881 1.1089 110.8921 1.0027 100.2735 1.0403 104.0347

1.5 1.4687 97.9190 1.5040 100.2687 1.4776 98.51022 1.8540 92.7025 1.9986 99.9335 1.8454 92.2741

0.5 0.5715 114.3031 0.4956 99.1338 0.5101 102.03001 1.0916 109.1667 0.9958 99.5839 1.1001 110.0100

1.5 1.6622 110.8183 1.4833 98.8883 1.4901 99.34002 1.8372 91.8644 2.0178 100.8914 1.9264 96.3243

0.5 0.4997 99.9499 0.5159 103.1835 0.4953 99.07601 1.0230 102.3072 0.9833 98.3381 0.9207 92.0777

1.5 1.4101 94.0082 1.5106 100.7127 1.4586 97.24582 1.9869 99.3483 1.9876 99.3804 1.9780 98.9009

0.5 0.4052 81.0420 0.4866 97.3241 0.4811 96.22201 0.9766 97.6618 1.0105 101.0555 0.9608 96.0856

1.5 1.5686 104.5735 1.4992 99.9514 1.5110 100.73332 1.9606 98.0332 1.9926 99.6336 2.0247 101.2398

0.5 0.4485 89.7027 0.4832 96.6479 0.4864 97.28041 1.0143 101.4323 1.0263 102.6329 1.1096 110.9600

1.5 1.5105 100.7020 1.4951 99.6774 1.5004 100.03192 2.0764 103.8222 1.9984 99.9206 1.8860 94.3014

0.5 0.4407 88.1400 0.5044 100.8939 0.5008 100.17201 1.0777 107.7761 1.0006 100.0615 0.9885 98.8520

1.5 1.4601 97.3420 1.4846 98.9741 1.4706 98.04392 2.0347 101.7354 2.0192 100.9624 2.0142 100.7141

2.5 2.5009 100.0373 2.4939 99.7592 2.5314 101.2595Mean 99.8947 Mean 99.9961 Mean 99.1139

93

Table 4.21 Estimated Concentrations of Valsartan in the prediction setunder optimized PCR model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5237 104.7516 0.5226 104.5214 0.5140 102.80631 1.0197 101.9741 1.0275 102.7595 1.0285 102.8558

1.5 1.4854 99.0320 1.5087 100.5861 1.6044 106.96132 1.8896 94.4807 1.8966 94.8335 1.9592 97.96341 1.0128 101.2805 1.0956 109.5674 1.0544 105.44901 1.0388 103.8891 1.0606 106.0621 1.0719 107.1925

0.5 0.5074 101.4882 0.5349 106.9926 0.5093 101.87631 1.0103 101.0326 1.0060 100.6045 1.0152 101.5259

1.5 1.5373 102.4880 1.5964 106.4289 1.6241 108.27783 3.1030 103.4335 3.0456 101.5232 3.0246 100.8206

Mean 101.3850 Mean 103.3879 Mean 103.5729

Table 4.22 Estimated concentrations of Hydrochlorothiazide in theprediction set under optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0440 102.2017 2.0645 103.2289 2.0101 100.50802 2.0965 104.8268 2.0178 100.8903 2.0186 100.9330

2.5 2.3501 94.0047 2.4961 99.8470 2.5036 100.14572.5 2.3974 95.8965 2.4470 97.8816 2.4358 97.43250.5 0.5015 100.3031 0.4812 96.2522 0.4948 98.96461 1.0916 109.1667 0.9211 92.1187 0.9921 99.2194

2.5 2.3581 94.3274 2.4736 98.9456 2.4901 99.60402.5 2.4589 98.3578 2.4934 99.7396 2.5264 101.05942.5 2.4456 97.8273 2.4731 98.9258 2.6250 105.00213 2.8263 94.2104 2.8930 96.4360 2.8549 95.1645

Mean 99.1122 Mean 98.4266 Mean 99.8033

94

Figure 4.9 Plots of Calibration and predictions sets of Valsartan andHydrochlorothiazide in both PLS and PCR models

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PLS-CAL-D0

y = 0.9999x+0.0002

R² = 0.9999

00.5

11.5

22.5

33.5


VAL-PLS-CAL-D1

y = 0.999x + 0.001R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PLS-CAL-D2

y = 1.034x - 0.017R² = 0.995

00.5

11.5

22.5

33.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PLS-PRE-D0

y = 0.986x + 0.047R² = 0.993

00.5

11.5

22.5

33.5

0 1 2 3

Amount Present

VAL-PLS-PRE-D1

y = 1.009x + 0.013R² = 0.998

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Amount Present

VAL-PLS-PRE-D2

y = 0.994x + 0.007R² = 0.994

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0y = 0.999x + 9E-05

R² = 0.999

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-CAL-D1

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-CAL-D2

95

Figure 4.9 Continued

y = 0.983x + 0.056R² = 0.988

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.9646x +0.0887R² = 0.9917

00.5

11.5

22.5

33.5


HCZ-PLS-PRE-D1

y = 0.991x + 0.049R² = 0.998

00.5

11.5

22.5

33.5


HCZ-PLS-PRE-D2

y = 0.998x + 0.001R² = 0.998

00.5

11.5

22.5

33.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PCR-CAL-D0

y = 0.998x + 0.003R² = 0.998

00.5

11.5

22.5

33.5


VAL-PCR-CAL-D1

y = 0.998x + 0.002R² = 0.998

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PCR-CAL-D2

y = 1.0104x-0.0006

R² = 0.9952

00.5

11.5

22.5

33.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PCR-PRE-D0

y = 0.985x + 0.049R² = 0.994

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PCR-PRE-D1

y = 0.997x + 0.043R² = 0.995

0

0.51

1.52

2.53

3.5


VAL-PCR-PRE-D2

96


y = 0.983x + 0.021R² = 0.983

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D1

y = 0.978x + 0.014R² = 0.991

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D2

y = 0.913x + 0.139R² = 0.989

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 0.993x - 0.010R² = 0.996

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PCR-PRE-D1

y = 0.984x + 0.027R² = 0.992

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PCR-PRE-D2

97

Table 4.23 Statistical Parameters calculated from application of PCRmodel to the spectral data of calibration and prediction sets


Parameters D0 D1 D2

Cal

ibra

tion

Set

VAL HCZ VAL HCZ VAL HCZ

Mean (%)100.012

899.894

7100.165

899.996

199.9036

99.1139

SD 2.0940 7.6861 2.4259 1.4396 2.3856 4.6103

R2 0.9989 0.9837 0.9983 0.9996 0.9987 0.9916

RMSEC 0.2851 0.0765 0.0357 0.0595 0.0315 0.0571

Pred

ictio

nSe

t

Mean (%)101.385

099.112

2103.387

998.426

6103.572

999.803

3

SD 2.9195 5.0583 4.2362 3.0215 3.2905 2.5542

R2 0.9952 0.9894 0.9944 0.9961 0.9958 0.9923

RMSEP 0.0521 0.1037 0.0612 0.0517 0.0619 0.0649


Suitably diluted samples of formulations were analysed for the drugcontent using optimized PLS and PCR models. The results are tabulated inTable 4.24. Estimated amount and percentage of recovery are mean values ofsix estimations. The percentage recovery was found to vary in the range of99.17 – 100.96 for VAL and 98.72 – 101.28 for HCZ respectively. The resultsshow good reproducibility of the proposed methods.

98

Table 4.24 Assay results from application of PLS and PCR models inPharmaceutical preparations

Method Tablet Method

VAL HCZAmountFound*

(mg/tab)± SD

%recovery

AmountFound*

(mg/tab)± SD

%recovery

PLS T1

D080.44 ±

0.11100.550

12.58 ±0.08

100.640

D179.86 ±

0.0899.825

12.61 ±0.05

100.880

D279.74 ±

0.0499.675

12.46 ±0.003

99.680

PCR T1

D078.67 ±

0.2298.337

12.45 ±0.06

99.601

D179.46 ±

0.1199.325

12.38 ±0.03

99.040

D280.64 ±

0.07100.800

12.62 ±0.002

100.960

* Average of six determinations

T1 – Valent H – Lupin Ltd, Mumbai

Valsartan – 80 mg, Hydrochlorothiazide – 12.5 mg

99

Figure 4.10 Comparison of Mean Recovery of PLS and PCR models forFundamental (D0), First Derivative (D1) and SecondDerivative (D2) Spectra

96

98

100

102

104

D0 D1 D2

% R

ecov

ery

Valsartan - PLS


96

98

100

102

104

D0 D1 D2%

Rec

over

y

Valsartan - PCR


96

98

100

102

104

D0 D1 D2

% R

ecov

ery



96

98

100

102

104

D0 D1 D2

% R

ecov

ery



100

4.2.3 Binary Mixture 3 – Telmisartan (TEL) andHydrochlorothiazide (HCZ)


Figures 4.11, 4.12 and 4.13 show overlain spectra of both the drugsin fundamental, first derivative and second derivative modes respectively.

Figure 4.11 Fundamental Spectra of Telmisartan, Hydrochlorothiazideand mixture

Figure 4.12 First Derivative Spectra of Telmisartan, Hydrochlorothiazideand mixture

101

Figure 4.13 Second Derivative Spectra of Telmisartan,Hydrochlorothiazide and mixture

4.2.3.2 Building Calibration set

A calibration set of 25 different laboratory mixtures were preparedwith the concentration range of 1-5 µg/ml and 0.5-2 µg/ml of TEL and HCZrespectively.

4.2.3.3 Optimum conditions for PLS model


Data Points : 111 nm ( 1 nm)

Preprocessing : Autoscale, Mean Centre, orthogonal signalcorrection, autoscale



102

Table 4.25 Concentrations of Calibration and Prediction sets in µg/ml

MixtureNumber

Calibration Set (µg/ml) Prediction Set (µg/ml)

TEL HCZ TEL HCZ

M1 1 0.5 1 2

M2 1 1 2 2.5

M3 1 1.5 3.5 0.5

M4 1 2 4 2

M5 2 0.5 5 2

M6 2 1 1.5 2

M7 2 1.5 2.5 2.5

M8 2 2 3.5 1.5

M9 3 2.5 3 2.5

M10 3 2 1.5 1.5

M11 3 1.5 - -

M12 3 1 - -

M13 4 0.5 - -

M14 4 1 - -

M15 4 1.5 - -

M16 4 2 - -

M17 5 2.5 - -

M18 5 1.5 - -

M19 5 0.5 - -

M20 5 1 - -

M21 1.5 1 - -

M22 2.5 2 - -

M23 3.5 1 - -

M24 4.5 1 - -

M25 3.5 2 - -

103

Table 4.26 Estimated Concentrations of Telmisartan in the calibrationset under optimized PLS model


FundamentalMode

First Derivativemode


Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 1.0344 103.4401 1.0538 105.3833 0.9754 97.54791 1.0193 101.9311 1.0388 103.8897 1.1063 110.63491 1.0114 101.1401 0.9288 92.8869 1.0902 109.02061 1.0140 101.400 1.0110 101.1046 0.9723 97.23482 1.9974 99.8702 2.0289 101.4456 1.9772 98.86452 1.9110 95.5501 1.9988 99.9439 1.9878 99.39002 2.1311 106.5550 2.0393 101.9671 2.1949 109.74502 1.9986 99.9300 2.0393 101.9671 2.2948 114.74293 2.9896 99.6533 2.9711 99.0369 3.0949 103.16333 2.9776 99.2533 3.0575 101.9177 3.0086 100.28753 2.9722 99.0733 2.9306 97.6873 2.9795 99.31873 2.9586 98.6203 2.9502 98.3412 2.9954 99.84694 4.0868 102.1703 4.0089 100.2230 3.9811 99.52964 4.0065 100.1625 3.9635 99.0890 4.0395 100.98784 3.9929 99.8225 4.0021 100.0547 4.0000 100.00104 4.0268 100.6700 3.9757 99.3928 4.0251 100.62935 4.9725 99.4512 5.0067 100.1356 5.0150 100.30105 4.9811 99.6221 4.9998 99.9964 4.9587 99.17475 4.8851 97.7021 4.9627 99.2544 5.0151 100.30295 5.0316 100.6321 5.0210 100.4219 5.0165 100.3316

1.5 1.4938 99.5866 1.4285 95.2342 1.4502 96.68332.5 2.5244 100.9763 2.5466 101.8648 2.5241 100.96763.5 3.4999 99.9971 3.4559 98.7418 3.5251 100.71764.5 4.5838 101.8622 4.5921 102.0483 4.4762 99.47253.5 3.5019 100.0543 3.4872 99.6369 3.5250 100.7169

Mean 100.3648 Mean 100.0666 Mean 101.5845

104



FundamentalMode



Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4789 95.7801 0.5146 102.9389 0.5112 102.24361 0.9302 93.0201 0.9737 97.3749 0.9612 96.1262

1.5 1.4966 99.7733 1.4945 99.6340 1.5152 101.01572 2.0341 101.7051 2.0085 100.4282 2.0049 100.2464

0.5 0.4756 95.1201 0.4339 86.7932 0.5188 103.76351 1.0433 104.3302 1.0465 104.6569 1.0199 101.9937

1.5 1.4562 97.0811 1.4800 98.6666 1.4974 99.82662 2.0355 101.7751 1.8800 94.0001 1.9974 99.8723

2.5 2.5136 100.5441 2.5351 101.4059 2.4974 99.89602 1.9596 97.9811 1.9691 98.4555 1.9587 97.9363

1.5 1.4906 99.3733 1.5467 103.1162 1.5210 101.40421 1.0679 106.7912 0.9439 94.3923 1.0159 101.5944

0.5 0.5113 102.2612 0.4920 98.4150 0.4923 98.46101 1.0284 102.8411 1.0387 103.8737 0.9908 99.0817

1.5 1.5177 101.1801 1.3986 93.2435 1.4817 98.78002 1.9886 99.4300 1.9513 97.5683 2.0030 100.1547

2.5 2.4729 98.9160 2.5004 100.0165 2.5339 101.35731.5 1.5343 102.2867 1.5130 100.8689 1.4694 97.96560.5 0.4959 99.1821 0.5610 112.2047 0.5485 109.70151 0.9939 99.3932 1.0125 101.2525 0.9862 98.62771 1.0938 109.3823 1.0550 105.5054 0.9973 99.73822 1.9169 95.8452 1.9450 97.2513 1.9957 99.78871 1.0325 103.2521 0.9672 96.7210 0.9790 97.90641 0.9509 95.0931 1.0290 102.9037 0.9952 99.52522 2.0096 100.4822 2.0087 100.4383 2.0368 101.8444

Mean 100.1119 Mean 99.6850 Mean 100.3540

105

Table 4.28 Estimated Concentrations of Telmisartan in the predictionset under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 0.9660 96.6001 0.9606 96.0600 0.9854 98.54262 1.8958 94.7900 2.0032 100.1618 2.0581 102.9050

3.5 3.7801 108.0029 3.5163 100.4666 3.4966 99.90504 4.1426 103.5650 4.1109 102.7739 4.0727 101.81945 5.2721 105.4421 5.0972 101.9445 5.0706 101.4125

1.5 1.5174 101.1611 1.5200 101.3349 1.5107 100.71532.5 2.3818 95.2721 2.4922 99.6908 2.5054 100.21753.5 3.6720 104.9143 3.5103 100.2968 3.5201 100.57633 2.9896 99.6533 2.9828 99.4283 3.0278 100.9288

1.5 1.4652 97.6801 1.4736 98.2451 1.5060 100.4064Mean 100.7079 Mean 100.0431 Mean 100.7429

Table 4.29 Estimated Concentrations of Hydrochlorothiazide in theprediction set under PLS model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 1.9545 97.7250 2.0113 100.5675 2.0146 100.73472.5 2.4919 99.6761 2.5024 100.0969 2.4750 99.00190.5 0.5118 102.3611 0.5194 103.8923 0.4769 95.39202 2.0762 103.8101 2.0568 102.8423 1.9974 99.87432 1.7970 89.8511 1.8828 94.1429 2.0187 100.93642 1.7937 89.6851 1.9470 97.3511 2.0434 102.1720

2.5 2.4016 96.0642 2.5165 100.6604 2.5022 100.08941.5 1.4637 97.5823 1.4950 99.6673 1.5063 100.42572.5 2.5136 100.5441 2.5061 100.2451 2.4804 99.21881.5 1.4652 97.6801 1.4858 99.0567 1.5165 101.1057

Mean 97.4974 Mean 99.8523 Mean 99.8951

106

4.2.3.4 Statistical Parameters of PLS model

Statistical parameters are represented in Table 4.30. The R2 valuewas found to be better for both the drugs in the first and second derivativemodes than fundamental mode. The SD, RMSEC and RMSEP values are alsofound to be low in derivative modes than fundamental mode for the predictionset whereas reverse in case of calibration set.

Table 4.30 Statistical Parameters calculated from application of PLSmodel to the spectral data of calibration and prediction sets

Partial Least SquaresParameters D0 D1 D2

Cal

ibra

tion

Set

TEL HCZ TEL HCZ TEL HCZMean(%)

100.3648

100.1119

100.0666

99.6850

101.5845

100.3540

SD 2.0171 3.7459 2.5203 4.8749 4.4987 2.5803R2 0.9987 0.9955 0.9990 0.9941 0.9969 0.9987

RMSEC

0.0495 0.0406 0.0432 0.0473 0.0814 0.0219

Pred

ictio

nSe

t

Mean(%)

100.7079

97.4974 100.0403

99.8523

100.7949

99.8951

SD 4.6357 4.7044 1.9031 2.7113 1.1679 1.8345R2 0.9955 0.9776 0.9995 0.9941 0.9970 0.9988

RMSEP

0.1516 0.0104 0.0501 0.0456 0.0416 0.0207

4.2.3.5 Optimized conditions for PCR model



Preprocessing : Autoscale, Mean Centre, orthogonal signal

correction, autoscale


Number of PCs : 8

107

Table 4.31 Estimated Concentrations of Telmisartan in the calibrationset under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 1.0321 103.2100 1.0000 100.0049 1.0452 104.52521 1.0025 100.2510 1.0625 106.2564 1.0933 109.33651 1.0129 101.2911 0.9430 94.3097 1.0533 105.33981 0.9985 99.8520 0.9653 96.5333 1.0057 100.57962 1.9791 98.95521 2.0325 101.6251 2.0278 101.39482 1.9156 95.7811 1.9608 98.0402 1.9582 97.91062 2.1431 107.1550 2.1127 105.6362 2.2443 112.21842 2.0064 100.3201 2.1127 105.6362 2.2443 112.21843 2.9956 99.8533 3.0094 100.3151 3.1444 104.81333 3.0047 100.1567 3.0639 102.1324 3.0693 102.31163 2.9778 99.2611 2.9678 98.9293 2.9729 99.09963 2.9606 98.6866 3.0046 100.1559 2.9816 99.38864 4.1013 102.5325 4.0201 100.5042 3.9713 99.28464 4.0005 100.0125 3.9703 99.2595 4.1183 102.95834 4.0009 100.0225 4.0460 101.1522 4.0082 100.20704 4.0419 101.0475 3.9731 99.3276 4.0038 100.09525 4.9411 98.8221 4.9738 99.4776 4.9676 99.35395 4.9717 99.4341 4.9157 98.3148 4.9752 99.50485 4.8784 97.5681 4.8967 97.9352 4.9669 99.33855 5.0281 100.5621 4.9701 99.4035 5.0287 100.5752

1.5 1.3954 93.0266 1.3383 89.2215 1.3806 92.04062.5 2.5239 100.9561 2.4773 99.0940 2.6089 104.35963.5 3.5124 100.3543 3.5264 100.7557 3.4813 99.46604.5 4.5831 101.8467 4.6711 103.8024 4.4917 99.81623.5 3.4912 99.7485 3.4846 99.5614 3.5558 101.5968

Mean 100.0279 Mean 99.8954 Mean 101.9093

108

Table 4.32 Estimated Concentrations of Hydrochlorothiazide in thecalibration set under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4932 98.6400 0.5234 104.6841 0.5258 105.16401 0.9243 92.4301 0.8869 88.6921 0.9179 91.7909

1.5 1.4466 96.4411 1.5135 100.9001 1.5297 101.98002 2.0769 103.8451 2.0461 102.3051 2.1012 105.0645

0.5 0.4763 95.2612 0.5097 101.9501 0.4835 96.71401 1.0339 103.3922 1.0818 108.1812 1.1248 112.4800

1.5 1.4442 96.2811 1.4800 98.6728 1.4873 99.15332 2.0203 101.0150 1.7801 89.0051 1.9473 97.3650

2.5 2.6038 104.1521 2.5271 101.0811 2.5473 101.89202 1.9514 97.5720 2.0628 103.1445 2.0706 103.5300

1.5 1.4853 99.0221 1.4652 97.6801 1.4429 96.19381 0.8658 86.5811 0.8855 88.5598 0.8506 85.0687

0.5 0.5099 101.9812 0.5111 102.2341 0.4791 95.83001 1.0187 101.8702 1.0471 104.7127 0.9884 98.8497

1.5 1.5058 100.3867 1.4117 94.1184 1.4814 98.76172 1.8924 94.6222 1.9000 95.0026 1.9861 99.3061

2.5 2.4525 98.1110 2.3666 94.6655 2.4594 98.37871.5 1.5138 100.9221 1.4682 97.8839 1.4669 97.79330.5 0.4997 99.9421 0.5123 102.4621 0.5532 110.65001 1.0651 106.5112 1.0399 103.9919 1.1566 115.66431 1.0321 103.2121 1.0041 100.4152 0.9619 96.19292 1.9495 97.4751 1.8225 91.1260 2.1014 105.07091 1.0712 107.1202 1.1378 113.7871 1.0185 101.85961 0.9874 98.7423 1.0629 106.2937 0.9278 92.780562 2.0763 103.8151 1.9830 99.1508 2.0096 100.4835

Mean 99.5723 Mean 99.6279 Mean 100.3207

109

Table 4.33 Estimated Concentrations of Telmisartan in the predictionset under optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 0.9587 95.8700 0.9396 93.9659 0.9879 98.79612 1.8892 94.4600 1.9938 99.6939 2.0467 102.3396

3.5 3.7772 107.9200 3.5145 100.4163 3.5139 100.39744 4.1259 103.1475 4.0456 101.1407 4.1471 103.67935 5.2408 104.8160 5.0946 101.8938 5.1554 103.1085

1.5 1.5842 105.6133 1.5492 103.2861 1.5105 100.70362.5 2.2948 91.7920 2.5093 100.3745 2.5179 100.71903.5 3.6661 104.7457 3.5077 100.2209 3.6257 103.59393 2.9956 99.8533 3.0445 101.4863 3.0290 100.9670

1.5 1.4633 97.5533 1.4853 99.0204 5.0284 100.5681Mean 100.5771 Mean 100.1499 Mean 101.4873

Table 4.34 Estimated Concentrations of Hydrochlorothiazide in theprediction set under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 1.9474 97.3701 2.0113 100.5675 2.0100 100.50262.5 2.4741 98.9640 2.5024 100.0969 2.4630 98.52230.5 0.5191 103.8110 0.5194 103.8923 0.4675 93.51302 2.1335 106.6751 2.0568 102.8423 2.0645 103.22502 1.9676 98.3800 1.8828 94.1429 2.0341 101.70612 1.8625 93.1251 1.9470 97.3511 1.9975 99.8791

2.5 2.3464 93.8560 2.5165 100.6604 2.5784 103.13731.5 1.6166 107.7733 1.4950 99.6673 1.5169 101.13122.5 2.6038 104.1521 2.5061 100.2451 2.6018 104.07541.5 1.4324 95.4933 1.4858 99.0567 1.5149 100.9977

Mean 99.9588 Mean 99.8523 Mean 100.6690

110

4.2.3.6 Statistical Parameters of PCR model

Statistical parameters are represented in Table 4.35. The R2 valuewas found to be better for both the drugs in fundamental mode than inderivative modes for calibration set whereas reverse in case of prediction set.The SD, RMSEC and RMSEP values are also found to be low in fundamentalmode than in derivative modes for calibration set whereas reverse in case ofprediction set.



Parameters D0 D1 D2

Cal

ibra

tion

Set

TEL HCZ TEL HCZ TEL HCZ

Mean

(%)

100.0279 99.5723 99.8954 99.6279 101.9093 100.3207

SD 2.5260 4.5731 3.5451 6.2722 4.4530 6.5567

R2 0.9982 0.9910 0.9973 0.9821 0.9962 0.9877

RMSEC 0.0572 0.0581 0.0698 0.0861 0.0917 0.0689

Pred

ictio

n

Set

Mean

(%)

100.5771 99.9588 100.1499 99.8523 101.4873 100.6690

SD 5.4533 5.2975 2.4832 2.7113 1.6106 3.0201

R2 0.9928 0.9726 0.9995 0.9907 0.9988 0.9963

RMSEP 0.1558 0.0970 0.0441 0.0621 0.0813 0.0498

111

Figure 4.14 Plots of Calibration and predictions sets of Telmisartan andHydrochlorothiazide in both PLS and PCR models

y = 0.995x + 0.018R² = 0.998

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

TEL-PLS-CAL-D0

y = 0.999x + 0.003R² = 0.999

0

1

2

3

4

5

6


TEL-PLS-CAL-D1

y = 0.985x + 0.072R² = 0.996

0

1

2

3

4

5

6


TEL-PLS-CAL-D2

y = 1.09x - 0.189R² = 0.995

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amont Present

TEL-PLS-PRE-D0

y = 1.030x - 0.067R² = 0.999

0

1

2

3

4

5

6


TEL-PLS-PRE-D1

y = 1.014x - 0.014R² = 0.999

0

1

2

3

4

5

6


TEL-PLS-PRE-D2

y = 0.992x + 0.011R² = 0.995

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0y = 0.984x + 0.013

R² = 0.994

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-CAL-D1

y = 0.998x + 0.002R² = 0.998

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-CAL-D2

112


y = 0.976x - 0.007R² = 0.977

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.995x + 0.000R² = 0.994

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-PRE-D1

y = 1.001x + 0.000R² = 0.998

0

0.5

1

1.5

2

2.5

3


HCZ-PLS-PRE-D2

y = 0.9982x+0.0054

R² = 0.9982

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

TEL-PCR-CAL-D0

y = 0.997x + 0.008R² = 0.997

0

1

2

3

4

5

6


TEL-PCR-CAL-D1

y = 0.982x + 0.088R² = 0.996

0

1

2

3

4

5

6


TEL-PCR-CAL-D2

y = 1.080x - 0.171R² = 0.992

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

TEL-PCR-PRE-D0

y = 1.024x - 0.049R² = 0.999

0

1

2

3

4

5

6


TEL-PCR-PRE-D1

y = 1.028x - 0.031R² = 0.998

0

1

2

3

4

5

6


TEL-PCR-PRE-D2

113



Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.36. The percentage recoverywas found to be from 99.15 – 101.32 for TEL and 99.60 – 100.64 for HCZrespectively.

y = 1.005x - 0.011R² = 0.991

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.945x + 0.056R² = 0.982

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D1

y = 1.012x - 0.012R² = 0.987

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-CAL-D2

y = 0.970x + 0.046R² = 0.972

0

0.5

1

1.5

2

2.5

3

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 0.969x + 0.033R² = 0.990

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-PRE-D1

y = 1.038x - 0.047R² = 0.996

0

0.5

1

1.5

2

2.5

3


HCZ-PCR-PRE-D2

114

Table 4.36 Assay results for PLS and PCR models in PharmaceuticalPreparation

Method Tablet Mode

TEL HCZ

Amount

Found*

(mg/tab)

± SD

Mean

%

recovery

Amount

Found*

(mg/tab)

± SD

Mean

%

recovery

PLS T1

D040.53 ±

0.08101.32

12.55 ±

0.09100.40

D140.34 ±

0.04100.85

12.57 ±

0.06100.56

D239.78 ±

0.0399.45

12.45 ±

0.0299.60

PCR T1

D039.66 ±

0.0899.15

12.48 ±

0.0499.84

D140.21±

0.05100.52

12.58 ±

0.03100.64

D239.76 ±

0.0299.40

12.47 ±

0.0299.76

* Average of six estimations

T1 – Telista H – Lupin Ltd, Mumbai

Telmisartan – 40 mg and Hydrochlorothiazide – 12.5 mg

115


96

98

100

102

104

D0 D1 D2

Telmisartan - PLS


96

98

100

102

104

D0 D1 D2

Telmisartan - PCR


96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2



116

4.2.4 Ternary Mixture 1– Irbesartan (IRB), Hydrochlorothiazide(HCZ) and Ramipril (RAM)

4.2.4.1 Spectral characteristics

The spectra of all three drugs were overlapped in fundamental(Figure 4.16), first derivative (Figure 4.17) and second derivative mode(Figure 4.18) to study the spectral characteristics. They reveal a completeoverlap throughout the UV region of 200-400 nm. For the multivariateanalysis, 25 different synthetic mixtures were prepared in the concentrationranges of 0.5-3 µg/ml of IRB and HCZ, 1-6 µg/ml of RAM respectively(Table 4.37). The range was fixed so that absorbance value does not exceed1.5 in fundamental spectral mode.

Figure 4.16 Fundamental absorption spectra of Irbesartan,Hydrochlorothiazide, Ramipril and mixture

117

Figure 4.17 First derivative spectra of Irbesartan, Hydrochlorothiazide,Ramipril and mixture

Figure 4.18 Second derivative spectra of Irbesartan,Hydrochlorothiazide, Ramipril and mixture

118


Calibration Set (µg/ml) Prediction Set (µg/ml)IRB HCZ RAM IRB HCZ RAM0.5 0.5 5 2 0.5 1.51 0.5 4 2 1 2.5

1.5 0.5 3 1 1.5 22 0.5 2 1.5 2 2.5

0.5 1 5 0 2.5 1.51 1 4 0.5 3 4

1.5 1 3 1.5 3 42 1 2 0 0 3

0.5 0.5 5 0 1 61 1.5 4 1 2 1.5

1.5 1.5 3 - - -2 1.5 2 - - -0 2 0 - - -

0.5 2 5 - - -1 2 4 - - -

1.5 2 3 - - -0.5 2.5 5 - - -1 2.5 4 - - -

1.5 2.5 3 - - -2 2.5 2 - - -

0.5 3 1 - - -1 3 1.5 - - -0 0 6 - - -3 0 0 - - -

1.5 3 2 - - -

4.2.4.2 Optimized conditions for PLS model



Preprocessing : Mean Centre and orthogonal signal correction



119

Table 4.38 Estimated concentrations of Irbesartan in the calibration setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4571 91.4335 0.4991 99.8345 0.5644 112.89971 0.9813 98.1340 1.0608 106.0821 1.0403 104.0379

1.5 1.5635 104.2387 1.4943 99.6263 1.4770 98.46722 2.0235 101.1783 2.0154 100.7732 2.0238 101.1919

0.5 0.4605 92.1016 0.4508 90.1643 0.5677 113.55531 1.1385 113.8513 1.0516 105.1623 0.9165 91.6597

1.5 1.4885 99.2385 1.5717 104.7834 1.5736 104.91102 1.9588 97.9446 1.8729 93.6471 1.9628 98.1406

0.5 0.4852 97.0580 0.4910 98.2136 0.4389 87.78641 1.0485 104.8505 1.0440 104.4041 1.0042 100.4269

1.5 1.5099 100.6608 1.4453 96.3587 1.5308 102.05502 1.8984 94.9237 2.0047 100.2396 2.0205 101.02800 0.0462 0 0.0433 0 0.0530 0

0.5 0.4753 95.0767 0.4498 89.9709 0.3819 76.38451 0.9890 98.9043 1.0246 102.4633 1.0371 103.7146

1.5 1.5589 103.9293 1.5348 102.3238 1.5245 101.63590.5 0.4723 94.4668 0.4164 83.2828 0.5005 100.10101 1.0788 107.8811 1.0093 100.9383 1.0097 100.9709

1.5 1.4260 95.0677 1.5826 105.5076 1.4124 94.16012 2.0247 101.2376 2.0184 100.9231 2.0022 100.1143

0.5 0.5087 101.7501 0.4549 90.9875 0.4248 84.97481 0.9201 92.0157 1.0248 102.4819 1.0743 107.43840 0.0173 0 0.0074 0 0.0147 03 2.9719 99.0666 2.9527 98.4251 2.9318 97.7291

1.5 1.5305 102.0358 1.4781 98.5423 1.5409 102.7284Mean 99.4367 Mean 98.9190 Mean 99.3962

120

Table 4.39 Estimated Concentrations of Hydrochlorothiazide in thecalibration set under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4608 92.1641 0.4794 95.8978 0.4829 96.58000.5 0.4860 97.2080 0.5026 100.5211 0.4922 98.44400.5 0.5057 101.1511 0.5755 115.1008 0.5078 101.56110.5 0.4943 98.8715 0.5020 100.4141 0.4891 97.82411 1.0767 107.6778 0.9566 95.6622 0.9518 95.18011 0.9282 92.8228 0.9376 93.7684 1.0829 108.29501 1.0373 103.7351 1.0059 100.5910 0.9106 91.06531 0.9741 97.4178 1.0089 100.8957 1.0615 106.1562

0.5 0.5272 105.4401 0.5479 109.5976 0.6740 134.80661.5 1.5135 100.9036 1.5062 100.4194 1.4648 97.65331.5 1.6098 107.3217 1.4286 95.2459 1.4777 98.51331.5 1.4919 99.4645 1.5520 103.4709 1.4884 99.22662 1.9864 99.3210 1.9864 99.3247 1.9989 99.94942 1.9189 95.9461 2.0426 102.1310 2.0346 101.73072 1.9870 99.3514 1.9735 98.6798 1.8908 94.54312 2.0572 102.8602 2.0545 102.7276 2.0795 103.9786

2.5 2.5472 101.8897 2.5769 103.0767 2.4582 98.33022.5 2.4446 97.7865 2.5110 100.4408 2.5022 100.08952.5 2.4916 99.6648 2.3880 95.5232 2.6018 104.07462.5 2.4525 98.1030 2.5251 101.0072 2.4085 96.34083 3.0218 100.7283 3.0197 100.6594 2.9638 98.79653 3.0023 100.0793 2.9804 99.3497 2.9751 99.17290 0.0076 0 0.0227 0 0.0154 00 0.0033 0 0.0026 0 0.0234 03 2.9882 99.6085 2.9627 98.7594 3.0331 101.1047

Mean 99.9790 Mean 100.5767 Mean 101.0181

121

Table 4.40 Estimated concentrations of Ramipril in the calibration setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 5.0226 100.4525 4.9013 98.0260 5.1337 102.67584 3.9425 98.5633 3.9399 98.4977 4.1564 103.91123 3.1302 104.3404 3.0032 100.1088 2.8260 94.20092 1.8487 92.4356 1.9256 96.2820 2.0351 101.75505 4.9996 99.9936 5.1685 103.3707 5.0618 101.23704 4.0629 101.5736 4.2284 105.7106 4.0825 102.06293 3.0187 100.6245 2.8677 95.5904 2.9726 99.08802 2.0139 100.6994 2.1334 106.6747 1.8963 94.81505 5.0435 100.8702 4.9897 99.7940 4.9120 98.24134 3.8438 96.0953 4.0016 100.0409 4.0253 100.63343 3.0895 102.9858 2.9686 98.9538 3.0703 102.34422 2.0728 103.6426 1.8939 94.6996 2.1413 107.06880 0.0012 0 0.0057 0 0.0145 05 4.9439 98.8798 5.0557 101.1152 4.8564 97.12874 3.9611 99.0291 3.8252 95.6312 3.9745 99.36453 3.1486 104.9564 2.9325 97.7524 2.8325 94.41745 4.8580 97.1605 4.6921 93.8437 4.8212 96.42494 3.9262 98.1573 4.1406 103.5174 3.9681 99.20293 3.0824 102.7499 2.9910 99.7004 3.0485 101.61762 1.8568 92.8403 2.0164 100.8222 2.0460 102.30181 0.9740 97.4031 0.8477 84.7763 0.8739 87.3900

1.5 1.5096 100.6430 1.5402 102.6825 1.4990 99.93336 6.0363 100.6056 6.0452 100.7542 6.0343 100.57320 0.0651 0 -0.0083 0 0.0126 02 2.1572 107.8613 2.2931 114.6569 2.1296 106.4808

Mean 100.1114 Mean 99.6957 Mean 99.6899

122

Table 4.41 Estimated concentrations of Irbesartan in the prediction setunder optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 1.9841 99.2086 1.9556 97.7833 2.0449 102.24862 1.9808 99.0407 1.9133 95.6681 2.0306 101.53351 1.1089 110.8900 1.0282 102.8223 1.0807 108.0795

1.5 1.6380 109.2045 1.4897 99.3158 2.0056 100.28020 0.3005 0 0.0607 0 0.0027 0

0.5 0.4949 98.9889 0.4893 97.8663 0.4994 99.89191.5 1.4659 97.7296 1.5153 101.0216 1.5527 103.51460 0.0243 0 0.0054 0 0.0876 00 -0.3073 0 0.0794 0 0.0908 01 0.9609 96.0950 0.9645 96.4522 0.9821 98.2181

Mean 101.5939 Mean 98.7042 Mean 101.9666

Table 4.42 Estimated Concentrations of Hydrochlorothiazide in theprediction set under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5536 110.7276 0.5352 107.0481 0.5302 106.05301 1.0161 101.6124 1.0647 106.4768 1.0325 103.2542

1.5 1.5621 104.1417 1.4630 97.5339 1.5284 101.89672 2.1003 105.0179 2.1139 105.6952 1.9737 98.6867

2.5 2.3898 95.5945 2.5112 100.4484 2.4765 99.06023 3.1298 104.3288 3.2374 107.9152 3.0775 102.58363 2.9652 98.8402 3.0827 102.7575 3.0847 102.82530 0.0452 0 0.0614 0 0.0430 01 1.0659 106.5950 0.9937 99.3759 0.9365 93.65212 1.9343 96.7162 1.9228 96.1442 1.8797 93.9864

Mean 102.6194 Mean 102.5995 Mean 100.2220

123

Table 4.43 Estimated Concentrations of Ramipril in the prediction setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1.5 1.5051 100.3404 1.4695 97.9730 1.4734 98.22982.5 2.4370 97.4837 2.4436 97.7446 2.5037 100.15132 2.0715 103.5780 2.0832 104.1611 2.0230 101.1548

2.5 2.5131 100.5255 2.7339 109.3564 2.5354 101.41851.5 1.5735 104.9047 1.4859 99.0653 1.5248 101.65554 4.2603 106.5085 3.9566 98.9162 3.9007 97.51874 4.0876 102.1901 3.9350 98.3754 3.9676 99.19213 3.0050 100.1687 3.0440 101.4695 3.0036 100.12326 6.0580 100.9668 6.0885 101.4761 6.0896 101.4933

1.5 1.5042 100.2807 1.5536 103.5760 1.5329 102.1999Mean 101.6947 Mean 101.2114 Mean 100.3137

4.2.4.3 Statistical Parameters for PLS model

The mean % recovery ranges from 98.919 to 101.018 for all thethree drugs in the calibration set and from 98.704 to 102.619 in the predictionset. The SD values were found to increase with increasing spectral mode inthe calibration set but seem to decrease with increasing spectral mode in theprediction set. R2 value ranges from 0.9932 – 0.9979 in the calibration set andfrom 0.9635 – 0.9988 in the prediction set. RMSEC value ranges from 0.0431– 0.1302 for all three drugs in the calibration set. RMSEP value ranges from0.0469 – 0.1481 for all three drugs in the prediction set.

124

Table 4.44 Statistical Parameters calculated from application of PLSmodel to the spectral data of calibration and prediction sets


Drug Mean SD R2 RMSEC Mean SD R2 RMSEP

IRB-D0

99.4367 5.3837 0.9943 0.5298 101.5939 5.8949 0.9635 0.1481

IRB-D1

98.9190 5.8347 0.9950 0.0494 98.7042 2.5344 0.9969 0.0469

IRB-D2

99.3962 8.3026 0.9940 0.0546 101.9666 3.1945 0.9966 0.0534

HCZ-D0

99.9790 3.8467 0.9979 0.0431 102.6194 4.8994 0.9948 0.0761

HCZ-D1

100.5767 4.6254 0.9978 0.0445 102.5995 4.4022 0.9940 0.0963

HCZ-D2

101.0181 8.2895 0.9957 0.0624 100.2220 4.2442 0.9962 0.0612

RAM-D0

100.1114 3.6052 0.9968 0.0892 101.6947 2.6470 0.9971 0.0961

RAM-D1

99.6957 5.5833 0.9932 0.1302 101.2114 3.6695 0.9960 0.0920

RAM-D2

99.6899 4.3270 0.9960 0.1006 100.3137 1.5689 0.9988 0.0481

4.2.4.4 Optimum Conditions for PCR model



Preprocessing : Mean Centre, orthogonal signal correction

and autoscale


Number of PCs : 13

125

Table 4.45 Estimated Concentrations of Irbesartan in the calibrationset under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5579 111.5880 0.5538 110.7641 0.4975 99.51401 1.0632 106.3283 1.0281 102.8111 1.0909 109.0900

1.5 1.4953 99.6890 1.5483 103.2211 1.4647 97.64662 1.9342 96.7128 1.9745 98.7253 2.0118 100.5913

0.5 0.5154 103.0968 0.5003 100.0780 0.5045 100.90401 1.0846 108.4612 0.9933 99.3300 0.8872 88.7280

1.5 1.4815 98.7666 1.5035 100.2333 1.4810 98.73332 1.8220 91.1001 1.8971 94.8550 1.8808 94.0432

0.5 0.5098 101.9680 0.5420 108.4040 0.5401 108.03801 0.9689 96.8992 0.8997 89.9759 1.0629 106.2900

1.5 1.4470 96.4713 1.5392 102.6133 1.4840 98.93332 2.1267 106.3385 2.1821 109.1083 2.1107 105.53600 0.0852 0 0.0879 0 0.0045 0

0.5 0.4376 87.5380 0.5513 110.2780 0.5077 101.55401 0.7942 79.4284 1.0384 103.8427 0.8834 88.3478

1.5 1.5530 103.5392 1.5096 100.6400 1.4659 97.72660.5 0.5235 104.7012 0.5568 111.3700 0.4904 98.08401 0.9591 95.9155 1.0574 105.7400 1.0115 101.1500

1.5 1.5807 105.3816 1.5131 100.8733 1.3864 92.43112 2.1168 105.8425 1.9294 96.4742 1.9440 97.2032

0.5 0.6962 139.2489 0.5104 102.0840 0.6141 122.83351 0.6092 60.9251 0.9051 90.5160 0.9582 95.82000 0.01830 0 0.0079 0 0.0093 03 2.7605 92.0176 2.8489 94.9645 2.8939 96.4651

1.5 1.4946 99.6435 1.6612 110.7480 1.5224 101.4933Mean 99.6348 Mean 102.0716 Mean 99.7634

126

Table 4.46 Estimated Concentrations of Hydrochlorothiazide in thecalibration set under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4591 91.8350 0.5277 105.5440 0.4757 95.15670.5 0.5129 102.5924 0.4753 95.0728 0.4870 97.40600.5 0.5024 100.4825 0.5075 101.5080 0.5376 107.52530.5 0.5025 100.5000 0.4861 97.2340 0.5072 101.44271 1.1648 116.4848 0.7905 79.0552 0.8533 85.33791 0.8425 84.2530 0.8285 82.8546 0.9024 90.24291 1.0100 101.0070 0.9809 98.0988 0.8875 88.75931 0.9037 90.3783 0.9267 92.6786 0.9275 92.7541

0.5 0.5221 104.4220 0.5315 106.3120 0.5027 100.54001.5 1.4995 99.9699 1.3978 93.1927 1.4205 94.70171.5 1.5367 102.4466 1.5360 102.4021 1.5570 103.80001.5 1.5219 101.4622 1.6229 108.1997 1.4972 99.81562 2.1169 105.8486 2.0459 102.2994 2.0886 104.43202 1.7831 89.1599 2.0011 100.0575 1.9642 98.21002 1.8839 94.1950 1.8122 90.6123 1.8975 94.87502 2.0913 104.5685 2.1015 105.0750 2.0990 104.9534

2.5 2.5591 102.3640 2.5409 101.6370 2.5087 100.34802.5 2.4381 97.5258 2.4926 99.7040 2.5174 100.69602.5 2.5271 101.0870 2.3943 95.7743 2.5058 100.23202.5 2.5337 101.3496 2.5015 100.0600 2.5106 100.42403 2.9520 98.4025 3.0542 101.8075 2.9500 98.33623 2.9715 99.0528 2.8941 96.4705 2.8991 96.66390 0.0492 0 0.0287 0 0.0492 00 0.0501 0 0.0367 0 0.0443 03 2.8631 95.4389 3.0511 101.7057 2.9068 96.8946

Mean 99.3403 Mean 98.1459 Mean 97.9803

127

Table 4.47 Estimated Concentrations of Ramipril in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 5.1260 102.5212 4.7456 94.9125 4.7802 95.60444 3.8964 97.4109 3.9700 99.2509 4.1449 103.62393 3.2535 108.4520 3.1927 106.4256 3.1566 105.22242 1.9563 97.8199 1.9306 96.5325 1.8860 94.30005 5.0648 101.2976 5.1870 103.7403 5.2683 105.36714 4.0861 102.1538 4.1343 103.3575 4.1300 103.25173 2.8755 95.8500 2.7119 90.3980 3.3836 112.78832 1.9717 98.5880 2.0274 101.3700 1.9291 96.45505 4.8077 96.1545 4.9961 99.9237 4.9692 99.38404 3.8407 96.0175 3.9587 98.9678 3.9117 97.79393 3.1707 105.6921 3.3289 110.9664 3.2056 106.85332 1.9181 95.9084 1.8672 93.3637 2.0874 104.37090 0.0887 0 0.0371 0 0.0128 05 4.8904 97.8081 4.9789 99.5781 5.0386 100.77204 4.3552 108.8824 3.8741 96.8525 3.9194 97.98503 3.3733 112.4447 3.0161 100.5378 2.9992 99.97655 4.5534 91.0697 4.9273 98.5460 4.9426 98.85204 3.8474 96.1850 3.8813 97.0333 3.9337 98.34253 2.9222 97.4079 2.7005 90.0178 2.9300 97.66922 1.9117 95.5890 2.0915 104.5754 1.9046 95.23111 1.0016 100.1601 0.8438 84.3844 1.0127 101.2700

1.5 1.5119 100.7996 1.5446 102.9733 1.5822 105.48426 6.1247 102.0791 6.1293 102.1563 6.2083 103.47190 0.1196 0 -0.2014 0 0.0839 02 2.0705 103.5250 2.0258 101.2900 2.0459 102.2950

Mean 100.1659 Mean 99.0067 Mean 101.1463

128

Table 4.48 Estimated concentrations of Irbesartan in the prediction setunder optimized PCR model


D0 D1 D2Amount found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.1509 107.5455 1.8982 94.9140 1.8936 94.68232 1.8245 91.2287 1.9797 98.9879 2.0238 101.19071 1.0317 103.1798 1.0985 109.8507 1.0677 106.7704

1.5 1.4765 98.4348 1.4961 99.7444 1.9055 95.27590 0.0531 0 0.0813 0 0.0394 0

0.5 0.4886 97.7235 0.5163 103.2670 0.4706 94.12281.5 1.4616 97.4439 1.4820 98.8015 1.5373 102.48790 0.0181 0 0.0046 0 0.0045 00 0.0741 0 -0.0778 0 0.0094 01 0.9908 99.0875 1.0837 108.3774 1.0195 101.95862 Mean 99.0195 Mean 101.7793 Mean 99.4984



D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4473 89.4717 0.5096 101.9360 0.4747 94.94341 0.8522 85.2247 1.0152 101.5250 1.0389 103.8906

1.5 1.4766 98.4412 1.4709 98.0613 1.5029 100.19512 2.0479 102.3992 2.0843 104.2191 2.0722 103.6100

2.5 2.4599 98.3998 2.4476 97.9078 2.5383 101.53243 3.1047 103.4912 3.1086 103.6208 3.0792 102.64173 2.9147 97.1592 3.2052 106.8412 2.8761 95.87240 0.0003 0 0.0127 0 0.0404 01 1.0294 102.9457 0.9818 98.1896 1.0063 100.63762 1.8545 92.7294 1.9627 98.1353 1.9411 97.0599

Mean 96.6958 Mean 101.1596 Mean 100.0426

129

Table 4.50 Estimated Concentrations of Ramipril in the prediction setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1.5 1.5647 104.3140 1.5074 100.4970 1.4853 99.02212.5 2.2843 91.3747 2.5066 100.2653 2.5011 100.04762 2.0118 100.5935 2.0840 104.2032 2.0440 102.2018

2.5 2.5384 101.5388 2.5270 101.0828 2.6102 104.41131.5 1.4337 95.5817 1.4758 98.3887 1.4119 94.12864 4.1010 102.5273 3.8325 95.8143 3.9036 97.59014 3.8676 96.6923 3.8875 97.1880 4.0855 102.13963 3.0470 101.5689 3.0141 100.4727 3.0049 100.16586 6.1601 102.6686 6.3702 106.1715 6.0234 100.3905

1.5 1.5011 100.0743 1.5356 102.3761 1.4750 98.3389Mean 99.6934 Mean 100.6460 Mean 99.8436

4.2.4.5 Statistical Parameters for PCR model


130




IRB-D0 99.6348 13.873 0.9685 0.1262 99.0195 4.4397 0.9889 0.0808

IRB-D1 102.0716 6.2388 0.9893 0.0766 101.7793 5.4545 0.9932 0.0638

IRB-D2 99.7634 7.2508 0.9915 0.0675 99.4984 4.8417 0.9960 0.0544

HCZ-D0 99.3403 6.5716 0.9919 0.0857 96.6958 6.3584 0.9940 0.0832

HCZ-D1 98.1459 7.0496 0.9922 0.0867 101.1596 3.2896 0.9958 0.0817

HCZ-D2 97.9803 5.3299 0.9954 0.0681 100.0426 3.3386 0.9963 0.0596

RAM-D0 100.1659 5.0919 0.9877 0.1761 99.6934 3.7579 0.9952 0.1059

RAM-D1 99.0067 5.8100 0.9912 0.1538 100.6460 2.9489 0.9926 0.1370

RAM-D2 101.1463 4.4451 0.9929 0.1408 99.8436 2.6985 0.9980 0.0631


Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.52. The percentage recoverywas found to be from 99.21 – 100.81 for IRB, 99.04 – 100.88 for HCZ and99.60 – 101.20 for RAM respectively. These methods can be successfullyapplied for formulations containing either two or three drugs.

131

Figure 4.19 Plots of calibration and prediction sets of Irbesartan,Hydrochlorothiazide and Ramipril in both PLS and PCRmodels

y = 0.994x + 0.006R² = 0.994

-0.5

00.5

1

1.52

2.5

33.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

IRB-PLS-CAL-D0

y = 0.995x + 0.005R² = 0.995

0

0.5

1

1.5

2

2.5

3

3.5


IRB-PLS-CAL-D1

y = 0.994x + 0.007R² = 0.994

-0.5

0

0.51

1.5

22.5

3

3.5


IRB-PLS-CAL-D2

y = 0.999x + 0.017R² = 0.994

0

0.5

1

1.5

2

2.5

0 1 2

Am

ount

Pre

dict

ed

Amount Present

IRB-PLS-PRE-D0

y = 0.999x + 0.017R² = 0.994

0

0.5

1

1.5

2

2.5

0 1 2Amount Present

IRB-PLS-PRE-D0

y = 1.016x + 0.002R² = 0.996

-0.5

0

0.5

1

1.5

2

2.5

0 1 2

Amount Present

IRB-PLS-PRE-D2

y = 0.997x + 0.003R² = 0.997

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0

y = 0.997x + 0.003R² = 0.997

-0.5

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PLS-CAL-D1

y = 0.987x + 0.021R² = 0.995

00.5

11.5

22.5

33.5


HCZ-PLS-CAL-D2

132


y = 0.984x + 0.052R² = 0.994

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 1.029x + 9E-05R² = 0.994

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PLS-PRE-D1

y = 1.006x - 0.003R² = 0.996

-0.5

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PLS-PRE-D2

y = 0.996x + 0.01R² = 0.996

-101234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-CAL-D0

y = 0.993x + 0.021R² = 0.993

-101234567


RAM-PLS-CAL-D1

y = 0.998x + 0.001R² = 0.996

-101234567


RAM-PLS-CAL-D2

y = 1.022x - 0.012R² = 0.997

01234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-PRE-D0

y = 1.000x + 0.027R² = 0.996

01234567


RAM-PLS-PRE-D1

y = 1.002x - 0.002R² = 0.998

01234567


RAM-PLS-PRE-D2

133


y = 0.953x + 0.038R² = 0.968

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

IRB-PCR-CAL-D0

y = 0.961x + 0.059R² = 0.989

0

0.5

11.5

22.5

33.5


IRB-PCR-CAL-D1

y = 0.971x + 0.019R² = 0.991

-0.50

0.51

1.52

2.53

3.5


IRB-PCR-CAL-D2

y = 0.997x - 0.008R² = 0.988

-0.5

0

0.5

1

1.5

2

2.5

0 1 2

Am

ount

Pre

dict

ed

Amount Present

IRB-PCR-PRE-D0

y = 0.977x + 0.025R² = 0.995

-0.5

0

0.5

1

1.5

2

2.5

0 1 2Amount Present

IRB-PCR-PRE-D1

y = 0.974x + 0.020R² = 0.996

-0.5

0

0.5

1

1.5

2

2.5

0 1 2

Amount Present

IRB-PCR-PRE-D2

y = 0.987x + 0.007R² = 0.991

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 1.005x - 0.027R² = 0.992

-0.50

0.51

1.52

2.53

3.5


HCZ-PCR-CAL-D1

y = 0.996x - 0.018R² = 0.995

-0.50

0.51

1.52

2.53

3.5


HCZ-PCR-CAL-D2

134


y = 1.010x - 0.048R² = 0.994

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 1.038x - 0.034R² = 0.995

-1

0

1

2

3

4

0 1 2 3

Amount Present

HCZ-PCR-PRE-D1

y = 0.990x + 0.022R² = 0.996

00.5

11.5

22.5

33.5


HCZ-PCR-PRE-D2

y = 0.992x + 0.023R² = 0.987

-101234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-CAL-D0

y = 1.012x - 0.061R² = 0.991

-101234567


RAM-PCR-CAL-D1

y = 1.012x - 0.006R² = 0.992

-1

0

1

2

3

4

5

6

7


RAM-PCR-CAL-D2

y = 1.027x - 0.078R² = 0.995

0

1

2

3

4

5

6

7

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-PRE-D0

y = 1.038x - 0.086R² = 0.992

0

1

2

3

4

5

6

7


RAM-PCR-PRE-D1

y = 1.008x - 0.019R² = 0.998

0

12

34

56

7


RAM-PCR-PRE-D2

135

Table 4.52 Assay results for PLS and PCR models in PharmaceuticalPreparations

Met

hod

Tablet Mode

IRB HCZAmountFound*

(mg/tab) ±SD

%recover

y

AmountFound*

(mg/tab) ± SD

%recovery

PLS

T1D0 150.45 ± 0.21 100.30 12.57 ± 0.11 100.56D1 150.32 ± 0.11 100.21 12.61 ± 0.13 100.88D2 149.88 ± 0.08 99.92 12.66 ± 0.13 101.28

T2D0 148.82 ± 0.05 99.21 12.57 ± 0.05 100.56D1 149.76 ± 0.12 99.84 12.59 ± 0.11 100.72D2 150.43 ± 0.04 100.28 12.47 ± 0.03 99.76

PC

R

T1D0 149.11 ± 0.11 99.40 12.46 ± 0.04 99.68D1 150.21 ± 0.23 100.14 12.38 ± 0.13 99.04D2 150.08 ± 0.06 100.05 12.55 ± 0.12 100.40

T2D0 150.42 ± 0.03 100.28 12.56 ± 0.06 100.48D1 151.22 ± 0.07 100.81 12.34 ± 0.23 98.72D2 149.53 ± 0.03 99.68 12.57 ± 0.04 100.56

etho

d

Tablet ModeRAM HCZ

AmountFound*

(mg/tab) ±

%recovery

AmountFound*

(mg/tab) ± SD

%recovery

PLS T3

D0 2.53 ± 0.04 101.20 12.58 ± 0.11 100.64D1 2.47 ± 0.03 98.80 12.58 ± 0.03 100.64D2 2.51 ± 0.03 100.40 12.48 ± 0.04 99.84

PCR T3

D0 2.49 ± 0.04 99.60 12.46 ± 0.03 99.68D1 2.53 ± 0.03 101.20 12.56 ± 0.02 100.48D2 2.52 ± 0.02 100.80 12.46 ± 0.04 99.68


T1 – IROVEL H – Sun Pharmaceuticals, J&K


T2 – XARB H – Hetero Labs, HP


T3 – Cardace # 2.5 – Aventis Pharma, Goa

Ramipril – 2.5 mg and Hydrochlorothiazide – 12.5 mg

136


96

98

100

102

104

D0 D1 D2

Irbesartan - PLS


96

98

100

102

104

D0 D1 D2

Irbesartan - PCR


96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2

Ramipril -PLS


96

98

100

102

104

D0 D1 D2

Ramipril - PCR


137

4.2.5 Ternary Mixture 2– Valsartan (VAL), Hydrochlorothiazide(HCZ) and Ramipril (RAM)


The spectra of all three drugs were overlapped in fundamental(Figure 4.21), first derivative (Figure 4.22) and second derivative (Figure4.23) to study the spectral characteristics. They reveal a complete overlapthroughout the UV region of 200-400 nm. For multivariate analysis, 25different synthetic mixtures were prepared in the concentration ranges of 0-3µg/ml of VAL and HCZ, 0-6 µg/ml of RAM respectively (Table 4.53). Theconcentration range starts from 0 in order to meet the requirement of utilizingthe analysis of any two drugs mentioned in the combination

Figure 4.21 Fundamental Spectra of Valsartan, Hydrochlorothiazide,Ramipril and mixture

138

Figure 4.22 First Derivative Spectra of Valsartan, Hydrochlorothiazide,Ramipril and mixture

Figure 4.23 Second Derivative Spectra of Valsartan,Hydrochlorothiazide, Ramipril and mixture

139


Calibration Set (µg/ml) Prediction Set (µg/ml)VAL HCZ RAM VAL HCZ RAM0.5 0.5 5 3 0.5 2.51 0.5 4 2.5 1 2.5

1.5 0.5 3 1 1.5 2.52 0.5 2 2 2 2

0.5 1 5 0 3 01 1 4 1 3 2

1.5 1 3 2 3 12 1 2 0 0.5 4

0.5 0.5 5 1 0.5 61 1.5 4 0 2 0

1.5 1.5 3 - - -2 1.5 2 - - -0 2 0 - - -

0.5 2 5 - - -1 2 4 - - -

1.5 2 3 - - -0.5 2.5 5 - - -1 2.5 4 - - -

1.5 2.5 3 - - -2 2.5 2 - - -

0.5 3 1 - - -1 3 1.5 - - -0 0 6 - - -3 0 0 - - -

1.5 3 2 - - -







140

Table 4.54 Estimated Concentrations of Valsartan in the calibration setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4455 89.1097 0.5115 102.3100 0.4252 85.05601 1.0488 104.8806 1.1258 112.5844 1.0357 103.5743

1.5 1.5370 102.4711 1.5286 101.9066 1.5301 102.01162 2.0243 101.2196 2.1087 105.4350 2.0104 100.5249

0.5 0.4476 89.5246 0.5395 107.9087 0.5132 102.65891 1.0109 101.0940 0.9577 95.7770 0.9963 99.6396

1.5 1.4737 98.2517 1.5085 100.5666 1.5482 103.21722 1.9324 96.6208 1.9085 95.4250 1.9471 97.3577

0.5 0.5124 102.4859 0.5261 105.2220 0.5179 103.59351 1.0627 106.2790 0.9888 98.8890 0.9531 95.3117

1.5 1.4582 97.2157 1.5401 102.6772 1.4989 99.92672 2.0239 101.1992 1.9073 95.3650 2.0315 101.57590 0.0128 0 0.0792 0 0.0356 0

0.5 0.4616 92.3329 0.5323 106.4686 0.4629 92.58711 1.0546 105.4644 1.0091 100.9100 1.0333 103.3313

1.5 1.4154 94.3627 1.6334 108.8979 1.4608 97.39310.5 0.5598 111.9791 0.4940 98.8020 0.5140 102.81011 0.9920 99.2055 1.0184 101.8144 1.0758 107.5852

1.5 1.5099 100.6633 1.5627 104.1824 1.4310 95.40622 2.0022 100.1128 1.9751 98.7562 1.9713 98.5676

0.5 0.4968 99.3660 0.5057 101.1584 0.4577 91.55241 1.0249 102.4956 1.0712 107.1285 0.9992 99.92440 0.0059 0 0.0460 0 0.0110 03 3.0126 100.4202 2.9692 98.9733 2.9936 99.7872

1.5 1.4728 98.1881 1.4820 98.8016 1.5448 102.9919Mean 99.7801 Mean 102.1721 Mean 99.4384

141

Table 4.55 Estimated Concentrations of Hydrochlorothiazide in thecalibration set under optimized PLS model


FundamentalMode



Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4823 96.4651 0.5205 104.1060 0.4560 91.21280.5 0.5065 101.3019 0.5093 101.8620 0.5680 113.60890.5 0.5195 103.9084 0.4610 92.2060 0.5535 110.71200.5 0.4916 98.3260 0.5185 103.7160 0.4527 90.54291 0.9675 96.7522 0.9745 97.4510 0.9579 95.79221 1.0547 105.4796 1.0491 104.9118 0.9490 94.90061 1.0060 100.6047 0.9812 98.1284 1.0695 106.95651 0.9736 97.3667 0.9812 98.1284 0.9664 96.6419

0.5 0.5440 108.8039 0.5221 104.4300 0.6044 120.89921.5 1.4517 96.7858 1.3790 91.9385 1.3899 92.66421.5 1.4700 98.0018 1.4253 95.0232 1.4438 96.25541.5 1.4731 98.2104 1.4944 99.6268 1.5375 102.50632 2.0095 100.4765 1.9941 99.7066 2.0481 102.40512 2.0352 101.7614 1.9715 98.5790 1.9618 98.09152 1.9897 99.4893 2.0474 102.3745 2.0092 100.46072 2.0071 100.3594 2.0853 104.2687 2.0206 101.0317

2.5 2.4775 99.1009 2.4724 98.8989 2.5317 101.26802.5 2.4984 99.9394 2.5556 102.2258 2.5636 102.54602.5 2.5015 100.0601 2.4545 98.1809 2.4429 97.71622.5 2.5370 101.4828 2.5095 100.3814 2.4594 98.37853 2.9839 99.4637 3.0284 100.9486 2.8735 95.78373 3.0030 100.1025 3.0031 100.1042 3.1377 104.59190 0.0047 0 0.0121 0 0.0018 00 0.0080 0 0.0142 0 0.0119 03 3.0124 100.4137 2.9646 98.8210 2.9919 99.7315

Mean 100.2024 Mean 100.5805 Mean 99.3347

142



D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 4.9786 99.5738 4.9867 99.7351 4.9918 99.83644 4.0500 101.2518 3.9875 99.6888 4.0277 100.69413 3.0328 101.0953 3.0213 100.7117 3.0029 100.09712 1.9918 99.5928 2.0058 100.2931 1.9792 98.96185 4.8722 97.4452 5.0517 101.0359 4.9579 99.15864 3.9528 98.8201 3.9436 98.5924 3.8944 97.36113 3.0995 103.3197 2.8235 94.1166 3.1424 104.74942 2.0146 100.7318 2.0235 101.1750 1.9777 98.88885 5.0577 101.1542 4.9434 98.8691 4.9784 99.56814 3.9494 98.7360 4.0114 100.2866 4.0104 100.26203 2.9110 97.0355 3.0358 101.1955 3.0647 102.15902 1.9651 98.2576 2.0586 102.9305 2.0100 100.50400 0.0014 0 0.0001 0 0.0055 05 4.9676 99.3534 4.9266 98.5326 4.9757 99.51424 4.0236 100.5900 3.9295 98.2389 3.9786 99.46603 2.9691 98.9707 3.0527 101.7598 2.8738 95.79525 5.0799 101.5986 5.0966 101.9328 5.0955 101.91104 4.0085 100.2133 4.0614 101.5353 3.9471 98.67773 2.9805 99.3503 2.8788 95.9628 3.0459 101.53062 2.0019 100.0955 1.9561 97.8071 1.9636 98.18001 0.8166 81.6624 0.9827 98.2728 0.9042 90.4298

1.5 1.6534 110.2332 1.5379 102.5291 1.6331 108.87576 6.0216 100.3610 6.0079 100.1329 6.0301 100.50290 0.0081 0 0.0167 0 0.0132 02 2.0275 101.3763 1.9824 99.1233 1.9650 98.2544

Mean 99.6008 Mean 99.7590 Mean 99.7990

143

Table 4.57 Estimated concentrations of Valsartan in the prediction setunder optimized PLS model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

3 2.9122 97.0739 2.9430 98.1022 2.8848 96.16252.5 2.4793 99.1720 2.4858 99.4339 2.5569 102.27641 0.9759 97.5932 0.9396 93.9631 1.0354 103.54832 1.9164 95.8202 1.0085 100.8521 1.0376 103.76200 0.0084 0 0.0047 0 0.0039 01 0.9652 96.5221 0.9656 96.5619 0.9693 96.93822 1.9013 95.0654 1.9273 96.3663 1.9691 98.45820 0.0088 0 0.0045 0 0.0098 01 1.0857 108.5741 0.9711 97.1169 0.9889 98.89120 0.0085 0 0.0042 0 0.0058 0

Mean 98.5458 Mean 97.4852 Mean 100.0052

Table 4.58 Estimated concentrations of Hydrochlorothiazide in theprediction set under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5010 100.2063 0.4843 96.8658 0.4868 97.37221 1.0027 100.2776 1.0785 107.8580 1.0131 101.3126

1.5 1.5082 100.5515 1.4956 99.7072 1.5117 100.78642 2.0398 101.9940 1.8652 93.2626 1.9678 98.39323 3.0073 100.2462 3.0111 100.3712 3.0877 102.92333 3.1287 104.2917 2.9627 98.7587 3.0065 100.21693 3.1905 106.3517 2.9468 98.2286 2.9931 99.7730

0.5 0.4999 99.9923 0.4846 96.9263 0.5108 102.17071 0.9826 98.2650 0.9851 98.5116 0.9889 98.89702 2.0156 100.7840 2.0358 101.7905 2.0365 101.8284

Mean 101.4409 Mean 99.4838 Mean 100.1670

144

Table 4.59 Estimated Concentrations of Ramipril in the prediction setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2.5 2.4250 97.0017 2.4938 99.7559 2.4697 98.78892.5 2.5081 100.3251 2.4381 97.5242 2.4831 99.32402.5 2.5578 102.3156 2.4682 98.7284 2.4773 99.09492 1.8669 93.3490 2.0732 103.6607 2.0151 100.75630 0.0002 0 0.0032 0 0.0080 02 1.9208 96.0446 2.0591 102.9580 1.9573 97.86591 0.9823 98.2322 1.0254 102.5446 0.9875 98.75974 3.8737 96.8439 3.8918 97.2965 3.8809 97.02446 6.0248 100.4149 6.0607 101.0132 6.1097 101.82840 0.0084 0 0.0039 0 0.0035 0

Mean 98.0659 Mean 100.4352 Mean 99.1803



145

Table 4.60 Statistical Parameters calculated from application of PLSmodel to spectral data of calibration and prediction sets



VAL-D0

99.7801 5.2405 0.9968 0.0934 98.5458 4.6128 0.9979 0.0615

VAL-D1

102.1721 4.5095 0.9940 0.0600 97.4852 2.2419 0.9986 0.0481

VAL-D2

99.4384 4.4362 0.9970 0.0387 100.0052 2.1525 0.9978 0.0495

HCZ-D0

100.2024 2.8717 0.9993 0.0247 101.4409 2.3582 0.9979 0.0742

HCZ-D1

100.5805 2.5303 0.9980 0.0427 99.4838 3.8118 0.9970 0.0553

HCZ-D2

99.3347 2.1613 0.9955 0.0629 100.1670 1.7762 0.9992 0.0329

RAM-D0

99.6008 4.6785 0.9981 0.0699 98.0659 2.8702 0.9988 0.0755

RAM-D1

99.7590 2.0842 0.9986 0.0605 100.4352 2.4857 0.9989 0.0593

RAM-D2

99.7990 3.3044 0.9984 0.0640 99.1803 1.5226 0.9991 0.0556






Number of PCs : 13

146

Table 4.61 Estimated concentrations of Valsartan in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.3590 71.8177 0.4862 97.2520 0.4608 92.16201 1.1112 111.1233 1.1197 111.9785 1.1037 110.3700

1.5 1.4909 99.3933 1.5365 102.4333 1.4671 97.80662 2.0771 103.8563 2.1049 105.2450 2.1017 105.0850

0.5 0.4772 95.4542 0.5024 100.4820 0.5236 104.72671 0.9457 94.5762 0.8883 88.8339 0.9837 98.3750

1.5 1.4541 96.9400 1.4823 98.8200 1.5109 100.73022 1.9590 97.9544 1.8923 94.6150 1.9724 98.6239

0.5 0.5552 111.0429 0.5100 102.0180 0.4829 96.58201 1.0367 103.6764 0.9606 96.0620 0.9631 96.3110

1.5 1.4360 95.7391 1.5124 100.8266 1.4428 96.19222 1.9579 97.8967 1.8809 94.0450 2.0227 101.13800 0.0758 0 0.0858 0 0.0592 0

0.5 0.5078 101.5700 0.5155 103.1053 0.5144 102.89401 1.1449 114.4915 1.1039 110.3969 1.0993 109.9376

1.5 1.3615 90.7684 1.4798 98.6533 1.4802 98.68110.5 0.5207 104.1400 0.5143 102.866 0.4838 96.77201 0.9868 98.6853 1.0308 103.0838 1.0558 105.5867

1.5 1.5441 102.9422 1.5109 100.7267 1.5362 102.41962 1.9734 98.6723 1.9904 99.5215 1.9474 97.3723

0.5 0.4827 96.5489 0.4696 93.9213 0.5111 102.22001 1.0330 103.3038 1.0900 109.0004 1.0500 105.00490 0.0073 0 0.0067 0 0.1084 03 2.9749 99.1642 2.8792 95.9733 2.8739 95.7966

1.5 1.4408 96.0535 1.4960 99.7355 1.4833 98.8926Mean 99.3831 Mean 100.4172 Mean 100.2109

147

Table 4.62 Estimated concentrations of Hydrochlorothiazide in thecalibration set under optimized PCR model


FundamentalMode



Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4705 94.1172 0.4898 97.9660 0.4869 97.39000.5 0.4008 80.1749 0.4600 92.0060 0.4686 93.72800.5 0.5309 106.1980 0.5345 106.9120 0.5575 111.50000.5 0.5199 103.9980 0.5212 104.2480 0.5020 100.40201 1.0734 107.3451 0.9267 92.6780 0.9888 98.88001 0.8900 89.0010 1.1402 114.0221 0.9788 97.88521 1.0143 101.4348 1.0022 100.2209 1.0531 105.31651 0.9807 98.0700 1.0022 100.2209 0.9283 92.8324

0.5 0.4671 93.4380 0.4912 98.2400 0.5530 110.60401.5 1.4506 96.7066 1.4613 97.4200 1.3564 90.43061.5 1.4893 99.2913 1.5076 100.5066 1.5046 100.30661.5 1.5302 102.0141 1.5320 102.1333 1.4746 98.30662 2.0143 100.7165 1.9800 99.0042 2.1085 105.42922 2.0685 103.4265 1.9872 99.3615 2.1058 105.29082 1.9794 98.97035 1.9078 95.3938 1.8344 91.72262 1.9855 99.2782 2.0437 102.1874 1.8697 93.4886

2.5 2.4290 97.1617 2.5498 101.9931 2.4805 99.22162.5 2.5880 103.5229 2.6349 105.3997 2.4826 99.30402.5 2.5237 100.9493 2.3893 95.5743 2.3971 95.88692.5 2.4631 98.5252 2.4639 98.5589 2.2988 91.95213 3.0036 100.1213 3.0290 100.9675 2.7740 92.46923 3.0764 102.5475 2.9127 97.0921 3.0448 101.49400 0.0013 0 0.0085 0 0.0078 00 0.0097 0 0.0068 0 0.0016 03 2.8314 94.3801 3.0036 100.1209 3.1109 103.6983

Mean 98.7560 Mean 100.0968 Mean 99.0234

148

Table 4.63 Estimated concentrations of Ramipril in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 4.9729 99.4598 4.9547 99.0947 4.8712 97.42544 3.9870 99.6769 3.8655 96.6384 4.0450 101.12683 3.0621 102.0738 3.0826 102.7550 3.1470 104.90082 2.0214 101.0731 2.1305 106.5279 2.0705 103.52785 4.7969 95.9397 4.8375 96.7502 4.8177 96.35534 3.9780 99.4507 3.8403 96.0098 3.7784 94.46043 3.2126 107.0873 2.8390 94.6333 3.1802 106.00782 2.0175 100.8770 2.0390 101.9500 2.0190 100.95025 4.9705 99.4113 4.9037 98.0756 5.0413 100.82774 3.9714 99.2859 3.9774 99.4361 3.9440 98.60053 2.9695 98.9859 3.0592 101.9758 3.0374 101.24922 1.9734 98.6723 2.1029 105.1485 2.0745 103.72760 0.0086 0 0.0020 0 0.0008 05 4.9729 99.4597 4.9782 99.5650 4.9667 99.33434 3.9452 98.6324 3.8326 95.8150 3.8525 96.31473 2.9052 96.8423 3.0863 102.8794 2.8940 96.46705 5.1131 102.2620 5.2019 104.0398 5.1635 103.27164 4.0651 101.6277 4.1478 103.6964 3.9266 98.16703 2.9783 99.2776 2.8794 95.9830 3.1329 104.43312 1.9503 97.5174 1.9113 95.5698 1.8839 94.19531 0.9389 93.8930 0.9571 95.7140 0.9764 97.6441

1.5 1.7049 113.6633 1.4897 99.3133 1.4953 99.68666 6.0668 101.1139 6.1927 103.2132 6.1098 101.83000 0.0423 0 0.0934 0 0.0915 02 2.0285 101.4291 1.8808 94.0439 1.8817 94.0850

Mean 100.2652 Mean 99.5143 Mean 99.7647

149

Table 4.64 Estimated concentrations of Valsartan in the prediction setunder optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

3 2.8213 94.0438 2.8925 96.4199 2.9426 98.08952.5 2.4576 98.3060 2.4537 98.1497 2.4873 99.49511 0.9881 98.8101 0.9799 97.9911 1.0181 101.81822 1.9123 95.6189 1.0535 105.3503 1.0213 102.13620 0.0095 0 0.0043 0 0.0067 01 0.9650 96.5030 0.9706 97.0631 0.9688 96.88862 1.8951 94.7568 1.9750 98.7526 1.9240 96.20160 0.0059 0 0.0010 0 0.0089 01 1.0771 107.7160 0.9553 95.5358 1.0303 103.03820 0.0444 0 0.0091 0 0.0005 0

Mean 97.9649 Mean 98.4661 Mean 99.6668



D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4833 96.6674 0.4818 96.3742 0.5039 100.79661 1.0201 102.0177 1.0424 104.2437 1.0365 103.6530

1.5 1.4676 97.8412 1.5447 102.9849 1.5234 101.56112 2.1966 109.8316 1.9631 98.1557 2.0204 101.02313 2.9989 99.9637 3.1189 103.9658 3.0830 102.76773 3.1861 106.2057 2.8467 94.8907 2.8830 96.10043 3.1234 104.1159 2.9381 97.9389 2.8921 96.4056

0.5 0.4752 95.0470 0.5109 102.1800 0.5048 100.96470.5 0.4699 93.9834 0.5024 100.4860 0.5187 103.75082 2.0964 104.8205 2.0678 103.3929 2.0326 101.6305

Mean 101.7163 Mean 100.2703 Mean 100.8543

150

Table 4.66 Estimated concentrations of Ramipril in the prediction setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2.5 2.5589 102.3572 2.4823 99.2927 2.5060 100.2425

2.5 2.5122 100.4912 2.6466 105.8647 2.4947 99.7907

2.5 2.5554 102.2161 2.4943 99.7739 2.4605 98.4206

2 1.9014 95.0717 2.0524 102.6247 2.0968 104.8424

0 0.0014 0 0.0086 0 0.0037 0

2 1.9687 98.4391 2.1097 105.4869 2.0604 103.0214

1 0.9896 98.9632 1.0504 105.0429 1.0053 100.5313

4 3.8413 96.0345 3.9617 99.0437 3.9676 99.1912

6 6.0627 101.0459 6.2046 103.4114 6.1262 102.1047

0 0.0049 0 0.0086 0 0.0095 98.3389

Mean 99.3274 Mean 102.5676 Mean 101.0181



151

Table 4.67 Statistical Parameters calculated from application of PCRmodel to spectral data of calibration and prediction sets



VAL-D0

99.3831 8.2907 0.9912 0.0665 97.9649 4.6407 0.9976 0.0796

VAL-D1

100.4172 5.4928 0.9919 0.0667 98.4661 3.2285 0.9975 0.0596

VAL-D2

100.2109 4.6325 0.9944 0.0575 99.6668 2.7183 0.9992 0.0553

HCZ-D0

98.7560 5.8817 0.9959 0.0620 101.7163 5.2165 0.9957 0.1005

HCZ-D1

100.0968 4.7200 0.9961 0.0586 100.2703 3.3984 0.9950 0.0719

HCZ-D2

99.0234 3.9266 0.9908 0.0949 100.8543 2.6545 0.9968 0.0599

RAM-D0

100.2652 3.8644 0.9969 0.0895 99.3274 2.7178 0.9983 0.0749

RAM-D1

99.5143 3.7343 0.9950 0.1149 102.5676 2.6581 0.9985 0.0918

RAM-D2

99.7647 3.5678 0.9951 0.1112 101.0181 2.1463 0.9993 0.0567


Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.68. The percentage recoverywas found to be from 99.21 – 100.81 for VAL, 99.04 – 100.88 for HCZ and99.60 – 101.20 for RAM respectively. These methods can be successfullyapplied for formulations containing either two or three drugs.

152

Figure 4.24 Plots of calibration and prediction sets of Valsartan,Hydrochlorothiazide and Ramipril in both PLS and PCRmodels

y = 0.996x + 0.003R² = 0.996

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PLS-CAL-D0

y = 0.975x + 0.049R² = 0.994

0

0.5

1

1.5

2

2.5

33.5

0 1 2 3

Amount Present

VAL-PLS-CAL-D1

y = 0.997x + 0.003R² = 0.997

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PLS-CAL-D2

y = 0.969x + 0.011R² = 0.997

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PLS-PRE-D0y = 0.982x - 0.005

R² = 0.998

-0.50

0.51

1.5

22.5

33.5

0 1 2 3

Amount Present

VAL-PLS-PRE-D1

y = 0.994x - 0.009R² = 0.997

-0.5

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PLS-PRE-D2

y = 0.999x + 0.001R² = 0.999

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0y = 0.998x - 0.000

R² = 0.998

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Amount Present

HCZ-PLS-CAL-D1

y = 0.995x + 0.006R² = 0.995

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Amount Present

HCZ-PLS-CAL-D2

153


y = 1.0471x -0.0447

R² = 0.9979

0

0.5

1

1.52

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.985x + 0.010R² = 0.997

00.5

11.5

22.5

33.5


HCZ-PLS-PRE-D1

y = 1.009x - 0.003R² = 0.999

00.5

11.5

22.5

33.5


HCZ-PLS-PRE-D2

y = 0.998x + 0.006R² = 0.998

-101234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-CAL-D0

y = 0.999x - 0.006R² = 0.998

-101234567

0 2 4 6

Amount Present

RAM-PLS-CAL-D1

y = 0.998x + 0.005R² = 0.998

01234567


RAM-PLS-CAL-D2

y = 1.006x - 0.059R² = 0.998

-1

0

1

2

3

4

5

6

7

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-PRE-D0

y = 0.992x + 0.026R² = 0.998

01234567

0 2 4 6

Amount Present

RAM-PLS-PRE-D1

y = 1.009x - 0.036R² = 0.999

-101234567

0 1 2 3 4 5 6

Amount Present

RAM-PLS-PRE-D2

154


y = 0.982x + 0.016R² = 0.991

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PCR-CAL-D0

y = 0.961x + 0.047R² = 0.991

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PCR-CAL-D1

y = 0.964x + 0.051R² = 0.994

00.5

11.5

22.5

33.5

0 1 2 3

Amount Present

VAL-PCR-CAL-D2

y = 0.948x + 0.027R² = 0.997

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

VAL-PCR-PRE-D0

y = 0.977x - 6E-05R² = 0.997

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Amount Present

VAL-PCR-PRE-D1

y = 0.953x + 0.066R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


VAL-PCR-PRE-D2

y = 1.005x - 0.019R² = 0.995

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.992x + 0.010R² = 0.996

-0.5

0

0.51

1.5

2

2.5

3

3.5

0 1 2 3

Amount Present

HCZ-PCR-CAL-D1

y = 0.972x + 0.019R² = 0.990

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Amount Present

HCZ-PCR-CAL-D2

155


y = 1.057x - 0.049R² = 0.995

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 0.985x + 0.026R² = 0.995

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PCR-PRE-D1

y = 0.977x + 0.038R² = 0.996

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PCR-PRE-D2

y = 0.989x + 0.042R² = 0.996

-101234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-CAL-D0

y = 1.005x - 0.034R² = 0.995

-101234567

0 2 4 6

Amount Present

RAM-PCR-CAL-D1

y = 0.997x + 0.000R² = 0.995

-101234567

0 1 2 3 4 5 6

Amount Present

RAM-PCR-CAL-D2

y = 1.008x - 0.039R² = 0.998

-101234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-PRE-D0

y = 1.02x + 0.007R² = 0.998

-101234567

0 2 4 6

Amount Present

RAM-PCR-PRE-D1

y = 1.015x - 0.015R² = 0.999

-101234567


RAM-PCR-PRE-D2

156

Table 4.68 Assay results of PLS and PCR models in PharmaceuticalPreparations

Met

hod

Tablet Mode

VAL HCZAmountFound*

(mg/tab) ± SD

%recovery

AmountFound*

(mg/tab) ± SD

%recovery

PLS T1

D0 80.32 ± 0.06 100.40 12.62 ± 0.12 100.96D1 80.43 ± 0.03 100.53 12.58 ± 0.23 100.64D2 80.21 ± 0.03 100.26 12.47 ± 0.11 99.76

PCR T1

D0 78.33 ± 0.04 97.91 12.43 ± 0.08 99.44D1 78.51 ± 0.12 98.13 12.59 ± 0.13 100.72D2 80.30 ± 0.05 100.37 12.53 ± 0.04 100.24

Met

hod

Tablet Mode

RAM HCZAmountFound*

(mg/tab) ±SD

%recovery

AmountFound*

(mg/tab) ±SD

%recovery

PLS T2

D0 2.46 ± 0.03 98.40 12.45 ± 0.11 99.60D1 2.52 ± 0.04 100.80 12.58 ± 0.03 100.64D2 2.47 ± 0.02 98.80 12.48 ± 0.04 99.84

PCR T2

D0 2.45 ± 0.05 98.00 12.51 ± 0.02 100.08D1 2.51 ± 0.04 100.40 12.58 ± 0.02 100.64D2 2.52 ± 0.03 100.80 12.48 ± 0.02 99.84

* Average of Six estimations

T1 – Valent H – Lupin Pharmaceuticals, Mumbai

Valsartan – 80 mg and Hydrochlorothiazide – 12.5 mg

T2 – Cardace # 2.5 – Aventis Pharma, Goa

Ramipril – 2.5 mg and Hydrochlorothiazide – 12.5 mg

157


96

98

100

102

104

D0 D1 D2

Valsartan - PLS


96

98

100

102

104

D0 D1 D2

Valsartan - PCR


96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2

Ramipril -PLS


96

98

100

102

104

D0 D1 D2

Ramipril - PCR


158

4.2.6 Ternary Mixture 3– Telmisartan (TEL), Hydrochlorothiazide

(HCZ) and Ramipril (RAM)


The spectra of all three drugs were overlapped in fundamental(Figure 4.26), first derivative (Figure 4.27) and second derivative mode(Figure 4.28) to study the spectral characteristics. They reveal a completeoverlap throughout the UV region of 200-400 nm. For multivariate analysis,25 different synthetic mixtures were prepared in the concentration ranges of0-3 µg/ml of TEL and HCZ, 0-6 µg/ml of RAM respectively (Table 4.69).The concentration range starts from zero in order to meet the requirement ofutilizing the analysis of any two drugs mentioned in the combination

Figure 4.26 Fundamental Spectra of Telmisartan, Hydrochlorothiazide,Ramipril and mixture

159

Figure 4.27 First Derivative Spectra of Telmisartan,Hydrochlorothiazide, Ramipril and mixture

Figure 4.28 Second Derivative Spectra of Telmisartan,Hydrochlorothiazide, Ramipril and mixture

160


Calibration Set (µg/ml) Prediction Set (µg/ml)TEL HCZ RAM TEL HCZ RAM0.5 0.5 5 2 0.5 11 0.5 4 2.5 1 1.5

1.5 0.5 3 1 1.5 2.52 0.5 2 2 2 2

0.5 1 5 0 2.5 01 1 4 1 3 2

1.5 1 3 2 3 12 1 2 0 0 3

0.5 1.5 5 1 1 61 1.5 4 0 2 0

1.5 1.5 3 - - -2 1.5 2 - - -0 2 0 - - -

0.5 2 5 - - -1 2 4 - - -

1.5 2 3 - - -0.5 2.5 5 - - -1 2.5 4 - - -

1.5 2.5 3 - - -2 2.5 2 - - -

0.5 3 1 - - -1 3 1.5 - - -0 0 6 - - -3 0 0 - - -

1.5 3 2 - - -







161

Table 4.70 Estimated Concentrations of Telmisartan in the calibrationset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4753 95.0720 0.4887 97.7455 0.4949 98.98501 1.0627 106.2711 1.0199 101.9968 1.0230 102.3039

1.5 1.5469 103.1313 1.5245 101.6366 1.5154 101.02742 1.9481 97.4099 1.9628 98.1436 1.9886 99.4344

0.5 0.4975 99.5024 0.4625 92.5177 0.4917 98.34031 1.0199 101.9938 1.0267 102.6710 1.0073 100.7379

1.5 1.5052 100.3525 1.5204 101.3652 1.5068 100.45372 1.9571 97.8550 1.9721 98.6095 1.9867 99.3371

0.5 0.5003 100.0691 0.5207 104.1482 0.4909 98.18361 1.0004 100.0473 0.9967 99.6759 0.9921 99.2166

1.5 1.5094 100.6323 1.4929 99.5317 1.5049 100.32922 1.9915 99.5776 1.9958 99.7903 2.0007 100.03500 0.0075 0 0.0013 0 0.0007 0

0.5 0.5172 103.4501 0.5023 100.4760 0.5175 103.51181 0.9815 98.1580 1.0043 100.4351 0.9739 97.3944

1.5 1.4932 99.5502 1.5091 100.6132 1.4791 98.60750.5 0.4868 97.3676 0.4839 96.7850 0.4996 99.93531 0.9646 96.4646 0.9963 99.6357 1.0178 101.7874

1.5 1.5363 102.4212 1.5062 100.4134 1.5109 100.72852 2.0002 100.0122 2.0030 100.1549 2.0011 100.0570

0.5 0.5084 101.6930 0.5130 102.6050 0.5025 100.51581 0.9736 97.3647 0.9878 98.7858 0.9953 99.53420 0.0057 0 0.0013 0 0.0057 03 3.0062 100.2072 3.0088 100.2953 3.0017 100.0597

1.5 1.5248 101.6568 1.5003 100.0232 1.5016 100.1103Mean 100.0114 Mean 99.9154 Mean 100.0298

162



D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4698 93.9782 0.4911 98.2278 0.4895 97.90800.5 0.5026 100.5362 0.4783 95.6688 0.4841 96.82080.5 0.5345 106.9091 0.5083 101.6795 0.5042 100.84600.5 0.4828 96.5762 0.5050 101.0047 0.4861 97.23801 1.0102 101.0237 0.9738 97.3899 0.9658 96.58321 1.0330 103.3099 1.0127 101.2750 0.9879 98.79101 0.9935 99.3549 1.0035 100.3536 1.0298 102.98521 0.9605 96.0504 0.9784 97.8421 0.9687 96.8702

1.5 1.4858 99.0551 1.5217 101.4480 1.5079 100.52661.5 1.5294 101.9604 1.5231 101.5466 1.5057 100.38001.5 1.4769 98.4600 1.4718 98.1214 1.4922 99.48551.5 1.4904 99.3609 1.4875 99.1711 1.5426 102.84002 1.9958 99.7944 1.9941 99.7093 1.9826 99.13002 2.0229 101.1466 1.9959 99.7954 2.0028 100.14022 2.0232 101.1601 2.0623 103.1172 2.0280 101.40202 2.0250 101.2507 2.0366 101.8309 2.0625 103.1267

2.5 2.4864 99.4576 2.4831 99.3271 2.4880 99.52002.5 2.4297 97.1907 2.4428 97.7125 2.4924 99.69602.5 2.5218 100.8743 2.5069 100.2774 2.5025 100.10252.5 2.5055 100.2202 2.4985 99.9419 2.5077 100.31083 3.0384 101.2833 3.0140 100.4694 3.0196 100.65513 2.9584 98.6133 2.9976 99.9200 2.9862 99.54200 0.0006 0 0.0025 0 0.0064 00 0.0011 0 0.0066 0 0.0027 03 3.0107 100.3598 2.9927 99.7593 2.9933 99.7780

Mean 99.9098 Mean 99.8082 Mean 99.7686

163



D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 4.9492 98.9859 5.0141 100.2829 5.0138 100.27694 3.9336 98.3405 3.9575 98.9383 3.9553 98.88313 3.0114 100.3802 2.9585 98.6177 3.0097 100.32472 1.9847 99.2369 2.0219 101.0993 2.0304 101.52495 4.9539 99.0785 5.0407 100.8147 5.0260 100.52084 4.0906 102.2651 4.0369 100.9239 3.9708 99.27153 3.0491 101.6387 3.0670 102.2346 3.0370 101.23632 1.9137 95.6888 2.0104 100.5204 1.9838 99.19245 5.0271 100.5424 4.9929 99.8585 4.9669 99.33934 4.0704 101.7607 3.8968 97.4213 3.9856 99.64093 2.9940 99.8013 2.9712 99.0420 2.9695 98.98392 2.0055 100.2772 2.0222 101.1126 2.0019 100.09500 0.0085 0 0.0026 0 0.0019 05 4.9903 99.8062 5.0595 101.1909 4.9392 98.78454 4.0277 100.6947 3.9689 99.2227 4.0200 100.50073 3.0091 100.3038 3.0475 101.5834 2.9922 99.74095 4.9614 99.2294 5.0306 100.6135 5.0458 100.91784 3.9835 99.5898 3.9414 98.5373 3.9821 99.55253 2.9559 98.5312 2.9803 99.3449 3.0559 101.86532 2.0375 101.8758 2.0020 100.1014 1.9830 99.15151 1.0549 105.4977 1.0589 105.8979 1.0076 100.7697

1.5 1.4581 97.2127 1.4208 94.7209 1.4829 98.86316 6.0342 100.5702 5.9803 99.6723 6.0263 100.43930 0.0020 0 0.0021 0 0.0046 02 1.9897 99.4864 2.0235 101.1771 2.0159 100.7980

Mean 100.0163 Mean 100.1273 Mean 100.0292

164

Table 4.73 Estimated concentrations of Telmisartan in the predictionset under optimized PLS model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0977 104.8886 2.0254 101.2741 2.0148 100.74242.5 2.4181 96.7253 2.4894 99.5788 2.4768 99.07481 0.9019 90.1998 1.0662 106.6227 1.0381 103.81972 1.8328 91.6412 2.0448 102.2417 1.9108 95.54360 0.0049 0 0.0076 0 0.0006 01 0.9885 98.8577 1.0184 101.8430 0.9963 99.63502 1.8635 93.1794 2.0653 103.2680 1.9732 98.66050 0.0030 0 0.0072 0 0.0040 01 0.9268 92.6838 1.0004 100.0409 0.9809 98.09960 0.1621 0 0.0037 0 0.0077 0

Mean 95.5437 Mean 102.1242 Mean 99.3680



D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5232 104.6570 0.5286 105.7354 0.5057 101.14711 1.0763 107.6328 0.9879 98.7985 1.0227 102.2708

1.5 1.4910 99.4051 1.4812 98.7482 1.4678 97.85512 1.9635 98.1792 1.9793 98.9698 2.0693 103.4658

2.5 2.4526 98.1071 2.5263 101.0525 2.4609 98.43973 2.9716 99.0559 2.8638 95.4610 3.0252 100.84093 3.0831 102.7704 2.8923 96.4108 3.0581 101.93670 0.0089 0 0.0066 0 0.0016 01 0.9220 92.2081 0.9642 96.4206 1.0673 106.73572 1.8618 93.0921 1.9605 98.0263 1.9476 97.3826

Mean 99.4565 Mean 98.8470 Mean 101.1194

165

Table 4.75 Estimated concentrations of Ramipril in the prediction setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 1.0762 107.6204 1.0962 109.6266 1.0340 103.4032

1.5 1.5916 106.1118 1.5133 100.8883 1.4853 99.0242

2.5 2.3231 92.9278 2.5390 101.5631 2.4831 99.3277

2 2.0527 102.6364 2.0814 104.0741 2.0958 104.7902

0 0.0056 0 0.0004 0 0.0005 0

2 2.0648 103.2419 2.1112 105.5601 2.0641 103.2093

1 1.0512 105.1270 0.8921 89.2149 0.9983 99.8323

3 3.1295 104.3199 3.1748 105.8283 3.0283 100.9449

6 6.3084 105.1412 6.1416 102.3616 6.0574 100.9581

0 0.0090 0 0.0077 0 0.0087 0

Mean 103.3908 Mean 102.3896 Mean 101.4362



166




TEL-D0 100.0113 2.4938 0.9986 0.0260 95.4357 4.7389 0.9936 0.1162

TEL-D1 99.9154 2.2777 0.9994 0.0169 102.1242 2.1771 0.9989 0.0476

TEL-D2 100.0298 1.3344 0.9997 0.0116 99.3680 2.3482 0.9988 0.0489

HCZ-D0 99.9098 2.5394 0.9992 0.0265 99.4565 4.7322 0.9957 0.0672

HCZ-D1 99.8082 1.6946 0.9993 0.0234 98.8470 2.9117 0.9981 0.0606

HCZ-D2 99.7686 1.8157 0.9994 0.0223 101.1194 2.8071 0.9981 0.0443

RAM-D0

100.0163 1.8752 0.9993 0.0428 103.3908 4.2175 0.9958 0.1471

RAM-D1

100.1273 1.9912 0.9993 0.0429 102.3896 5.6302 0.9986 0.0980

RAM-D2

100.0292 0.8865 0.9997 0.0282 101.4362 1.9864 0.9996 0.0471






Number of PCs : 13

167

Table 4.77 Estimated concentration of Telmisartan in the calibrationset under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4786 95.7303 0.4746 94.9297 0.4949 98.99361 1.0377 103.7789 1.0361 103.6106 1.1417 114.1700

1.5 1.5316 102.1129 1.5367 102.4467 1.5013 100.08662 1.9740 98.7016 1.9480 97.4035 1.8927 94.6350

0.5 0.4825 96.5036 0.4647 92.9457 0.5069 101.39401 1.0295 102.9582 1.0312 103.1263 0.9824 98.2400

1.5 1.5244 101.6280 1.5174 101.1632 1.5252 101.68242 1.9455 97.2756 1.9727 98.6396 2.0183 100.9199

0.5 0.5015 100.3173 0.5218 104.3615 0.5170 103.40441 1.0053 100.5317 1.0012 100.1250 1.0461 104.6102

1.5 1.5193 101.2906 1.5232 101.5481 1.4387 95.91572 1.9919 99.5979 1.9849 99.2452 2.0604 103.02000 0.0073 0 0.0056 0 0.0604 0

0.5 0.5284 105.6924 0.5301 106.0375 0.4794 95.88001 0.9751 97.5113 1.0085 100.8508 0.9800 98.0030

1.5 1.4961 99.7448 1.5018 100.1214 1.5102 100.68200.5 0.4965 99.3145 0.4830 96.6119 0.5117 102.35601 0.9392 93.9244 0.9508 95.0899 0.9393 93.9323

1.5 1.5411 102.7419 1.5107 100.7165 1.4855 99.03332 1.9939 99.6979 2.0027 100.1369 2.0393 101.9691

0.5 0.5174 103.4965 0.5096 101.9248 0.4821 96.43601 0.9674 96.7452 0.9779 97.7993 0.9833 98.33100 0.0008 0 0.0106 0 -0.0009 03 3.0045 100.1504 3.0066 100.2228 3.0460 101.5336

1.5 1.5210 101.4035 1.5098 100.6573 1.5274 101.8266Mean 100.0369 Mean 99.9876 Mean 100.4049

168

Table 4.78 Estimated concentrations of Hydrochlorothiazide in thecalibration set under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4780 95.6105 0.4704 94.0918 0.4916 98.32260.5 0.5104 102.0978 0.4815 96.3083 0.4841 96.83570.5 0.5191 103.8217 0.5073 101.4657 0.4979 99.58610.5 0.5161 103.2207 0.4931 98.6215 0.4706 94.12091 1.0305 103.0572 0.9629 96.2967 0.9270 92.70061 0.9974 99.7460 1.0266 102.6623 1.0117 101.17231 0.9486 94.8657 0.9569 95.6952 1.0601 106.01781 0.9494 94.9480 1.0126 101.2628 0.9499 94.9993

1.5 1.4932 99.5525 1.5340 102.2672 1.5071 100.47331.5 1.5697 104.6509 1.5546 103.6417 1.5258 101.72051.5 1.5209 101.3946 1.5453 103.0248 1.4902 99.34871.5 1.5294 101.9617 1.4773 98.4889 1.5010 100.06662 2.0111 100.5576 2.0020 100.1047 1.9510 97.55002 2.0493 102.4668 2.0860 104.3005 2.0577 102.88642 1.9932 99.6626 2.0574 102.8730 2.0170 100.85202 2.0055 100.2795 2.0386 101.9301 2.0797 103.9850

2.5 2.4870 99.4822 2.4608 98.4350 2.4740 98.96372.5 2.3387 93.5495 2.3519 94.0765 2.3258 93.03402.5 2.5645 102.5812 2.4953 99.8158 2.5802 103.21002.5 2.4950 99.8023 2.5055 100.2229 2.4963 99.85533 3.0705 102.3505 3.0361 101.2058 2.9842 99.47433 2.9329 97.7658 2.9686 98.9556 3.0375 101.25170 0.0051 0 0.0057 0 0.0263 00 0.0059 0 0.0034 0 0.0135 03 2.9976 99.9216 2.9765 99.2179 2.9488 98.2957

Mean 100.1455 Mean 99.7811 Mean 99.3358

169

Table 4.79 Estimated concentrations of Ramipril in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

5 5.2840 105.6819 5.0510 101.0200 5.0044 100.08844 3.8347 95.8675 4.0286 100.7170 4.0636 101.59003 2.8989 96.6303 3.0989 103.2991 3.0606 102.02112 2.0611 103.0550 1.9304 96.5214 1.8801 94.00505 4.8565 97.1312 4.8819 97.6380 5.0580 101.16004 4.0204 100.5117 3.8952 97.3807 3.9087 97.71753 2.8465 94.8836 2.7564 91.8817 3.0268 100.89502 1.9724 98.6200 1.9197 95.9871 2.0573 102.86885 5.2131 104.2625 5.0372 100.7440 5.0366 100.73324 4.2051 105.1276 4.1045 102.6145 4.1649 104.12443 3.0809 102.6979 2.8956 96.5200 2.8011 93.37182 2.0495 102.4750 2.0748 103.7400 2.2722 113.61000 0.0076 0 0.0022 0 0.0022 05 4.9103 98.2070 4.9790 99.5810 4.9495 98.99004 3.8568 96.4204 3.9008 97.5203 3.9370 98.42623 2.7756 92.5231 2.9730 99.1015 3.1143 103.81145 5.2546 105.0921 5.0635 101.2700 5.1693 103.38674 3.7539 93.8475 3.9109 97.7725 3.8519 96.29753 2.7820 92.7356 2.7107 90.3584 2.8435 94.78352 2.0411 102.0578 1.9956 99.7822 2.1149 105.74661 1.1967 119.6782 1.2218 122.1870 1.0283 102.8300

1.5 1.4561 97.0733 1.3965 93.1040 1.5274 101.82666 5.8316 97.1939 5.7269 95.4486 5.9097 98.49560 0.0015 0 0.0065 0 0.0094 02 1.9712 98.5648 1.8574 92.8727 2.0076 100.3800

Mean 100.0147 Mean 99.0027 Mean 100.7640

170

Table 4.80 Estimated concentrations of Telmisartan in the prediction set under optimized PCR model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.1617 108.0883 2.0749 103.7494 2.0486 102.43052.5 2.4218 96.8730 2.5197 100.7900 2.4642 98.56941 0.9642 96.4299 1.1327 113.2753 1.0001 100.01192 1.8256 91.2847 1.9553 97.7671 1.9317 96.58950 0.0020 0 0.0090 0 0.0081 01 0.9775 97.7577 1.0643 106.4327 1.0150 101.50142 1.9485 97.4263 1.9646 98.2333 2.0022 100.11410 0.0091 0 0.0011 0 0.0142 01 0.8898 88.9866 1.1048 110.4883 1.0372 103.72840 -0.0066 0 0.0074 0 0.0066 0

Mean 96.6924 Mean 104.3909 Mean 103.7284



D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4734 94.6907 0.5168 103.3776 0.4896 97.92681 1.0297 102.9779 1.1099 110.9908 1.0255 102.5574

1.5 1.4742 98.2844 1.5878 105.8544 1.4881 99.21022 1.9236 96.1838 2.0615 103.0770 2.0050 100.2538

2.5 2.4087 96.3505 2.5233 100.9327 2.4834 99.33823 2.9411 98.0396 3.0896 102.9875 3.0653 102.17893 3.0400 101.3357 3.1670 105.5671 3.0009 100.03150 0.0091 0 0.0047 0 0.0019 01 0.9175 91.7532 1.0883 108.8341 1.0415 104.15672 1.8055 90.2776 1.8928 94.6405 1.8901 94.5083

Mean 96.6548 Mean 104.0291 Mean 100.0180

171

Table 4.82 Estimated concentration of Ramipril in the prediction setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

1 1.0665 106.6509 0.9958 99.5885 0.9834 98.3422

1.5 1.3547 90.3171 1.5366 102.4464 1.5398 102.6583

2.5 2.5442 101.7684 2.4913 99.6534 2.5036 100.1449

2 2.0988 104.9416 2.0993 104.9674 1.8954 94.7745

0 0.0079 0 0.0070 0 0.0086 0

2 2.1251 106.2571 2.0624 103.1215 2.0616 103.0821

1 1.0354 103.5405 1.0865 108.6529 1.0440 104.4042

3 3.1619 105.3967 3.1363 104.5457 2.8999 96.6636

6 6.1880 103.1349 6.2260 103.7673 6.1763 102.9386

0 0.0061 0 0.0036 0 0.0062 0

Mean 102.7509 Mean 103.3429 Mean 100.3760



172




TEL-D0 100.0369 2.8002 0.9986 0.0265 96.6924 5.6094 0.9921 0.0962

TEL-D1 99.9876 3.8025 0.9988 0.0246 104.3909 5.5478 0.9975 0.0803

TEL-D2 100.4049 4.0944 0.9954 0.0485 103.7284 2.5096 0.9985 0.0426

HCZ-D0 100.1455 2.9691 0.9973 0.0474 96.6548 3.8777 0.9957 0.0871

HCZ-D1 99.7811 2.9005 0.9976 0.0450 104.0291 4.4397 0.9951 0.0899

HCZ-D2 99.3358 3.3134 0.9968 0.0523 100.0180 2.6697 0.9981 0.0438

RAM-D0

100.0147 5.7594 0.9908 0.1537 102.7509 4.9451 0.9941 0.1691

RAM-D1

99.0027 6.0707 0.9943 0.1274 103.3429 2.7618 0.9993 0.0992

RAM-D2

100.7460 4.2182 0.9953 0.1103 100.3760 3.2533 0.9983 0.0770


Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.84. The percentage recoverywas found to be from 98.32 – 101.90 for TEL, 99.44 – 101.04 for HCZ and98.80 – 102.40 for RAM respectively. These methods can be successfullyapplied for formulations containing either two or three drugs.

173

Table 4.84 Assay results of PLS and PCR models in Pharmaceuticalpreparations

Met

hod

Tablet Mode

TEL HCZAmountFound*

(mg/tab) ±SD

%recovery

AmountFound*

(mg/tab) ±SD

%recovery

PLS T1

D0 40.54 ± 0.05 101.35 12.43 ± 0.11 99.44D1 40.43 ± 0.06 101.07 12.61 ± 0.07 100.88D2 40.76 ± 0.03 101.90 12.59 ± 0.03 100.72

PCR T1

D0 38.92 ± 0.12 97.30 12.63 ± 0.12 101.04D1 39.43 ± 0.07 98.57 12.54 ± 0.04 100.32D2 40.23 ± 0.04 100.57 12.54 ± 0.02 100.32

Met

hod

Tablet Mode

TEL RAMAmountFound*

(mg/tab) ± SD

%recovery

AmountFound*

(mg/tab) ± SD

%recovery

PLS T2

D0 40.46 ± 0.33 101.15 4.94 ± 0.08 98.80D1 40.43 ± 0.12 101.07 5.08 ± 0.06 101.60D2 39.77 ± 0.05 99.42 5.03 ± 0.05 100.60

PCR T2

D0 40.68 ± 0.11 101.70 5.12 ± 0.04 102.40D1 39.33 ± 0.21 98.32 4.96 ± 0.03 99.20D2 40.62 ± 0.06 101.55 5.03 ± 0.03 100.60

* Average of Six estimations

T1 – Telista H – Lupin Pharmaceuticals, Mumbai

Telmisartan – 40 mg and Hydrochlorothiazide – 12.5 mg

T2 – Tazloc*R – Hetero Drugs, HP

Telmisartan – 40 mg and Ramipril – 5 mg

174

Figure 4.29 Plots of calibration and prediction sets of Telmisartan,Hydrochlorothiazide and Ramipril in both PLS and PCRmodels

y = 0.998x + 0.001R² = 0.998

-0.5

0

0.5

11.5

2

2.5

33.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

TEL-PLS-CAL-D0

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


TEL-PLS-CAL-D1

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


TEL-PLS-CAL-D2

y = 1.025x - 0.116R² = 0.993

-0.5

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

TEL-PLS-PRE-D0

y = 0.989x + 0.047R² = 0.998

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5Amount Present

TEL-PLS-PRE-D1

y = 0.957x + 0.057R² = 0.998

0

0.5

1

1.5

2

2.5

3


TEL-PLS-PRE-D2

y = 0.999x + 0.001R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PLS-CAL-D1

y = 1.001x + 0.001R² = 0.999

00.5

11.5

22.5

33.5


HCZ-PLS-CAL-D2

175


y = 1.003x - 0.025R² = 0.995

-0.5

00.5

11.5

2

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.972x + 0.011R² = 0.998

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Amount Present

HCZ-PLS-PRE-D1

y = 1.006x - 0.000R² = 0.998

-0.5

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PLS-PRE-D2

y = 0.999x + 0.002R² = 0.999

01234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-CAL-D0

y = 0.999x + 0.002R² = 0.999

-101234567


RAM-PLS-CAL-D1

y = 0.999x + 0.001R² = 0.999

01234567


RAM-PLS-CAL-D2

y = 1.020x + 0.052R² = 0.995

0

1

2

3

4

5

6

7

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PLS-PRE-D0

y = 1.029x - 0.000R² = 0.998

0

1

2

3

4

5

6

7


RAM-PLS-PRE-D1

y = 1.003x + 0.025R² = 0.999

0

1

2

3

4

5

6

7


RAM-PLS-PRE-D2

176


y = 0.998x + 0.001R² = 0.998

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

TEL-PCR-CAL-D0

y = 0.998x + 0.001R² = 0.998

0

0.5

1

1.5

2

2.5

3

3.5


TEL-PCR-CAL-D1

y = 1.005x - 0.002R² = 0.995

-0.5

0

0.5

1

1.5

2

2.5

3

3.5


TEL-PCR-CAL-D2

y = 1.011x - 0.064R² = 0.992

-0.5

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

TEL-PCR-PRE-D0

y = 0.960x + 0.103R² = 0.997

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Amount Present

TEL-PCR-PRE-D1

y = 0.974x + 0.041R² = 0.998

0

0.5

1

1.5

2

2.5

3


TEL-PCR-PRE-D2

y = 0.997x + 0.004R² = 0.997

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.997x + 0.003R² = 0.997

-0.50

0.51

1.52

2.53

3.5


HCZ-PCR-CAL-D1

y = 0.995x + 0.002R² = 0.996

0

0.5

1

1.5

2

2.5

3

3.5


HCZ-PCR-CAL-D2

177


y = 1.003x - 0.064R² = 0.995

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 1.025x + 0.007R² = 0.995

-0.50

0.51

1.52

2.53

3.5


HCZ-PCR-PRE-D1

y = 1.001x - 0.003R² = 0.998

-0.50

0.51

1.52

2.53

3.5

0 1 2 3

Amount Present

HCZ-PCR-PRE-D2

y = 1x - 0.015R² = 0.990

01234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-CAL-D0

y = 0.981x + 0.017R² = 0.994

0

12

3

45

67


RAM-PCR-CAL-D1

y = 0.988x + 0.052R² = 0.995

01234567


RAM-PCR-CAL-D2

y = 0.997x + 0.116R² = 0.994

01234567

0 2 4 6

Am

ount

Pre

dict

ed

Amount Present

RAM-PCR-PRE-D0y = 1.028x + 0.019

R² = 0.999

01234567


RAM-PCR-PRE-D1

y = 1.018x - 0.021R² = 0.998

-101234567


RAM-PCR-PRE-D2

178


94

96

98

100

102

104

106

D0 D1 D2

Telmisartan - PLS


94

96

98

100

102

104

106

D0 D1 D2

Telmisartan - PCR


94

96

98

100

102

104

106

D0 D1 D2



94

96

98

100

102

104

106

D0 D1 D2



94

96

98

100

102

104

106

D0 D1 D2

Ramipril -PLS


94

96

98

100

102

104

106

D0 D1 D2

Ramipril - PCR


179

4.2.7 Ternary Mixture 4 – Trandolapril (TRA), Verapamil (VER) and Manidipine (MAN)


The spectra of all three drugs were overlapped in fundamental(Figure 4.31), first derivative (Figure 4.32) and second derivative mode(Figure 4.33) to study the spectral characteristics. They reveal a completeoverlap throughout the UV region of 200-400 nm. For multivariate analysis,25 different synthetic mixtures were prepared in the concentration ranges of0-10 µg/ml of TRA and MAN, 0-5 µg/ml of VER respectively (Table 4.85).The concentration range starts from zero in order to meet the requirement ofutilizing the analysis of any two drugs mentioned in the combination

Figure 4.31 Fundamental Spectra of Trandolapril, Verapamil,Manidipine and mixture

180

Figure 4.32 First derivative spectra of Trandolapril, Verapamil,Manidipine and mixture

Figure 4.33 Second derivative spectra of Trandolapril, Verapamil,Manidipine and mixture

181


Calibration Set (µg/ml) Prediction Set (µg/ml)TRA VER MAN TRA VER MAN

2 0 2 10 0 24 0 4 8 4 26 1 6 8 3 108 0 8 2 4 210 0 10 8 0 80 1 2 6 1 12 1 0 10 5 102 2 0 6 4 010 2 2 8 1 102 1 2 6 0 04 2 4 - - -6 3 6 - - -8 4 8 - - -6 2 6 - - -4 4 4 - - -2 3 2 - - -10 3 2 - - -2 3 10 - - -4 0 8 - - -8 0 4 - - -0 5 10 - - -10 5 0 - - -0 0 10 - - -10 4 8 - - -0 0 6 - - -




Preprocessing : Mean Centre, auto scale, orthogonal signalcorrection

Cross Validation : Leave one out and Latent Variables -13

182

Table 4.86 Estimated concentrations of Trandolapril in the calibrationset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0011 100.0591 2.0615 103.0775 1.9343 96.71774 4.0202 100.5064 3.9315 98.2899 4.0845 102.11376 6.0223 100.3721 5.9370 98.9514 5.9103 98.50588 7.9939 99.9242 8.1356 101.6953 8.0878 101.0981

10 9.9948 99.9488 9.9092 99.0922 9.9415 99.41510 0.0040 0 0.0061 0 0.0006 02 2.0044 100.2225 2.1605 108.0285 2.0008 100.04442 2.0033 100.1678 1.9743 98.7153 1.9982 99.9148

10 10.0019 100.0192 10.0539 100.5394 10.012 100.12142 2.0231 101.1577 1.9377 96.8893 2.0468 102.34454 4.0171 100.4285 4.0758 101.8955 4.0205 100.51466 5.9644 99.4078 6.0143 100.2383 5.9127 98.54598 8.0311 100.3897 8.1729 102.1618 8.1180 101.47606 6.0309 100.5151 5.5910 93.1833 5.8969 98.28284 4.0651 101.6285 4.1158 102.8966 4.0382 100.95582 1.9786 98.9315 2.0721 103.6081 2.0511 102.5570

10 9.9618 99.6189 9.9570 99.5709 10.1305 101.30582 2.0331 101.6595 2.1136 105.6843 2.0355 101.77954 3.9723 99.3086 4.1102 102.7560 4.0932 102.33248 7.9655 99.5692 8.0800 101.0001 7.9257 99.07160 0.00235 0 0.0077 0 0.0006 0

10 9.9966 99.9664 9.9137 99.1375 9.9210 99.21050 0.0086 0 0.0609 0 0.0067 0

10 10.0008 100.0081 9.9343 99.3437 9.9376 99.37690 0.00435 0 0.0018 0 0.0005 0

Mean 100.1814 Mean 100.7979 Mean 100.1092

183

Table 4.87 Estimated concentrations of Verapamil in the calibration setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0 0.0083 0 0.0023 0 0.0058 00 0.0016 0 0.0055 0 0.0073 01 1.0672 106.7238 1.0793 107.9399 1.0652 106.52470 0.0023 0 0.0078 0 0.0046 00 0.0077 0 0.0034 0 0.0055 01 1.0828 108.2804 1.0703 107.0327 1.0702 107.02801 1.0043 100.4396 0.9984 99.8414 1.0117 101.17422 2.0068 100.3446 2.0122 100.6120 2.0035 100.17512 1.9971 99.8595 2.0489 102.4458 1.9995 99.97971 0.9308 93.0859 0.9481 94.8188 0.9599 95.99652 2.0039 100.1994 1.9672 98.3622 1.9705 98.52713 3.0023 100.0782 3.0048 100.1616 2.9912 99.70764 4.0283 100.7091 4.0280 100.7019 4.0074 100.18622 2.0431 102.1586 2.0517 102.5881 2.0574 102.87214 4.0210 100.5273 3.9907 99.7695 4.0166 100.41693 2.9273 97.5777 2.9889 99.6323 2.9618 98.72683 3.0628 102.0947 3.0308 101.0271 3.0547 101.82473 3.0093 100.3113 3.0166 100.5541 3.0212 100.70710 0.0040 0 0.0048 0 0.0043 00 0.0055 0 0.0050 0 0.0007 05 4.9753 99.5076 4.9624 99.2482 4.9694 99.38845 4.9500 99.0003 4.9517 99.0343 4.9602 99.20530 0.0068 0 0.0049 0 0.0001 04 3.9861 99.6545 3.9851 99.6293 3.9921 99.80270 0.0033 0 -0.0065 0 0.0089 0

Mean 100.6207 Mean 100.7882 Mean 100.7202

184

Table 4.88 Estimated concentrations of Manidipine in the calibrationset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0921 104.6073 2.2340 111.7034 1.9557 97.78584 3.8818 97.0460 3.9655 99.1385 3.9449 98.62466 5.9497 99.1624 5.9174 98.6242 5.9050 98.41708 8.0281 100.3523 8.0636 100.7954 7.9892 99.8659

10 10.0284 100.2824 10.0267 100.2671 9.9862 99.86242 2.0278 101.3925 2.1170 105.8540 1.7859 89.29940 0.0091 0 0.0003 0 0.0060 00 0.0067 0 0.0047 0 0.0011 02 2.0673 103.3684 2.0906 104.5337 1.8679 93.39792 2.0100 100.5011 2.0263 101.3191 1.9284 96.42114 3.9104 97.7622 3.7541 93.8532 3.6834 92.08526 5.9685 99.4766 5.7209 95.3494 6.0231 100.38508 7.9558 99.4483 7.9422 99.2778 8.0697 100.87196 5.9329 98.8822 6.3165 105.2750 6.0541 100.90264 4.0275 100.6877 3.6273 90.6830 4.1209 103.02392 1.9949 99.7480 1.9310 96.5542 1.9894 99.47142 1.9109 95.5487 1.9865 99.3291 1.8725 93.6258

10 10.1515 101.5155 10.3233 103.2335 9.9841 99.84148 7.9213 99.0165 7.9806 99.7577 7.9769 99.71144 4.1326 103.3167 3.9243 98.1095 4.1866 104.6657

10 10.0001 100.0011 10.1199 101.1997 9.9440 99.44070 0.0088 0 0.0054 0 0.0044 0

10 9.9724 99.7249 9.7125 97.1258 10.077 100.77068 8.0197 100.2471 8.0303 100.3791 8.0489 100.61176 5.9740 99.5677 5.9578 99.2973 6.0835 101.3924

Mean 100.4529 Mean 100.0754 Mean 98.6579

185

Table 4.89 Estimated concentrations of Trandolapril in the predictionset under optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

10 10.1940 101.9408 10.0132 100.1320 10.3861 103.86188 8.1179 101.4737 8.0066 100.0831 7.9352 99.19028 8.1425 101.7821 8.0514 100.6429 8.2594 103.24312 2.0458 102.2915 2.1389 106.9483 2.0167 100.83948 7.9948 99.9360 8.0813 101.0166 8.2555 103.19406 5.7971 96.6187 6.0760 101.2679 6.0509 100.849610 9.6724 96.7244 10.0441 100.4415 9.9544 99.54416 6.1614 102.6901 6.0961 101.6017 6.0915 101.52558 8.3736 104.6703 8.2262 102.8286 8.1583 101.97906 5.9148 98.5813 6.2027 103.3798 6.0999 101.6655

Mean 100.6709 Mean 101.8342 Mean 101.5892

Table 4.90 Estimated concentrations of Verapamil in the prediction setunder optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0 0.0034 0 0.0073 0 0.0099 04 4.2282 105.7067 4.1518 103.7950 3.9861 99.65463 3.1341 104.4718 3.0697 102.3253 3.0299 100.99714 4.0691 101.7295 4.0074 100.1856 3.8509 96.27370 0.0077 0 0.0093 0 0.0027 01 1.0284 102.8422 1.0394 103.9445 0.9866 98.66755 5.3130 106.2613 5.1647 103.2958 5.0669 101.33864 3.9297 98.2425 3.8920 97.3018 3.9066 97.66531 1.0024 100.2491 1.0173 101.7350 0.9864 98.64030 0.0066 0 0.0046 0 0.0070 0

Mean 102.7862 Mean 101.7976 Mean 99.0339

186

Table 4.91 Estimated concentrations of Manidipine in the predictionset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 1.7191 85.9556 2.0471 102.3578 2.1523 107.6194

2 1.9823 99.1193 2.0670 103.3543 2.0719 103.5973

10 10.1013 101.0132 10.2163 102.1631 10.1188 101.1886

2 2.1343 106.7171 2.1040 105.2035 2.1376 106.8829

8 8.0282 100.3530 7.8094 97.6183 7.9166 98.9582

1 1.0259 102.5975 1.0884 108.8495 0.9686 96.8673

10 9.9497 99.4972 10.0592 100.5922 10.1661 101.6615

0 0.0093 0 0.0013 0 0.0013 0

10 10.2684 102.6844 9.7833 97.8330 10.2477 102.4773

0 0.0008 0 0.0072 0 0.0011 0

Mean 99.7422 Mean 102.2465 Mean 102.4066



187




TRA-D0

100.1814 0.6705 0.9999 0.0278 100.6709 2.5118 0.9924 0.1988

TRA-D1

100.7979 3.0734 0.9988 0.1230 101.8342 1.9974 0.9992 0.1175

TRA-D2

100.1092 1.5456 0.9996 0.0679 101.5892 1.4725 0.9968 0.1828

VER-D0

100.6207 3.1860 0.9995 0.0385 102.7862 2.7162 0.9978 0.1360

VER-D1

100.7882 2.9530 0.9995 0.3619 101.7976 2.2018 0.9986 0.0846

VER-D2

100.7202 2.6323 0.9996 0.0347 99.0339 1.6618 0.9985 0.0793

MAN-D0

100.4529 1.9792 0.9996 0.0651 99.7422 5.6785 0.9992 0.1384

MAN-D1

100.0754 4.3067 0.9974 0.1670 102.2465 3.4861 0.9999 0.1303

MAN-D2

98.7569 3.5683 0.9983 0.1366 102.4066 3.4140 0.9995 0.1288






Number of PCs : 13

188

Table 4.93 Estimated Concentrations of Trandolapril in the calibrationset under optimized PCR models


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0617 103.0894 2.0068 100.3429 2.0200 101.00264 4.0588 101.4722 4.0116 100.2907 3.9701 99.25406 6.1216 102.0277 5.9223 98.7051 5.9780 99.63408 7.9994 99.9926 8.1059 101.3239 8.1921 102.402210 9.8657 98.6570 9.9444 99.4447 9.8477 98.47730 0.0022 0 0.0040 0 0.0096 02 2.0257 101.2856 2.0170 100.8541 1.8783 93.91872 2.0387 101.9378 1.9036 95.1845 1.9917 99.588710 10.1431 101.4319 9.9961 99.9613 10.185 101.85502 1.9812 99.0612 2.1506 107.5342 2.0604 103.02224 4.0323 100.8089 4.2105 105.2648 4.0176 100.44216 6.0369 100.6160 6.1385 102.3087 6.2228 103.71418 8.1250 101.5634 8.1284 101.6054 8.1403 101.75426 6.0484 100.8070 5.6194 93.6567 5.7375 95.62574 4.1070 102.6760 4.0936 102.3420 4.0299 100.74832 1.7393 86.9676 1.8066 90.3315 1.9688 98.444310 9.8944 98.9444 9.9933 99.9331 9.8567 98.56722 2.0428 102.1432 2.2148 110.7440 1.8746 93.73274 3.8585 96.4632 4.0835 102.0884 4.1971 104.92938 7.9189 98.9870 7.9847 99.8098 7.8436 98.04620 0.0028 0 0.0002 0 0.0049 010 9.9249 99.2498 9.9422 99.4223 10.0056 100.05610 0.0018 0 0.0021 0 0.0054 010 9.9902 99.9021 9.9200 99.2009 9.9190 99.19070 0.0031 0 0.0064 0 0.0085 0

Mean 99.9088 Mean 100.4928 Mean 99.7336

189

Table 4.94 Estimated concentrations of Verapamil in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0 0.0028 0 0.0018 0 0.0062 00 0.0002 0 0.0099 0 0.0063 01 1.0594 105.9499 1.0603 106.0384 1.0519 105.19380 0.0006 0 0.0030 0 0.0034 00 0.0086 0 0.0339 0 0.0099 01 1.0551 105.5124 1.0703 107.0305 1.0353 103.53891 1.0107 101.0720 0.9936 99.3673 1.0216 102.16012 2.0082 100.4145 2.0532 102.6625 2.0262 101.31112 2.0446 102.2331 1.9929 99.6456 1.9882 99.41291 0.9433 94.3384 1.0037 100.3797 0.9570 95.70212 1.9973 99.8654 2.0108 100.5435 1.9847 99.23653 2.9729 99.0987 2.9858 99.5298 2.9991 99.97224 4.0486 101.2157 4.0294 100.7358 3.9960 99.90152 2.0276 101.3844 2.0521 102.6054 2.0331 101.65574 3.9950 99.8754 3.9923 99.8090 3.9850 99.62743 2.9503 98.3477 2.9382 97.9431 2.9782 99.27483 3.0242 100.8078 3.0542 101.8075 3.0827 102.75923 3.0324 101.0822 3.0031 100.1055 2.9760 99.20000 0.0034 0 0.0077 0 0.0058 00 -0.0540 0 0.0019 0 0.0015 05 4.9867 99.7351 4.9721 99.4421 5.0024 100.04885 4.9504 99.0080 4.9582 99.1653 4.9681 99.36250 -0.0277 0 -0.0204 0 0.0056 04 3.9857 99.6443 3.9850 99.6261 3.9906 99.76550 -0.0201 0 0.0332 0 0.0036 0

Mean 100.5638 Mean 100.9669 Mean 100.4778

190

Table 4.95 Estimated concentrations of Manidipine in the calibrationset under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 2.0905 104.5250 2.1401 107.0050 2.0450 102.25454 4.0375 100.9375 4.1006 102.5150 3.7298 93.24516 5.9895 99.8250 6.0853 101.4216 6.1711 102.85168 7.9473 99.3418 7.8797 98.4962 8.0235 100.2937

10 9.7618 97.6183 10.1236 101.2360 9.9486 99.48602 2.1003 105.0150 2.0592 102.9600 2.1080 105.40000 0.0010 0 0.04972 0 -0.0877 00 0.0064 0 0.0719 0 0.0534 02 1.9488 97.4400 2.4865 124.3251 2.0595 102.97502 2.0308 101.5448 2.1826 109.1344 2.0485 102.42504 4.1329 103.3236 4.1365 103.4125 3.8420 96.05006 6.1305 102.1765 6.3226 105.3766 5.6842 94.73668 8.1367 101.7087 8.0685 100.8562 8.1110 101.38756 6.2486 104.1434 6.1086 101.8100 6.1169 101.94834 4.2334 105.8365 4.0967 102.4175 4.0767 101.91752 2.1027 105.1350 2.1508 107.5400 1.9700 98.50272 1.7777 88.8882 2.0895 104.4784 2.0827 104.1350

10 10.0204 100.2049 9.8728 98.7280 9.8062 98.06208 7.5761 94.7016 7.8287 97.8598 8.1233 101.54154 4.2833 107.0843 3.9782 99.4550 4.0382 100.9550

10 9.6628 96.6282 9.7685 97.6850 9.4957 94.95780 0.0082 0 -0.0156 0 0.0059 0

10 10.1638 101.6380 9.6318 96.3184 9.5504 95.50418 7.8113 97.6419 7.9155 98.9447 8.0023 100.02966 5.9267 98.7784 6.0673 101.1216 5.1942 86.5713

Mean 100.6426 Mean 102.8680 Mean 99.3286

191

Table 4.96 Estimated concentrations of Trandolapril in the predictionset under optimized PCR model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

10 9.8520 98.5204 10.1176 101.1760 10.0600 100.60048 8.1789 102.2363 8.1172 101.4656 8.2542 103.17768 8.2550 103.1885 8.0760 100.9509 8.0744 100.93052 2.0636 103.1843 2.0582 102.9129 2.1023 105.11508 7.8657 98.3213 8.2009 102.5118 8.0894 101.11776 5.8237 97.0625 6.2042 103.4049 5.9855 99.7583

10 10.3747 103.7475 9.7678 97.6780 10.3491 103.49196 5.9012 98.3533 6.4449 107.4164 6.2022 103.37168 8.0573 100.7163 8.2298 102.8735 8.0668 100.83546 6.0609 101.0156 6.1186 101.9770 5.9783 99.6396

Mean 100.6346 Mean 102.2367 Mean 101.8038

Table 4.97 Estimated concentrations of Verapamil in the prediction setunder optimized PCR model


D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0 0.0075 0 0.0056 0 0.0092 04 4.0735 101.8388 3.9590 98.9770 3.9456 98.64213 3.0608 102.0290 2.9267 97.5569 3.0064 100.21514 4.0066 100.1668 3.8361 95.9037 3.8114 95.28640 0.0086 0 0.0059 0 0.0054 01 1.0416 104.1696 0.9819 98.1947 0.9863 98.63925 5.0749 101.4990 4.9744 99.4883 4.9505 99.01194 3.8699 96.7481 3.8934 97.3370 3.7792 94.48241 1.0084 100.8492 1.0327 103.2787 0.9850 98.50310 0.0047 0 0.0012 0 0.0026 0

Mean 101.0429 Mean 98.6766 Mean 97.8258

192

Table 4.98 Estimated concentration of Manidipine in the prediction setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

2 1.9628 98.1414 2.0644 103.2233 2.0845 104.2294

2 1.9378 96.8917 2.0164 100.8206 2.0300 101.5042

10 9.9364 99.3641 10.0695 100.6955 9.9108 99.1082

2 2.0362 101.8105 1.9682 98.4100 2.0738 103.6944

8 7.7618 97.0229 8.0807 101.0097 8.0959 101.1992

1 1.0661 106.6167 1.0296 102.9621 0.9717 97.1763

10 10.0931 100.9313 10.3249 103.2492 9.7513 97.5135

0 0.0265 0 0.0102 0 0.0007 0

10 10.0081 100.0819 9.6877 96.8776 9.8109 98.1092

0 0.0045 0 0.0002 0 0.0095 0

Mean 100.1076 Mean 100.9060 Mean 100.3168



193




TRA-D0

99.9088 3.2858 0.9993 0.0993 100.6346 2.3068 0.9943 0.1814

TRA-D1

100.4928 4.2266 0.9987 0.1273 102.2367 2.3098 0.9950 0.2092

TRA-D2

99.7336 2.8226 0.9988 0.1208 101.8038 1.7506 0.9978 0.1613

VER-D0

100.5638 2.5288 0.9995 0.0355 101.0429 2.0990 0.9988 0.0667

VER-D1

100.9669 2.3551 0.9996 0.0343 98.6766 2.1678 0.9990 0.0799

VER-D2

100.4778 2.0798 0.9997 0.0288 97.8258 1.9467 0.9987 0.0989

MAN-D0

100.6426 4.1102 0.9969 0.1992 100.1076 2.9627 0.9996 0.0923

MAN-D1

102.8680 5.7177 0.9980 0.1759 100.9060 2.1681 0.9988 0.1487

MAN-D2

99.3286 4.2545 0.9955 0.2416 100.3168 2.5729 0.9995 0.1157


Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.100. The percentage recoverywas found to be from 99.60 – 101.20 for TRA, 99.00 – 100.40 for VER and99.70 – 102.10 for MAN respectively. These methods can be successfullyapplied for formulations containing either two or three drugs.

194

Table 4.100 Assay results of PLS and PCR models in Pharmaceuticalpreparations

Met

hod

Mode

TRA VER MAN

AmountFound*

(mg/tab)± SD

Mean%

recovery

AmountFound*

(mg/tab)± SD

Mean%

recovery

AmountFound*

(mg/tab)± SD

Mean%

recovery

PLS

D0 10.12± 0.11 101.20 5.02

± 0.06 100.40 10.21± 0.08 102.10

D1 9.98± 0.08 99.80 4.98

± 0.03 99.60 10.11± 0.05 101.10

D2 10.08± 0.05 100.80 5.01

± 0.02 100.20 9.97± 0.03 99.70

PCR

D0 9.96± 0.12 99.60 4.95

± 0.04 99.00 9.97± 0.05 99.70

D1 10.08± 0.07 100.80 5.01

± 0.01 100.20 10.07± 0.04 100.70

D2 9.98± 0.06 99.80 4.98

± 0.002 99.60 10.11± 0.02 101.10


Synthetic Mixture – Each 50 mg contains

Trandalopril – 10 mg

Verapamil - 10 mg

Manidipine – 5 mg

195

Figure 4.34 Plots of calibration and prediction sets of Trandolapril,Verapamil and Manidipine in both PLS and PCR models

y = 0.999x +0.000

R² = 0.999

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

TRA-PLS-CAL-D0

y = 0.998x + 0.005R² = 0.998

-2

0

2

4

6

8

10

12


TRA-PLS-CAL-D1

y = 0.999x + 0.001R² = 0.999

-2

0

2

4

6

8

10

12


TRA-PLS-CAL-D2

y = 1.000x + 0.035R² = 0.992

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

TRA-PLS-PRE-D0

y = 0.985x + 0.198R² = 0.999

0

2

4

6

8

10

12


TRA-PLS-PRE-D1

y = 1.021x - 0.033R² = 0.996

0

2

4

6

8

10

12


TRA-PLS-PRE-D2

y = 0.999x + 0.001R² = 0.999

-1

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

VER-PLS-CAL-D0

y = 0.999x + 0.000R² = 0.999

-1

0

1

2

3

4

5

6


VER-PLS-CAL-D1

y = 0.999x + 0.000R² = 0.999

-1

0

1

2

3

4

5

6

0 1 2 3 4 5

Amount Present

VER-PLS-CAL-D2

196

Figure 4.34Continued

y = 1.034x + 0.001R² = 0.997

-1

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

VER-PLS-PRE-D0

y = 1.014x + 0.004R² = 0.998

-1

0

1

2

3

4

5

6


VER-PLS-PRE-D1

y = 0.999x - 0.027R² = 0.998

-1

0

1

2

3

4

5

6


VER-PLS-PRE-D2

y = 0.999x + 0.001R² = 0.999

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

MAN-PLS-CAL-D0

y = 0.997x + 0.012R² = 0.997

0

2

4

6

8

10

12


MAN-PLS-CAL-D1

y = 0.998x + 0.008R² = 0.998

0

2

4

6

8

10

12


MAN-PLS-CAL-D2

y = 1.015x - 0.061R² = 0.999

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

MAN-PLS-PRE-D0

y = 0.997x + 0.016R² = 0.999

-2

0

2

4

6

8

10

12


MAN-PLS-PRE-D1

y = 1.011x + 0.019R² = 0.999

-2

0

2

4

6

8

10

12


MAN-PLS-PRE-D2

197


y = 0.999x + 0.003R² = 0.999

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

TRA-PCR-CAL-D0

y = 0.998x + 0.006R² = 0.998

-2

0

2

4

6

8

10

12


TRA-PCR-CAL-D1

y = 0.998x + 0.005R² = 0.998

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Amount Present

TRA-PCR-CAL-D2

y = 1.017x - 0.079R² = 0.994

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

TRA-PCR-PRE-D0

y = 0.978x + 0.285R² = 0.995

0

2

4

6

8

10

12


TRA-PCR-PRE-D1

y = 1.016x - 0.000R² = 0.997

0

2

4

6

8

10

12


TRA-PCR-PRE-D2

y = 0.999x + 0.000R² = 0.999

-1

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

VER-PCR-CAL-D0

y = 0.999x + 0.000R² = 0.999

-1

0

1

2

3

4

5

6


VER-PCR-CAL-D1

y = 0.999x + 0.000R² = 0.999

-1

0

1

2

3

4

5

6


VER-PCR-CAL-D2

198


y = 1.001x + 0.012R² = 0.998

-1

0

1

2

3

4

5

6

0 1 2 3 4 5

Am

ount

Pre

dict

ed

Amount Present

VER-PCR-PRE-D0

y = 0.981x - 0.000R² = 0.999

-1

0

1

2

3

4

5

6


VER-PCR-PRE-D1

y = 0.974x + 0.000R² = 0.998

-1

0

1

2

3

4

5

6


VER-PCR-PRE-D2

y = 0.976x + 0.143R² = 0.996

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

MAN-PCR-CAL-D0

y = 0.972x + 0.183R² = 0.998

-2

0

2

4

6

8

10

12


MAN-PCR-CAL-D1

y = 0.975x + 0.048R² = 0.995

-2

0

2

4

6

8

10

12


MAN-PCR-CAL-D2

y = 0.998x - 0.021R² = 0.999

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Am

ount

Pre

dict

ed

Amount Present

MAN-PCR-PRE-D0

y = 1.003x + 0.004R² = 0.998

-2

0

2

4

6

8

10

12


MAN-PCR-PRE-D1

y = 0.985x + 0.033R² = 0.999

-2

0

2

4

6

8

10

12

0 2 4 6 8 10

Amount Present

MAN-PCR-PRE-D2

199

Figure 4.35 Comparison of Mean Recovery of PLS and PCR models forfundamental (D0), First Derivative (D1) and SecondDerivative (D2) Spectra

96

98

100

102

104

D0 D1 D2

Trandolapril - PLS


96

98

100

102

104

D0 D1 D2

Trandolapril - PCR


96

98

100

102

104

D0 D1 D2

Verapamil - PLS


96

98

100

102

104

D0 D1 D2

Verapamil - PCR


96

98

100

102

104

D0 D1 D2

Manidipine -PLS


96

98

100

102

104

D0 D1 D2

Manidipine - PCR

Calibration set

Prediction set

200

4.2.8 Ternary Mixture 5 – Losartan (LOS), Hydrochlorothiazide(HCZ) and Amlodipine besylate (AML)


The spectra of all three drugs were overlapped in fundamental(Figure 4.36), first derivative (Figure 4.37) and second derivative mode(Figure 4.38) to study the spectral characteristics. They reveal a completeoverlap throughout the UV region of 200-400 nm. For multivariate analysis,25 different synthetic mixtures were prepared in the concentration ranges of0.2-3.2 µg/ml of LOS, 0.1-1.6 µg/ml of HCZ and 0.5-2.5 µg/ml of AMLrespectively (Table 4.101). The concentration range starts from zero in orderto meet the requirement of utilizing the analysis of any two drugs mentionedin the combination

Figure 4.36 Fundamental Spectra of Losartan, Hydrochlorothiazide,Amlodipine and mixture

201

Figure 4.37 First derivative spectra of Losartan, Hydrochlorothiazide,Amlodipine and mixture

Figure 4.38 Second derivative spectra of Losartan, Hydrochlorothiazide,Aamlodipine and mixture

202


Calibration Set (µg/ml) Prediction Set (µg/ml)LOS HCZ AML LOS HCZ AML0.2 0.1 0.5 0.2 0.2 0.50.4 0.1 0.5 0.4 0.1 1.50.8 0.1 0.5 0.4 0.4 21.6 0.1 0.5 0.8 0.8 13.2 0.1 0.5 3.2 0.8 10.2 0.2 1 1.6 0.2 20.4 0.2 1 1.6 1.6 10.8 0.2 1 0.2 0.4 2.51.6 0.2 1 0.4 0.8 1.53.2 0.2 1 0.8 1.6 20.2 0.4 1.5 - - -0.4 0.4 1.5 - - -0.8 0.4 1.5 - - -1.6 0.4 1.5 - - -3.2 0.4 1.5 - - -0.2 0.8 2 - - -0.4 0.8 2 - - -0.8 0.8 2 - - -1.6 0.8 2 - - -3.2 0.8 2 - - -0.2 1.6 2.5 - - -0.4 1.6 2.5 - - -0.8 1.6 2.5 - - -1.6 1.6 2.5 - - -3.2 1.6 2.5 - - -




Preprocessing : Mean Centre, auto scale, orthogonal signal

correction



203

Table 4.102 Estimated concentrations of Losartan in the calibration setunder optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.2009 100.4994 0.2187 109.3710 0.1910 95.50400.4 0.3802 95.0654 0.3633 90.8411 0.4114 102.86900.8 0.8114 101.4254 0.8238 102.9772 0.7886 98.57831.6 1.5929 99.5622 1.5913 99.4607 1.5931 99.57313.2 3.1994 99.9821 3.1908 99.7148 3.2148 100.46510.2 0.2073 103.6989 0.2162 108.1364 0.1921 96.08280.4 0.4035 100.8989 0.3981 99.5376 0.4227 105.68490.8 0.7941 99.2693 0.8039 100.4984 0.8237 102.97121.6 1.6081 100.5067 1.5984 99.9019 1.5563 97.26943.2 3.2159 100.4971 3.2107 100.3356 3.2091 100.28460.2 0.1953 97.6675 0.1872 93.6396 0.1896 94.81170.4 0.3953 98.8368 0.4053 101.3276 0.4072 101.80620.8 0.8033 100.4162 0.8036 100.4591 0.8034 100.42931.6 1.6094 100.5877 1.5854 99.0902 1.5968 99.80243.2 3.1897 99.6791 3.1956 99.8638 3.1943 99.82380.2 0.1984 99.2165 0.2086 104.3381 0.2299 114.98690.4 0.4243 106.0816 0.4095 102.3825 0.3879 96.98940.8 0.7767 97.0942 0.7806 97.5795 0.7862 98.28141.6 1.5968 99.8034 1.6052 100.3256 1.6146 100.91683.2 3.1905 99.7042 3.2041 100.1308 3.1888 99.65010.2 0.1974 98.7038 0.1998 99.9122 0.2002 100.10300.4 0.4074 101.8628 0.3788 94.7148 0.3999 99.99130.8 0.7968 99.6069 0.8176 102.2080 0.7963 99.53751.6 1.6020 100.1292 1.5983 99.8993 1.5881 99.25763.2 3.2019 100.0597 3.2039 100.1223 3.2129 100.4061

Mean 100.0342 Mean 99.8281 Mean 100.2478

204



D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.1 0.0975 97.5572 0.0958 95.8600 0.0970 97.08840.1 0.0986 98.6808 0.0995 99.5714 0.1046 104.62970.1 0.1029 102.9268 0.1043 104.3601 0.1000 100.03080.1 0.1031 103.1974 0.1033 103.3901 0.1007 100.70160.1 0.1017 101.7755 0.0961 96.1464 0.1002 100.22140.2 0.1959 97.9700 0.2014 100.7149 0.2000 100.00890.2 0.2063 103.1521 0.2003 100.1789 0.2003 100.16790.2 0.1988 99.4029 0.2022 101.1388 0.1943 97.17830.2 0.1965 98.2875 0.1980 99.0392 0.2012 100.60890.2 0.1928 96.4033 0.2032 101.6232 0.2001 100.09980.4 0.4027 100.6904 0.3961 99.0382 0.3999 99.99130.4 0.3996 99.9212 0.3986 99.6716 0.4014 100.36390.4 0.3977 99.4394 0.4009 100.2409 0.4019 100.49040.4 0.4017 100.4426 0.3909 97.7431 0.3955 98.88380.4 0.4012 100.3019 0.4034 100.8609 0.3995 99.88650.8 0.8133 101.6739 0.8002 100.0300 0.8088 101.10900.8 0.7940 99.2551 0.7940 99.26056 0.8049 100.61880.8 0.7981 99.7638 0.8028 100.3615 0.7911 98.89170.8 0.8019 100.2435 0.8055 100.6940 0.8019 100.24810.8 0.7974 99.6758 0.8042 100.5307 0.7975 99.69621.6 1.6014 100.0899 1.6075 100.4728 1.5998 99.98921.6 1.5963 99.7728 1.5980 99.8806 1.5933 99.58721.6 1.5953 99.7070 1.6030 100.1880 1.6011 100.07071.6 1.5948 99.6756 1.5957 99.7316 1.5987 99.91961.6 1.6094 100.5887 1.5936 99.6046 1.6051 100.3219

Mean 100.0238 Mean 100.2557 Mean 99.8721

205

Table 4.104 Estimated concentrations of Amlodipine in the calibrationset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4963 99.2730 0.4649 92.9978 0.4928 98.56910.5 0.4978 99.5645 0.5034 100.6812 0.5320 106.40060.5 0.5050 101.0106 0.5288 105.7673 0.5185 103.70980.5 0.4979 99.5876 0.5201 104.0250 0.4686 93.72370.5 0.4980 99.6101 0.4844 96.8985 0.4967 99.35261 1.0113 101.1367 1.0011 100.1182 1.0104 101.04011 0.9867 98.6789 1.0011 100.1135 0.9919 99.19771 1.0028 100.2888 0.9892 98.9291 0.9884 98.84751 1.0014 100.1404 1.0157 101.5726 1.0034 100.34261 1.0082 100.8238 0.9961 99.6153 0.9983 99.8322

1.5 1.4973 99.8219 1.5064 100.4302 1.5041 100.27701.5 1.5002 100.0135 1.4920 99.4719 1.4906 99.37771.5 1.5018 100.0135 1.5036 99.4719 1.4955 99.37771.5 1.5018 100.1201 1.4968 99.7911 1.4889 99.26141.5 1.4973 99.8204 1.4997 99.9851 1.5169 101.12732 1.9915 99.5799 1.9923 99.6164 1.9992 99.96282 2.0016 100.0831 1.9930 99.6509 1.9892 99.46072 1.9983 99.9168 1.9975 99.8797 2.0025 100.12722 1.9959 99.7962 2.0124 100.6208 2.0110 100.55442 2.0044 100.2246 1.9973 99.8692 2.0003 100.0188

2.5 2.5016 100.0677 2.5123 100.4954 2.4996 99.98472.5 2.5151 100.6050 2.5111 100.4477 2.4924 99.69762.5 2.4920 99.6837 2.4836 99.3473 2.5161 100.64462.5 2.5032 99.6837 2.4907 99.3473 2.4906 100.64472.5 2.4914 99.6573 2.5050 100.2012 2.5011 100.0441

Mean 99.9240 Mean 99.9934 Mean 100.1743

206

Table 4.105 Estimated concentrations of Losartan in the prediction setunder optimized PLS model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.1889 94.4629 0.2073 103.6849 0.1998 99.94140.4 0.4141 103.5400 0.4017 100.4460 0.4088 102.21160.4 0.4081 102.0411 0.3938 98.4724 0.4056 101.42210.8 0.8296 103.7096 0.8243 103.0495 0.8074 100.93343.2 3.3404 104.3884 3.1762 99.2582 3.1884 99.63761.6 1.5511 96.9440 1.5756 98.4804 1.5838 98.99051.6 1.5386 96.1657 1.5600 97.5042 1.6108 100.68120.2 0.1815 90.7967 0.2005 100.2936 0.2075 103.79960.4 0.3856 96.4188 0.4099 102.4788 0.4088 102.21550.8 0.7826 97.8305 0.8066 100.8305 0.8060 100.7606

Mean 98.6298 Mean 100.4499 Mean 101.0593



D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.2071 103.5653 0.2023 101.1797 0.2057 102.88970.1 0.1056 105.6490 0.1003 100.3898 0.1006 100.63190.4 0.4117 102.9362 0.4067 101.6848 0.4000 100.01160.8 0.8180 102.2506 0.8151 101.8894 0.8052 100.65670.8 0.8089 101.1235 0.7890 98.6326 0.8052 100.66150.2 0.2103 105.1847 0.2062 103.1244 0.2061 103.06771.6 1.6160 101.0012 1.5717 98.2323 1.6137 100.85640.4 0.3869 96.7361 0.3803 95.0767 0.3984 99.62280.8 0.8078 100.9823 0.7895 98.6949 0.8067 100.84421.6 1.6106 100.6656 1.6194 101.2149 1.5833 98.9562

Mean 102.1057 Mean 99.8703 Mean 100.8130

207

Table 4.107 Estimated concentrations of Amlodipine in the predictionset under optimized PLS model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5152 103.0598 0.5200 104.0140 0.4897 97.9550

1.5 1.5081 100.5428 1.5159 101.0629 1.4896 99.3071

2 2.0697 103.4870 2.0432 102.1626 2.0931 104.6577

1 0.9805 98.0567 1.0876 108.7672 0.9961 99.6104

1 0.9933 99.3363 1.0176 101.7621 0.9902 99.0295

2 2.1169 105.8499 2.0649 103.2494 1.9840 99.2026

1 1.0040 100.4034 1.0331 103.3160 0.9875 98.7590

2.5 2.4840 99.3633 2.4791 99.1675 2.5106 100.4259

1.5 1.4977 99.8486 1.4773 98.4879 1.4921 99.4736

2 1.8984 94.9216 1.9388 96.9446 2.0082 100.4148

Mean 101.3231 Mean 102.8528 Mean 99.7494



208




LOS-D0

100.0342 2.0122 0.9999 0.0101 98.6298 4.3344 0.9977 0.0529

LOS-D1

99.8281 3.7559 0.9998 0.0135 100.4499 1.9848 0.9998 0.0189

LOS-D2

100.2478 3.8072 0.9998 0.0153 101.0593 1.3455 0.9999 0.0092

HCZ-D0

100.0238 1.6146 0.9999 0.0040 102.1057 2.4319 0.9998 0.0115

HCZ-D1

100.2557 1.7549 0.9999 0.0040 99.8703 2.2392 0.9992 0.0145

HCZ-D2

99.8721 1.3226 0.9999 0.0037 100.8130 1.2241 0.9998 0.0080

AML-D0

99.9240 0.5256 0.9999 0.0061 102.3231 2.9137 0.9918 0.0546

AML-D1

99.9934 2.1462 0.9997 0.0128 102.8528 3.1740 0.9954 0.0453

AML-D2

100.1743 2.0387 0.9997 0.0125 99.7494 1.7359 0.9979 0.0310






Number of PCs : 13

209

Table 4.109 Estimated concentrations of Losartan in the calibration setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.2258 112.9328 0.1781 89.0762 0.2001 100.09380.4 0.3018 75.4716 0.3132 78.3166 0.3706 92.65740.8 0.8514 106.4333 0.8187 102.3435 0.7967 99.58971.6 1.5887 99.2963 1.5758 98.4914 1.5663 97.89783.2 3.2011 100.0372 3.3055 103.2989 3.2842 102.63320.2 0.2319 115.9883 0.2065 103.2763 0.1979 98.99720.4 0.3836 95.9005 0.3967 99.1791 0.4111 102.79040.8 0.8232 102.9028 0.7622 95.2850 0.7525 94.07241.6 1.6119 100.7496 1.5638 97.7425 1.5224 95.15423.2 3.1963 99.8862 3.2186 100.5813 3.1841 99.50470.2 0.1861 93.0774 0.2058 102.9033 0.1891 94.59260.4 0.4221 105.5454 0.4105 102.6431 0.4152 103.81870.8 0.8114 101.4296 0.7752 96.9042 0.8009 100.12271.6 1.5293 95.5873 1.5671 97.9462 1.6061 100.38653.2 3.2170 100.5333 3.1696 99.0524 3.1917 99.74330.2 0.2069 103.4968 0.2157 107.8991 0.2178 108.93630.4 0.4259 106.4815 0.4315 107.8783 0.3817 95.43700.8 0.7744 96.8124 0.7391 92.3959 0.7824 97.81041.6 1.6171 101.0737 1.5600 97.5050 1.5959 99.74673.2 3.2038 100.1205 3.2060 100.1893 3.1894 99.67140.2 0.1899 94.9607 0.1843 92.1901 0.1941 97.08880.4 0.3777 94.4265 0.3952 98.8139 0.4372 109.31100.8 0.8125 101.5724 0.8138 101.7331 0.7745 96.81641.6 1.6375 102.3454 1.6398 102.4875 1.6002 100.01683.2 3.1712 99.1020 3.1460 98.3129 3.2063 100.1969

Mean 100.2465 Mean 98.6159 Mean 99.7309

210

Table 4.110 Estimated concentrations of Hydrochlorothiazide in thecalibration set under PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.1 0.0995 99.5694 0.0958 95.8720 0.1004 100.42290.1 0.0893 89.3521 0.1010 101.0301 0.1017 101.77010.1 0.1083 108.3253 0.1056 105.6667 0.0973 97.35070.1 0.1022 102.2736 0.1055 105.5910 0.0956 95.68950.1 0.1022 102.2288 0.1093 109.3663 0.1040 104.01670.2 0.2033 101.6558 0.2033 101.6851 0.2047 102.37450.2 0.2039 101.9801 0.2083 104.1931 0.2052 102.63390.2 0.2041 102.0656 0.1961 98.0835 0.1933 96.68820.2 0.1950 97.5056 0.1924 96.2281 0.2005 100.29530.2 0.1882 94.1168 0.1956 97.8435 0.2005 100.26910.4 0.3993 99.8402 0.4010 100.2666 0.3971 99.27680.4 0.4048 101.2099 0.4026 100.6695 0.4013 100.34550.4 0.3983 99.5934 0.3989 99.7360 0.4016 100.40170.4 0.3912 97.8092 0.3874 96.8725 0.3967 99.19620.4 0.4091 102.2919 0.4072 101.8042 0.3984 99.60540.8 0.8172 102.1573 0.8114 101.4362 0.8083 101.04010.8 0.7903 98.7969 0.7881 98.5134 0.8065 100.81320.8 0.7977 99.7186 0.8049 100.6127 0.7878 98.48250.8 0.8053 100.6686 0.7971 99.6404 0.8011 100.14800.8 0.7925 99.0745 0.7983 99.7895 0.7972 99.65731.6 1.5997 99.9832 1.6061 100.3850 1.5982 99.89291.6 1.5909 99.4369 1.5938 99.6142 1.5988 99.92871.6 1.6011 100.0730 1.6225 101.4082 1.5966 99.78921.6 1.5925 99.5330 1.5923 99.5204 1.6010 100.06371.6 1.6128 100.8044 1.5842 99.0138 1.6049 100.3123

Mean 100.0026 Mean 100.1842 Mean 99.9211

211

Table 4.111 Estimated concentrations of Amlodipine in the calibrationset under optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.4314 86.2801 0.4903 98.0744 0.4887 97.74550.5 0.5208 104.1605 0.5004 100.0937 0.5252 105.04720.5 0.5325 106.5095 0.5089 101.7854 0.5192 103.85450.5 0.4962 99.2497 0.5012 100.2496 0.4942 98.84520.5 0.5351 107.0212 0.5555 111.1084 0.5136 102.73761 1.0478 104.7851 0.9921 99.2142 1.0480 104.80681 1.0322 103.2216 1.0048 100.4806 0.9507 95.07611 1.0118 101.1868 0.9582 95.8287 0.9402 94.02071 0.9686 96.8685 0.9882 98.8272 0.9458 94.58731 0.9957 99.5724 0.9973 99.7314 0.9554 95.5462

1.5 1.5028 100.1871 1.5058 100.3928 1.5031 100.20811.5 1.4771 98.4771 1.5414 102.7641 1.4613 97.42131.5 1.5124 100.8319 1.4828 98.8589 1.5244 101.63001.5 1.5303 102.0200 1.4930 99.5384 1.4892 99.28011.5 1.4484 96.5643 1.5087 100.5834 1.4915 99.43612 1.9050 95.2503 1.8979 94.8982 2.0186 100.93332 1.9852 99.2602 2.0004 100.0217 2.0023 100.11772 2.0338 101.6943 2.0535 102.6794 1.9904 99.52102 2.0485 102.4298 1.9225 96.1294 2.0180 100.90392 1.9418 97.0916 1.9642 98.2114 2.0296 101.4823

2.5 2.4772 99.0901 2.5489 101.9590 2.5076 100.30762.5 2.5312 101.2517 2.5081 100.3267 2.5555 102.22362.5 2.5395 101.5826 2.5605 102.4207 2.4449 97.79742.5 2.4267 97.0719 2.4234 96.9396 2.5214 100.85912.5 2.5668 102.6759 2.5205 100.8234 2.4599 98.3986

Mean 98.8014 Mean 100.2411 Mean 100.0667

212

Table 4.112 Estimated concentrations of Losartan in the prediction setunder optimized PCR model


D0 D1 D2Amount

found(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.2078 103.9339 0.1971 98.5960 0.2067 103.36330.4 0.4083 102.0968 0.4181 104.5349 0.4092 102.30090.4 0.3858 96.4502 0.4086 102.1570 0.4087 102.18270.8 0.7918 98.9835 0.7955 99.4497 0.8040 100.50373.2 3.2433 101.3531 3.2688 102.1521 3.2026 100.08181.6 1.6130 100.8183 1.5791 98.6988 1.6156 100.97831.6 1.6113 100.7087 1.5785 98.6562 1.5933 99.58740.2 0.2052 102.6167 0.2028 101.4452 0.2091 104.57460.4 0.4028 100.7077 0.3834 95.8570 0.3940 98.51000.8 0.8017 100.2141 0.8096 101.2017 0.7894 98.6859

Mean 100.7883 Mean 100.2749 Mean 101.0769



D0 D1 D2Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.2 0.2048 102.4075 0.2078 103.9074 0.2133 106.65600.1 0.1069 106.9872 0.1032 103.2060 0.1058 105.88820.4 0.3862 96.5708 0.3978 99.4548 0.4101 102.53050.8 0.8004 100.0548 0.8041 100.5142 0.7841 98.01460.8 0.8038 100.4806 0.7932 99.1615 0.8009 100.11600.2 0.2025 101.2738 0.2043 102.1844 0.2047 102.35691.6 1.6048 100.3032 1.6080 100.5002 1.6093 100.58680.4 0.3976 99.4004 0.4069 101.7484 0.3898 97.46380.8 0.7958 99.4836 0.8142 101.7851 0.7922 99.02591.6 1.6223 101.3978 1.6046 100.2894 1.6133 100.8336

Mean 101.5820 Mean 101.3419 Mean 100.8381

213

Table 4.114 Estimated concentration of Amlodipine in the prediction setunder optimized PCR model


D0 D1 D2

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

Amountfound

(µg/ml)

%recovery

0.5 0.5070 101.4176 0.5055 101.1179 0.4881 97.6200

1.5 1.5038 100.2590 1.4901 99.3454 1.4802 98.6821

2 2.0771 103.8550 2.0865 104.3293 2.0057 100.2892

1 0.9748 97.4892 1.0681 106.8174 1.0052 100.5208

1 1.0186 101.8600 1.0376 103.7640 1.0262 102.6228

2 2.0993 104.9664 2.0419 102.0985 2.0932 104.6631

1 1.0538 105.3880 1.0272 102.7214 1.0112 101.1285

2.5 2.5135 100.5403 2.4486 97.9450 2.5153 100.6129

1.5 1.5085 100.5694 1.5090 100.6016 1.5008 100.0549

2 2.1338 106.6903 1.9875 99.3766 1.9731 98.6599

Mean 101.9756 Mean 102.5994 Mean 100.6977


The mean % recovery ranges from 98.615 to 100.246 for all thethree drugs in the calibration set and from 100.274 to 102.599 in theprediction set. The SD values were found to decrease with increasing spectralmode in both calibration set and prediction set. R2 value ranges from 0.9964 –0.9999 in the calibration set and from 0.9955 – 0.9999 in the prediction set.RMSEC value ranges from 0.0074 – 0.0425 for all three drugs in thecalibration set. RMSEP value ranges from 0.0070 – 0.0615 for all three drugsin the prediction set.

214




LOS-D0

100.2465 7.2717 0.9991 0.0321 100.7883 1.9413 0.9999 0.0161

LOS-D1

98.6159 5.9842 0.9988 0.0387 100.2749 2.3571 0.9995 0.0253

LOS-D2

99.7309 3.9065 0.9993 0.0292 101.0769 1.8983 0.9999 0.0086

HCZ-D0

100.0026 3.2566 0.9998 0.0074 101.5820 2.5293 0.9998 0.0091

HCZ-D1

100.1842 3.0030 0.9998 0.0083 101.3419 1.4800 0.9999 0.0070

HCZ-D2

99.9211 1.7286 0.9999 0.0043 100.8381 2.9222 0.9996 0.0100

AML-D0

98.8014 4.1154 0.9964 0.0425 101.9756 2.6983 0.9955 0.0615

AML-D1

99.9211 3.0385 0.9968 0.0401 102.5994 2.5610 0.9959 0.0436

AML-D2

100.0667 2.9122 0.9979 0.0327 100.6977 1.9368 0.9973 0.0332


Optimized PLS and PCR models were applied to commercialtablets and the results are tabulated in Table 4.116. The percentage recoverywas found to be from 97.96 – 100.68 for LOS, 99.44 – 100.40 for HCZ and98.60 – 100.80 for AML respectively. These methods can be successfullyapplied for formulations containing three drugs.

215

Table 4.116 Assay results of PLS and PCR models in PharmaceuticalPreparations

Met

hod

Mode

LOS HCZ AML

AmountFound*

(mg/tab)

± SD

Mean

%recovery

AmountFound*

(mg/tab)

± SD

Mean

%

recovery

AmountFound*

(mg/tab)

± SD

Mean

%recovery

PLS

D050.12

± 0.12100.24

12.52

± 0.09100.16

5.04

± 0.03100.80

D150.34

± 0.11100.68

12.46

± 0.0599.68

5.02

± 0.03100.40

D249.54

± 0.0799.08

12.43

± 0.0399.44

4.96

± 0.0299.20

PCR

D048.98

± 0.1297.96

12.43

± 0.0899.44

4.93

± 0.0398.60

D150.33

± 0.07100.66

12.55

± 0.06100.40

4.94

± 0.0398.80

D250.33

± 0.06100.66

12.46

± 0.0399.68

4.96

± 0.0299.20

Formulation – Trilopace* - Akums drugs & Pharmaceuticals, Uttaranchal

Losartan Potassium – 50 mg

Hydrochlorothiazide – 12.5 mg

Amlodipine Besylate – 5 mg

216

Figure 4.39 Plots of calibration and prediction sets of LOS, HCZ andAML in both PLS and PCR models

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5

0 0.8 1.6 2.4 3.2

Am

ount

Pre

dict

ed

Amount Present

LOS-PLS-CAL-DO

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.52

2.5

3

3.5

0 0.8 1.6 2.4 3.2Amount Present

LOS-PLS-CAL-D1

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PLS-CAL-D2

y = 1.032x - 0.029R² = 0.997

00.5

11.5

22.5

33.5

4

0 0.8 1.6 2.4 3.2

Am

ount

Pre

dict

ed

Amount Present

LOS-PLS-PRE-D0

y = 0.986x + 0.008R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PLS-PRE-D1

y = 0.993x + 0.008R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PLS-PRE-D2

y = 0.999x + 3E-05R² = 0.999

0

0.5

1

1.5

2

0 0.4 0.8 1.2 1.6

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-CAL-D0

y = 0.999x + 3E-05R² = 0.999

0

0.5

1

1.5

2


HCZ-PLS-CAL-D1

y = 1x + 3E-05R² = 1

0

0.5

1

1.5

2


HCZ-PLS-CAL-D2

217


y = 1.006x +0.004

R² = 0.999

0

0.5

1

1.5

2

0 0.4 0.8 1.2 1.6

Am

ount

Pre

dict

ed

Amount Present

HCZ-PLS-PRE-D0

y = 0.996x + 0.000R² = 0.999

0

0.5

1

1.5

2


HCZ-PLS-PRE-D1

y = 0.997x + 0.004R² = 0.999

0

0.5

1

1.5

2


HCZ-PLS-PRE-D2

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

AML-PLS-CAL-D0

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3


AML-PLS-CAL-D1

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

2.5

3


AML-PLS-CAL-D2

y = 1.006x - 0.002R² = 0.991

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

AML-PLS-PRE-D0

y = 0.975x + 0.054R² = 0.995

0

0.5

1

1.5

2

2.5

3


AML-PLS-PRE-D1

y = 1.021x - 0.028R² = 0.997

0

0.5

1

1.5

2

2.5

3


AML-PLS-PRE-D2

218


y = 0.999x + 0.001R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5

0 0.8 1.6 2.4 3.2

Am

ount

Pre

dict

ed

Amount Present

LOS-PCR-CAL-D0

y = 1.005x - 0.014R² = 0.998

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PCR-CAL-D1

y = 1.003x - 0.009R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PCR-CAL-D2

y = 1.013x - 0.005R² = 0.999

00.5

11.5

22.5

33.5

0 0.8 1.6 2.4 3.2

Am

ount

Pre

dict

ed

Amount Present

LOS-PCR-PRE-D0

y = 1.015x - 0.010R² = 0.999

0

0.5

1

1.5

2

2.5

3

3.5


LOS-PCR-PRE-D1

y = 0.999x + 0.004R² = 0.999

00.5

11.5

2

2.53

3.5

0 0.8 1.6 2.4 3.2

Amount Present

LOS-PCR-PRE-D2

y = 0.999x + 0.000R² = 0.999

0

0.5

1

1.5

2

0 0.4 0.8 1.2 1.6

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-CAL-D0

y = 0.999x + 0.001R² = 0.999

0

0.5

1

1.5

2


HCZ-PCR-CAL-D1

y = 0.999x + 4E-05R² = 0.999

0

0.5

1

1.5

2


HCZ-PCR-CAL-D2

219


y = 1.008x - 0.003R² = 0.999

0

0.5

1

1.5

2

0 0.4 0.8 1.2 1.6

Am

ount

Pre

dict

ed

Amount Present

HCZ-PCR-PRE-D0

y = 1.001x + 0.003R² = 0.999

0

0.5

1

1.5

2


HCZ-PCR-PRE-D1

y = 1.002x + 0.000R² = 0.999

0

0.5

1

1.5

2


HCZ-PCR-PRE-D2

y = 0.996x + 0.005R² = 0.996

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

AML-PCR-CAL-D0

y = 0.996x +0.002

R² = 0.996

0

0.5

1

1.5

2

2.5

3


AML-PCR-CAL-D1

y = 1.004x - 0.010R² = 0.997

0

0.5

1

1.5

2

2.5

3


AML-PCR-CAL-D2

y = 1.039x - 0.02R² = 0.995

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Am

ount

Pre

dict

ed

Amount Present

AML-PCR-PRE-D0

y = 0.981x + 0.048R² = 0.995

0

0.5

1

1.5

2

2.5

3


AML-PCR-PRE-D1

y = 1.012x - 0.008R² = 0.997

0

0.5

1

1.5

2

2.5

3


AML-PCR-PRE-D2

220


96

98

100

102

104

D0 D1 D2

Losartan - PLS


96

98

100

102

104

D0 D1 D2

Losartan - PCR


96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2



96

98

100

102

104

D0 D1 D2

Amlodipine-PLS


96

98

100

102

104

D0 D1 D2

Amlodipine - PCR


CHAPTER 4 CHEMOMETRICS ASSISTED QUANTITATIVE...

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