Analytical method development and validation by chemometric approach for the marketed formulations.

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ANALYTICAL METHOD DEVELOPMENT AND

VALIDATION BY CHEMOMETRIC APPROACH FOR THE

MARKETED FORMULATIONS

THESIS Submitted to

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY,

Guindy, Chennai-32, Tamil Nadu, India.

In partial fulfilment of the requirements for the award of the Degree of

DOCTOR OF PHILOSOPHY

[Faculty of Pharmacy]

Submitted by

Mrs. YAMUNA.S, M Pharm.,

Under the Guidance of

Prof. Dr. A. SHANTHA, B Pharm, M.Sc(Pharm), Ph.D.,

HEAD

DEPARTMENT OF PHARMACEUTICAL ANALYSIS, C. L. BAID METHA COLLEGE OF PHARMACY,

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LIST OF ABBREVIATIONS USED

AR Analytical Reagent BP British Pharmacopoeia cm

CS

Centimetre

Calibration Set

CV Cross Validation

˚C Degree centigrade

EU GMP European Union Good Manufacturing Practice FDA Food and Drug Administration

GC/MS Gas Chromatography/Mass Spectroscopy GLC Gas Liquid Chromatography

GMP Good Manufacturing Practice

g Grams

g mL-1 Grams Per Millilitre

HPTLC High Performance Thin Layer Chromatography ICH International Conference on Harmonisation IP

ISTD

Indian Pharmacopoeia

Internal Standard

l Litre

LC/MS Liquid Chromatography/Mass Spectroscopy LOD Limit Of Detection

LOQ Limit Of Quantification LLOD Lower Limit Of Detection LLOQ Lower Limit Of Quantification

λmax Lamda max, Absorption Maximum

mL Millilitre

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µL Microlitre

µg Microgram

µm Micrometre

MLR Multiple Linear Regression mL min-1 Millilitre Per Minute µL min-1 Microlitre Per Minute mg mL-1 Milligram Per Millilitre µg mL-1 Microgram Per Millilitre

ng Nanogram

nm Nanometre

OVAT One Variable At A Time PC Prinicipal Component

% Percentage

PCR Principal Component Regression PLS Partial Least Squares Regression PRESS

PS

Predicted Residual Error Sum of Squares

Prediction Set

QSAR Quantitative Structure Activity Relationship r Regression Coefficient

RMSEC Root Mean Squared Error of Calibration RMSECV Root Mean Squared Error of Cross validation RMSEP Root Mean Squared Error of Prediction

Rt Retention Time

Rf Retardation Factor

Rs Resolution

RP-HPLC Reverse Phase High Performance Liquid Chromatography RP-UHPLC Reverse Phase Ultra High Performance Liquid Chromatography RSD Relative Standard Deviation

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SD Standard Deviation

SE Standard Error

SS Sum of Squares

SEC Standard Error of Calibration SEP Standard Error of Prediction

σ Standard Deviation

S Slope of the calibration curve

USP-NF United States Pharmacopoeia National Formulary UV-Vis Ultra - Violet Visible Spectrophotometry

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7 RESULTS AND DISCUSSION

7.1 CHEMOMETRICS-ASSISTED

UV SPECTROPHOTOMETRY

140

7.2 RP-UHPLC WITH FACTORIAL

DESIGN

147

7.3 HPTLC WITH FACTORIAL DESIGN 153

8 SUMMARY AND CONCLUSION 156

9 BIBLIOGRAPHY I

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LIST OF SPECTRAS

S.No. TITLE 1 Overlay UV spectrum of the calibration set of cefixime and ornidazole 2 Overlay UV spectrum of the prediction set of cefixime and ornidazole 3 Overlay UV absorbance spectrum of assay of cefixime and ornidazole 4 Overlay UV spectrum of recovery studies of cefixime and ornidazole 5 Overlay UV spectrum illustrating overlapping of peaks of cefixime and

ornidazole

6 Overlay UV spectrum of the calibration set of cefixime and ofloxacin 7 Overlay UV spectrum of the prediction set of cefixime and ofloxacin 8 Overlay UV absorbance spectrum of assay of cefixime and ofloxacin 9 Overlay UV spectrum of recovery studies of cefixime and ofloxacin 10 Overlay UV spectrum illustrating overlapping of peaks of cefixime and

Ofloxacin

11 Overlay UV spectrum of the calibration set of imipramine hydrochloride and chlordiazepoxide hydrochloride

12 Overlay UV spectrum of the prediction set of imipramine hydrochloride and chlordiazepoxide hydrochloride

13 Overlay UV absorbance spectrum of assay of imipramine hydrochloride and chlordiazepoxide hydrochloride

14 Overlay UV spectrum of recovery studies of imipramine hydrochloride and chlordiazepoxide hydrochloride

15 Overlay UV spectrum illustrating overlapping of peaks of imipramine hydrochloride and chlordiazepoxide hydrochloride

16 Overlay UV spectrum of the calibration set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

17 Overlay UV spectrum of the prediction set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

18

Overlay UV absorbance spectrum of assay of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

19 Overlay UV spectrum of recovery studies of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

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21 Overlay UV spectrum of the calibration set of montelukast sodium and bambuterol hydrochloride

22 Overlay UV spectrum of the prediction set of montelukast sodium and bambuterol hydrochloride

23 Overlay UV absorbance spectrum of assay of montelukast sodium and bambuterol hydrochloride

24 Overlay UV spectrum of recovery studies of montelukast sodium and bambuterol hydrochloride

25 Overlay UV spectrum illustrating overlapping of peaks of montelukast sodium and bambuterol hydrochloride

26 Overlay UV spectrum of the calibration set of montelukast sodium and fexofenadine hydrochloride

27 Overlay UV spectrum of the prediction set of montelukast sodium and fexofenadine hydrochloride

28 Overlay UV absorbance spectrum of assay of montelukast sodium and fexofenadine hydrochloride

29 Overlay UV spectrum of recovery studies of montelukast sodium and fexofenadine hydrochloride (100%)

30 Overlay UV spectrum illustrating overlapping of peaks of montelukast sodium and fexofenadine hydrochloride

31 Overlay UV spectrum of the calibration set of montelukast sodium and rupatadine fumarate

32 Overlay UV spectrum of the prediction set of montelukast sodium and rupatadine fumarate

33 Overlay UV absorbance spectrum of assay of montelukast sodium and rupatadine fumarate

34 Overlay UV spectrum of recovery studies of montelukast sodium and rupatadine fumarate (100%)

35 Overlay UV spectrum illustrating overlapping of peaks of montelukast sodium and rupatadine fumarate

36 Overlay UV spectrum of the calibration set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

37 Overlay UV spectrum of the prediction set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

38 Overlay UV absorbance spectrum of assay of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

39 Overlay UV spectrum of recovery studies of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

40 Overlay UV spectrum illustrating overlapping of peaks of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride (100%)

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42 Overlay UV spectrum of the prediction set of salbutamol sulphate, ambroxol hydrochloride and theophylline

43 Overlay UV absorbance spectrum of assay of salbutamol sulphate, ambroxol hydrochloride and theophylline

44 Overlay UV spectrum of recovery studies of salbutamol sulphate, ambroxol hydrochloride and theophylline

45 Overlay UV spectrum illustrating overlapping of peaks of salbutamol sulphate, ambroxol hydrochloride and theophylline

46 Overlay UV spectrum of the calibration set of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride 47 Overlay UV spectrum of the prediction set of pseudoephedrine

hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride 48 Overlay UV absorbance spectrum of assay of pseudoephedrine

hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride 49 Overlay UV spectrum of recovery studies of pseudoephedrine

hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride 50 Overlay UV spectrum illustrating overlapping of peaks of

pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride

51 Overlay UV spectrum of the calibration set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

52 Overlay UV spectrum of the prediction set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

53 Overlay UV absorbance spectrum of assay of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

54 Overlay UV spectrum of recovery studies of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

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LIST OF CHROMATOGRAMS

Method I Development and validation of RP-UHPLC method for the simultaneous determination of montelukast sodium and bambuterol hydrochloride (Combination-1) and montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride (Combination-2) in oral solid dosage forms 1 Chromatogram of blank

2 Chromatogram of bambuterol hydrochloride (Standard) 3 Chromatogram of levocetirizine dihydrochloride (Standard) 4 Chromatogram of ambroxol hydrochloride (Standard) 5 Chromatogram of montelukast sodium (Standard) 6 Chromatogram of mixed standards

7 Chromatogram of sample formulation containing montelukast sodium and bambuterol hydrochloride

8 Chromatogram of sample formulation containing montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride 9 Chromatogram of system suitability studies

10 Chromatogram of mixed standards - linearity level 1 (70%) 11 Chromatogram of mixed standards - linearity level 2 (80%) 12 Chromatogram of mixed standards - linearity level 3 (90%) 13 Chromatogram of mixed standards - linearity level 4(100%) 14 Chromatogram of mixed standards - linearity level 5 (110%) 15 Chromatogram of mixed standards - linearity level 6 (120%) 16 Chromatogram of mixed standards - linearity level 7 (130%)

17 Chromatogram of assay (montelukast sodium and bambuterol hydrochloride - combination 1)

18 Chromatogram of recovery study of montelukast sodium and bambuterol hydrochloride at 80% level

21 Chromatogram of assay (montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride - combination 2)

22 Chromatogram of recovery study of montelukast sodium,

levocetirizine dihydrochloride and ambroxol hydrochloride at 80% level

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levocetirizine dihydrochloride and ambroxol hydrochloride at 120% level

25 Chromatogram of system precision

26 Chromatogram of method precision (montelukast sodium and bambuterol hydrochloride - combination 1)

27 Chromatogram of method precision (montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride - combination 2)

28 Chromatogram of robustness (Standard) - R9 29 Chromatogram of robustness (Standard) - R7 30 Chromatogram of robustness (Standard) - R2 31 Chromatogram of robustness (Standard) - R5 32 Chromatogram of robustness (Standard) - R8 33 Chromatogram of robustness (Standard) - R1 34 Chromatogram of robustness (Standard) - R6 35 Chromatogram of robustness (Standard) - R3 36 Chromatogram of robustness (Standard) - R4

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Method II Development and validation of RP-UHPLC method for the simultaneous determination of chlordiazepoxide hydrochloride and imipramine hydrochloride in tablet dosage form

55 Chromatogram of blank

56 Chromatogram of chlordiazepoxide hydrochloride (Standard) 57 Chromatogram of imipramine hydrochloride (Standard) 58 Chromatogram of mixed standards

59 Chromatogram of sample formulation 60 Chromatogram of system suitability studies

61 Chromatogram of mixed standards - linearity level 1 (70%) 62 Chromatogram of mixed standards - linearity level 2 (80%) 63 Chromatogram of mixed standards - linearity level 3 (90%) 64 Chromatogram of mixed standards - linearity level 4(100%) 65 Chromatogram of mixed standards - linearity level 5 (110%) 66 Chromatogram of mixed standards - linearity level 6 (120%) 67 Chromatogram of mixed standards - linearity level 7 (130%) 68 Chromatogram of assay of the combination-chlordiazepoxide

hydrochloride and imipramine hydrochloride

69 Chromatogram of recovery study of chlordiazepoxide hydrochloride and imipramine hydrochloride at 80% level

72 Chromatogram of system precision 73 Chromatogram of method precision

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85 Chromatogram of robustness (Sample) - R10 86 Chromatogram of robustness (Sample) - R2 87 Chromatogram of robustness (Sample) - R4 88 Chromatogram of robustness (Sample) - R8 89 Chromatogram of robustness (Sample) - R7 90 Chromatogram of robustness (Sample) - R6

Method III Development and validation of RP-UHPLC method for the simultaneous determination of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride in tablet dosage form

92 Chromatogram of pseudoephedrine hydrochloride (Standard) 93 Chromatogram of levocetirizine dihydochloride (Standard) 94 Chromatogram of ambroxol hydochloride (Standard) 95 Chromatogram of mixed standards

98 Chromatogram of mixed standards - linearity level 1 (70%) 99 Chromatogram of mixed standards - linearity level 2 (80%) 100 Chromatogram of mixed standards - linearity level 3 (90%) 101 Chromatogram of mixed standards - linearity level 4(100%) 102 Chromatogram of mixed standards - linearity level 5 (110%) 103 Chromatogram of mixed standards - linearity level 6 (120%) 104 Chromatogram of mixed standards - linearity level 7 (130%) 105 Chromatogram of assay of the combination-pseudoephedrine

hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride

106 Chromatogram of recovery study of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride at 80% level

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111 Chromatogram of robustness (Standard) - R1 112 Chromatogram of robustness (Standard) - R8 113 Chromatogram of robustness (Standard) - R11 114 Chromatogram of robustness (Standard) - R4 115 Chromatogram of robustness (Standard) - R3 116 Chromatogram of robustness (Standard) - R9 117 Chromatogram of robustness (Standard) - R10 118 Chromatogram of robustness (Standard) - R6 119 Chromatogram of robustness (Standard) - R2 120 Chromatogram of robustness (Sample) - R1 121 Chromatogram of robustness (Sample) - R8 122 Chromatogram of robustness (Sample) - R11 123 Chromatogram of robustness (Sample) - R4 124 Chromatogram of robustness (Sample) - R3 125 Chromatogram of robustness (Sample) - R9 126 Chromatogram of robustness (Sample) - R10 127 Chromatogram of robustness (Sample) - R6 128 Chromatogram of robustness (Sample) - R2

Method IV Development and validation of RP-UHPLC method for the simultaneous determination of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol in tablet dosage form

130 Chromatogram of levocetirizine dihydochloride (Standard) 131 Chromatogram of ambroxol hydochloride (Standard) 132 Chromatogram of phenylephrine hydochloride (Standard) 133 Chromatogram of paracetamol (Standard)

134 Chromatogram of mixed standards 135 Chromatogram of sample formulation 136 Chromatogram of system suitability studies

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143 Chromatogram of mixed standards - linearity level 7 (130%) 144 Chromatogram of assay of the combination-levocetirizine

dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

145 Chromatogram of recovery study of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and

paracetamol at 80% level

Method V Development and validation of RP-UHPLC method for the simultaneous determination of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride in tablet dosage form

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175 Chromatogram of mixed standards - linearity level 1 (70%) 176 Chromatogram of mixed standards - linearity level 2 (80%) 177 Chromatogram of mixed standards - linearity level 3 (90%) 178 Chromatogram of mixed standards - linearity level 4(100%) 179 Chromatogram of mixed standards - linearity level 5 (110%) 180 Chromatogram of mixed standards - linearity level 6 (120%) 181 Chromatogram of mixed standards - linearity level 7 (130%) 182 Chromatogram of assay of the combination-trihexyphenidyl

hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

183 Chromatogram of recovery study of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride at 80% level

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201 Chromatogram of robustness (Sample) - R3 202 Chromatogram of robustness (Sample) - R1 203 Chromatogram of robustness (Sample) - R10 204 Chromatogram of robustness (Sample) - R4 205 Chromatogram of robustness (Sample) - R5

Method VI Development and validation of RP-UHPLC method for the simultaneous determination of salbutamol sulphate, ambroxol hydrochloride and theophylline in tablet dosage form

207 Chromatogram of salbutamol sulphate (Standard) 208 Chromatogram of ambroxol hydrochloride (Standard) 209 Chromatogram of theophylline (Standard)

213 Chromatogram of mixed standards - linearity level 1 (70%) 214 Chromatogram of mixed standards - linearity level 2 (80%) 215 Chromatogram of mixed standards - linearity level 3 (90%) 216 Chromatogram of mixed standards - linearity level 4(100%) 217 Chromatogram of mixed standards - linearity level 5 (110%) 218 Chromatogram of mixed standards - linearity level 6 (120%) 219 Chromatogram of mixed standards - linearity level 7 (130%) 220 Chromatogram of assay of the combination-salbutamol sulphate,

ambroxol hydrochloride and theophylline

221 Chromatogram of recovery study of salbutamol sulphate, ambroxol hydrochloride and theophylline at 80% level

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231 Chromatogram of robustness (Standard) - R10 232 Chromatogram of robustness (Standard) - R2 233 Chromatogram of robustness (Standard) - R8 234 Chromatogram of robustness (Standard) - R1 235 Chromatogram of robustness (Sample) - R5 236 Chromatogram of robustness (Sample) - R4 237 Chromatogram of robustness (Sample) - R9 238 Chromatogram of robustness (Sample) - R3 239 Chromatogram of robustness (Sample) - R11 240 Chromatogram of robustness (Sample) - R10 241 Chromatogram of robustness (Sample) - R2 242 Chromatogram of robustness (Sample) - R8 243 Chromatogram of robustness (Sample) - R1 Method

VII Development and validation of RP-UHPLC method for the simultaneous determination of montelukast sodium and rupatadine in tablet dosage form using fexofenadine hydrochloride as internal standard

245 Chromatogram of montelukast sodium (Standard) 246 Chromatogram of rupatadine (Standard)

247 Chromatogram of fumaric acid (Standard)

248 Chromatogram of fexofenadine hydrochloride (Internal Standard) 249 Chromatogram of mixed standards

252 Chromatogram of mixed standards - linearity level 1 (70%) 253 Chromatogram of mixed standards - linearity level 2 (80%) 254 Chromatogram of mixed standards - linearity level 3 (90%) 255 Chromatogram of mixed standards - linearity level 4(100%) 256 Chromatogram of mixed standards - linearity level 5 (110%) 257 Chromatogram of mixed standards - linearity level 6 (120%) 258 Chromatogram of mixed standards - linearity level 7 (130%)

259 Chromatogram of assay of the combination-montelukast sodium and rupatidine

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261 Chromatogram of recovery study of montelukast sodium and rupatadine at 100% level

262 Chromatogram of recovery study of montelukast sodium and rupatadine at 120% level

265 Chromatogram of robustness (Standard) - R6 266 Chromatogram of robustness (Standard) - R2 267 Chromatogram of robustness (Standard) - R11 268 Chromatogram of robustness (Standard) - R10 269 Chromatogram of robustness (Standard) - R5 270 Chromatogram of robustness (Standard) - R1 271 Chromatogram of robustness (Standard) - R3 272 Chromatogram of robustness (Standard) - R8 273 Chromatogram of robustness (Standard) - R4 274 Chromatogram of robustness (Sample) - R6 275 Chromatogram of robustness (Sample) - R2 276 Chromatogram of robustness (Sample) - R11 277 Chromatogram of robustness (Sample) - R10 278 Chromatogram of robustness (Sample) - R5 279 Chromatogram of robustness (Sample) - R1 280 Chromatogram of robustness (Sample) - R3 281 Chromatogram of robustness (Sample) - R8 282 Chromatogram of robustness (Sample) - R4 Method

VIII

Development and validation of RP-UHPLC method for the simultaneous determination of cefixime and ofloxacin in tablet dosage form using ornidazole as internal standard

284 Chromatogram of cefixime (Standard) 285 Chromatogram of ofloxacin (Standard)

286 Chromatogram of ornidazole (Internal Standard) 287 Chromatogram of mixed standards

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292 Chromatogram of mixed standards - linearity level 3 (90%) 293 Chromatogram of mixed standards - linearity level 4(100%) 294 Chromatogram of mixed standards - linearity level 5 (110%) 295 Chromatogram of mixed standards - linearity level 6 (120%) 296 Chromatogram of mixed standards - linearity level 7 (130%) 297 Chromatogram of assay of the combination-cefixime and ofloxacin 298 Chromatogram of recovery study of cefixime and ofloxacin at

80% level

299 Chromatogram of recovery study of cefixime and ofloxacin at 100% level

300 Chromatogram of recovery study of cefixime and ofloxacin at 120% level

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LIST OF DENSITOGRAMS

Method I Development and validation of HPTLC method for the simultaneous determination of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride in oral solid dosage forms

1 Overlay densitogram of specificity studies 2 Overlay densitogram of linearity studies 3 Overlay densitogram of assay

4 Overlay densitogram of recovery studies 5 Overlay densitogram of system precision 6 Overlay densitogram of method precision 7 Densitogram of robustness (Standard) - R1 8 Densitogram of robustness (Standard) - R2 9 Densitogram of robustness (Standard) - R3 10 Densitogram of robustness (Standard) - R4 11 Densitogram of robustness (Standard) - R5 12 Densitogram of robustness (Standard) - R7 13 Densitogram of robustness (Standard) - R9 14 Densitogram of robustness (Standard) - R10 15 Densitogram of robustness (Sample) - R1 16 Densitogram of robustness (Sample) - R2 17 Densitogram of robustness (Sample) - R3 18 Densitogram of robustness (Sample) - R4 19 Densitogram of robustness (Sample) - R5 20 Densitogram of robustness (Sample) - R7 21 Densitogram of robustness (Sample) - R9 22 Densitogram of robustness (Sample) - R10

Method II Development and validation of HPTLC method for the simultaneous determination of chlordiazepoxide hydrochloride and imipramine hydrochloride in tablet dosage form 23 Overlay densitogram of specificity studies

24 Overlay densitogram of linearity studies 25 Overlay densitogram of assay

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28 Overlay densitogram of method precision 29 Densitogram of robustness (Standard) - R1 30 Densitogram of robustness (Standard) - R2 31 Densitogram of robustness (Standard) - R3 32 Densitogram of robustness (Standard) - R6 33 Densitogram of robustness (Standard) - R7 34 Densitogram of robustness (Standard) - R8 35 Densitogram of robustness (Standard) - R9 36 Densitogram of robustness (Standard) - R10 37 Densitogram of robustness (Sample) - R1 38 Densitogram of robustness (Sample) - R2 39 Densitogram of robustness (Sample) - R3 40 Densitogram of robustness (Sample) - R6 41 Densitogram of robustness (Sample) - R7 42 Densitogram of robustness (Sample) - R8 43 Densitogram of robustness (Sample) - R9 44 Densitogram of robustness (Sample) - R10

Method III Development and validation of HPTLC method for the simultaneous determination of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol in tablet dosage form

45 Overlay densitogram of specificity studies 46 Overlay densitogram of linearity studies 47 Overlay densitogram of assay

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60 Densitogram of robustness (Sample) - R2 61 Densitogram of robustness (Sample) - R4 62 Densitogram of robustness (Sample) - R5 63 Densitogram of robustness (Sample) - R6 64 Densitogram of robustness (Sample) - R7 65 Densitogram of robustness (Sample) - R8 66 Densitogram of robustness (Sample) - R9

Method IV Development and validation of HPTLC method for the simultaneous determination of cefixime & ofloxacin (Combination-1) and cefixime and ornidazole (Combination-2) in tablet dosage form

67 Overlay densitogram of specificity studies 68 Overlay densitogram of linearity studies

69 Overlay densitogram of assay of cefixime and ofloxacin (Combination-1)

70 Overlay densitogram of recovery studies of cefixime and Ofloxacin 71 Overlay densitogram of assay of cefixime and ornidazole

(Combination-2)

72 Overlay densitogram of recovery studies of cefixime and ornidazole

73 Overlay densitogram of system precision

74 Overlay densitogram of method precision of cefixime and ofloxacin (Combination-1)

75 Overlay densitogram of method precision of cefixime and ornidazole (combination-2)

76 Densitogram of robustness (Standard) - R1 77 Densitogram of robustness (Standard) - R2 78 Densitogram of robustness (Standard) - R3 79 Densitogram of robustness (Standard) - R4 80 Densitogram of robustness (Standard) - R7 81 Densitogram of robustness (Standard) - R9 82 Densitogram of robustness (Standard) - R10 83 Densitogram of robustness (Standard) - R11

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LIST OF TABLES

S.No. CHEMOMETRICS-ASSISTED UV SPECTROPHOTOMETRY 1 Factorial design for the calibration set of double combination

2 Factorial design for the calibration set of triple combination 3 Factorial design for the calibration set of tetra combination 4 Factorial design for the prediction set of triple combination 5 Factorial design for the prediction set of tetra combination

6 Composition of the calibration set of cefixime and ornidazole for PLS and PCR

7 Reference and predicted values of the validation set of cefixime and ornidazole by PLS and PCR

8 Statistical parameters of PLS and PCR for the calibration and prediction set of cefixime and ornidazole

9 Assay results of the combination-cefixime and ornidazole by PLS and PCR

10 Recovery study results of the combination-cefixime and ornidazole by PLS and PCR

11 Composition of the calibration set of cefixime and ofloxacin for PLS and PCR

12 Reference and predicted values of the validation set of cefixime and ofloxacin by PLS and PCR

13 Statistical parameters of PLS and PCR for the calibration and prediction set of cefixime and ofloxacin

14 Assay results of the combination-cefixime and ofloxacin by PLS and PCR 15 Recovery study results of the combination-cefixime and ofloxacin by PLS

and PCR

16 Composition of the calibration set of chlordiazepoxide hydrochloride and imipramine hydrochloride for PLS and PCR

17 Reference and predicted values of the validation set of chlordiazepoxide hydrochloride and imipramine hydrochloride by PLS and PCR

18 Statistical parameters of PLS and PCR for the calibration and prediction set of chlordiazepoxide hydrochloride and imipramine hydrochloride 19 Assay results of the combination-chlordiazepoxide hydrochloride and

imipramine hydrochloride by PLS and PCR

20 Recovery study results of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by PLS and PCR

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21 Composition of the calibration set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride & trifluoperazine hydrochloride for PLS and PCR

22 Reference and predicted values of the validation set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PLS

23 Reference and predicted values of the validation set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PCR

24 Statistical parameters of PLS and PCR for the calibration and prediction set of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

25 Assay results of the combination-trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PLS and PCR

26 Recovery study results of the combination-trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PLS and PCR

27 Composition of the calibration set of montelukast sodium and bambuterol hydrochloride for PLS and PCR

28 Reference and predicted values of the validation set of montelukast sodium and bambuterol hydrochloride by PLS and PCR

29 Statistical parameters of PLS and PCR for the calibration and prediction set of montelukast sodium and bambuterol hydrochloride

30 Assay results of the combination-montelukast sodium and bambuterol hydrochloride by PLS and PCR

31 Recovery study results of the combination-montelukast sodium and bambuterol hydrochloride by PLS and PCR

32 Composition of the calibration set of montelukast sodium and fexofenadine hydrochloride for PLS and PCR

33 Reference and predicted values of the validation set of montelukast sodium and fexofenadine hydrochloride by PLS and PCR

34 Statistical parameters of PLS and PCR for the calibration and prediction set of montelukast sodium and fexofenadine hydrochloride

35 Assay results of the combination-montelukast sodium and fexofenadine hydrochloride by PLS and PCR

36 Recovery study results of the combination-montelukast sodium and fexofenadine hydrochloride by PLS and PCR

37 Composition of the calibration set of montelukast sodium and rupatadine fumarate for PLS and PCR

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39 Statistical parameters of PLS and PCR for the calibration and prediction set of montelukast sodium and rupatadine fumarate

40 Assay results of the combination-montelukast sodium and rupatadine fumarate by PLS and PCR

41 Recovery study results of the combination-montelukast sodium and rupatadine fumarate by PLS and PCR

42 Composition of the calibration set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride for PLS and PCR

43 Reference and predicted values of the validation set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS

44 Reference and predicted values of the validation set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PCR

45 Statistical parameters of PLS and PCR for the calibration and prediction set of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

46 Assay results of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS and PCR

47 Recovery study results of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS and PCR

48 Composition of the calibration set of salbutamol sulphate, ambroxol hydrochloride and theophylline for PLS and PCR

49 Reference and predicted values of the validation set of salbutamol sulphate, ambroxol hydrochloride and theophylline by PLS

50 Reference and predicted values of the validation set of salbutamol sulphate, ambroxol hydrochloride and theophylline by PCR

51 Statistical parameters of PLS and PCR for the calibration and prediction set of salbutamol sulphate, ambroxol hydrochloride and theophylline 52 Assay results of the combination-salbutamol sulphate, ambroxol

hydrochloride and theophylline by PLS and PCR

53 Recovery study results of the combination-salbutamol sulphate, ambroxol hydrochloride and theophylline by PLS and PCR

54 Composition of the calibration set of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride for PLS and PCR

55 Reference and predicted values of the validation set of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS

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56 Reference and predicted values of the validation set of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PCR

57 Statistical parameters of PLS and PCR for the calibration and prediction set of pseudoephedrine hydrochloride , levocetirizine dihydrochloride and ambroxol hydrochloride

58 Assay results of the combination-pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS and PCR

59 Recovery study results of the combination-pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS and PCR

60 Composition of the calibration set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol for PLS and PCR

61 Statistical parameters of PLS and PCR for the calibration and prediction set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

62 Reference and predicted values of the validation set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by PLS

63 Reference and predicted values of the validation set of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by PCR

64 Assay results of the combination- levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by PLS and PCR

65 Recovery study results of the combination- levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by PLS and PCR

REVERSE PHASE-ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

66 System suitability parameters of bambuterol hydrochloride, levocetirizine dihydrochloride, ambroxol hydrochloride and montelukast sodium by RP-UHPLC

67 Linearity studies of bambuterol hydrochloride, levocetirizine dihydrochloride, ambroxol hydrochloride and montelukast sodium by RP-UHPLC

68 Assay results of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

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70 Assay results of the combination-montelukast sodium and bambuterol hydrochloride by RP-UHPLC

71 Recovery study results of the combination-montelukast sodium and bambuterol hydrochloride by RP-UHPLC

72 System precision of bambuterol hydrochloride, levocetirizine dihydrochloride, ambroxol hydrochloride and montelukast sodium by RP-UHPLC

73 Method precision of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

74 Method precision of the combination-montelukast sodium and bambuterol hydrochloride by RP-UHPLC

75 LOD and LOQ of bambuterol hydrochloride, levocetirizine dihydrochloride, ambroxol hydrochloride and montelukast sodium by RP-UHPLC

76 Factorial design table of montelukast sodium, bambuterol hydrochloride, levocetirizine dihydrochloriede and ambroxol hydrochloride by RP-UHPLC

77 Factorial response table of the combination-montelukast sodium and bambuterol hydrochloride by RP-UHPLC

78 Results of DOE analysis of the combination-montelukast sodium and bambuterol hydrochloride by RP-UHPLC

79 Factorial response table of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

80 Results of DOE analysis of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

81 System suitability parameters of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

82 Linearity studies of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

83 Assay results of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

84 Recovery study results of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

85 System precision of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

86 LOD and LOQ of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

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88 Factorial design table of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

89 Factorial response table of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

90 Results of DOE analysis of chlordiazepoxide hydrochloride and imipramine hydrochloride by RP-UHPLC

91 System suitability parameters of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

92 Linearity studies of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

93 Assay results of the combination-pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

94 Recovery study results of the combination-pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

95 System precision of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

96 Method precision of the combination-pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

97 LOD and LOQ of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

98 Factorial design table of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

99 Factorial response table of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

100 Results of DOE analysis of pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by RP-UHPLC

101 System suitability parameters of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

102 Linearity studies of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

103 Assay results of the combination-levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

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105 System precision of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

106 LOD and LOQ of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

107 Method precision of the combination-levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

108 Factorial design table of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

109 Factorial response table of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

110 Results of DOE analysis of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by RP-UHPLC

111 System suitability parameters of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

112 Linearity studies of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

113 Assay results of the combination-trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

114 Recovery study results of the combination-trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

115 LOD and LOQ of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

116 System precision of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

117 Method precision of the combination-trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

118 Factorial design table of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

119 Factorial response table of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

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120 Results of DOE analysis of trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by RP-UHPLC

121 System suitability parameters of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

122 Linearity studies of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

123 Assay results of the combination-salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

124 Recovery study results of the combination-salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

125 System precision of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

126 Method precision of the combination-salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

127 LOD and LOQ of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

128 Factorial design table of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

129 Factorial response table of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

130 Results of DOE analysis of salbutamol sulphate, ambroxol hydrochloride and theophylline by RP-UHPLC

131 System suitability parameters of montelukast sodium, rupatadine, fumaric acid and fexofenadine hydrochloride (ISTD) by RP-UHPLC

132 Linearity studies of montelukast sodium and rupatadine by RP-UHPLC 133 Assay results of the combination-montelukast sodium and rupatadine by

RP-UHPLC

134 Recovery study results of the combination-montelukast sodium and rupatadine by

RP-UHPLC

135 System precision of montelukast sodium and rupatadine by RP-UHPLC 136 LOD and LOQ of montelukast sodium and rupatadine by RP-UHPLC 137 Method precision of the combination-montelukast sodium and rupatadine

by RP-UHPLC

138 Factorial design table of montelukast sodium and rupatadine by RP-UHPLC

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140 Results of DOE analysis of montelukast sodium and rupatadine by RP-UHPLC

141 System suitability parameters of cefixime, ofloxacin and ornidazole (ISTD) by RP-UHPLC

142 Linearity studies of cefixime and ofloxacin by RP-UHPLC

143 Assay results of the combination-cefixime and ofloxacin by RP-UHPLC 144 Recovery study results of the combination-cefixime and ofloxacin by

RP-UHPLC

145 System precision of cefixime and ofloxacin by RP-UHPLC 146 LOD and LOQ of cefixime and ofloxacin by RP-UHPLC

147 Method precision of the combination-cefixime and ofloxacin by RP-UHPLC

148 Factorial design table of cefixime and ofloxacin by RP-UHPLC 149 Factorial response table of cefixime and ofloxacin by RP-UHPLC 150 Results of DOE analysis of cefixime and ofloxacin by RP-UHPLC

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY 151 System suitability parameters of montelukast sodium, levocetirizine

dihydrochloride and ambroxol hydrochloride by HPTLC

152 Linearity studies of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

153 Assay results of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

154 Recovery study results of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC 155 LOD and LOQ of montelukast sodium, levocetirizine dihydrochloride and

ambroxol hydrochloride by HPTLC

156 System precision of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

157 Method precision of the combination-montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

158 Factorial design table of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

159 Factorial response table of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

160 Results of DOE analysis of montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by HPTLC

161 System suitability parameters of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

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162 Linearity studies of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

163 LOD and LOQ of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

164 Assay results of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

165 Recovery study results of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

166 System precision of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

167 Method precision of the combination-chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

168 Factorial design table of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

169 Factorial response table of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

170 Results of DOE analysis of chlordiazepoxide hydrochloride and imipramine hydrochloride by HPTLC

171 System suitability parameters of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 172 Linearity studies of levocetirizine dihydrochloride, ambroxol

hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 173 Assay results of the combination-levocetirizine dihydrochloride,

ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC

174 Recovery study results of the combination-levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC

175 LOD and LOQ of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 176 System precision of levocetirizine dihydrochloride, ambroxol

hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 177 Factorial design table of levocetirizine dihydrochloride, ambroxol

hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 178 Method precision of the combination-levocetirizine dihydrochloride,

ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC

179 Factorial response table of levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by HPTLC 180 Results of DOE analysis of levocetirizine dihydrochloride, ambroxol

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181 System suitability parameters of cefixime, ofloxacin and ornidazole by HPTLC

182 Linearity studies of cefixime, ofloxacin and ornidazole by HPTLC 183 Assay results of the combination-cefixime and ofloxacin by HPTLC 184 Assay results of the combination-cefixime and ornidazole by HPTLC 185 Recovery study results of the combination-cefixime and ofloxacin by

HPTLC

186 Recovery study results of the combination-cefixime and ornidazole by HPTLC

187 Method precision of the combination-cefixime and ofloxacin by HPTLC 188 System precision of cefixime, ofloxacin and ornidazole by HPTLC 189 LOD and LOQ of cefixime, ofloxacin and ornidazole by HPTLC

190 Method precision of the combination-cefixime and ornidazole by HPTLC 191 Factorial design table of cefixime, ofloxacin and ornidazole by HPTLC 192 Factorial response table of the combination-cefixime and ofloxacin by

HPTLC

193 Factorial response table of the combination-cefixime and ornidazole by HPTLC

194 Results of DOE analysis of the combination-cefixime and ofloxacin by HPTLC

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LIST OF GRAPHS

CHEMOMETRICS-ASSISTED UV SPECTROPHOTOMETRY

Combination

I Cefixime and ornidazole

1 PLS calibration and prediction curves of cefixime 2 PLS calibration and prediction curves of ornidazole 3 PCR calibration and prediction curves of cefixime 4 PCR calibration and prediction curves of ornidazole

5 RMSEP values generated for cefixime and ornidazole by PLS 6 RMSEP values generated for cefixime and ornidazole by PCR Combination

II Cefixime and ofloxacin

7 PLS calibration and prediction curves of cefixime 8 PLS calibration and prediction curves of Ofloxacin 9 PCR calibration and prediction curves of cefixime 10 PCR calibration and prediction curves of Ofloxacin

11 RMSEP values generated for cefixime and ofloxacin by PLS 12 RMSEP values generated for cefixime and ofloxacin by PCR Combination

III Imipramine hydrochloride and chlordiazepoxide hydrochloride 13 PLS calibration and prediction curves of chlordiazepoxide

hydrochloride

14 PLS calibration and prediction curves of imipramine hydrochloride

15 PCR calibration and prediction curves of chlordiazepoxide hydrochloride

16 PCR calibration and prediction curves of imipramine hydrochloride

17 RMSEP values generated for imipramine hydrochloride and chlordiazepoxide hydrochloride by PLS

18 RMSEP values generated for imipramine hydrochloride and chlordiazepoxide hydrochloride by PCR

Combination

IV Trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride 19 PLS calibration and prediction curves of trihexyphenidyl

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20 PLS calibration and prediction curves of chlorpromazine hydrochloride

21 PLS calibration and prediction curves of trifluoperazine hydrochloride

22 PCR calibration and prediction curves of trihexyphenidyl hydrochloride

23 PCR calibration and prediction curves of chlorpromazine hydrochloride

24 PCR calibration and prediction curves of trifluoperazine hydrochloride

25 RMSEP values generated for trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PLS

26 RMSEP values generated for trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride by PCR

Combination

V Montelukast sodium and bambuterol hydrochloride 27 PLS calibration and prediction curves of montelukast sodium 28 PLS calibration and prediction curves of bambuterol

hydrochloride

29 PCR calibration and prediction curves of montelukast sodium 30 PCR calibration and prediction curves of bambuterol

hydrochloride

31 RMSEP values generated for montelukast sodium and bambuterol hydrochloride by PLS

32 RMSEP values generated for montelukast sodium and bambuterol hydrochloride by PCR

Combination

VI Montelukast sodium and fexofenadine hydrochloride 33 PLS calibration and prediction curves of montelukast sodium 34 PLS calibration and prediction curves of fexofenadine

hydrochloride

35 PCR calibration and prediction curves of montelukast sodium 36 PCR calibration and prediction curves of fexofenadine

hydrochloride

37 RMSEP values generated for montelukast sodium and fexofenadine hydrochloride by PLS

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Combination

VII Montelukast sodium and rupatadine fumarate

39 PLS calibration and prediction curves of montelukast sodium 40 PLS calibration and prediction curves of rupatadine fumarate 41 PCR calibration and prediction curves of montelukast sodium 42 PCR calibration and prediction curves of rupatadine fumarate 43 RMSEP values generated for montelukast sodium and

rupatadine fumarate by PLS

44 RMSEP values generated for montelukast sodium and rupatadine fumarate by PCR

Combination VIII

Montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

45 PLS calibration and prediction curves of montelukast sodium 46 PLS calibration and prediction curves of levocetirizine

dihydrochloride

47 PLS calibration and prediction curves of ambroxol hydrochloride

48 PCR calibration and prediction curves of montelukast sodium 49 PCR calibration and prediction curves of levocetirizine

dihydrochloride

50 PCR calibration and prediction curves of ambroxol hydrochloride

51 RMSEP values generated for montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS

52 RMSEP values generated for montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride by PCR

Combination IX

Salbutamol sulphate, ambroxol hydrochloride and theophylline

53 PLS calibration and prediction curves of salbutamol sulphate 54 PLS calibration and prediction curves of ambroxol

hydrochloride

55 PLS calibration and prediction curves of theophylline

56 PCR calibration and prediction curves of salbutamol sulphate 57 PCR calibration and prediction curves of ambroxol

hydrochloride

58 PCR calibration and prediction curves of theophylline

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60 RMSEP values generated for salbutamol sulphate, ambroxol hydrochloride and theophylline by PCR

Combination

X Pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride 61 PLS calibration and prediction curves of pseudoephedrine

hydrochloride

62 PLS calibration and prediction curves of levocetirizine dihydrochloride

64 PCR calibration and prediction curves of pseudoephedrine hydrochloride

65 PCR calibration and prediction curves of levocetirizine dihydrochloride

67 RMSEP values generated for pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PLS

68 RMSEP values generated for pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride by PCR

Combination

XI Levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol 69 PLS calibration and prediction curves of levocetirizine

dihydrochloride

71 PLS calibration and prediction curves of phenylephrine hydrochloride

72 PLS calibration and prediction curves of paracetamol

73 PCR calibration and prediction curves of levocetirizine dihydrochloride

75 PCR calibration and prediction curves of phenylephrine hydrochloride

76 PCR calibration and prediction curves of paracetamol

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78 RMSEP values generated for levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol by PCR

REVERSE PHASE - ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Method I Bambuterol hydrochloride, levocetirizine dihydrochloride, ambroxol hydrochloride and montelukast sodium

79 Calibration curve of bambuterol hydrochloride by RP-UHPLC 80 Calibration curve of levocetirizine dihydrochloride by

RP-UHPLC

81 Calibration curve of ambroxol hydrochloride by RP-UHPLC 82 Calibration curve of montelukast sodium by RP-UHPLC 83 Residual plot of bambuterol hydrochloride by RP-UHPLC 84 Residual plot of levocetirizine dihydrochloride by RP-UHPLC 85 Residual plot of ambroxol hydrochloride by RP-UHPLC 86 Residual plot of montelukast sodium by RP-UHPLC

Method II Chlordiazepoxide hydrochloride and imipramine hydrochloride

87 Calibration curve of chlordiazepoxide hydrochloride by RP-UHPLC

88 Calibration curve of imipramine hydrochloride by RP-UHPLC 89 Residual plot of chlordiazepoxide hydrochloride by RP-UHPLC 90 Residual plot of imipramine hydrochloride by RP-UHPLC

Method III Pseudoephedrine hydrochloride, levocetirizine dihydrochloride and ambroxol hydrochloride

91 Calibration curve of pseudoephedrine hydrochloride by RP-UHPLC

92 Calibration curve of levocetirizine dihydrochloride by RP-UHPLC

93 Calibration curve of ambroxol hydrochloride by RP-UHPLC 94 Residual plot of pseudoephedrine hydrochloride by RP-UHPLC 95 Residual plot of levocetirizine dihydrochloride by RP-UHPLC 96 Residual plot of ambroxol hydrochloride by RP-UHPLC

Method IV Levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

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98 Calibration curve of ambroxol hydrochloride by RP-UHPLC 99 Calibration curve of phenylephrine hydrochloride by

RP-UHPLC

100 Calibration curve of paracetamol by RP-UHPLC

101 Residual plot of levocetirizine dihydrochloride by RP-UHPLC 102 Residual plot of ambroxol hydrochloride by RP-UHPLC 103 Residual plot of phenylephrine hydrochloride by RP-UHPLC 104 Residual plot of paracetamol by RP-UHPLC

Method V Trihexyphenidyl hydrochloride, chlorpromazine hydrochloride and trifluoperazine hydrochloride

105 Calibration curve of trihexyphenidyl hydrochloride by RP-UHPLC

106 Calibration curve of chlorpromazine hydrochloride by RP-UHPLC

107 Calibration curve of trifluoperazine hydrochloride by RP-UHPLC

108 Residual plot of trihexyphenidyl hydrochloride by RP-UHPLC 109 Residual plot of chlorpromazine hydrochloride by RP-UHPLC 110 Residual plot of trifluoperazine hydrochloride by RP-UHPLC Method VI Salbutamol sulphate, ambroxol hydrochloride and

theophylline

111 Calibration curve of salbutamol sulphate by RP-UHPLC 112 Calibration curve of ambroxol hydrochloride by RP-UHPLC 113 Calibration curve of theophylline by RP-UHPLC

114 Residual plot of salbutamol sulphate by RP-UHPLC 115 Residual plot of ambroxol hydrochloride by RP-UHPLC 116 Residual plot of theophylline by RP-UHPLC

Method VII Montelukast sodium and rupatadine fumarate using fexofenadine hydrochloride as internal standard

117 Calibration curve of montelukast sodium by RP-UHPLC 118 Calibration curve of rupatadine by RP-UHPLC

119 Residual plot of montelukast sodium by RP-UHPLC 120 Residual plot of rupatadine by RP-UHPLC

Method VIII Cefixime and ofloxacin using ornidazole as internal standard 121 Calibration curve of cefixime by RP-UHPLC

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124 Residual plot of ofloxacin by RP-UHPLC

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Method I Montelukast sodium, levocetirizine dihydrochloride and ambroxol hydrochloride

125 Calibration curve of montelukast sodium by HPTLC

126 Calibration curve of levocetirizine dihydrochloride by HPTLC 127 Calibration curve of ambroxol hydrochloride by HPTLC 128 Residual plot of montelukast sodium by HPTLC

129 Residual plot of levocetirizine dihydrochloride by HPTLC 130 Residual plot of ambroxol hydrochloride by HPTLC

Method II Chlordiazepoxide hydrochloride and imipramine hydrochloride

131 Calibration curve of chlordiazepoxide hydrochloride by HPTLC 132 Calibration curve of imipramine hydrochloride by HPTLC 133 Residual plot of chlordiazepoxide hydrochloride by HPTLC 134 Residual plot of imipramine hydrochloride by HPTLC

Method III Levocetirizine dihydrochloride, ambroxol hydrochloride, phenylephrine hydrochloride and paracetamol

135 Calibration curve of levocetirizine dihydrochloride by HPTLC 136 Calibration curve of ambroxol hydrochloride by HPTLC 137 Calibration curve of phenylephrine hydrochloride by HPTLC 138 Calibration curve of paracetamol by HPTLC

139 Residual plot of levocetirizine dihydrochloride by HPTLC 140 Residual plot of ambroxol hydrochloride by HPTLC 141 Residual plot of phenylephrine hydrochloride by HPTLC 142 Residual plot of paracetamol by HPTLC

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1.

INTRODUCTION

Global healthcare market has highly evolved over the years. A number of medications to treat diseases and infections have grown at a tremendous pace. Still, the use of a single drug to treat the patient completely has not sufficed the requirements. Since the patient may present with a number of symptoms, the requirement to treat these symptoms with different medications still persists. This necessity has generated the idea of combination products (multi-component formulations)1 which have found acceptance from the doctors and currently, the multi-component formulations are also growing at a tremendous rate. The major reasons for the enormous growth of multi-component formulations are greater patient acceptability, multiple actions, increased potency, quicker relief and fewer side effects2.

The most prevalent as well as reference analytical methods currently available for the determination of various multi-component formulations are different chromatographic methods like HPLC, HPTLC, and GLC3-5. However, these methods are generally time-consuming, complex in nature, demands the need of expensive instruments and ultra-pure solvents. On the other hand, Spectrophotometry is a simple, cost effective and fast analytical method for performing quantitative analysis6-8_{. However, this method is not selective and} demands that the analytes in a given sample to have different spectrum with less degree of overlapping. Therefore, the analysis of the multi-component formulations demands simple, fast, accurate, precise and cost-effective analytical methods and therefore finding an alternative analytical technique or procedure has become crucial.

With the emergence of computer and statistics in chemistry, now the analytical chemists are able to solve the above analytical problems of complex mixtures through Chemometrics9.

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In an ideal world, the amplitude of the information should be much greater than the amplitude of the noise and in a more simple way, the information in the data can be related to the properties of the samples from which the data is collected. However, in the realistic world, the analysts are frequently forced to work with data that has almost as much noise as information or data whose information is related in complex way to the properties of interest which are not readily recognisable by a simple inspection of the data. The chemometric techniques help the analysts to do something worth with such kind of data.

These chemometric techniques help the analysts in many ways; 1. The noise can be removed as much as possible from the data.

2. Helps in extracting information as much as possible from the collected data.

3. Enable in making accurate predictions about unknown drug samples from the information11_.

As coined by Svante Wold in 1972, ‘Chemometrics is a mathematical and statistical tool for extracting information from large tables of measured chemical data or for designing chemical experiment to gain maximum information out of as few experiments as possible’12. The applications of chemometrics in analytical

chemistry are now enormous and its applications have revealed in spectroscopy13-14_{, chromatography}15-16_{, Near IR}17-18_{, spectrofluorimetry}19-20_and

other disciplines of analytical chemistry 21-22.

1.1 MULTIVARIATE CALIBRATION AND PREDICTION

Multivariate calibration has nowadays gained a notable position in modern analytical chemistry. Probably, it is the segment within chemometrics which has fascinated the most interest so far [for example, the authors like Marten and Naes, Brown and Sundberg have devoted text books and articles on it] 23.

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concentration levels of the different chemical components in the stream from UV measurements. The methods are therefore simply powerful10.

Multivariate calibration provides numerous benefits over univariate approaches: a. The multiple components can be analysed simultaneously. This is

impossible with univariate analyses. Thereby, the amount of time needed for method development can be reduced drastically.

b. It can also improve precision in prediction. Statistics show that when a measurement is repeated n times and calculation of a mean value will result in a reduction in the standard deviation of the mean by a factor of √n. This is commonly called as signal averaging.

c. Fault detection is also facilitated by multivariate calibration whereas this is impossible with univariate analysis. When an unknown interferent (another component) is present, errors occur since the instruments have a significant sensitivity to it. The presence of interferent results in the overestimation of the concentration of the analyte of interest. Additionally, the analyst would be unaware of the problem. There are two ways to avoid interferent problems in univariate measurement techniques; separation of the analytes of interest physically from all interferents and/or using of selective measurements (as in derivative spectroscopy). This classical approach may not work always for complex samples, or may become impractical in terms of time or cost required for analysis.

The presence of an interferent can be detected by means of multivariate techniques. But, the interferents’ detection can fail if, for example, an interfering species has the same relative response on the

Analytical method development and validation by chemometric approach for the marketed formulations.