cm bhaskar reddy

Dr C.M. BHASKAR REDDY

RAYALASEEMA UNIVERSITY KURNOOL

AP

INDIA MOBILE : 9966476194

SPECTROPHOTOMETRIC STUDIES ON SOME ADVANCED DRUGS AND PHARMACEUTICALS A thesis submitted to the RAYALASEEMA UNIVERSITY KURNOOL For the award of

DOCTOR OF PHILOSOPHY in FACULTY OF CHEMISTRY By

C.M. BHASKAR REDDY [Reg. No : PP CHE 0016 ] Under the Guidance of

Dr G.V. SUBBA REDDY PROFESSOR OF CHEMISTRY, HEAD & VICE-PRINCIPAL , JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR COLLEGE OF ENGINEERING ,PULIVENDULA 516 390,A.P, INDIA

RAYALASEEMA UNIVERSITY KURNOOL-518007 , A.P, INDIA MAY - 2017



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DECLARATION I declare that the thesis entitled “SPECTROPHOTOMETRIC STUDIES ON SOME ADVANCED DRUGS AND PHARMACEUTICALS" has been prepared by me under the Supervision of Dr.G G.V. Subba Reddy Professor & Head of Chemistry and Vice Principal, JNTUA College of Engineering, Pulivendula, YSR (Kadapa), District, Andhra Pradesh (India). No part of this thesis has formed the basis for the award of any degree or fellowship previously.

C.M. BHASKAR REDDY RESEARCH SCHOLAR DEPARTMENT OF CHEMISTRY RAYALASEEMA UNIVERSITY KURNOOL-518007, A.P, INDIA



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RESEARCH SUPERVISOR’S REPORT This is to certify that the thesis entitled SPECTROPHOTOMETRIC STUDIES ON SOME ADVANCED DRUGS AND PHARMACEUTICALS submitted to RAYALASEEMA

UNIVERSITY,

KURNOOL,

ANDHRA

PRADESH, by Mr. C.M. BHASKAR REDDY for the award of degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY is a record of bonafied research work carried out by him under my supervision. The thesis has reached the standard of the regulations for the degree and it has not been previously formed the basis for the award of any degree, diploma, associate ship, fellowship or any other similar title to the candidate or any other person(s).

SUPERVISOR Dr G. V. Subba Reddy Professor of Chemistry , Head & Vice Principal JNTUA College of Engineering Pulivendula -516 390 YSR Kadapa (Dist), A.P , INDIA

SIGNATURE OF RESEARCH SUPERVISOR



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ACKNOWLEDGEMENTS I express my deep sense of gratitude and sincere appreciation to Prof. G.V. Subba Reddy, Head and Vice-Principal, Department of Chemistry, JNTUACE Pulivendula, who is a open door to all the students for his inspiring guidance, Moral support, continuous encouragement. I am gratified by his guidance provided to me all the way through my research work and the time spent to listen and correspond to various ideas and thoughts. I sincerely thank the management of samskruti College of engineering & Technology, Hyderabad for providing exceptional infrastructural facilities. In this context, I appreciate the freedom of research given by Management of samskruti Group of institutions , Hyderabad I thank my father late sri C.C Muni Reddy who is always there for me, my mother C. Venkata Lakshumma for their constant encouragement and inspiration, my wife, K. Haritha, and daughters C.M. Vyshnavi Reddy and C.M. chandana Reddy for her patience in arranging all the activities on time and unreasonable support provided by my family members without that I could not have completed my project. I thank my daughters C.M. Vyshnavi Reddy and C.M. chandana Reddy for their cute smiles, through which my mind used to get refresh whenever I get tired. I thankfully acknowledge the support from my friends L.Tarunendu Sekhar principal cum correspondent of RTD Degree college, Proddatur, K.Rama mohan Reddy, K.V. Narasimha Reddy and Dr. M.L.N. sarma of C.B.I.T, Proddatur, Mr Raja Reddy correspondent of Bhavana jr. college, Proddatur, Mr Maheboob Sharief of Rajupalem. My heartfelt thanks to staff of Apollo Pharmacy, Hyderabad for his support. Last and for forever, I am thankful to the Almighty of god being with me and for his blessings.


RAYALASEEMA UNIVERSITY KURNOOL MOBILE : 9966476194

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ABSTRACT This present research work mostly focusing on the important analytical technique namely U.V. spectrophotometric studies of advanced and medicinally important compounds and its pharmaceutical formulations confiding in the present pharmaceutical era. This research work is entitled to develop precise, accurate, sensitive, and low cost U.V. spectrophotometric method . It is a fact that there is no pharma industry can develop or manufacture pure bulk drug without involvement of analytical techniques. The therapeutic activity of any advanced medicinally important bulk drug is exclusively depends on its purity. If the medicinal compound is not up to mark in its purity which develops side effects to the people who are treated with drugs. Therefore analytical techniques play a key role to overcome problems with impurities present in medicinal compounds in present pharmaceutical world. The current research work of author confronts nine advanced medicinally important drugs with comprehensive therapeutic activity in their pharmaceutical domain. Those drugs are Roxythromycin, Flucloxacillin, Ceftriaxone, Ganciclovir, Zanamvir, Emtricitabine, Efavirenz, Vinblastin and Nitazoxanide . These drugs were selected for U.V. Spectrophotometric analysis based on their pharmacological activity. The therapeutic and medicinal importance of the selective drugs for thr proposed research work is tabulated in table.1. Table:1 Name and the Medicinal importance of proposed drugs for the present investigation S.No

Name of the drug

Medicinal importance

1

Roxythromycin

Semi synthetic and macrolide anti biotic

2

Flucloxacillin

beta - lactam antibiotic

3

Ceftriaxone

cephalosporin antibiotic

4

Ganciclovir

antiviral agent

5

Zanamvir

first neuraminidase inhibitor

6

Emtricitabine

Anti Retroviral agent

7

Efavirenz

Anti Retroviral agent

8

Vinblastin

antineoplastic agent

9

Nitazoxanide

Anti Protozoan agent I



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drugs used in the present investigation are Roxythromycin is a advanced synthetic and macrolitic agent,

Flucloxacillin is the broad spectrum beta - lactam antibiotic,

Ceftriaxone is a semi synthetic and broad-spectrum 3rd generation cephalosporin antibiotic compound , Ganciclovir is an antiviral agent used to prevent number of viral diseases caused by a notorious virus called cytomegalovirus. Zanamvir is the first inhibitor of neuroaminadase. Emtricitabine is an Anti Retroviral agent, Efavirenz is also an excellent Anti Retroviral organic drug.

Vinblastin is an anti-mitotic,

antineoplastic organic compound and is used to cure some varieties of cancer in the human body. Nitazoxanide is the new anti protozoan drug and is used to eliminate, Helminthic parasites in the various organs of the human body.

Extensive Literature survey has been done on nine medicinally important drugs. It revealed that most of the reported methods with different techniques were time consuming, less accurate, expensive and not economical. All developed methods are simple, reliable, good reproducibility and validated according to latest guidelines of International Conference on Harmonization. Methods developed by author are routine check, quantification and purity of above said nine drugs. These methods are comprehensively used in pharma industry, quality control institutions, testing laboratories of pharmaceutical dosage forms, and other novel research disciplines like drug delivery systems in human beings etc.

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PREFACE Generally Active pharmaceutical Ingredient (API) is synthesized from industry or extract from plant or animal product, it should be having therapeutic activity without impurities. Active pharmaceutical Ingredient is converted into a pharmaceutical formulation form and which is more acceptable material as pharmaceutical dosage form to cure number of diseases through injection or swallowing by human beings. Basically drugs having medicinal value which influences pharmacological activities on body of human beings.

The peculiarity of

particular drug purity is depends on various pharmacological factors namely synthetic route, process of manufacturing, way of storage and transportation. In the present scenario no one can manufacture or synthesize 100% pure form of API or synthesis of pharmaceutical formulation from drugs get contaminated with various toxic materials, they can impact tremendously on their therapeutic activity and create more side effects to the human beings . To overcome this type of problems, analytical techniques given number of solutions to get pure pharmaceutical formulation from the pharma industry through research and development. Majority of drugs and pharmaceutical formulations play an important role to cure ,controlling and prevention of number of diseases of human beings. A common thing is observed, it is a fact a single drug is manufactured by various companies with different trade names. This type of system influences micro changes in drug composition and will have an adverse effect on various biochemical and physiological activities of the patient. It is more painful to the present R&D of pharma industry . In pharma industry quality play a pivotal role in every new product .Quality control is a systematic concept to develop new drug from pharma industry by using series of measurements to remove error and design new compounds through production, finally particular drug is send out with a symbol of OK stamp, which indicated standard of the drug. The quality of new drug or pharmaceutical formulation is systematically assured by quality control methods. To develop good laboratory practice in pharma industry needs some precise analytical methods to avoid contamination and maintain quality of the new drug. Many new drugs are entering into pharma market with impurities which causes side effects to the human beings. The author was evaluated through thorough investigation III



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of purity of various drugs after releasing into the market. The author was made an comprehensive

survey

of

reviewing

Pharmacopeias

like

United

States

pharmacopeia(USP), European pharmacopeia (UP), British pharmacopeia (BP) and Indian pharmacopeia (IP). A comprehensive literature survey was performed for analytical abstracts related to the proposed drugs and pharmaceuticals by utilising important websites of science direct, scirus, scopus, nature, dekker and various printed journals of national and international of repute. Further, some referred books namely

spectrophotometric method development and method validation

and

Guidelines namely ICH Stability guidelines (Q1), Method validation (Q2), and Impurities guideline (Q3) were also taken into consideration for better evaluation and justification. Quantitative analysis of medicinal compounds are performed to maintain and balance the proportion of the common essential components of drugs which is often referred as the “Related Substances” and “Assaying’’. The new Drug substances and finished pharmaceutical formulation form is subjected to methods of

quality control to

ascertain that its essential constituents of medicinally active drugs are within the specification limits. For specific determination of drugs it is therefore very much important to have a sensitive, precise , accurate and acceptable form of ‘’VALIDATED ANALYTICAL METHODS’’. The initial stage of drug processing is, the finalised finished product will be subjected to accelerated authentic stability conditions to know the life time of the medicinally active drug and to provide stability indicating methods within the given range . It is mandatory to determine a specific drug to meet all the requirements of ICH. Analytical methods play a key role for validation in the present scenario. It is the course of action by the analytical procedure shall be useful for its confirmation, specificity and suitability of its intended purpose. As per the directions of ICH Various variety of analytical parameters were given such as sensitivity, selectivity, precision, accuracy, linearity, specificity, ruggedness and robustness. This data shall also be a documentary evidence of every drug for the advanced technology transfer from any Research and Development laboratory of analysis to laboratory of Quality Control .

IV


Literature


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survey of medicinal compounds revealed that number of analytical

techniques had been available for the purity and assay of organic drugs namely spectrophotometric determination of pencillin by using ammonium vanadate by Abdul kalek1,indirect spectrophotometric determination of antibiotics by Sastry et al2 with haematoxylin as new reagent, induced gamma radiation effect on ampicillin by dessnekey3, selective calorimetric method of paracetamol with indophenol by Fogg4, simultaneous colorimetric determination of paracetamol &ibuprofen by Singhal5, extraction spectrophotometric determination of ascorbic acid byBiryuk6, DC polarographic determination of tetracyclins by Chatten et al7, electrochemical behaviour of diazepines by Fatmaimi and Sengun et al8,catalytic polarographic behaviour of 2-mercaptobenzothiazole by Dityan seva et al9, pulse polarography of cystin and its derivatives was developed by Mairessa10, HPLC method of adefovir dipivoxil with human plasma by Sparidans et al11, Electrochemical method for Levocetrizine by Paw et al12,Thin layer chromatography of silymarin by Abdel-salam et al13,Potentiometric determination of Enalapril maleate by Aboul Enein et al14, Capillary Electrophoresis of Gemifloxacin Mesylate by Elbashir et al

15

, Tritrimetric

analysis of Pantoprazole by N.v.Pimpodkar et al16 ,Fluorimetric analysis of Cetrizine by Melvaki and Seetharamappa et al17,Differential pulse polarographic analysis of Salicylic acid and Ascorbic acid by Matsuda and Kouji et al18, Derivative spectrophotometric analysis of Pencillin Derivatives by Yesilada and Akgul et al19,fluorimetry, colorimetry, elution techniques, HPLC, RP-HPLC, Electro analytical techniques, tritrimetric methods, potentiometric methods, gas chromatographic methods, bio-analytical methods, LCMS/MS, electrochemical methods, thin layer chromatography, and flotation processes are utilised for various drugs in literature in most of the journals. These methods are not reproducible and expensive .In the present investigation the author has taken to determine new methods of various selected advanced organic compounds by using U. V. SPECTROPHOTOMETRY. It is an advanced technique to analyse purity and assay of the selected drugs. This is versatile and reproducible. The sensitivity and selectivity of spectrophotometric method is depending on the nature of chemical reactions with solvent and reagent .

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TABLE OF CONTENTS Chapter No.

Name of the Chapter

Page No.

1.

Introduction

1-20

2.

Literature Survey

21-26

3.

Experimental Investigations

27-55

4.

Results and Discussion

56-108

5

Summary and conclusion

109-111

References

112-118

Research papers published

119-121

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CONTENTS IN DETAIL S. No.

Description Chapter – I

Page No.

General Introduction

Introduction- Basic Principles and Discovery of U.V. Spectrophotometry

1

Fig :1

U.V.Double beam Spectrophotometer

2

Fig :2

Schematic Representation of U.V. Spectrophotometry

3

Fig :3

Absorption cells for U. V. Spectrophotometry

4

1.2

Development of Analytical Procedure &Validation

5

Fig :4

Schematic Representation of Analytical Validation

5

1.2.2

Development of Analytical Method

6

Fig :5

Schematic Representation of Process of Analytical Validation

7

1.2.5

Analytical Parameters

8

1.2.6

Characters of Analytical Validation

8

1.2.7

Summary of Analytical Validation Procedure

10

Table : 2

Parameters & Types of Analysis of Analytical Validation

8

Table : 3

Types of Validation for Various Tests

10

Definition of Drugs

12

History of Drugs

12

Classification of Drugs

18

1.1

Chapter – II

Literature Survey

2.1

Roxythromycin

21

2.2

Flucloxacillin

22

2.3

Ceftriaxone

23

2.4

Zanamvir

23

2.5

Ganciclovir

24

2.6

Emtricitabine

24 VII



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2.7

Efavirenz

25

2.8

Vinblastin

25

2.9

Nitazoxanide

26

Chapter – III Experimental Investigation 3.I.1 Fig :3.I.1.1 Table : 3.I.1.1 3.I.1.6 3.I.1.7 Table :3.I.1.2

Roxythromycin

27

Detailed Chemical Structure of Roxythromycin

27

Detailed Chemical Data of Roxythromycin

28

Preparation of Standard Solutions of Roxythromycin Preparation of Sample Solutions of Roxythromycin

29 29

Optical Parameters of Roxythromycin

30

Flucloxacillin

30

Detailed Chemical Structure of Flucloxacillin

31

Detailed Chemical Data of Flucloxacillin

31

3.I.2.6

Preparation of Standard Solutions of Flucloxacillin

32

3.I.2.7

Preparation of Sample Solutions of Flucloxacillin

32

Optical Parameters of Flucloxacillin

33

Ceftriaxone

33

Detailed Chemical Structure of Ceftriaxone

33

Detailed Chemical Data of Ceftriaxone

34

3.I.3.6

Preparation of Standard Solutions of Ceftriaxone

35

3.I.3.7

Preparation of Sample Solutions of Ceftriaxone

35

Optical Parameters of Ceftriaxone

36

Zanamvir

36

Detailed Chemical Structure of Zanamvir

37

Detailed Chemical Data of Zanamvir

37

3.II.1.6

Preparation of Standard Solutions of Zanamvir

38

3.II.1.7

Preparation of Sample Solutions of Zanamvir

39

Optical Parameters of Zanamvir

39

Ganciclovir

40

Detailed Chemical Structure of Ganciclovir

40

3.I.2 Fig :3.I.2.1 Table :3.I.2.1

Table :3.I.2.2 3.I.3 Fig :3.I.3.1 Table :3.I.3.1

Table :3.I.3.2 3.II.1 Fig :3.II.1.1 Table :3.II.1.1

Table :3.II.1.2 3.II.2 Fig :3.II.2.1

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Table :3.II.2.1


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Detailed Chemical Data of Ganciclovir

41

3.II.2.6

Preparation of Standard Solutions of Ganciclovir

41

3.II.2.7

Preparation of Sample Solutions of Ganciclovir

42

Optical Parameters of Ganciclovir

42

Emtricitabine

43

Detailed Chemical Structure of Emtricitabine

43

Detailed Chemical Data of Emtricitabine

44

3.III.1.6

Preparation of Standard Solutions of Emtricitabine

44

3.III.1.7

Preparation of Sample Solutions of Emtricitabine

45

Optical Parameters of Emtricitabine

45

Efavirenz

46

Detailed Chemical Structure of Efavirenz

46

Detailed Chemical Data of Efavirenz

46

3.III.2.6

Preparation of Standard Solutions of Efavirenz

47

3.III.2.7

Preparation of Sample Solutions of Efavirenz

47

Optical Parameters of Efavirenz

48

Vinblastin

49

Detailed Chemical Structure of Vinblastin

50

Detailed Chemical Data of Vinblastin

50

3.IV.1.6

Preparation of Standard Solutions of Vinblastin

51

3.IV.1.7

Preparation of Sample Solutions of Vinblastin

51

Optical Parameters of Vinblastin

52

Nitazoxanide

52

Detailed Chemical Structure of Nitazoxanide

53

Detailed Chemical Data of Nitazoxanide

53

3.IV.2.6

Preparation of Standard Solutions of Nitazoxanide

54

3.IV.2.7

Preparation of Sample Solutions of Nitazoxanide

54

54Table :3.IV.2.2

Optical Parameters of Nitazoxanide

55

Table :3.II.2.2 3.III.1 Fig :3.III.1.1 Table :3.III.1.1

Table :3.III.1.2 3.III.2 Fig :3.III.2.1 Table :3.III.2.1

Table :3.III.2.2 3.IV.1 Fig :3.IV.1.1 Table :3.IV.1.1

Table :3.IV.1.2 3.IV.2 Fig :3.IV.2.1 Table :3.IV.2.1

Chapter – IV Result and Discussion 4.I.1

Roxythromycin

56 IX



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4.I.1.1

Determination of λ Max

56

4.I.1.2

Preparation of Calibration Curve

58

4.I.1.3

Validation of Method of Roxythromycin

60

4.I.1.8

Recovery studies of Marketed Formulations

61

Fig : 4.I.1.1

U.V.Spectrum of Roxythromycin

57

Fig : 4.I.1.2

Calibration Curve of Roxythromycin

59

Table : 4.I.1.1

Pecision & Accuracy of Roxythromycin

60

Table : 4.I.1.2

LOD and LOQ of Roxythromycin

61

Table : 4.I.1.3


61

Flucloxacillin

61

4.I.2.1


61

4.I.2.2


63

4.I.2.3

Validation of Method of the Drug

65

4.I.2.8


66

Fig : 4.I.2.1

U.V.Spectrum of Flucloxacillin

62

Fig : 4.I.2.2

Calibration Curve of Flucloxacillin

64

Table : 4.I.2.1

Pecision & Accuracy of Flucloxacillin

65

Table : 4.I.2.2

LOD and LOQ of Flucloxacillin

66

Table : 4.I.2.3


66

Ceftriaxone

67

4.I.3.1


67

4.I.3.2


69

4.I.3.3


71

4.I.3.8


72

Fig : 4.I.3.1

U.V.Spectrum of Ceftriaxone

68

Fig : 4.I.3.2

Calibration Curve of Ceftriaxone

70

Table : 4.I.3.1

Precision & Accuracy of Ceftriaxone

71

Table : 4.I.3.2

LOD and LOQ of Ceftriaxone

72

Table : 4.I.3.3


72

Zanamvir

73


73

4.I.2

4.I.3

4.II.1 4.II.I.1

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4.II.I.2


75

4.II.I.3


77

4.II.I.8


78

Fig : 4.II.I.1

U.V.Spectrum of Zanamvir

74

Fig : 4.II.I.2

Calibration Curve of Zanamvir

76

Table : 4.II.I.1

Precision & Accuracy of Zanamvir

77

Table : 4.II.I.2

LOD and LOQ of Zanamvir

78

Table : 4.II.I.3


78

Ganciclovir

79

4.II.2.1


79

4.II.2.2


81

4.II.2.3


83

4.II.2.8


84

Fig : 4.II.2.1

U.V.Spectrum of Ganciclovir

80

Fig : 4.II.2.2

Calibration Curve of Ganciclovir

82

Table: 4.II.2.1

Precision & Accuracy of Ganciclovir

83

Table: 4.II.2.2

LOD and LOQ of Ganciclovir

84

Table: 4.II.2.3


84

Emtricitabine

85

4.III.1.1


85

4.III.1.2


87

4.III.1.3


89

4.III.1.8


90

Fig : 4.III.1.1

U.V.Spectrum of Emtricitabine

86

Fig : 4.III.1.2

Calibration Curve of Emtricitabine

88

Table: 4.III.1.1

Precision & Accuracy of Emtricitabine

89

Table: 4.III.1.2

LOD and LOQ of Emtricitabine

90

Table: 4.III.1.3


90

Efavirenz

91

4.III.2.1


91

4.III.2.2


93

4.II.2

4.III.1

4.III.2

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4.III.2.3


95

4.III.2.8


96

Fig : 4.III.2.1

U.V.Spectrum of Efavirenz

92

Fig : 4.III.2.2

Calibration Curve of Efavirenz

94

Table: 4.III.2.1

Precision & Accuracy of Efavirenz

95

Table: 4.III.2.2

LOD and LOQ of Efavirenz

96

Table: 4.III.2.3


96

Vinblastin

97

4.IV.1.1


97

4.IV.1.2


99

4.IV.1.3


101

4.IV.1.8


102

Fig : 4.IV.1.1

U.V.Spectrum of Vinblastin

98

Fig : 4.IV.1.2

Calibration Curve of Vinblastin

100

Table: 4.IV.1.1

Precision & Accuracy of Vinblastin

101

Table: 4.IV.1.2

LOD and LOQ of Vinblastin

102

Table: 4.IV.1.3


102

Nitazoxanide

103

4.IV.2.1


103

4.IV.2.2


105

4.IV.2.3


107

4.IV.2.8


108

Fig : 4.IV.2.1

U.V.Spectrum of Nitazoxanide

104

Fig : 4.IV.2.2

Calibration Curve of Nitazoxanide

106

Table: 4.IV.2.1

Precision & Accuracy of Nitazoxanide

107

Table: 4.IV.2.2

LOD and LOQ of Nitazoxanide

108

Table: 4.IV.2.3


108

4.IV.1

4.IV.2

Chapter – V Summary and Conclusion

109-111

References

112-118

Research Papers Published

119-121

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CHAPTER I INTRODUCTION

CHAPTER 1 INTRODUCTION

1.1 Introduction and Basic principles of U.V. Spectrophotometry The term spectrophotometry is explained by the committee of optical chemical society of united states of America, is a measurement of exact radiant energy and utility of wavelength. The energy is frankly from emitting sources or transmitting absorbing chemicals. Spectrophotometry is the division of analytical chemistry very much mistreated by analytical scientist. It recompense in its simplicity of technique with accurate and precise results. Mainly some exceptions of the analytical researcher is concerned in the quantity of light engrossed by the Active Pharmaceutical Ingredient (API) and quantified the API by adopting optimization technique. Discovery of “U.V. Spectrophotometry: The spectrophotometer was discovered by American chemist A.O. Beckmann in the year 1939. It is a key instrument in pharma industry to detect impurities and functional groups of medicinal compounds generally used for therapeutically. U.V. spectrophotometry is an important

analytical

technique is based on the

measurement of the light absorbed or transmitted by a chromogen placed between a light source and light intensity measuring device U.V . spectrophotometry obeys the common law called beer-lambert’s law. According to this law, if a grin of monochromatic illumination is sending throughout a homogeneous solution of enthralling medium, the pace of drop off the strength of radiation with thickness of the absorbing medium is relative to the incident emission as well as the absorption of the solution. Lambert’s-beer principle is given by Absorbance=log(IO/I) =E.c.L here, IO = strength of incident brightness I= amount of light absorbed A= absorbance C=concentration of the solute used (mol dm-3) L= length of the sample used in the cell in cm E = Molar absorptivity (dm3 mol-1 cm-1) 1

As per the directions of this law, larger the amount of molecules competent of enthralling light of a specified wave length, the larger the scope of brightness absorption, so that it is the common essential principle of U.V. spectrophotometry. •

The analytic absorption spectroscopy in U.V. region of electromagnetic spectrum is extensively used for quantitative analysis of the field of pharmaceutical and biomedical analysis.

•

The prescribed unit used in u.v. visible spectroscopy is wave length expressed as nanometers (nm) .

•

The location of utmost absorbance of a height is chosen as λ max.

•

wave length in this region is divide into two ranges i.e 200-400 (ultraviolet) and about 400-800 nm ( visible region).

•

Measurement of chromophore in visible region has been extensively used.

•

Most of the chemical species do not posses suitable chromogenic properties and are made to react with a suitable reagent giving rise to coloured species.

Fig.1: Shimadzu U.V. double beam spectrophotometer

2

Fig.2: Schematic diagram of U.V. spectrophotometry Authenticity of Lambert’s- Beer Law ➢ The strength of the Lambert‘s-beer law is pretentious by quantity of factors . ➢ For single ray instruments the absorbance array for specific measurement should be below 0.3-0.6 units of absorbance, the most favourable range is o.45 units of absorbance. ➢ For double beam instruments the optimum range lies between 0.6 and 1.2 absorbance units.

Absorption cells or cuvetts ➢ For the visible area (400-700nm) harmonized couple of glass curvets are used. ➢ For U.V. region, fused form of silica cells have elevated transmittance in the array of 190-1000nm were engaged (glass has poor transmission properly in the ultraviolet region). ➢ In systematic analytical exertion, cells of

1 cm lane length were used,

however, cells of longer wave length (up to 10 cm) are available and employed in the case where the concentration of the substance to be determined is too low or the substance has low absorbance values. ➢ Cells to be used for spectrophotometric analysis have optical surface opposite to each other. 3

➢ For correct identification each pair of matched cells should be similarly marked i.e. highly concentrated absorbing cuvette as sample cell and former being implied as a reference cell and this orientation should be maintained during actual analysis Cleaning of cells ➢ If the cell contained aqueous solutions, it can be rapidly cleaned by repeated using with distilled water and then with methanol or methanol. ➢ Periodically the cells and stopper should be soaked in fresh solution of chromic acid followed by thorough rinsing with distilled water to restore their matched performance. ➢ Optical side of the cells should not be touched, preferably cleaned with soft Muslin cloth or photographic lens tissue.

Fig.3: Absorption cells for U.V. spectrophotometry

Solvents •

eminence of spectral capacity is directly pretentious by the variety and transparency of solvents were used.

•

Distilled water, alcohol, (methanol ,ethanol),chloroform and dilute solutions of acid and alkali are most commonly used solvent for spectrophotometric analysis . 4

•

The solvent should be free from UV absorbing contaminants and should not interact with the substance to be analysed.

•

Analytical grade of solvents namely Distilled water, alcohol, (methanol, ethanol) and chloroform were used throughout the analysis of selected drugs.

Buffer Phosphate buffer of pH 7.4 is used throughout the analysis. 1.2 General introduction on Development of Analytical Procedure and Validation20

1.2.1 Selection of Analytical Method The main aim of analyst is to develop ideal analytical method to the pharma industry. First stage in the selection or development of method is to be established. The selected analytical method must have the following parameters. ➢ As simple as possible. ➢ A method should be specific. ➢ Most productive, economical, and more convenient. ➢ As accurate and precise as required. ➢ analytical method to be fully optimised before transfer for validation of its characteristics such as accuracy,precision,sensitivity and ruggedness etc.

Fig.4: Schematic representation of analytical validation

5

1.2.2 Development of Analytical Method The fundamental ideas of for the development of analytical method as follows. ➢ The new medicinally compound or pharmaceutical formulation or dosage form may not be official in all the pharmacopoeias ➢ Use personal experience and knowledge of analytical chemistry. ➢ Try out well established procedures for the development of new method. ➢ Regarding survey of literature ,unfortunately in most of the reported methods, the information

about

various

parameters

of

the

method

(precision,accuracy,LOD,LOQ and interference).

1.2.3

Validation

A route involving authentication by laboratory study that procedure give precise and reproducible outcome for proposed analytical relevance in a verified and reputable sequence. The Analytical monitor of a pharmaceutical ingredient is indispensable to certify its safety or worth all over all phase of the ridge life, which means storage, use and distribution. Analytical method

of validation ensure that the selective and precise,more

advanced analytical method can give excellent and consistent results passable for intentional use. These principles are applicable both for pharmacoepial and pharmacoepial procedures being used. They are also applicable for examining physiochemical as well as biological procedures.

6

Fig.5: Schematic Representation of process of Analytical validation

1.2.4 Purpose for validation ➢ It enables scientists to communicate scientifically and effectively on technical matters. ➢ Setting standards of evaluation procedures for checking compliance and taking remedial measures. ➢ Closer interaction with pharmacopoeial forum to address analytical problems. ➢ Validation shall provide adequacy and reliability of a method or a procedure to convene the encoded criterion providing elevated extent of assurance of the identical level of excellence is constantly build keen on each limit of completed merchandise from lot to lot. ➢ Retrospective validation is useful for trend comparison of results.

7

1.2.5

Analytical parameters

Accuracy It relate to the proximity of experiment grades to the factual assessment i.e. appraise of correctness of analytic method. It is articulated as % resurgence by the evaluate of well-known /supplementary quantity of analyte in the linearity assortment. It is easily resolute by compare the grades with individuals obtain by means of an substitute technique which has already been validated. Precision It expresses as measure of concurrence among individual analysis grades when procedure /process is functional to a identical trial usually articulated as SD. It is a measure of degree of repeatability or reproducibility under normal conditions. Different characteristics employed for analytical validation given in table no: 2.

1.2. 6 CHARACTERISTICS OF ANALYTICAL VALIDATION Table.2: Parameters and Types of Analysis of Analytical Validation TYPES OF ANALYSIS Parameters

Assay

QT

QL

ASSAY

Precision

YES

YES

NO

YES

Accuracy

YES

YES

---------

-----------

Specificity

YES

YES

YES

------------

Linearity

YES

YES

NO

-----------

Range

YES

------------

-----------

Ruggedness

YES

YES

YES

YES

LOD

NO

NO

YES

-----------

LOQ

NO

YES

NO

------------

YES

Repeatability (under same conditions) accuracy of the system when continual by the same person, same analysis under same position of laboratory circumstances (any reagent or equipment ) surrounded by a short break of time , the only divergence being the trial. The exact repeatability is endorsed to any test modus operandi is vital to be confident by haulage out whole

8

break up purpose on divide sample of the identical consignment of substance under usual laboratory state of affairs . this is the criteria for compendia assay procedures. Reproducibility (under different conditions) Whatever the area under discussion is conceded out by poles apart analyst in diverse research

centres or labs by means of changed available equipments,chemical

reagents and laboratory establishments and on poles apart days and variability of analytical results as function of analyst,day-to-day tec. The prime requirement of reproducibility test results are ➢ The test must be based on test methods which do not allow different procedures or interpretation. ➢ The accuracy /precision of the test methods must be known in advance. This requires the adoption of of test methods which have been properly validated. ➢ Proficiency tests are required on a continuing basis to ensure that laboratories are maintaining satisfactory level of performance and may be used to generate reliable reproducibility data . Intermediate precision Within the same laboratory but different days ,analysts, equipments and reagents. Linearity provision of the

mode to reduce test grades that are steadfastly relative to the

attentiveness of anlyte.

Range ➢ Lowest and highest level of analyte that the method can determine with reasonable accuracy and precision in the range of 80/100/120% of the claim. ➢ Determination of least square method /regression line, slope of curve will provide RSD. ➢ Use of reference standard ➢ Analysis in duplicate ➢ Response –concentration curve on graph paper to establish Beer’s law --reaction to be linear on at slightest five to six point. ➢ Three point linearity curve in the concentration range of 80-120% of the claim gives an error of ± 1.0 % and may be resorted to. ➢ Slope of regression line - - measure of linearity ➢ Y intercept – measure of potential assay bias. 9

Ruggedness Extent of reproducibility of investigation grades obtain by analysing by the similar trial beneath array of customary test circumstances such as poles apart ➢ Days ➢ Analysts in the pharma industry ➢ Instruments used in industry ➢ Reagents used at present i.e., need of authority of ecological variables on the process. Assessment of reproducibility of analysis grades to the exactitude of examine is the nonstop gauge of ruggedness of the scheme. The summary of the analytical validation given in the table no: 3.

1.2.7 SUMMARY OF ANALYTICAL VALIDATION PROCEDURE Table:3. Types of validation for various Tests TYPE OF VALIDATION

TEST FOR

SPECIFICITY

INTERFERENCE

ACCURACY

LINEARITY AND RECOVERY

SENSITIVITY

LOD AND LOQ

PRECISION

REPEATABILITY,REPRODUCIBILITY RUGGEDNESS

Robustness It is the assess of the ability of the methodical method to stay on unaltered by tiny but premeditated variation in procedure. It provides a hint about changeability of the scheme during usual laboratory circumstances. Sensitivity Provision of the analysis route to trace little variation in concentration.

Specificity /selectivity Capacity of the process to determine exactly and in particular the analyte of attention in existence of matrix and other workings possible to be nearby in the trial milieu and 10

impurity, humiliation goods and other associated substance. Degree of agreement among results will explain specificity of the method. Limit of detection (LOD) Tiniest absorption of the analyte in the trial that the mode can notice but not essentially enumerate beneath the stated tentative circumstances simply indicates that the sample is below or above certain level. Limit test prescribed as percentage or as parts per million (ppm-mcg/ml). The Limit of detection not depends on the modus operandi of testing but also on the form of tool. A . Instrumental •

S/N 2 :1 or 3:1

•

2-3 times of SD of blank response

B. Non instrumental •

One has to establish the smallest level at which analytic are unfailingly detected ---usually Limit of detection is 2-3 times lower than LOQ.

•

Obviously LOD is influenced by absolute value of the blank and SD of the method of analysis.

The LOD is calculate by by means of the equation is LOD = of the SD,

3.s k

here, S = intercept

K = The slope of the calibration curve (mean).

Limit of quantitation (LOQ) It is define as the smallest attentiveness of the substance in a trial that can be estimated quantitatively with tolerable precision, accuracy and trustworthiness by a specified scheme underneath stated investigational conditions. The procedure usually followed is to analyse sample contain decreasing recognized quantity of the analytic material and determine the least level at which tolerable level of accuracy or precision is attained. However, it is preferable to validate the method at or near LOD. •

Instrumental: usually 10 times of standard deviation of blank response; 2-3 times higher than LOD.

•

Non Instrumental: one has to establish the level experimentally depending on the method of analysis employed.

The limits of quantitation (LOQ), is calculate by by means of the equation LOQ = 10 S K

here, S = intercept of the standard divergence K = the gradient of the calibration

curve (mean) 11

Definition of drugs The drugs play a key role in spectrophotometry, chemically drug means dry herb, it is a French word obtained from drogue. it is defined as “a substance is used for the prevention ,curing of various diseases in human beings as well as animals”. The world health organisation given a relevant definition for the drug is “an ingredient or chemical is utilized to correct various physiological, pathological problems of human beings. The ideal nature of drug is it should correct and satisfy, without side effects to an individual, without toxicity, should not injure other parts of the infected area, should not damage natural physiological system and functions of human body and work on particular, specific part of the human body only. In the present scenario only few drugs satisfy regulations and conditions of WHO. History of drugs In India ayurveda play a key role to cure number of diseases by using extracts of various plants,for example a product psoralea is obtained from plant namely psoralea corifolia to cure leucoderm, reserpine obtained from rawolfia serpentiana. India is the birth place of earliest ayurvedic scientists namely susruta,charaka and dhanwantari, and vaghbhatta they discovered number of ayurvedic medicines from Rig-Veda to cure variety of infections including liver problems extracted from plants. Iron is present in haemoglobin at very first Greek people utilised iron to cure anaemia and body weakness. in Egypt people of babylonia and asyria followed western type of medical system.Chinese people were used some medical preparations from their material medica during 2500-3000 B.C . In 450 B.C the discovery of first allopathic medical technique to cure diseases goes to Hippocrates a great Greek famous physician. He was the father of modern medicine. After 600 years Roman people namely the Gale of Pergamon conducted, comprehensive investigation related to animal physiology and anatomy by using various herbal extracts. They were also used some common elements like ores of cu, zn sulphates of iron and oxides of some cadmium salts, introduced the system of assaying of medical preparations to control number of diseases implemented dosage terminology. Later innovation of common alkaloid opium pills to cure coughs and to cure Gout disease with colchicum by Persian eminent scientists namely Avnnia and Rhazes, now this medication is still implemented in modern medicine also. Later years a noted Botanical scientist discovered tincture of morphine namely laudanum to get relief from pain, still it is used to cure deadly schistosomiasis. In 17th century 12

Europe people Jesuit and their allies used extracts of bark of cinchona officianalis to cure malaria; it was notified in Indian Ayurveda in the earliest. In 18th century England people used an useful extract obtained from plant namely fox-glove to cure dropsy disease,it is a heart related disease. In 19th century advanced medicine came to fight against number of unknown diseases, immunisation technique started against diseases like small pox, method of giving anaesthesia during surgery and introduction of antiseptics. End of 19th century most of the allopathic medical scientists discovered organisms responsible for diseases like typhoid, malaria, cholera, plague and dysentery. In chemistry, organic chemistry is driving force to study hormones, vitamins and synthesis of drugs to cure number of unknown diseases. Tremendous work has done in the field of chemotherapy, at the beginning sulpha drugs and some common antibiotics were developed. After the discovery of number of drugs and pharmaceuticals in the world, Scientists were optimised various physical, chemical, analytical, biological, biochemical and microbiological techniques for the therapeutic use. Enzymes play a key role in the field of drug action in human body, for past 30 years new therapeutic agents manufactured by various organic chemists, more magnificent advances were done in pharma industry. To overcome number of diseases in nature every individual using number of drugs discovered in pharma industry with knowledge of organic chemist. The systematic research has been carried out in various pharmaceutical laboratories tend to the development of numerous modern drugs in the present era. The research work related to discovery of new drugs is a continuous and spontaneous process in various research centres, universities and pharma industries.

Nature of synthetic drugs Chemical substances make a change in biological functions in human body through series of chemical reactions. Actually various molecules of drugs interacts with a specific site or molecule of body biological system. Some osmotic agents directly react with water molecules of animal body. Hormones also drugs they can synthesize directly in animal body.

Physical state of drugs and their nature Based on physical state of the drug they are of three types namely gaseous drugs(budacortand nitrous oxide),liquid drugs(menthol,chloroform,nicotine) and solid 13

drugs (tropine alkaloids,acetyl salicylic acid). For asthalic people some liquid drugs like theophylline and budacort prescribed in vapour state to inhale .most of the drugs are weak bases or acids. Some drugs sending into body through intravenously, some are intramuscularly administered, based on the patient condition drug may be given through injection or through swallowing. Size of the drug Drug size play a key role during prescription generally it varies from microgram (very small) to grams (very large ) the molecular weight of the drug between 0.5gms(ex :alprazolam-a sleep inducer) and 1000gms(ex : paracetamol-antipyretic). The lower limit of the drug in narrow range generally adjusted by as per the requirements of specific action on the exact site of the animal or human body by chemists .a specific drug have excellent fit to only one type of unique receptor of the physiological system. The shape and charge of ideal drug has unique to avoid its reaction to other receptos of the body. Peculiarity of the drug depends on molecular weight units, generally a drug molecule having hundred molecular weight units in size is acceptable in present pharma industry .any drug having more than thousand grams should not dissolve and diffuse readily in various organs of the human body. Some drugs reacts with food particles of the large intestine, to avoid this sending of drugs directly through intravenous or intramuscularly.

Shape of the drugs The chemical activity is depending on drug size, any drug molecule it should interact on particular receptor site, shape of any drug molecule is complementary to that of human body receptor site, by this way complementary locking takes place during physiological action of the drug under the influence of human body fluids. Reactivity of drugs and their receptors Physiological nature of human body is every foreign material interacts or reacts chemically or biochemically with receptors under the influence of chemical forces or chemical bonds between drug and human body. Chemical forces are of three types in human body they are covalent forces, forces of electrostatic attraction and hydrophobic forces. Covalent bonds are very strong in biological systems and also reversible. During interaction of receptor of the body and drug is electrostatic attractions are common because they can form strong chain linkages between charged ionic molecules due to presence of hydrogen bonds, weak dipole interactions like 14

vanderwaals forces. Generally electrostatic attractions are much weaker than the covalent bonds of the particular site of the human body. Interaction of some lipid soluble drugs

having hydrophobic ,they

are very weak .in human body cell

membranes of lipids interacts very closely with the lower surface of pockets of receptors . The peculiarity of the synthetic drug with their bonding is not much importance DRUGS-SOURCES 1. Drugs in plants Generally drugs are extracted from various parts of medicinal plant to cure number of diseases in human beings. According to a famous Greek physician Galen, preparation of drugs from plants are called ‘galenicals’ because plants which contain biologically active ingredients namely alkaloids, glycosides, gums and resins.

a. Alkaloids Organic compounds having plant bases contain nitrogen they can form organic salts in presence of acids, insoluble in polar solvent like water. Generally acidic salts of any alkaloid are easily dissolved in water. Right now some thousands of alkaloids are extracted from plant alkaloids. For ex : an anti cholinergic drug namely quinine derived from leaves of atropa species, a bronchodilator namely ephedrine obtained from ephedra vulgaris, an antihypertensive drug reserpine is extracted from roots of rawolfia serpentiana, a famous narcotic drug is derived from unripe capsule of papaver sominiferum.

b. Glycosides Plants which contain sugar and non sugar moiety combination are called glycosides with nucleus of cyclopentane-phenanthrene steroid. Heating of glycoside with any mineral acid undergo hydrolysis and splits off the sugar moiety. A glycoside which contains non residual part is called aglycone. For ex: digoxin is a famous glycoside to treat cardiac problems derived from the leaves of digitalis lantana. c. Oils Naturally oils are insoluble in water but easily soluble in organic solvents like menthol, chloroform,ethanol. Different oils extracted from different plants having 15

medicinal character can be divided into two division’s namely mineral volatile oils and fixed oils. 1. Volatile oils: oils derived from terpenoids are called volatile oils. Ex: ginger oil, coriander oil, eucalyptus oil, clove oil is used to cure pain from tooth decay. 2. Fixed oils: oils which contain esters of fatty acids and glycerol. Ex: mustard oil, ground nut oil, olive oil, castor oil d . Resins Organic compounds which obtain when oxidation of volatile oils. They are toxic in nature. Ex: colocynth and podophyllum e . Gums: Organic compounds derived from plants having colloidal nature and used as emulsifying agent. Ex: gum acacia, gum guggal, and agar-agar f. Drugs in tannins: Organic compounds derived from plants containing non-nitrogenous substances due to their action on mucous membrane with protective action.

2.

Drugs in animals: Some of the drugs derived from animals namely pepsin is an enzyme obtained from stomach of pigs, insulin is obtained from pancreas of pigs and sheep, thyroxine is an hormone obtained from thyroid gland of oxen.

3.

Drugs in human : Some of the drugs derived from human beings also. For Ex: growth hormones are derived from anterior side of pituitary gland, female hormone namely gonadotrophine is derived from pregnant women and immunoglobulins are derived from human blood.

4.

Drugs in microbes : Micro organisms are chief sources of antibiotics For Ex: famous antibiotic penicillin was derived from fungus namely pencillium notatum and p. chrysogenum by Alexander Fleming, common Antibiotics like neomycin, actinomycin and streptomycin is derived from family namely actinomycetaceae . 16

Drugs in minerals: number of metals and non-metals play a key role for the growth of human body and correct some diseases present in human beings. For Ex: sulphur and arsenic are used to cure leucoderm, iron is more effective element to retard anaemia ,for gastric problems aluminium and magnesium hydroxide, radio iodine , radium is used to cure cancer. Drugs through synthesis: most of the drugs obtained through R&D in

pharma

industry For Ex: ofloxacin, paracetamol,norfloxacin,nitazoxanide etc , some drugs derived from natural sources For Ex: pencillin, vinblastin, vincrystine, vapol-A, etc . Drugs – dosage forms: Synthesis of pharmaceutical formulations in pharma industry and supplied in various ways of administration to patients are called dosage forms. Different types of dosage forms as follows. Tablets Drugs available to the patients in the form of disc shaped solid pharmaceutical preparation form is called tablet. Generally not more than 0.5g, For Ex: paracetamol available in the trade names like crocin, metacin and ultragin. Tablets are of two types namely sugar –coated and enteric coated. Capsule Capsule is prepared from water soluble polymer called PHBV. Drugs available in powdered form are filled in capsule. For Ex: novomax is a capsule which contains amoxicillin. Injections Drugs available in liquid form for intramuscular or intravenous administration or parental administrations are called injections. Generally injections supplied in glass vials, plastic vials or bottles having large infusion. Some drugs available in powdered form diluted with suitable solvent. For Ex: for new born babies’ novomax available in injection form. Ointments Drugs available in semi solid pharmaceutical formulation are called ointment. Most of the ointments are antiseptic, analgesic. The common base in ointment is theobroma or gelatin of glycerine. For Ex: zincoderm is a common skin ointment which contains drug namely clobetasol and base.

17

Syrup 66gms of sugar in 100 ml of water is called syrup. Generally plant flavour is used as a vehicle for active ingredients. Most of the syrups are prepared for below 10 years children. For Ex: benadril is a common cough expectorant for people of all ages, kolQ is a common cold expectorant for children. Tinctures Drugs of vegetative origin which contains alcohol are called tinctures. Generally only 10% of the drug present in tincture. Inhalations Drugs are used to treat respiratory problems through nebuliser or inhaled are called inhalations. Lotions Drugs are used for topical applications in liquid form are called lotions. For Ex: ascobol is a common lotion used for skin problems. Enema Drugs are used to treat constipation problems in liquid form is called enema. For Ex: barium sulphate is used as liquid for enema. Generally every time only 100-200ml is acceptable for enema. CLASSIFICATION OF DRUGS Antibiotics Drugs derived from living fungal organisms are called antibiotics .While using in low concentrations they destroy DNA or RNA of microorganisms. Ex: levofloxacin, amoxicillin, flucloxacillin, ceftriaxone and roxythromycin. Antifungal Drugs are used against fungal infections of human body are called antifungal. Ex: ketoconazole, miconazole, clotrimazole, itraconazole , fluconazole. Antiparasitic drugs Drugs are used to prevent the growth of parasites present in human body. Ex: crotamiton, benzyl benzoate, albendazole, bephenium, niclosamide, nitazoxanide. Antibacterials Drugs which destroy DNA or RNA of

bacteria are called antibacterials.

Ex:clindamycin, clarithromycin, daptomycin, telavancin, vancomycin.

18

Antivirals Drugs which destroy DNA or RNA of virus are called antivirals. Ex: acyclovir, zanamvir, abcavir, foscarnet, trifluridine.

Anticancer drugs Drugs which destroy abnormal growth of multiple irregular cell divisionsof human beings. Ex: ambochlorin, anastrozole, bexarotene, busulfan, carmustin, vinblastine,vincristine. Cardiovascular drugs Drugs are used to influence mechanism of heart either depress or stimulate and work on cardiovascular system of human body. Ex: agrylin, ticagrelor , cardizem , corlopam , valsartan, atorostatin,rosostatin, enalapril, clopidogrel. Antiretrovirals Drugs are not kill Hiv virus but to prevent the destruction of immunity system of human

beings

are

called

antiretrovirals.

Ex:

lamivudine

,emtricitabine,

ritonauvir,lopinavir,stauvudine, efavirenz.

Anti inflammatory drugs Drugs are purely non steroidal and used to minimise pain and fever of human beings. Ex: naproxen, paracetamol, diclofenac sodium, etoricoxib, piroxicam, ibuprofen, nimsulide, thiocolchicide. CNS drugs Drugs are used to influence and work on spinal cord and brains of central nervous system are called drugs of central nervous system. Ex:

alprazolam ,aolpidem,

triazolam, ambobarbital, Phenobarbital. Hormones Chemical substances secreted to influence biochemical metabolism and regulate entire organs of human body are called hormones. Ex: estrogen,vasopressin, insulin, thyroxin,oxytocin.

19

Vitamins Chemical substances obtained from food to the growth of human body are called vitamins. Except D vitamin all other water soluble and fat soluble vitamins obtained from organic food. Ex: ergosterol , colcalciferol, ascorbic acid, retinol. Diagnostic agents Chemical substances are used to diagnosise and detect abnormalities of human body through scanning and x-rays are called diagnostic agents. Ex: phosphocol, corticotrophin.

20

loxilan, sincalide,

CHAPTER

II

LITERATURE SURVEY

CHAPTER

2

LITERATURE SURVEY The main objective of drug industry is to manufacture and analyse new compounds without side effects to human beings. Drug industry is mainly depending on qualitative analysis of advanced medicinal compounds with utmost care. In the present scenario advanced organic compounds can be analysed by new spectrophotometric methods by using Linear Regression analysis and Standard addition method. Most of the researchers used number of methods to analyse various medicinally important compounds and reported number of validated techniques in present pharmaceutical industry

2.1 ROXITHROMYCIN Reddy, M. N etal.21 developed a method for the determination of Roxithromycin by using potassium permanganate as reagent at 412 nm in the conc of 10-75 μg /mL. Jiang, H., etal.22

estimated a method for Roxithromycin by using 7, 7, 8, 8-

tetracyanoquinodimethane (TCNQ) as reagent u.v. detection at 743 nm in the conc of 0-55 μg/ mL. Li,H etal.23developed another method for Roxithromycin by using Alizarin as reagent in the conc of 0.2-18.0 μg/ mL maximum absorbance at 428 nm. Rajasekaran, A etal.24 determined a spectrophotometric method for Roxithromycin by using cresol red as reagent at 456 nm in the conc of 0.80 μg /mL. Suhagia, B. Etal.25 estimated a spectrophotometric method for Roxithromycin by using Marquis reagent at 495 nm in the conc of 15-25 μg/ mL. Zhao, G. Z.etal.26 developed a spectrophotometric method with charge transfer reaction for Roxithromycin by using Purpurin at 544 nm in the conc of 0-120 μg /mL. Shobana, E. etal.27 determined various new spectrophotometric methods

for

Roxithromycin with variety of reagents including Supracen violet 3B at 590nm in the conc of 5.0-60 μg/ mL, Vanillin at 500nm in the conc of 5.0-5.0 μg/ mL ,Tropaeolin at 490nm in the conc of 5.0-4.0 μg/ mL and

p-dimethylamino

benzaldehyde (PDAB) in the conc of 5.0-5.0 μg mL/at 500 nm. Sastry. C. S.etal.28 given a two new spectrophotometric methods for Roxithromycin in the conc of 2.5-12.45 μg/ mLat 760 nm by using Folin-Ciocalteu (FC) as reagent, 21

and Ferric chloride & 1,10-phenanthroline as reagent at 520nm in the conc of 2.54.0 μg /mL. Abdelmageed. O. H29 estimated spectrophotometric method for Roxithromycin in the conc of 1.0-10 μg/ mLat 542-544 nm by using Eosin Y as reagent in aqueous buffered medium.1Z Ayman A. Gouda etal30 developed a spectrophotometric method for Roxithromycin with Haematoxylin as reagent &Boric Acid at 587nm in the conc of 4-8 μg /mL. Swapna etal31 has done a spectrophotometric method for Roxithromycin at 410nm in the conc of 1-5 μg /mLin presence of reagent Ferric Chloride with solvents namely Distilled Water, 1N Hcl. Neif Rahmann Ahamad32 estimated a spectrophotometric method for Roxithromycin with Sulphuric Acid in the conc of 5-50 μg /mL at 485nm. Najma Sultana etal33 developed spectrophotometric method for Roxithromycin in the conc of 4-40μg /mL at 496nm in presence of reagent chloranilic acid. 2.2 FLUCLOXACILLIN A.S. Amin,etal34 developed a new simultaneous spectrophotometric method of flucloxacillin and dicloxacillin by using pyrocatechol violet as reagent. G.G. Mohamed, etal35 estimated spectrophotometrically two broad spectrum antibiotics namely Dicloxacillin and Flucloxacillin with potassium periodate as reagent. E.M. Abdel-Moety, etal36

determined a new spectrophotometric determination of

dicloxacillin and amoxicillin in binary mixture. A.

Ramirez

R.

Gutierrez,

etal37

estimated

High

performance

thin-

layerchromatography for the detection of erythromycin ,ampicillin, chloramphenicol, benzylpenicillin, flucloxacillin and in milk of cow. M. Fiorentino etal38 determined a spectrophotometric method of flucloxacillin by using ultra purified water as solvent and value of r2 is 50.0 to 100.0 μg /mL at 274nm and value of r2 is 0.9998. R. vijayalakshmi et al39 developed a spectrophotometric method of flucloxacillin by using bromo cresol green as reagent with chloroform in the conc of 0.5-2.5 μg/ mL at 433nm and value of r2 is 0.9996. Refat MS, El-Didamony et al40 determined a new Spectrophotometric method of flucloxacillin by using chloro anilic acid and DDQ as reagent at 476nm and value of r2 is 0.9979-0.9995, in the conc of16-80 μg/ mL. 22

M.Y.Emli et al 41 developed spectrophotometric method of flucloxacillin by using 2,4 - Dinitrophenol and 2,3- salicylic acid in the conc of 2.0-40 μg/mLat 446 nm, 435 value of r2 is 0.9992. Sudhasatyadey etal42 determined new spectrophotometric method of flucloxacillin by using Double distilled water and Caustic soda in the conc of 2-10 μg /mL r2 value of 0.9980 at 219nm. 2.3 CEFTRIAXONE S. A. Patel &Patel etal 43 developed a new spectrophotometric method of ceftriaxone by using Folin-Ciocalteu (FC) reagent in the conc of 2-36 μg /mL at 750 nm r2 value of 0.9926. M Jambulingam etal

44

estimated spectrophotometric method of ceftriaxone by using

5% Para dimethyl amino benzaldehyde as reagent with correlation coefficient of 0.9980 at 490.6nm in the concentration range of 5-25μg/ mL. C. Pasha, B. Narayana45 determined spectrophotometric method of ceftriaxone by using NaOH- Variamine blue as reagent in the conc of 0.2-0.7μg/ mL at 556 nm and r2 value of 0.9992. Ameen W. Qasim46 was given a new spectrophotometric method of ceftriaxone by usingFerric chloride as reagent at 485 nm r2 value of 0.9994 in the conc of 5-120μg/ mL. F.M.A. Rind etal47 determined spectrophotometric method of ceftriaxone by using 4dimethyl amino benzaldehyde as reagent at 397nm and r2 value of 0.9996 in the conc of 20-100μg /mL.

2.4 ZANAMVIR Sarisuta.N etal48 developed A Validated HPLC Method for Zanamivir by using Caco-2 cell culture model in presence of Acetonitrile as mobile phase &98%of double distilled water. G. D. Allen etal49 developed spectrometric technique of LCTM of zanamvir in the serum of human. Todd M. Baughman50 developed presence of zanamvir in plasma of monkey and rat by using HIC/TM technique of spectrometric method. M.Esinosa bosch et al51 developed colorimetric method of zanamvir Ravindra Reddy.Y et al52 developed a new RP-HPLC method to detect zanamvir in tablets by using ratio of Acetonitrile & potassium phosphate buffer pH 4.0. 23

Nevin E.R.K53 has developed

spectrophotometric method by using Acetonitrile

&water as solvent u.v. detection at 230nm. 2.5 GANCICLOVIR Pavagada J Ramesh etal54 developed Titrimetric analysis and Spectrophotometric method for

Ganciclovir Assay in Pharmaceuticl formulation by using P-

DMAB,cerium (iv) sulphate,perchloric acid in the conc of 2-10 μg /mL at460nm. Prakash,S.Sarsambi etal55 estimated ganciclovir by using Folin- ciocalteau reagent ,NaOH in the conc of 50.0-250.0 μg /mL at764,7nm. S. S. Prakash etal56 developed two New spectrophotometric methods for antiviral drug Ganciclovir by using p-dimethyl amino cinnamaldehyde at 524nm conc range of 10-50 μg/mL and another method by using

3- methyl-2-benzothiazolinone

hydrazone (MBTH) at 611.8 the concentration range of 50-250 μg mL-1 . T. Anil kumar etal57 determined selective and validated Spectrophotometric method of Ganciclovir by using Folin reagent &p-dimethyl amino benzaldehyde in the conc of 4-14 μg/mL &80-200 μg/mL

and u.v. detection at544nm,401

respectively. Usra I.S. Al-Neaimy etal58 developed a new Spectrophotometric method

of

Ganciclovir by using Quinalizarin reagent at560nm with conc of 1.0-20.0 μg /mL . 2.6

EMTRICITABINE

Q. B.Casal59 developed Chiral liquid chromatography with emtricitabine. S. Notari etal60 determined a new Liquid chromatographic analysis of emtricitabine. Syeda Kulsum etal61 were given a new spectrophotometric determination of Emtricitabine with Methanol-acetonitrile (70:30) in the conc of 2-20 μg/mL at 296nm. Heba. K.Ashour etal62

developed spectrophotometric method of Emtricitabine with

Distilled water at 228nm and 260nm in conc of 2-40 μg /mL . M.H. AbdelHay etal63 has given a spectrophotometric method of Emtricitabine with high purity distilled water in the conc 0.5-40 μg/ mL at 298.5nm . Nagaraju P.T etal64 has developed spectrophotometric analysis of Emtricitabine with methanol at 241.1 in the conc of5-30 μg/mL . P.Vishnu etal65 has estimated a spectrophotometric method of Emtricitabine with methanol in the conc. of 2-14 μg/mL&4-20 μg/mL at 302.17 and 306.88 respectively. K. D. Ingale etal66 developed a spectrophotometric method of Emtricitabine with methanol in the conc of 6-48 μg/ mL at281nm. 24

P. Ananda kumar etal67 determined spectrophotometric method of Emtricitabine with Double distilled water at 281nm in the conc of 4-24 μg m. 2.7 EFAVIRENZ B. Rajkumar etal68

determined the Reverse Phase-HPLC method to estimate

efavirenz. Sreelatha ,P.satish etal69 were developed spectrophotometric technique of Efavirenz with Beta naphthol,sodiumnitrite,methanol in the conc of 10.0-20.0μg /mL at 561nm. Dhara .S etal70developed a new spectrophotometric method of Efavirenz with methanol and phosphate buffer at254nm in the 247 conc of 2-12μg/mL Y.Anandakumar etal71 has determined a spectrophotometric method of Efavirenz with Methanol-sodium lauryl sulphate in the conc of 1-20μg /mL at 247nm. SomsubhraGhosh etal72 has given spectrophotometric determination of Efavirenz with DMSO at 264nm in the conc of 2-18μg/ mL. Prakash.V.Diwan etal73 estimated spectrophotometric method of Efavirenz with Mixture of water, acetonitrile, methanol, NaOH and Hcl in the conc of 10-40μg /mL at 247nm. Ajit .K. Nangare etal74 determined spectrophotometric method of Efavirenz with methanol at 248nm in the conc of 5-40μg /mL. Ajith etal75 estimated a new spectrophotometric method of Efavirenz with methanol in the conc of 5-40μg/ mL at 239nm. 2.8

VINBLASTIN

only one spectrophotometric method were reported byP.Nagaraja etal76 by using Diazotised dapsone (430 nm at conc range of 5-24)and ferric chloride in presence of Hydrochloric Acid (750 nm at conc range of 0.1-4). Gupta M.M etal77 developed high pressure liquid chromatographic method for the determination of mixture of drugs namely vinblastin,vincristine,vindoline and catharanthine with leaves of C.Roseus by using buffer of Glacial Acetic Acid of 0.5%, mixture of phosphate buffer of 0.1M and acetonitrile (MP) having c-18 columns,254 nm of U.V. observation. D.V. Singh etal78 developed another method using Rp-HPLC method of Catharanthus alkaloids with silica gel columns. Uniyal G.C etal79 A new hplc method developed using, Symmerty C18 column.

25

Zhou,

H.

etal80

developed

HPLC-mass

spectrometry

method

for

Vinca

Alkaloids.S.K.Volkov81 developed an innovative method of RP-HPLC for vinca species. H. I.Chu etal82 developed a new method of capillary zone electrophoresis for vincristine and vinblastine in plants of C.Roseus. Y. H.Choi etal83 developed an innovative method for the estimation vinblastine by using SFE technique from Catharanthus roseus. M .Verzele84 has developed conventional method of HPLC-UV for vinblastin. Y. Miura85 has developed conventional analytical method to remove impurities by using HPLC-UV for vinblastin . 2.9 NITAZOXANIDE Literature survey of Nitazoxanide reveals that Different spectrophotometric estimations were reported including by using Dimethyl Formamide &Phosphate Buffer by DeshPandey M.M etal86 , only Dimethyl Formamide by Shailendra Pandey etal87, Methanol by Madhuri. D .Game88, Singh H.P etal90 and Sunitha Sharma etal92 , Ethanol by Senthil Raja.M89 and Ferric Chloride & Ethanol by Sharma .S. etal91.

26

CHAPTER III EXPERIMENTAL

CHAPTER -III EXPERIMENTAL 3.I.1 ANTIBIOTICS Drugs are used to kill microorganisms like bacteria are called antibiotics. It is a natural compound derived from fungus. They are also called antimicrobial medicines, the first antibiotic penicillin discovered by Alexander Fleming in 1927. Ex : Roxithromycin , Flucloxacillin, Ceftriaxone, Ciprofloxacin, New Clarithromycin ,Cephalosporins, Cephalexin, Erythromycin ,Tetracycline, Amoxicillin 3. I.1. ROXITHROMYCIN 3. I.1.1 Introduction The chemical structure and chemical data of Roxithromycin depicted in Fig. 3.I.1.1 and in table 3.I.1.1 respectively.

Fig. 3.I.1.1 - Chemical structure of Roxithromycin Roxithromycin

is

a

semi-synthetic

antibiotic

14-membered-ring

macrolide

antibacterial drug, and it is clinically utilized to cure pneumonic problems, tonsillitis, allergic bronchitis in humans beings caused by Gram positive and negative bacteria. It is a Semi synthetic White solid and freely soluble in Methanol, Ethanol ,DMF, DMSO, and Deionised Water.

Roxithromycin was one of the new generation

erythromycin medicinal compounds. The amorphous Roxithromycin is synthesized by desolvation technique by using chloroform solvent. It is more effective to nullify common bacteria in human beings like legionella pneumophila.

27

Table 3.I.1.1 - Detailed chemical data of ROXITHROMYCIN (3R,4S,5S,6R,7R,9R,10E,11S,12R,13S,14R)-6{[(2S,3R,4S,6R)-4-(Dimethylamino)-3-hydroxy-6methyltetrahydro-2H-pyran-2-yl]oxy}-14-ethyl-7,12,13IUPAC Name

: trihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6dimethyltetrahydro-2H-pyran-2-yl]oxy}-10-{[(2methoxyethoxy)methoxy]imino}-3,5,7,9,11,13hexamethyloxacyclotetradecan-2-on .

Molecular formula Molecular mass

: C41H76N2O15

: 837.047 g/mol

Literature survey of Roxithromycin reveals that, almost all reported methods21-33were having tedious especially the present research work was more comfortable with the initial requirements.

The main aim of current research was to develop a new

spectrophotometric method of Roxithromycin by using solvent Deionised Water and phosphate buffer of pH 7.4. The developed method is validated according to LOD, LOQ, precision, linearity, accuracy, as per the requirements of ICH guidelines. 3.I.1.2 Experimental 3. I.1.2 Instrument and chemicals: Double beam

U.V. Spectrophotometer (1700-shimadzu) is used having matched 1

cm quartz cell. Chemicals were used in this proposed method are purely A.R. grade and procured from Merck India Ltd, Hyderabad. Pharmaceutical formulation of Roxithromycin

was

procured

from

Cipla

Pharmaceuticals

Ltd,Hyderabad.

Commercially available tablets namely Rotip(75mg) and Roxyfin(75mg) procured from Medwin pharmacy, Hyderabad, 3.I.1.4 Selection of Solvent: Deionised Water and phosphate buffer of pH 7.493 are used throughout the analysis.

28

3.I.1.5. Selection of Method and Wave Length: UV scan range of 380 nm to 500 nm was selected for the proposed method of Roxithromycin . The wavelength corresponding to maximum absorbance was found at 420 nm and the curve of calibration is taken at 420nm. The intercept of calibration line of this drug is estimated by using technique namely linear regression Analysis. 3.I.1.6 Preparation of standard stock solutions of Roxithromycin: The 100 milligrams of standard purified organic drug of Roxithromycin is weighed accurately and dissolved in 100ml Deionised water then transferred into 100 ml volumetric flasks to prepare 1000 μg/ mL94stock solution of Roxithromycin .Then to get different aliquots of 2ml to7 ml of standard stock solutions of this drug is transferred into sequence of six 10 ml volumetric flasks and prepared upto mark by adding De-ionized water to get the given concentration range of Beer’s law .To each flask 2mL of phosphate buffer of pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 380 nm to 500 nm to determine maximum absorbance of this method .The curve of calibration was plotted at the conc range of 20-70 μg/ mL. The wave length corresponding to maximum absorbance of Roxithromycin

measured at 420nm against Deionised

water as blank.

3. I.1.7. Preparation of sample solution of Roxithromycin: For the analysis of Roxithromycin two commercial brands namely Rotip (75mg) and Roxyfin (75mg) tablets were purchased from Medwin pharmacy, Hyderabad .Twenty tablets of the drug was weighed accurately and powdered, then 100 mg of the drug in powdered form dissolve in 100ml of Deionised water and sonicated for 5-10 minutes and filtered by using whatmann filter paper having code No.42. The filtrate of 10μg/mL concentration is taken in a six 10milli litre capacity volumetric flasks. After some time, to each 10 ml flask 2mL of phosphate buffer pH 7.4 solution is added. Then absorbance of Roxithromycin measured at 420nm against Deionised water as blank

29

Table 3.I.1.2 Optical Parameters of Roxithromycin S.N0

Parameter

Roxythromycin

1

λ Max (nm)

420

2

Beer-lambert’s Law Limit (µg/mL)

20-70

3

Correlatio coeffcient (r2)

0.9840

4

Regression Equation

0.017X-0.0860

5

Intercept (a)

0.0860

6

Slope (c)

0.017

7

SD

18.7082

8

Mean

45

9

Variance

350

10

LOD (%)

1.313

11

LOQ (%)

3.980

(Y=a+bc)

3.I.2. FLUCLOXACILLIN 3. I.2.1 Introduction Flucloxacillin is a broad spectrum antibiotic compound. Antibiotic, belonging to the isoxazolyl family of beta -lactam penicillins. It is an effective drug to cure diseases caused by pencillinase –resistant staphylococci. This is a crystalline white powdered compound and freely soluble in methanol, ethyl alcohol and water. The chemical formula, IUPAC name and calculated molecular weight of this drug is shown in table: 3.I.2.1 The molecular structure of Flucloxacillin was shown in fig: 3.I.2.1 Literature survey of Flucloxacillin reveals that, almost all reported methods34-42were having tedious especially the present research work was more comfortable with the initial requirements.


spectrophotometric method of Flucloxacillin by using chloroform, double distilled water (1:1) in presence of phosphate buffer of pH 7.4. The developed method is 30

validated according to LOD, LOQ, precision, linearity, accuracy, as per the requirements of ICH guidelines. All reported methods are not precise, simple, accurate and less expensive. Therefore, an attempt was made to develop a low cost, precise, economical, sensitive, routine and accurate U.V. spectrophotometric analysis for the quantification of Flucloxacillin in the bulk drugs and pharmaceutical ingredients.

Figure : 3.I.2.1 Structure of Flucloxacillin

Table: 3.I.2.1 - Detailed chemical data of Flucloxacillin IUPAC Name

: (2S, 5R, 6R)-6-({[3-(2-chloro-6-fluorophenyl)-5methylisoxazole -4-yl] carbonyl} amino)-3, 3-dimethyl-7oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid

Molecular

: C19 H17 Cl F N3 O5 S

formula Molecular

: 453.870 g/mol

mass

3. I.2.2 Experimental

3. I.2.3 Instruments and Apparatus : The spectrophotometric measurement of this drug was estimated by using UVdouble beam spectro photometer of 1700 shimadzu model having 1 cm matched Quartz cell , spectral band width is1 nm and is supported by UV win 5.0 software. 3.I.2.4 Reagents and Chemicals: All chemicals used in this analysis were AR grade. Chloroform

and double distilled water in presence of phosphate buffer of pH 7.4 31

were used as solvents and diluents throughout the analysis. Pharmaceutical formulation of flucloxacillin was supplied by M/S Sun pharmaceuticals, Mumbai (Maharastra) ,Chloroform, phosphate buffer with pH 7.4 and purified form of Double distilled water was purchased from M/S Merck India Ltd, Mumbai (Maharastra). This drug flucloxacillin was found commercially in the form of tablets namely Flox500 mg and Actinase 500mg were procured from Apollo pharmacy, Chennai. 3.1.2.3 Selection of Solvent:The buffer selected for Spectrophotometric estimation of flucloxacillin is phosphate buffer of pH 7.4, chloroform and double distilled water were used as solvents throughout the analysis. 3. I.2.5 Selection of Method and Wave Length: Analytical wavelength selected for the proposed method of flucloxacillin is 270 nm. The intercept of calibration line was determined by linear regression Analysis. 3. I.2.6 Preparation of Standard Solutions of Flucloxacillin: The 100 milli grams of standard (pure) form of flucloxacillin is weighed exactly and then transfer into 100 ml volumetric flasks to get 1000 μg/ mL of concentration by dissolving in chloroform solvent. The stock solutions of the drug is diluted with double distilled water to get 2, 4,6,8,10 and 12 μg/mL be taken in six 10 ml volumetric flasks and compose up volume by using double distilled water.To each flask 2mL of phosphate buffer of pH 7.4 solution is added. The curve of calibration was plotted in the conc of 2-12 μg/ mL for the determination of flucloxacillin in chloroform as a blank. UV scan range of lambda max (λmax) 200 nm to 800 nm was selected to determine maximum absorbance of the drug.

In this method the

wavelength corresponding to maximum absorbance was found at 270 nm. 3.I.2.7 Preparation of Sample Solutions of Flucloxacillin: For the analysis of flucloxacillin two commercial brands namely Flox (500 mg) and Actinase (500mg) tablets were procured from Apollo pharmacy, Chennai. Ten tablets of each brand of the drug was weighed accurately and powdered .100 mg of the drug in powdered form dissolve in 100 ml of chloroform and sonicated for some time and filtered by using whatmann filter paper No.42.The filtrate further treated with purified double distilled water to get10 μg/mL concentration of the drug is taken in a six 10 ml capacity volumetric flasks. To each volumetric flask 2mL of phosphate buffer with pH 7.4 solution is added then absorbance of flucloxacillin is measured at 270 nm

32

Table: 3.I.2.2 Optical Parameters of Flucloxacillin S.No

Parameter

Flucloxacillin

1

λMax (nm)

270nm

2

Limit of Beer’s Law (μg/ mL)

2-12

3

Correlation Coefficient(r2)

0.9730

4

Regression Equation (Y= a+bc)

Y=0.081X-0.054

5

Slope

(c)

0.0810

6

Intercept

(a)

0.0540

7

SD

3.3166

8

Mean

7

9

Variance

11

10

LOD (%)

0.122

11

LOQ(%)

0.409

3.1.3 CEFTRIAXONE 3. I.3.1 Introduction Ceftriaxone is a broad-spectrum Antibiotic, semi synthetic organic drug, and 3rd generation most curable cephalosporin. It is more useful for intravenous administration and excellent compound of intramuscular administration. It is more sophisticated compound to treat number of unwanted bacterial infections in human beings. It has a white coloured and yellowish crystalline powdered, freely dissolve in water. The coloured solution form of this drug is from light yellowish colour to light amber. The Iupac name, chemical formula and molecular weight of Ceftriaxone is shown in Table: 3.I.3.1. The molecular structure of Ceftriaxone was shown in Figure : 3.I.3.1

Figure: 3.I.3.1 Structure of Ceftriaxone 33

Table: 3.I.3.1 Detailed chemical data of CEFTRIAXONE (6R, 7R)-7-[2-(2-Amino-4-thiazolyl) glyoxylamido]-8-oxo3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-as-triazin-3IUPAC Name

: yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid, 72-(Z)-(O-methyloxime), disodium salt, sesquaterhydrate


: C18H16N8Na2O7S33.5 H2O

: 661.60 g/mol

Literature survey of Ceftriaxone reveals that, almost all reported methods43-47were having tedious especially the present research work was more comfortable with the initial requirements.


spectrophotometric method of Ceftriaxone by using chloroform, double distilled water (1:1) in presence of phosphate buffer of pH 7.4. The developed method is validated according to LOD, LOQ, precision, linearity, accuracy, as per the requirements of ICH guidelines. All reported methods are not precise, simple, accurate and less expensive. Therefore, an attempt was made to develop a low cost, precise, economical, sensitive, routine and accurate new U.V. spectrophotometric technique for the quantification of Ceftriaxone in the bulk drugs and many pharmaceutical formulations of the industry. 3. I.3.2 EXPERIMENTAL 3. I.3.3

Instruments and Apparatus:

The spectrophotometric analysis is done by U.V. double beam spectrophotometer of shimadzu (1700-model) with 1 cm matched quartz cell, spectral band width is1 nm and is supported by UV win 5.0 software.

3. I.3.4

Reagents and Chemicals: All chemicals used in this analysis were AR

grade. Chloroform and double distilled water in presence of phosphate buffer of pH 7.4 were used as solvents and diluents throughout the analysis. Pharmaceutical formulation of Ceftriaxone was supplied by M/S Sun pharmaceuticals, Mumbai 34

(Maharashtra), chloroform, Double distilled water, and phosphate buffer 7.4 pH was purchased from M/S Merck India Ltd, Mumbai (Maharastra). This drug was found commercially in the form of tablets namely Auxil (200 mg) and Belox (200 mg). 3. I.3.5 Selection of Solvent: The buffer selected for Spectrophotometric analysis of ceftriaxone was phosphate buffer of pH 7.4, chloroform and double distilled water were used as solvents throughout the analysis. 3. I.3.5 Selection of Method and Wave Length: Analytical wavelength of ceftriaxone is 290 nm is selected and suitable for the proposed method. The intercept of line of calibration is determined by linear regression Analysis. 3. I.3.6 Preparation of Standard Solution of Ceftriaxone: The 100 milli grams of standard (pure) form of ceftriaxone is weighed exactly and then transfer into 100 ml volumetric flasks to get 1000 μg/ mL of concentration by dissolving in chloroform solvent. The stock solution of the drug is diluted with double distilled water to get 4,6,8,10,12,14, and 16 μg/mL be in use in a seven 10 ml clean volumetric flasks and compose up volume with double distilled water. To each flask 2mL of phosphate buffer of pH 7.4 solution is added. The curve of calibration is plotted in the conc of 4-16μg/mL for determination of the drug in chloroform as a blank. UV scan range of lambda max (λmax) 200 nm to 800 nm was selected to determine maximum absorbance of the drug.

In this method the wavelength

corresponding to maximum absorbance was found at 290 nm.

3.I.3.7 Preparation of Sample Solutions of Ceftriaxone: For the analysis of ceftriaxone two commercial brands namely Auxil (200mg) and Belox (200mg) tablets were purchased from Apollo pharmacy, Chennai (TN).Ten tablets of each brand of the drug was weighed accurately and powdered .100 mg of the drug in powdered form dissolve in 100 ml of chloroform and sonicated for some time and filtered by using whatmann filter paper No.42. The filtrate formed is diluted with pure double distilled water to get 10μg/mL concentration of the drug. To each flask 2mL of fresh phosphate buffer (pH 7.4) solution is added, finally the absorbance of ceftriaxone measured at 290 nm.

35

Table: 3.I.3.2 Optical Parameters of Ceftriaxone S.No Parameter

Ceftriaxone

1

λMax (nm)

290nm

2


4-16

3

Correlation Coefficient(r2)

0.9880

4


Y=0.045X-0.049

5

Slope (c)

0.0450

6

Intercept (a)

0.0490

7

SD

3.8944

8

Mean

10

9

Variance

15.1666

10

LOD (%)

0.259

11

LOQ (%)

0.865

3.II ANTIVIRALS Drugs are used to cure viral infections of human beings are called Antivirals. Ex : Zanamvir,Ganciclovir,Acyclovir,Rimantidine,oseltamivir,Amantidine,Abcavir and Peramvir. 3.II.1 ZANAMVIR 3.II.1.1 INTRODUCTION Zanamivir is first neuroaminidase anti viral drug and excellent inhibitor to avoid number of viral infections in the human beings. The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.II.1.1. The colour of the drug is white. Crystalline in nature, freely soluble in polar solvent like water, phosphate buffer (pH 7.4) and methanol. The chemical structure of this drug is given in Fig: 3.II.1.1.

36

Fig : 3.II.1.1 The chemical structure of Zanamvir Literature survey of Zanamvir reveals that, almost all reported methods48-53 were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements.The main aim of current research was to develop a new spectrophotometric method of Zanamvir by using double distilled water (1:1) in presence of phosphate buffer of pH 7.4. The developed method is validated according to LOD, LOQ, precision, linearity, accuracy, as per the requirements of ICH guidelines Therefore, an attempt was made to investigate a low cost, more precise and more accurate spectrophotometric technique for the quantitative determination of Zanamivir in bulk and tablet form. Table: 3.II.1.1 Detailed chemical data of ZANAMVIR IUPAC Name Molecular Formula Molecular Weight

(2R,3R,4S)-3-(acetylamino)-4-Carbamimidamido-2: [(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran6-carboxylic acid : C12 H20 N4 O7

: 332.310 g/mol

37

3. II.1.2 Experimental

3. II.1.3. Instrument and Chemicals: The absorbance of the drug Zanamvir were carried out by using shimadzu company model 1700 UV spectrophotometer (double beam) having matched 1 cm quartz cell, 1 nm of spectral band width, supported by U.V win 5.0 software . All chemicals are AR grade, Double distilled Water, and phosphate buffer (pH 7.4) is used throughout the analysis and purchased from Merck India Ltd, Mumbai.

Pharmaceutical

formulation of Zanamvir was supplied byLupin India Ltd, Hyderabad. Commercially available tablets namely Rebetol and Virazol procured from Medwin

pharmacy,

Hyderabad 3.II.1.4

Selection of Solvent:

Phosphate buffer (pH 7.4) and Double distilled water are used throughout the analysis. 3.II.1.5 Selection of Method and Wave Length: UV scan range of 200 nm - 400 nm was selected for the proposed method of Zanamvir . The wavelength corresponding to maximum absorbance was found at 260 nm and curve of calibration is taken at 260 nm. The intercept of calibration line of the drug was determined by linear regression Analysis

3. II.1.6

Preparation of Standard Solutions of Zanamvir:

The 100 milli grams of standard (pure) form of Zanamvir is weighed accurately and dissolved in 100 ml Double distilled Water then transferred into 100 ml volumetric flasks to make 1000 μg/ mL Zanamvir stock solution .Then different aliquots of 2- 10 μg/mL were taken in eight 10 ml clean volumetric flasks .To each volumetric flask 2mL of phosphate buffer with pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 200 nm to 400 nm to determine maximum absorbance for this method .The curve of calibration is plotted in the conc of 2-10 μg/ mL. The wavelength corresponding to maximum absorbance of Zanamvir

measured at 260nm with Double distilled Water against

blank.

38

3. II.1.7 Preparation of Sample Solutions of Zanamvir : For the analysis of Zanamvir , two commercial brands of Rebetol(10mg) & Virazol (10mg),tablets are obtained from Medwin pharmacy of Hyderabad. Twenty tablets of the drug was weighed accurately and powdered, then 100 mg of the drug in powdered form dissolve in 100 ml of pure Double distilled Water and sonicated for some time, filtered by using whatmann filter paper No.42. The filtrate having 10μg/mL concentration then it is diluted with pure Double distilled Water then taken in a eight 10 ml clean volumetric flasks. To each 10 ml volumetric flask 2mL of phosphate buffer with pH 7.4 solution is added. Then absorbance of Zanamvir calculated at 260 nm with Double distilled Water against blank.

Table: 3.II.1.2 Optical Parameters of zanamvir S.no Parameter

Zanamvir

1

λMax (nm)

2

Limit of Beer’s Law

3

Correlation Coefficient

4

260 (μg/ mL)

2-10

(r2)

0.9999

Regression Equation

0.1005x-0.0002

(Y= a+bc)

5

Intercept

(a)

6

Slope

7

SD

8

Mean

9

Variance

7.5

10

LOD (%)

0.0059

11

LOQ (%)

0.0199

(c)

0.0002 0.1005 2.7386 6

39

3. II.2 GANCICLOVIR 3. II.2.1 INTRODUCTION Ganciclovir is an antiviral agent. It is used to cure people infected with cytomegalovirus. This drug is used to medicate bone marrow transplants. It is used to treat complications from HIV associated cytomegalovirus diseases in various organs of human body .The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.II.2.1. It has a white coloured and crystalline in nature and freely dissolve in polar solvent like water and chloroform. The complete chemical structure of this drug is given in Fig: 3.II.2.1. Literature survey of Ganciclovir reveals that, almost all reported methods54-58were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements. The main aim of current research was to develop a new spectrophotometric method of Ganciclovir by using chloroform, double distilled water in presence of phosphate buffer of pH 7.4.. The developed proposed method is validated according to LOD, LOQ, precision, linearity, accuracy, as per the requirements of ICH guidelines Therefore, an attempt was made to develop a low cost, precise, accurate new U.V.spectrophotometric technique for the determination of Ganciclovir in bulk drug and pharmaceutical formulation form.

Fig : 3.II.2.1 The chemical structure of Ganciclovir 40

Table: 3.II.2.1 Detailed chemical data of GANCICLOVIR IUPAC Name Molecular formula Molecular mass

:

9-[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine

: C9H13N504

: 255.23 g/mol

3. II.2.2 Experimental 3.II.2.3 Instruments and Chemicals : The absorbance of the drug were carried out by using shimadzu company model 1700 U.V.spectrophotometer with double beam having matched1 cm quartz cell , 1 nm spectral band width and supported by U.V. win 5.0 software . All chemicals are AR grade. Chloroform, double distilled water, phosphate buffer with pH 7.4 is used throughout the analysis. Pharmaceutical formulation of Ganciclovir was supplied by Natco pharma, Hyderabad. Chloroform, double distilled water and phosphate buffer 7.4 was purchased from Merck India Ltd, Mumbai. Commercially available tablets namely Natclovir (100mg) , (100mg),

Neoclovir

procured from Apollo pharmacy, Hyderabad,

3.II.2.4 Selection of Solvent: Phosphate buffer of pH 7.4, Chloroform and double distilled water are used throughout the analysis. 3. II.2.5

Selection of Method and Wave Length:

UV scan range of 200 nm to 800 nm was selected for the proposed method of Ganciclovir. The wavelength corresponding to maximum absorbance was found at 420 nm and curve of calibration is taken at 420 nm. The intercept of calibration line of the drug was determined by linear regression Analysis. 3. II.2.6 Preparation of Standard Solutions of Ganciclovir : The 100 mg of standard (pure) drug of Ganciclovir

is weighed accurately and

dissolved in 100 ml chloroform solvent then transferred into 100 ml volumetric flasks to prepare 1000 μg/ mL stock solution of the drug .Then μg/mL are in use in eight

different flasks of 2-16

10 ml clean volumetric flasks and compose up exact 41

volume with double distilled water .To each flask 2mL of phosphate buffer of pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 200 nm to 800 nm to determine maximum absorbance for this method .The curve of calibration was plot in the conc of 2-16 μg/ mL. The wavelength corresponding to maximum absorbance of Ganciclovir measured at 420nm against chloroform as blank.

3.II.2.7

Preparation of Sample Solutions of Ganciclovir :

For the analysis of Ganciclovir, two commercial brands namely Natclovir (100mg) and Neoclovir (100mg) tablets were purchased from Apollo pharmacy ,Hyderabad (Telangana) Twenty tablets of the drug was weighed accurately and powdered , then 100 mg of the drug in powdered form dissolve in 100ml of chloroform and sonication for few minutes then filtered by using filter paper of whatmann No.42. Then filtrate formed is again diluted with double distilled water to get 10μg/mL concentration, taken in a ten 10 ml clean volumetric flasks. To each fresh 10 mlvolumetric flask 2mL of phosphate buffer with pH 7.4 solution is added. Then absorbance of Ganciclovir measured at 420nm against chloroform as blank. Table: 3.II.2.2 S.NO

Optical Parameters of Ganciclovir

PARAMETER (nm)

GANCICLOVIR

1

λMax

2


2-16

3

Correlation Coeffcient(r2)

0.9970

4


0.026X+0.027

5

Intercept (a)

0.027

6

Slope (c)

0.026

7

SD

4.47214

8

Mean

9

9

Variance

20

10

LOD (%)

0.311

11

LOQ (%)

0.103

420nm

42

3.III ANTI RETROVIRALS Drugs are used to prevent the growth of the Hiv viral infections are called Anti Retrovirals. Several drugs were available under this category such as Emtricitabine, Efavirenz, Cobicistat, Lamivudine, Didanosine, Saquinqvir ,Zidovudine, Tenofovir and Stavudine. Out of which based on the significance, Emtricitabine and Efavirenz drugs were chosen for the present investigation. 3.III.1 EMTRICITABINE 3.III.1.1 INTRODUCTION Emtricitabine is an Anti Retroviral agent. It is a specific nucleoside of synthetic drug. It can work effectively against HIV of type 1 in human beings. This drug is most curable inhibitor of reverse transcriptase .The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.III.1.1. It is white in colour freely dissolve in water, chloroform and methanol. The complete chemical structure of this drug is given in Fig: 3.III.1.1. Literature survey of Emtricitabine reveals that, almost all reported methods59-67were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements. The main aim of current research was to develop a new spectrophotometric method of Emtricitabine by using chloroform, double distilled water in presence of phosphate buffer of pH 7.4. The developed method is more suitable to validate according to guidelines of ICH parameters namely LOD, LOQ, linearity, accuracy, precision, and range. Therefore, an attempt was made to develop a low cost precise, accurate new method of spectrophotometry to determine presence of the drug Emtricitabine in bulk form and pharmaceutical ingredient.

Fig : 3.III.1.1 The chemical structure of Emtricitabine 43

Table: 3.III.1.1 Detailed chemical data of Emtricitabine IUPAC

:

Name Molecular Formula Molecular mass

5-fluoro-1-(2R,5S)oxathiolan-5-

[2-(hydroxymethyl)-1,3

yl]cytosine

: C8 H10 F N3 O3 S

: 247.240 g/mol

3. III.1.2 Experimental 3.III.1.3 Instruments and Chemicals: The absorbance of the drug were carried out by using shimadzu company model 1700 double beam U.V. spectrophotometer 1 nm spectral band width, and matched 1 cm quartz cell, is, supported by U.V win 5.0 software .All chemicals are AR grade. Phosphate buffer with pH 7.4, Chloroform and double distilled water are used throughout the analysis. Pharmaceutical formulation of Emtricitabine was supplied by Emcure pharmaceuticals, Pune, (Maharastra). Chloroform, double distilled water and phosphate buffer 7.4 purchased from Merck India Ltd, Mumbai. Commercially available tablets namely Emtriva (100mg) , Atripla (100mg),

3.III.1.4

procured from Apollo pharmacy Chennai (Tamilnadu).

Selection of Solvent:

Phosphate buffer with pH 7.4 Chloroform and double distilled water are

used

throughout the analysis. 3.III.1.5 Selection of Method and Wave Length: UV scan range of 200-400nm was selected for the proposed method of Emtricitabine.The wavelength corresponding to maximum absorbance was found at 298 nm and calibration curve was taken at 298 nm. The intercept of calibration line of the drug was determined by linear regression Analysis. 3. III.1.6

Preparation of Standard Solutions of Emtricitabine :

The 100 mg of standard (pure) drug of Emtricitabine is weighed accurately and dissolved in 100 ml chloroform solvent then transferred into 100 ml volumetric flasks to prepare 1000 μg/ mL stock solution of the drug. The different aliquots of 2, 4,6,8,10,12,14,16,18, and 20 μg/mL are in use in ten 10 ml clean volumetric flasks 44

and compose up exact volume with double distilled water .To each flask 2mL of phosphate buffer of pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 200 nm to 400 nm to determine maximum absorbance for this method .The curve of calibration was plot in the conc of 2-20 μg/mL. The wavelength corresponding to maximum absorbance of Emtricitabine is calculated at 298 nm with chloroform against blank.

3.III.1.7

Preparation of Sample Solutions of Emtricitabine :

For the analysis of Emtricitabine two commercial brands namely Emtriva(100mg) and Atripla(100mg)

tablets were purchased from Apollo pharmacy , Chennai

(TN).Twenty tablets of the drug was weighed accurately and powdered , then 100 mg of the drug in powdered form dissolve in 100 ml of chloroform and sonication for10-15 minutes then filtered by using No.42 whatmann filter paper. Then filtrate formed is again diluted with double distilled water to get 10μg/mL concentration, taken in a ten 10 ml clean volumetric flasks. Toward each 10 ml volumetric flask 2mL phosphate buffer (pH 7.4) solution is added. Then absorbance of Emtricitabine calculated at 298nm with chloroform against blank.

Table : 3.III.1.2 Optical Parameters of Emtricitabine : S.No

Parameter

Emtricitabine

1

λMax (nm)

298

2


2.0-20.0

3


0.9910

4

Regression Equation

Y=0.013X+0.049

(Y= a+bc) 5

Intercept

(a)

0.0490

6

Slope

(c)

0.0130

7

SD

5.627

8

Mean

11

9

Variance

31.6666

10

LOD (%)

0.129

11

LOQ (%)

0.432

45

3.III.2 EFAVIRENZ 3.III.2.1 INTRODUCTION Efavirenz is a non –nucleosidic Anti Retroviral agent. This drug is a excellent human immuno virus inhibitor of type-1 and reverse transcriptase, because it spoils the working of reverse transcriptase. So that it is also called Reverse transcriptase inhibitor .The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.II.2.1. It has a white colour and easily dissolve in chloroform and methanol. The chemical structure of this drug is given in Fig: 3.III.2.1. Literature survey of Efavirenz reveals that, almost all reported methods68-75were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements. The main aim of current research was to develop a new spectrophotometric method of Efavirenz by using chloroform in presence of phosphate buffer of pH 7.4. The developed and proposed process is validating according to the requirements of ICH parameters namely LOD, LOQ, accuracy, linearity and precision .Therefore, an attempt was made to develop a low cost precise, accurate new U.V. spectrophotometry for the determination of the drug Efavirenz in bulk material and pharmaceutical formulation.

Fig : 3.III.2.1 The chemical structure of Efavirenz

Table: 3.III.2.1 Detailed chemical data of Efavirenz IUPAC Name Molecular formula Molecular mass

:

(4S)-6-chloro-4- (cyclopropylethynyl)- 1, 4-dihydro-4 -(trifluoromethyl)- 2H-3,1-benzoxazin- 2-one

: C14 H9 Cl F3 N O2

: 315.70 g/mol

46

3. III.2.2 Experimental 3. III.2.3 Instruments and Chemicals: The absorbance of the drug Efavirenz was carried out by using shimadzu company model 1700 U.V.spectrophotometer (double beam) containing1nm spectral band width and matched 1 cm quartz cell, is, supported by U.V win 5.0 software. All chemicals used in this analysis are AR grade. Chloroform and phosphate buffer 7.4 is used throughout the analysis. Pharmaceutical formulation of Efavirenz was supplied by Emcure pharmaceuticals, Pune, (MH). Chloroform and phosphate buffer of 7.4 procured from Merck india Ltd, Mumbai. Commercially available tablets namely Estiva (100mg) and Stocrin (100mg) procured from Apollo pharmacy Chennai (TN). 3.III.2.4

Selection of Solvent

Chloroform and phosphate buffer of pH of 7.4 are used throughout the analysis. 3. III.2.5

Selection of Method and Wave Length

UV scan range of 200 - 400 nm was selected for the proposed method of the drug and the wavelength corresponding to maximum absorbance was found at 256 nm and calibration curve was taken at 256 nm.. The intercept of calibration line of the drug was determined by linear regression Analysis. 3. III.2.6

Preparation of Standard Solutions of Efavirenz

The 100 milli grams of standard (pure) form of Efavirenz is weighed exactly and then transfer into 100 ml volumetric flasks to prepare 1000 μg/ mL20 stock solution of the drug. The drug dissolved in chloroform solvent, to get the desired concentration of stock solutions the drug were diluted with double distilled water to get different samples of the drug from 4- 28 μg/mL are in use in a seven 10 ml clean volumetric flasks and compose up exact volume with double distilled water .To each flask 2mL of phosphate buffer 7.4 solution is added, then all dilutions of the drug were scanned in the UV scan range of lambda max (λmax) 200 nm to 400 nm to determine maximum absorbance of the method. The calibration curve is plot in the conc of 4-28 μg/ mL for the determination of Efavirenz in chloroform as blank. The wavelength of Efavirenz corresponding to maximum absorbance was found at 256 nm

3. III.2.7 Preparation of Sample Solutions of Efavirenz For the analysis of Efavirenz two commercial brands namely Estiva (100mg) and Stocrin (100mg), tablets were procured from Apollo pharmacy, Chennai (TN).Ten tablets of the drug weighed accurately and powdered. 100 mg of the drug in powdered 47

form dissolve in 100 ml of chloroform and sonication for 10-15 minutes then filtered by using whatmann filter paper No.42. The filtrate formed is again watery with pure double distilled water to make 10μg/mL concentration of the drug is taken in a seven 10 ml very clean volumetric flasks. Toward each volumetric flask 2mL of phosphate buffer (pH 7.4) solution is added. Then all dilutions of the drug were scanned in the UV scan range of lambda max (λmax) 200 nm to 400 nm to determine maximum absorbance of the method. The absorbance of Efavirenz calculated at 256 nm with chloroform against blank.

Table: 3.III.2.2 Optical Parameters of Efavirenz S.NO

Parameter

Efavirenz

1

λMax (nm)

256nm

2


4-28

3

Coefficient of Correlation (r2)

0.9970

4

Equation of Regression (Y= a+bc)

Y=0.018X+0.012

5

Intercept

(a)

0.0120

6

Slope

(c)

0.0180

7

SD

8.1547

8

Mean

16.4

9

Variance

66.5

10

LOD

0.135

11

LOQ

0.453

48

3.IV

ANTI CANCERS & ANTI PROTOZOALS

ANTI CANCERS : Drugs are used to prevent the growth of the cancerous cells in human beings are called Anti cancer drugs. Ex : Vinblastin,Plicamycin,Cladribine,Mitomycin ,Carboplastin,Paclitaxel,Dacarbazin,Busulfan and Mitotane. ANTI PROTOZOALS: Drugs are used to prevent the infections of protozoans in human beings are called Anti Protozoal drugs. Ex :Nitazoxanide,Iodoquinol,Yodoxin,Eflomithine, Metronidazole,Pentamidine and Furazolidine

3.IV.1 VINBLASTIN 3.IV.1.1 INTRODUCTION Vinblastine is an anti-mitotic, antineoplastic agent. It is also called drug of antimicrotubule is used to nullify certain varieties of cancer. The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.IV.1.1. It is a white coloured crystalline powder and easily dissolve in DMSO ,methanol and water. The complete chemical structure of the drug vinblastin is shown in Fig : 3.IV.1.1

.

Literature survey of vinblastin reveals that, almost all reported methods76-85were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements. The main aim of current research was to develop a new spectrophotometric method of vinblastin by using methanol in presence of phosphate buffer of pH 7.4. The developed method is validate according to parameters of ICH guidelines namely accuracy,linearity,LOD,LOQ,precsion and range. Therefore, an attempt was made to develop a low cost precise, accurate new method of U.V.spectrophotometry to estimate vinblastin in the bulk drug and pharmaceutical ingredient in industry.

49

Fig: 3.IV.1.1 The chemical structure of Vinblastin

Table: 3.IV.1.1 Detailed chemical data of Vinblastin dimethyl (2β,3β,4β,5α,12β,19α)-15-[(5S,9S)-5-ethyl-5hydroxy- 9-(methoxycarbonyl)-1,4,5,6,7,8,9,10IUPAC Name

:

octahydro-2H-3,7-methanoazacycloundecino[5,4b]indol-9-yl]-3-hydroxy-16- methoxy-1-methyl-6,7didehydro aspidospermidine-3,4-dicarboxylate


:

:

C46 H58 N4 O9

810.975 g/mol

3. IV.1.2 Experimental 3. IV.1.3 Instrument and chemicals : The absorbance of the drug were carried out by using shimadzu company model 1700 U.V. spectrophotometer (double beam) having 1 nm spectral band width and matched 1 cm quartz cell, supported by U.V win 5.0 software .All chemicals are AR grade. Methanol and phosphate buffer with pH = 7.4 is used throughout the analysis. Pharmaceutical formulation of

Vinblastin was supplied by vinkem labs, Chennai.

Methanol and phosphate buffer 7.4 purchased from Merck India Ltd, Mumbai. Commercially available tablets namely velban and Gemzar are procured from Apollo pharmacy, Hyderabad.

3.IV.1.4 Selection of Solvent: Methanol and phosphate buffer of pH 7.4 are used throughout the analysis.

50

3. IV.1.5

Selection of Method and Wave Length:

UV scan range of 350 nm to 470 nm was selected for this method and the wavelength corresponding to maximum absorbance was found at 420 nm.

The intercept of

calibration curve of the drug was determined by Linear regression Analysis. 3. IV.1.6 Preparation of Standard Solutions of Vinblastin: The 100 mg of standard (pure) drug of Vinblastin is weighed correctly and dissolve in Methanol with volume100 ml solvent then transferred into 100 ml volumetric flasks to prepare 1000 μg/ mL stock solution of the drug .Then to get different aliquots of 0.60-1.60 μg/mL are in use in six

10 ml clean volumetric flasks and compose up

correct volume with same solvent.To each flask 2mL of phosphate buffer of pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 350 nm to 470 nm to determine maximum absorbance for proposed method .The curve of calibration is plot in the conc of 6-16 μg/ mL. The wavelength corresponding to maximum absorbance of Vinblastin measured at 420nm against methanol as blank. 3.IV.1.7

Preparation of Sample Solutions of Vinblastin :

For the analysis of Vinblastin and

, two commercial brands namely Gemzar (10mg)

Velban (10mg),tablets were purchased from Apollo pharmacy ,Hyderabad .

Twenty tablets of the drug Vinblastin was weighed accurately and powdered , then 100 mg of the drug in powdered form dissolve in 100ml of methanol and sonication for 10 minutes then filtered by using whatmann filter paper No.42. The filtrate formed having 10μg/mL concentration, taken in a six 10 ml very clean volumetric flasks. To each 10 ml flask 2mL of phosphate buffer (pH 7.4) solution is added. Then absorbance of Vinblastin measured at 420nm against methanol as blank.

51

Table: 3.IV.1.2 Optical parameters of Vinblastin S.no

Parameter

Vinblastin

1

λMax

(nm)

420

2

Limit of Beer’s Law (μg/

6-16

mL) 3


0.9997

4

Regression Equation

0.0388x-

(Y= a+bc)

0.0139

5

Intercept

(a)

0.0139

6

Slope

(c)

0.0388

7

SD

3.3166

8

Mean

11

9

Variance

11

10

LOD (%)

0.282

11

LOQ (%)

0.854

3.IV.2.2 NITAZOXANIDE 3.IV.2.1 INTRODUCTION Nitazoxanide is anti protozoan synthetic drug and also called Helminthic parasitic agent. The IUPAC name, chemical formula and molecular mass of this drug is given in Table: 3.IV.2.1. It has a light yellow in colour easily dissolve in solvents namely DMSO, methyl alcohol and ethyl alcohol. The chemical structure of this drug is given in Fig: 3.IV.2.1. Literature survey of Nitazoxanide reveals that, almost all reported methods86-92were having time consuming, expensive. Especially the present research work was more comfortable with the initial requirements. The main aim of current research was to develop a new spectrophotometric method of Nitazoxanide by using Methanol in presence of phosphate buffer of pH 7.4. The proposed new method is exclusively validated as per the concise requirements of parameters namely LOD, LOQ, precision,linearity,accuracy and ruggedness . Therefore, an attempt was made to develop a low cost precise, accurate new U.V.spectrophotometric technique to estimate Nitazoxanide in bulk material and pharmaceutical formulation. 52

Fig : 3.IV.2.1 The chemical structure of Nitazoxanide

Table: 3.IV.2.1 Detailed chemical data of Nitazoxanide IUPAC Name Molecular formula Molecular mass

:

[2-[(5-nitro-1,3-thiazol-2-yl) carbamoyl]

phenyl]

acetate

: C12 H9 N3 O5 S

: 307.280 g/mol

3.IV.2.2 Experimental 3.IV.2.3 Instrument and Chemicals: The absorbance of the drug were carried out by using shimadzu company model 1700 U.V. double beam spectrophotometer containing matched 1 cm quartz cell, with 1 nm spectral band width and supported by U.V win 5.0 software . All chemicals are AR grade. Methanol and phosphate buffer (pH = 7.4) is used throughout the analysis. Pharmaceutical formulation of Nitazoxanide was supplied by Mankind pharmaceuticals, Hyderabad. Methanol and phosphate buffer 7.4 purchased from Merck India Ltd, Mumbai. Commercially available tablets namely Zindax and Paramix procured from Medwin pharmacy, Hyderabad, 3.IV.2.4 Selection of Solvent: Methanol and phosphate buffer of pH 7.4. Are used throughout the analysis.

53

3.IV.2.5 Selection of Method and Wave Length: UV scan range of 300 nm to 400 nm was selected for the proposed method of Nitazoxanide . The wavelength corresponding to maximum absorbance was found at 340 nm and curve of calibration is engaged at 340nm. The intercept of calibration line of the drug is estimated by a method called linear regression Analysis. w3.IV.2.6 Preparation of Standard Solutions of Nitazoxanide : The 100 mg of standard (pure) drug of Nitazoxanide is weigh perfectly and dissolve in 100 ml of solvent methanol then transferred into 100 ml volumetric flasks to prepare 1000 μg/ mL stock solution of Nitazoxanide .Then to get different aliquots of Nitazoxanide was prepared by pipetting out 0.4,0.6,0.8,10,12,14,16,18 and 20 m L were taken in nine 10 ml clean volumetric flasks. Toward each volumetric flask 2mL of phosphate buffer of pH 7.4 solution is added, then all stock solutions of the drug were scanned in the UV scan range of lambda max (λmax) 300 nm to 400 nm to determine maximum absorbance of this method .The curve of calibration is plotted in the conc of 4.0-20.0 μg/ mL. The wavelength corresponding to maximum absorbance of Nitazoxanide measured at 340nm against methanol as blank.

3.IV.2.7

Preparation of Sample Solutions of Nitazoxanide :

For the analysis of Nitazoxanide two commercial brands namely Zindax (50mg) and Paramix

(50mg),tablets were purchased from Medwin pharmacy ,Hyderabad

.Twenty tablets of the drug was weighed accurately and powdered , then 100 mg of the drug in powdered form dissolve in 100 ml of methanol and sonication for 10-15 minutes then filtered by using No.42 whatmann filter paper. The filtrate of 10μg/mL concentration is taken in a nine 10 ml clean volumetric flasks. To each 10 ml flask 2mL of phosphate buffer (pH 7.4) solution is added. Then absorbance of Nitazoxanide measured at 340nm against Methanol as blank.

54

Table: 3.IV.2.2 S.no

Optical Parameters of Nitazoxanide Parameter

Nitazoxanide

1

λMax (nm)

340 nm

2

Limit of Beer’s Law (μg/

4-20

mL) 3


0.9920

4

Equation of Regression

0.047X-0.0580

(Y= a+bc) 5

Intercept (a)

0.0580

6

Slope (c)

0.047

7

SD

5.4772

8

Mean

12

9

Variance

30

10

LOD (%)

0.384

11

LOQ (%)

1.165

55

CHAPTER IV RESULTS & DISCUSSION

4.I.1 ROXITHROMYCIN 4.I.1.1 Determination of λ Max : UV scan range of 380 nm to 500 nm was selected to determine maximum absorbance by using 10 μg/ml solution, this compound in phosphate buffer at pH =7.4. The blank solution was run for U.V.spectra under similar experimental conditions without active ingredient to notice for any interference. From the spectra it reveals that no response found with the blank solution. The wave length corresponding to greatest absorbance is established at 420nm for drug under investigation. The spectrophotometric spectrum of Roxithromycin is shown in Fig. 4.I.1.1

56

Fig. 4.I.1.1: UV Spectrum of Roxithromycin in Water Concentration=10 pH= 7.4

57

4.I.1.2 Preparation of Calibration Curve : The main intention of this parameter was to define that total analytical procedure employs a linear retort in a specified conc of the analyte. As per the current methodology the linearity was derived from 20-70μg/ mL of the test solution concentration. On the basis of experimental results, calibration curve is plot at the conc of 20.0-70.0μg/ mL of six standard solutions of Roxithromycin in Deionised water at 420nm with phosphate buffer having pH =7.4. The correlation coefficient is calculated and creates more than 0.9840. The calibration plot is shown in fig: 4.I.1.2 .

58

CALIBRATION CURVE OF ROXITHROMYCIN 1.4 y = 0.017x - 0.0860 R² = 0.9840

1.2

1.2

ABSORBANCE

1

1

0.8

0.8

0.6

0.6

0.4

Series1 Linear (Series1)

0.4

0.2

0.2

0

0

-0.2 0

20

40

60

80

CONCENTRATION µg/mL

Fig. 4.I.1.2 Calibration Curve of Roxithromycin

59

4.I.1.3 Validation of Method : The spectrophotometric estimation of

Roxithromycin is validated according to the

directions of ICH to determine parameters like LOD, LOQ linearity, accuracy and precision of the proposed method. 4.I.1.4 Linearity and Range : Standard stock solution of Roxithromycin in appropriate dilution were assayed as per the proposed method According to Beer’s –Lambert’s law the concentration range of Roxithromycin is set up to be 20-70 μg/mL ,So that the curve of calibration

in

the Fig. 4.I.1.2 is linear at the given concentration of beer’s law range. 4.I.1.5 Precision : The proposed system of Roxithromycin is estimated by using drug concentration of Roxithromycin were analyzed . Analyses of the drug through intraday precision, estimated 6 times on the day where as 6 spontaneous days for interday.The analytical data is given in the Table 4.I.1.1 4.I.1.6 Accuracy : Accuracy of this proposed routine of Roxithromycin

is estimated through method of

standard addition .This process is carried out by addition of dissimilar amount namely 80, 100, and 120percent of the pure sample of the drugwith pre-analysed formulation. Accuracy data of this drug is revealed in the Table 4.I.1.1

Table 4.I.1.1 Precision & Accuracy of Roxithromycin

S.No

Name of the Sample

Labeled

%

Amount (mg/Capsule)

Level

Amount Found* (mg)

Precision

Inter

Intra

Day

Day

1

Rotip

75

120

74.23

0.0061

0.0068

2

Roxyfin

75

80

74.01

0.0075

0.0082

4.I.1.7. LOD and LOQ The LOQ and LOD of the drug Roxithromycin is estimated (Table : 4.I.1.2) through slope and response mechanism of SD according to rules and regulations of ICH.The

60

formulae of LOQ

and LOD were discussed already in I chapter of subsection

introduction of Analytical validation process. Table 4.I.1.2 LOD & LOQ of Roxithromycin S.no

Parameter

Roxythromycin

1

LOD (%)

1.313

2

LOQ (%)

3.980

4.I.1.8. Recovery Studies of Roxithromycin : Recovery analysis of Roxithromycin is execute to know the correctness of the projected method . This process is done by addition of identified capacity of fresh drug to the pre-analysed sample. The recovery percentage of the title compound found to be 98.68-98.97 shows accuracy of the method. The result of analysis of the drug is notified in Table4.I.1.3 Table4.I.1.3 Recovery studies of Marketed Formulations of Roxithromycin

S.No

4.I.1.9

Name of the Sample

Labeled Amount (mg/Capsule)

Amount

%

Found

Recovery

1

Rotip

75

74.23

98.97

2

Roxyfin

75

74.01

98.68

conclusion

The proposed technique is established to be easy, perceptive, exact, specific and economically viable for day to day investigation. The feasibility of the optimised method allows for its application in quality control research & development studies for the regular investigation of Roxithromycin in variety of pharmaceutical ingredients.

4.I.2. FLUCLOXACILLIN 4.I.2.1 Determination of λMax :UV scan range of 200 nm to 800 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug the 61

wavelength corresponding to maximum absorbance was found at 270 nm for drug under investigation. The spectrophotometric spectrum is shown in Figure : 4.I.2.1

Figure : 4.I.2.1 UV Visible Spectrum of Flucloxacillin Concentration=10

62

pH= 7.4

4.I.2.2 Preparation of Calibration Curve: The main aim of this parameter was to define that total analytical validation procedure employs a linear reply in a specified attention of the analyte. As per the requirements of current methodology the linearity was derived from 2-12μg/ mL of the test solution concentration. On the basis of experimental results, curve of calibration is developed in the conc of 2-12μg/ mL of six standard solutions of Flucloxacillin in chloroform at 270nm in phosphate buffer of pH = 7.4. The correlation coefficient be calculated and establish more than 0.9730. The calibration plot is shown in fig: 4.I.2.2 .

63

CALIBRATION CURVE OF FLUCLOXACILLIN 0.9 0.8

0.8

y = 0.081x - 0.054 R² = 0.9730 0.7

ABSORBANCE

0.6

0.6

0.5 0.4

Series1

0.4

Linear (Series1)

0.3 0.2

0.2

0.1 0

0 0

-0.1

2

4

6

8

10

12

CONCENTRATION

Figure : 4.I.2.2 Calibration Curve of Flucloxacillin

64

4.I.2.3 Validation of Method:The spectrophotometric analysis of flucloxacillin was validated according to the instructions and regulations of ICH to measure parameters namely LOD,LOQ, range,accuracy,ruggedness,linearity and precision of the proposed analyses.

4.I.2.4 Linearity and Range : Standard stock solutions of flucloxacillin in appropriate dilution were assayed as per the proposed method. According to Beer’s –Lambert’s law the concentration range is establish to be 2-12μg/ mL and curve of calibration given in Figure: 4.I.2.2 is linear in the given range. 4.I.2.5 Precision The exactness of the projected method of flucloxacillin is estimated by using concentrations of the drug were analyzed 6 times on a day is called intraday precision where as in interday precision for 6 incessant days .The analytical data of the drug have shown in the Table : 4.I.2.1 Table : 4.I.2.1 Determination of Accuracy and Precision of Flucloxacillin Name Of S.NO

The Sample

Labeled

%

Amount(mg/capsule) Level

Amount

Precision

found*

Inter

(mg)

day

Intraday

1

Flox

500

80

499.20

0.0078

0.0062

2

Actinase

500

100

499.99

0.0089

0.0069

(*average of six determinations) 4.I.2.6 Accuracy: The accurateness of the proposed process of flucloxacillin was estimated by by means of a method of standard addition. This process is carried out by addition of dissimilar amounts namely 80% and 100% of the pure sample of the compound to the pre-analyzed material. Accuracy data of the drug was shown in the Table : 4.I.2.1 4.I.2.7 LOD and LOQ: The LOQ and LOD of the drug flucloxacillin is estimated (Table : 4.I.2.2) through slope and response mechanism of SD according to rules and

65

regulations of ICH. The formulae of LOD and LOQ were discussed already in Ist chapter of subsection introduction of Analytical validation process.

Table 4.I.2.2 LOD & LOQ of Flucloxacillin S.No

Parameter

Flucloxacillin

1

LOD (%)

0.122

2

LOQ (%)

0.409

4.I.2.8 Recovery Studies of Flucloxacillin Recovery of flucloxacillin is calculated to know the exactness of the projected method. This process is done by addition of a identified mass of pure organic drug to a pre-analyzed sample. The result of analysis of the drug and recovery studies were notified in the Table: 4.I.2.3 . Table: 4.I.2.3 Recovery Analysis of Marketed Formulations of Flucloxacillin Name of

Label

Amount

the

claim

found

sample

(mg)

(mg)

1

Flox

500

499.20

99.840

2

Actinase

500

499.99

99.998

S.NO

4.I.2.9

% Recovery

Conclusion

This is a easy, specific and more economical U.V. visible spectro photometric determination of Flucloxacillin in bulk drug and pharmaceutical ingredient for industrial importance has been developed, it is validated according to the instructions and rules of as ICH. This discussion yielded a sensitive and reproducible assay procedure based on standard addition method and linear regression analysis; it is a sophisticated technique for various research institutions and universities for the systemic evaluation drug substances and constant monitoring to the pharma industry.

66

4.I.3 CEFTRIAXONE 4.I.3.1 Determination of λMax : UV scan range of 200 nm to 800 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wavelength corresponding to maximum absorbance was found at 290 nm for drug under investigation. The spectrophotometric spectrum was shown in Fig: 4.I.3.1 .

67

Figure : 4.I.3.1

UV Visible Spectrum of Ceftriaxone

Concentration=10

pH= 7.4

68

4.I.3.2 Preparation of Calibration Curve: The main objective of this parameter was to define that total analytical validation procedure employs a linear reply in a specified conc of the analyte. As per the requirements of current methodology the linearity was derived from 4-16μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is established in the conc of 4-16μg/mL of six samples standard solution of ceftriaxone in chloroform

at 290nm in phosphate buffer with pH =7.4. The

correlation coefficient is calculated and establish more than 0.9880. The calibration plot is shown in fig: 4.I.2.2 .

69

CALIBRATION CURVE OF CEFTRIAXONE 0.8

y = 0.045x - 0.049 R² = 0.9880

0.7 0.6

ABSORBANCE

0.5

0.7 0.6

0.5

0.4

0.4 Series1

0.3

0.3

0.2

0.2

0.1

0.1

0

0 0

-0.1

Linear (Series1)

5

10

15

20

CONCENTRATION

Figure : 4.I.3.2 Calibration Curve of Ceftriaxone

70

4.I.3.3 Validation of Method: The spectrophotometric analysis of ceftriaxone was determined and validate as per the regulations and rules of ICH and to determine validated parameters namely LOD,LOQ, linearity, accuracy, precision and range of the projected method.

4.I.3.4 Linearity and Range : Standard stock solutions of ceftriaxone in appropriate dilution were assayed as per the proposed method. According to Beer’s –Lambert’s law the concentration array is set up to be 4.0-16.0μg/mL for this drug and calibration plot in fig. 4.I.3.2 is linear in the given range. 4.I.3.5

Precision

The exactitude of the projected method of ceftriaxone be estimated through using drug concentrations of ceftriaxone were analyzed through intraday, that means 6 times in a day where as for inter day it was calculated for 6 spontaneous days.

The

analytical data of the drug have shown in the Table : 4.I.3.1. 4.I.3.6

Accuracy

The accurateness of the proposed technique of ceftriaxone is estimated by by means of method of standard addition. This process is conceded out by addition of dissimilar amounts namely 80% and 120% of a pure sample of the ceftriaxone to the preanalysed pharmaceutical formulation. Accuracy data of the drug shown within the Table : 4.I.3.1. Table : 4.I.3.1

Determination of Accuracy and Precision of Ceftriaxone

Name Of S.NO

The Sample

Labeled Amount (mg/capsule)

Amount %Level

Precision

found*

Inter

(mg)

day

Intraday

1

Auxil

200

120

199.88

0.0094

0.0082

2

Belox

200

80

199.90

0.0096

0.0098

(*average of six determinations)

4.I.3.7

LOD and LOQ 71

The LOD and LOQ of ceftriaxone was determined (Table: 4.I.3.2 ) by by means of standard divergence of the slope and response procedure as per the directions of ICH. The formulae of LOQ and LOD were already discussed in Ist chapter introduction subsection of Analytical validation process.

Table 4.I.3.2 LOD & LOQ of Ceftriaxone

4.I.3.8

S.No

Parameter

Ceftriaxone

1

LOD (%)

0.259

2

LOQ (%)

0.865

Recovery Studies of Ceftriaxone

Recovery of ceftriaxone is calculated to know the accurateness of the projected technique. This process is done by addition of a known magnitude of pure drug ceftriaxone to the pre-analyzed sample. The result of analysis of the drug and recovery studies are notified in the Table : 4.I.3.3. Table : 3.I.3.4 Recovery Analysis of Marketed Formulations of Ceftriaxone Name of S.NO

the sample

4.I.3.9

Label claim

Amount

(mg)

found (mg)

% Recovery

1

Auxil

200

199.88

99.94

2

Belox

200

199.99

99.99

Conclusion

It is a easy, specific and more economical U.V spectro photometric method is developed for the estimation of ceftriaxone in bulk and pharmaceutical formulation and validate according to rules and generalisations of ICH. It is more significant method for pharma world of developing nations, because it is more reproducible and encouraging in various areas for constant evaluation of the bulk drug substances.

72

4.II.1 ZANAMVIR 4.II.1.1 Determination of λ Max : UV scan range of 200 nm to 400 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wave length corresponding to maximum absorbance was found at 260 nm for the drug under invstigation. The spectrophotometric spectrum of Zanamvir is shown in Fig : 4.II.1.1.

73

Fig : 4.II.1.1

UV Spectrum of Zanamvir

Concentration : 10

74

Ph: 7.4

4.II.1.2 Preparation of Calibration Curve : The main aim of this parameter was to describe that total analytical validation procedure employs a linear reply in a specified concentration of analyte. As per the directions of current methodology the linearity was derived from 2-10μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is given in the Beer’s concentration range of 4.0-16.0μg mL of eight standard solutions of Zanamvir in Double distilled Water at 260nm in phosphate buffer of pH=7.4. The correlation coefficient was calculated and found more than 0.9999. The calibration plot is shown in fig: 4.I.2.2 .

75

calibration curve of zanamvir 6 5

5

y = 0.1005x -0.0002 R² = 0.9999

ABSORBANCE

4

4

3

3

2

2

FIG : 3.II.1.3 Calibration curve of Zanamvir 1

1

0 0

0.1

0.2 0.3 0.4 CONCENTRATION (µg/mL)

Fig : 4.II.1.2

0.5

calibration curve of Zanamvir

76

0.6

4.II.1.3 Validation of Method : The spectrophotometric estimation of Zanamvir

is validated according to the

directions of ICH to determine parameters namely linearity, LOQ ,LOD precision,range and the accuracy, of the projected system. 4.II.1.4 Linearity and Range : Standard stock solution of Zanamvir in appropriate dilution were assayed as per the proposed method According to Beer’s –Lambert’s law the concentration range of Zanamvir is establish to be 2-10 μg/mL ,So that the curve of calibration in the FIG : 4.II.1.2 is linear within the given concentration selection. 4.II.1.5 Precision : The correctness of the proposed process of Zanamvir

is estimated by using drug

concentration of Zanamvir were analyzed intraday precision through 6 times in a day where as inter day precision through 6 incessant days. Data is given in the Table : 4.II.1.1 . 4.II.1.6 Accuracy : The correctness of the proposed way of Zanamvir is estimated by by means of method of standard addition .This process is carried out by the addition of dissimilar amounts namely 80and120percent of the pure sample of the drug to the pre-analysed formulation. Correctness data of this drug is exposed in the Table: 4.II.1.1. Table : 4.II.1.1 Precision &Accuracy of Zanamvir Name Of S.NO

The Sample

Labeled

%

Amount(mg/capsule)

Level

Amount

Precision

found*

Inter

(mg)

day

Intraday

1

Rebetol

10

120

9.8

0.0072

0.0082

2

Virazol

10

80

9.9

0.0094

0.0081

(*average of six determinations) 4.II.1.7

LOD and LOQ

The LOQ and LOD of Zanamvir is determined (Table: 4.II.1.2 ) by utilising SD of the slope and response system as per the directions of ICH. The formulae of LOQ and LOD were discussed already in Ist chapter introduction subsection of Analytical validation process. 77

Table : 4.II.1.2 LOD & LOQ of Zanamvir S.No

Parameter

Zanamvir

1

LOD (%)

0.0059

2

LOQ (%)

0.0199

4.II.1.8 Recovery Studies of Zanamvir : Recovery of Zanamvir is estimated to know the truthfulness of the projected method. This process is done by the addition of a acknowledged amount of pure zanamvir to a pre-analysed material. The result of analysis of the drug is notified in the Table: 4.II.1.3 Table : 4.II.1.3 Recovery Analysis of Marketed Formulation of Zanamvir Name of S.NO

the sample

Label claim

Amount

(mg)

found (mg)

% Recovery

1

Rebetol

10

9.8

99

2

Virazol

10

9.9

99

4.II.1.9 Conclusion The proposed method is set up to be easy, sensitive, correct and more reliable method to estimate Zanamvir in bulk drug and in pharmaceutical formulation. The proposed method is more significant in terms of validation parameters and encouraging in various pharma industries for the quality control and systemic evaluation of the drug.

78

4.II.2

GANCICLOVIR

4.II.2.1 Determination of λ Max : UV scan range of 200 nm to 800 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wave length corresponding to maximum absorbance was found at 420 nm for the drug under investigation. The spectrophotometric spectrum is shown in Fig: 4.II.2.1.

79

Fig : 4.II.2.1 U V Visible Spectrum of Ganciclovir Concentration : 10

80

Ph : 7.4

4.II.2.2

Preparation of Calibration Curve :

The main aim of the present investigation is to describe that total analytical validation procedure employs a linear reply in a specified concentration of Beer’s range of the analyte. As per the directions of current methodology the linearity was derived from 2-16μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is shown in the Beer’s concentration range of 2-16μg/ mL of eight standard solutions of Ganciclovir in chloroform at 420nm in phosphate buffer of pH=7.4. The correlation coefficient is calculated and establish more than 0.9970. The calibration plot is shown in fig: 4.I.2.2 .

81

CALIBRATION CURVE OF GANCICLOVIR 0.9

y = 0.026x + 0.027 R² = 0.9970

0.8

0.8

0.7

0.7

ABSORBANCE

0.6

0.6

0.5

0.5

0.4

Series1

0.4

Linear (Series1) 0.3

0.3

0.2

0.2

0.1

0.1

0

0 0

10

20

30

40


Fig : 4.II.2.2 Calibration Curve of Ganciclovir

82

4.II.2.3 Validation of Method : The spectrophotometric estimation of Ganciclovir is validated according to the directions of ICH to determine statistical parameters like LOD,LOQ, accuracy, linearity, precision,of the projected method. 4.II.2.4 Linearity and Range : Standard stock solution of Ganciclovir in appropriate dilution were assayed as per the proposed method According to Beer’s –Lambert’s law the concentration range of Ganciclovir is set up to be 2-16μg/mL ,So that the curve of calibration in the Fig : 4.II.2.2 is linear within the given concentration of Beer’s range. 4.II.2.5 Precision : The exactitude of the projected method of Ganciclovir is estimated by using concentrations of the drug were analyzed through intraday precision that means 6 times in a day where as 6 incessant days through inter day precision. Data is given in the Table: 4.II.2.1 4.II.2.6 Accuracy : The truthfulness of the proposed technique of Ganciclovir is estimated by utilising method of standard addition .This process is carried out by the addition of dissimilar amount namely 80and100% of the pure sample of the Ganciclovir to the pre-analysed formulation. Accuracy data of the drug is shown in the Table: 4.II.2.1 Table: 4.II.2.1

S.NO

Determination of Accuracy and Precision of Ganciclovir

Labeled

Name of the

Amount

Sample

(mg/capsule)

Amount %Level

Precision

Found*

Inter

Intra

( mg)

Day

Day

1

NATCLOVIR

100

80

99.91

0.0086 0.0071

2

NEOCLOVIR

100

100

99.05

0.0093 0.0079

(*average of 6 determinations)

4.II.2.7

LOD and LOQ:

The LOQ and LOD of the Ganciclovir is calculated (Table: 4.II.2.2 ) by utilising slope and response procedure according to the directions of ICH. The formulae of 83

LOQ and LOD were already discussed in Ist chapter introduction subsection of Analytical validation process. Table : 4.II.2.2 LOD & LOQ of Ganciclovir S.No

Parameter

Ganciclovir

1

LOD (%)

0.311

2

LOQ (%)

0.103

4.II.2.8 Recovery Studies of Ganciclovir : Recovery analysis of Ganciclovir is determined to know the exactness of the projected method. This process is done by addition of a identified extent of pure Ganciclovir drug to a pre-analysed sample. The result of analysis of the drug is notified in the Table: 4.II.2.3 . Table: 4.II.2.3 Recovery studies of Marketed Formulations of Ganciclovir

S.NO

Name of the sample

Labeled

Amount

amount

found*

(mg/capsule)

( mg)

% Recovery

1

Natclovir

100

99.91

99.955

2

Neoclovir

100

99.05

99.525

4.II.2.9 Conclusion It is a low cost uncomplicated, precise and more economical UV visible spectrophotometric method to determine Ganciclovir in bulk drug and pharmaceutical material is investigated and validate as per the directions of International conference on Harmonisation. The performance of the current proposed method is reproducible in terms of LOD and LOQ indicates the significance of the proposed method for pharma industry.

84

4.III.1

EMTRICITABINE

4.III.1.1 Determination of λ Max : UV scan range of 200 nm to 400 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wave length corresponding to maximum absorbance was found at 298 nm for the drug under investigation. The spectrophotometric spectrum is shown in Fig: 4.III.1.1

85

Fig : 4.III.1.1 U V Visible Spectrum of Emtricitabine Concentration : 10

86

Ph : 7.4

4.III.1.2 Preparation of Calibration Curve : The main objective of the current investigation is to describe that total analytical validation procedure employs a linear reply in a specified concentration of Beer’s range of the analyte. As per the directions of current methodology the linearity was derived from 2-20μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is developed in the concentration of Beer’s range of 2-20μg/ mL of eight standard solutions of Emtricitabine in chloroform at 298 nm in phosphate buffer of pH=7.4. The correlation coefficient is calculated and set up more than 0.9910. The calibration plot is shown in fig: 4.I.2.2 .

87

CALIBRATION CURVE OF EMTRICITABINE 0.8

y = 0.013x + 0.049 R² = 0.9910

0.7

0.7

0.6

0.6

Absorbance

0.5

0.5

0.4

0.4

0.3

Series1 Linear (Series1)

0.3

0.2

0.2

0.1

0.1

0

0 0

10

20 30 (µg/mL) 40 concentration

50

60

Fig : 4.III.1.2 Calibration Curve of Emtricitabine

88

4.III.1.3 Validation of Method : The spectrophotometric estimation of Emtricitabine is validated according to the directions of ICH to determine LOD,LOQ, accuracy, linearity, precision, of the projected technique. 4.III.1.4 Linearity and Range : Standard stock solution of Emtricitabine in appropriate dilution were assayed as per the proposed method According to Beer’s –Lambert’s law the concentration range of Emtricitabine be established to be 2-20μg/mL ,So that the curve of calibration

in

the Fig : 4.III.1.2 explains linear in the given concentration of Beer’s law range. 4.III.1.5

Precision :

The correctness of the projected method of Emtricitabine is estimated by using concentrations of the drug were analyzed 6 times in a day through intraday precision where as analysed 6 spontaneous days through interday precision. Data is given in the Table : 4.III.1.1. 4.III.1.6 Accuracy : The accurateness of the proposed system of Emtricitabine is estimated by by means of method of standard addition .This process is carried out by the addition of unusual amounts namely 80percent and 100percent of the pure sample of the drug to the preanalysed pharmaceutical formulation. Accuracy data of the drug is given in the Table : 4.III.1.1.

Table : 4.III.1.1. Determination of Accuracy and Precision of Emtricitabine

S.NO

Name of the Sample

Labeled

Amount

Amount

%Level

mg/capsule)

Precision

Found*

Inter

Intra

( mg)

Day

Day

1

EMTRIVA

100

80

99.90

0.0082

0.0069

2

ATRIPLA

100

100

99.80

0.0094

0.0073

(*average of 6 determinations)

89

4.III.1.7 LOD and LOQ: The LOQ and LOD of Emtricitabine are calculated (Table : 4.III.1.2) through response and slope process of SD as per the directions ICH. The formulae of LOQ and LOD were already discussed in Ist chapter introduction subsection of Analytical validation process. Table 4.III.1.2 LOD & LOQ of Emtricitabine

4.III.1.8

S.No

Parameter

Emtricitabine

1

LOD (%)

0.129

2

LOQ (%)

0.432

Recovery Studies of Emtricitabine :

Recovery studies of Emtricitabine are calculated to know the correctness of the projected scheme. This process is done by the addition of a well-known amount of pure drug Emtricitabine to a pre-analysed sample. The result of analysis of the drug is notified in the Table : 4.III.1.3.

Table : 4.III.1.3 Recovery studies of Marketed Formulations of Emtricitabine

S.NO

4.III.1.9

Name of the Sample

Labeled

Amount

Amount

Found*

(mg/capsule)

( mg)

% Recovery

1

EMTRIVA

100

99.90

99.9

2

ATRIPLA

100

99.80

99.8

Conclusion

In this discussion a low cost simple, precise and more economical sophisticated technique namely UV visible spectrophotometric estimation of Emtricitabine in bulk and pharmaceutical ingredient is developed through Linear regression Analysis, standard addition method and validate according to the directions of ICH. It has utmost potential application in quality control evaluation and much more prospective investigation of the drug in R&D of various research institutions.

90

4.III.2 4.III.2.1

EFAVIRENZ Determination of λ Max

UV scan range of 200 nm to 400 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug and the wave length corresponding to maximum absorbance of Efavirenz was found at 256 nm for the drug under investigation. The spectrophotometric spectrum is shown in Fig : 4.III.2.1.

91

Fig : 4.III.2.1 UV Visible Spectrum of Efavirenz Concentration : 10

92

Ph : 7.4

4.III.2.2

Preparation of Calibration Curve:

The main intention of the current exploration is to describe that total analytical validation procedure employs a linear retort in a specified concentration of Beer’s range of the analyte. As per the directions of current methodology the linearity was derived from 4-28μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is designed in the concentration of Beer’s range of 4-28μg/mL of eight standard solutions of Efavirenz in chloroform at 256nm in phosphate buffer of pH = 7.4. The correlation coefficient is calculated and set up more than 0.9970. The calibration plot is shown in fig: 4.I.2.2 .

93

calibration curve of efavirenz 0.8

y = 0.018x + 0.012 R² = 0.9970

0.7

ABSORBANCE

0.6 0.5

0.7 0.6 0.5

0.4

0.4

0.3

Series1

0.3

0.2

Linear (Series1)

0.2

0.1

0.1

0

0 0

10

20

30

40


Fig : 4.III.2.2 Calibration curve of Efavirenz

94

4.III.2.3 Validation of Method The spectrophotometric analysis of the drug namely Efavirenz is validated according to the directions of ICH to determine LOD, LOQ, accuracy, linearity, precision, of the projected technique. 4.III.2.4 Linearity and Range Standard stock solutions of the drug namely Efavirenz in appropriate dilution were assayed as per the proposed method, According to Lambert’s-Beer law the range be establish to be 4-28μg/mL. So that the calibration curve of the drug Efavirenz (Fig : 4.III.2.2) is linear at the give range. 4.III.2.5 Precision The correctness of the projected method of Efavirenz is estimated by using concentrations of the drug were analyzed 6 times in a day through intraday precision where as analysed 6 spontaneous days through interday precision.Data of the drug shown in the Table: 4.III.2.1. 4.III.2.6 Accuracy The correctness of the proposed process of Efavirenz is estimated by utilising method of standard addition .This process is conceded out by addition of dissimilar amount namely 100 and 120 percent of the pure sample of the drug Efavirenz to the pre-analyzed formulation. Accuracy data of the drug shown in the Table: 4.III.2.1. Table: 4.III.2.1 Determination of Accuracy and Precision of Efavirenz S.

Name of the

Labeled Amount

NO

Sample

(mg/capsule)

1

ESTIVA

100

2

STOCRIN

100

4.III.2.7

% Level

Amount

Precision

Found* (mg)

Inter day

Intraday

100

98.20

0.0062

0.0064

120

98.14

0.0096

0.0078

LOD and LOQ

The LOD and LOQ of Efavirenz was determined (Table: 4.III.2.2) by utilising SD of the slope and response procedure as per the directions of ICH. The formulae of LOQ and LOD were noticed in Ist chapter introduction subsection of Analytical validation process.

95

Table 4.III.2.2 LOD & LOQ of Efavirenz S.No

Parameter

Efavirenz

1

LOD (%)

0.135

2

LOQ (%)

0.453

4.III.2.8 Recovery Studies of Efavirenz Recovery of Efavirenz is calculated to know the accurateness of the designed process. This process is done by addition of a branded amount of pure drug Efavirenz to a preanalysed sample. The result of analysis of the drug and revival study are notified in the Table: 4.III.2.3. Table: 4.III.2.3 Recovery Analysis of Marketed Formulations of Efavirenz S.NO

Name of The

Label Claim

Amount

%

Sample

In (mg)

Found (mg)

Recovery

1

ESTIVA

100

98.20

98.2

2

STOCRIN

100

98.10

98.1

4.III.2.9 Conclusion The present method of this drug is low cost simple, more economical U.V. spectro photometric method is defined for the determination of Efavirenz in pharmaceutical formulation and validated as per the directions of International conference on Harmonisation. It is more reliable and sensitive in terms of precision and accuracy in conditions of SD, LOD and LOQ of the current investigation is more consistent for the estimation of the bulk drug in various research stations.

96

4.IV.1 VINBLASTIN 4.IV.1.1 Determination of λ Max : UV scan range of 350 nm to 470 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wave length corresponding to maximum absorbance was found at 420 nm for the drug under investigation. The spectrophotometric spectrum of Vinblastin is shown in Fig: 4.IV.1.1.

97

Fig : 4.IV.1.1 UV spectrum of Vinblastin Concentration : 10

98

Ph : 7.4

4.IV.1.2

Preparation of Calibration Curve :

The main objective of the proposed method is to describe that total analytical validation process employs a linear rejoinder in a specified concentration of Beer’s range of an analyte. As per the directions of current methodology the linearity was derived from 6-16μg/ mL of the test solution concentration of the drug. On the basis of experimental results, calibration curve is given in the Beer’s range of 6-16μg/ mL of eigh standard solutions of Vinblastin in methanol at 470nm in phosphate buffer of pH=7.4. The correlation coefficient is calculated and establish more than 0.9997. The calibration plot is shown in fig: 4.I.2.2 .

99

25

y = 0.0388X-0.0139 R² = 0.9997

ABSORBANCE

20

20

15

15

10

10

5

5

0 0

0.2

0.4

0.6

0.8


Fig : 4.IV.1.2

Calibration curve of Vinblastin

100

1

4.IV.1.3 Validation of Method The spectrophotometric estimation of Vinblastin

is validated according to the

directions of ICH to determine LOQ, LOD, linearity, accuracy, and precision, of the projected technique. 4.IV.1.4

Linearity and Range

Standard stock solution of Vinblastin in appropriate dilution were assayed as per the proposed method According to Beer’s –Lambert’s law the concentration range of Vinblastin is establish to be 6-16μg/ mL ,So that the curve of calibration in the Fig : 4.IV.1.2 is linear at the given concentration of Beer’s range. 4.IV.1.5 Precision : The correctness of the projected method of vinblastin is estimated by using concentrations of the drug were analyzed 6 times in a day through intraday precision where as analysed 6 spontaneous days through interday precision. Data of the drug shown in the Table: 4.IV.1.1 4.IV.1.6 Accuracy : The correctness of the proposed technique of Vinblastin is estimated by utilising method of standard addition. This process is conceded out by addition of dissimilar amount namely 80 and 100 percent of the pure sample of the drug Vinblastin to the pre-analyzed formulation. Accuracy data of the drug shown in the Table: 4.IV.1.1

Table: 4.IV.1.1

S.NO

Name of the Sample

Precision &Accuracy of Vinblastin

Labeled Amount (mg/capsule)

% Level

Amount

Precision

Found* (mg)

Inter day

Intraday

1

Gemzar

10

80

9.1

0.0071

0.0085

2

Velban

10

100

9.0

0.0069

0.0074

4.IV.1.7

LOD and LOQ:

The LOQ and LOD of Vinblastin is calculated (Table: 4.IV.1.2) by utilising SD of the slope and response procedure as per the directions of ICH. The formulae of LOQ and 101

LOD were noticed in Ist chapter introduction subsection of Analytical validation process.

Table : 4.IV.1.2 LOD & LOQ of Vinblastin S.No

Parameter

Vinblastin

1

LOD (%)

0.282

2

LOQ (%)

0.854

4.IV.1.8 Recovery Studies of Marketed formulation of Vinblastin: Recovery of Vinblastin is determined to know the exactness of the projected method . This process is done by the addition of a branded amount of pure drug vinblastin to a pre-analysed sample. The result of analysis of the drug is notified in the Table: 4.IV.1.3 Table: 4.IV.1.3

Recovery studies of Marketed formulation of Vinblastin

Name

Labeled

Amount

of the

Amount

Found*

sample

(mg/capsule)

( mg)

1

Gemzar

10

9.1

91

2

Velban

10

9.0

90

S.no

% Recovery

4.IV.1.9 Conclusion The current method of this drug is trouble-free, specific and more inexpensive U.V. Spectrophotometric method is developed for the estimation of Vinblastin in bulk drug and pharmaceutical formulation and validate according to the directions of ICH. This developed method having distinct advantages over existing methods notified in literature survey.

102

4.IV.2

NITAZOXANIDE

4.IV.2.1 Determination of λ Max : UV scan range of 300 nm to 400 nm was selected to determine maximum absorbance by using 10 μg/ml solution of the drug, the wave length corresponding to maximum absorbance was found at 340 nm for the drug under investigation. The spectrophotometric spectrum Nitazoxanide is shown in Fig : 4.IV.2.1.

103

Fig : 4.IV.2.1 UV spectrum of Nitazoxanide Concentration : 10

104

PH : 7.4

4.IV.2.2 Preparation of Calibration Curve : The main objective of the proposed method is to describe that total analytical validation process employs a linear response in a specified concentration range of the analyte. As per the directions of current methodology the linearity was derived from 4-20μg/ mL of the test solution concentration of the drug. On the basis of experimental results, curve of calibration is given in the Beer’s concentration range of 4-20μg/ mL of eight standard solutions of Nitazoxanide in methanol at 340nm in phosphate buffer of pH =7.4. The correlation coefficient is calculated and establish more than 0.9920. The calibration plot is shown in fig: 4.I.2.2 .

105

CALIBRATION CURVE OF NITAZOXANIDE 1

y = 0.047x - 0.058 R² = 0.9920

0.8

0.9 0.8 0.7

ABSORBANCE

0.6

0.6

0.5 0.4

0.4

Series1

0.3 0.2

Linear (Series1)

0.2 0.1

0

0

-0.2 0

5

10

15

20

25


Fig : 4.IV.2.2 Calibration Curve of Nitazoxanide

106

4.IV.2.3 Validation of Method The spectrophotometric estimation of

Nitazoxanide is validated according to the

directions of ICH to determine LOQ, LOD, linearity, accuracy and precision, of the projected technique. 4.IV.2.4 Linearity and Range Standard stock solution of Nitazoxanide in appropriate dilution were assayed as per the proposed method According to Beer –Lambert’s law the concentration range of Nitazoxanide is set up to be 4-20 μg/ mL ,So that the curve of calibration

in the Fig

: 4.IV.2.2 is linear at the given Beer’s range. 4.IV.2.5

Precision

The correctness of the projected method of Nitazoxanide is estimated by using concentrations of the drug were analyzed 6 times in a day through intraday precision where as analysed 6 spontaneous days through interday precision. Data of the drug shown in the Table: 4.IV.2.1 4.IV.2.6

Accuracy

The correctness of the proposed technique of Nitazoxanide is estimated by utilising method of standard addition . This process is conceded out by addition of dissimilar amount namely 80 and 120 percent of the pure sample of the drug Nitazoxanide to the pre-analyzed formulation. Accuracy data of the drug shown in the Table: 4.IV.2.1. Table: 4.IV.2.1 Accuracy and Precision of Nitazoxanide

S.No

Name of the Sample

Labeled

Amount

Amount

%Level

(mg/capsule)

Precision

Found*

Inter

(mg)

day

Intraday

1

ZINDAX

50

120

49.2

0.0076

0.0072

2

PARAMIX

50

80

48.05

0.0083

0.0069

4.IV.2.7 LOD and LOQ The LOQ and LOD of Nitazoxanide is calculated (Table: 4.IV.2.2) by utilising SD of the slope and response procedure as per the directions of ICH. The formulae of LOQ and LOD were noticed in Ist chapter introduction subsection of Analytical validation process. 107

Table : 4.IV.2.2

LOD and LOQ OF Nitazoxanide

S.No

Parameter

Nitazoxanide

1

LOD (%)

0.384

2

LOQ (%)

1.165

4.IV.2.8 Recovery Studies of Nitazoxanide : Recovery of Nitazoxanide is calculated to know the accurateness of the projected process . This process is done by the addition of a well- known amount of pure drug Nitazoxanide to a pre-analysed sample. The result of analysis of the drug is notified in the Table: 4.IV.2.3 Table: 4.IV.2.3

S.NO

Recovery studies of Marketed Formulation of Nitazoxanide

Name of the sample

Labeled

Amount

Amount

Found*

(mg/capsule)

( mg)

% Recovery

1

ZINDAX

50

49.2

98.4

2

PARAMIX

50

48.05

96.1

4.IV.2.9 Conclusion To the most excellent of my information the current system of this drug is simple, sensitive, accurate and more economical UV visible spectrophotometric determination is incorporated for the continuous monitoring and estimation of Nitazoxanide in bulk drug and pharmaceutical formulation and validate according to the guidelines of ICH. It is a sophisticated technique particularly in R&D field in developing countries with limited budgets.

108

CHAPTER V

SUMMMARY & CONCLUSIONS

5.1 Summary, Conclusion and Recommendations Summary and Conclusion of this dissertation is given based on the work conceded by the author on validated spectrophotometric studies of advanced medicinal compounds. In this point of view, implementation of validated methods and determination of advanced medicinal compounds by using U.V. spectrophotometry. Assay, Calibration curve in the course of linear regression analysis and results were included in the present thesis. The Ist chapter of this thesis explains about basic principles of U.V. spectrophotometry, introduction of validation and development of analytical method procedures, analytical parameters and also highlighted the status of medicinally important advanced organic drugs and pharmaceuticals in the society to solve and treat various kinds of mankind problems. Chapter second is focused on the literature survey of various medicinal compounds selected to the present research programme. The comprehensive literature survey has done on selected advanced medicinal drugs related to the present research work. The methods observed in the literature survey of selected compounds are time consuming, expensive, and not coinciding as per the requirements of ICH. Hence the current research program was dedicated on Roxythromycin, Flucloxacillin, Ceftriaxone, Ganciclovir, Zanamvir, Emtricitabine, Efavirenz, Vinblastin and Nitazoxanide. The third chapter reveals that U.V. spectrometric determination of selected drugs with different solvents through

linear regression analysis and standard addition

method as per the requirements of International Conference on Harmonisation (ICH) The fourth chapter describes the result and discussion of selected drugs in the current research. •

The U.V Spectrum of Roxythromycin shows absorption maximum at 420 nm in pH 7.4. The linearity was observed between 20-70 μg/ mL with correlation coefficient of 0 .9840, LOD & LOQ were 1.313 and 3.980 respectively. The % recovery of drug & its formulations found to be in the range of 98.97-98.68 shows accuracy of the proposed method.

•

The U.V Spectrum of

Flucloxacillin shows absorption maximum at 270

nm , The linearity was observed between 2-12 μg/ mL with correlation coefficient of 0 .9730, LOD & LOQ were 0.122 and 0.409 respectively .The

109

% recovery of drug & its formulations found to be in the range of 99.84-99.99 shows accuracy of the proposed method. •

The U.V Spectrum of Ceftriaxone shows absorption maximum at 290 nm. The linearity was observed between 4-16 μg/ mL with correlation coefficient of 0 .9880, LOD & LOQ were 0.259 and 0.865 respectively. The % recovery of drug & its formulations found to be in the range of 99.94-99.995 shows accuracy of the proposed method.

•

The U.V Spectrum of Zanamvir shows absorption maximum at 260 nm .The linearity was observed between 2-10 μg/ mL with correlation coefficient of 0 .9999, LOD & LOQ were 0.0059 and 0.0199 respectively. The % recovery of drug & its formulations found to be in the range of 99-99 shows accuracy of the proposed method.

•

The U.V Spectrum of Ganciclovir shows absorption maximum at 420 nm . The linearity was observed between 2-16 μg/ mL with correlation coefficient of 0 .9970, LOD & LOQ were 0.311 and 0.103 respectively .The % recovery of drug & its formulations found to be in the range of 99.95-99.52 shows accuracy of the proposed method.

•

The U.V Spectrum of Emtricitabine shows absorption maximum at 298 nm. The linearity was observed between 2-20 μg/ mL with correlation coefficient of 0 .9910, LOD & LOQ were 0.129 and 0.432 respectively. The % recovery of drug & its formulations found to be in the range of 99.9-99.8 shows accuracy of the proposed method.

•

The U.V Spectrum of Efavirenz shows absorption maximum at 256 nm. The linearity was observed between 4-28 μg/ mL with correlation coefficient of 0 .9970, LOD & LOQ were 0.135 and 0.453 respectively. The % recovery of drug & its formulations found to be in the range of 98.2-98.1 shows accuracy of the proposed method.

•

The U.V Spectrum of Vinblastin shows absorption maximum at 420 nm. The linearity was observed between 6-16 μg/ mL with correlation coefficient of 0 .9997, LOD & LOQ were 0.282 and 0.854 respectively .The % recovery of drug & its formulations found to be in the range of 91-90 shows accuracy of the proposed method.

110

•

The U.V Spectrum of Nitazoxanide shows absorption maximum at 340 nm. The linearity was observed between 4-20 μg/ mL with correlation coefficient of 0 .9920, LOD & LOQ were 0.384 and 1.165 respectively then the % recovery of drug & its formulations found to be in the range of 98.4-96.1 shows accuracy of the proposed method

➢ The present thesis comprehensively delivers precise, more economical, accurate, more suitable and easy methods for the development of selected organic drugs to cure various diseases in the society. ➢ The methods given for selected compounds in this research implemented very easily for the quantification of selected drugs for bulk and pharmaceutical formulation forms. ➢ The proposed method of all selected drugs will fulfils all the latest trends in the pharma industry requirements of india and abroad to solve present problems. The latest methods can be used extensively in the advanced drug development of research stations and universities.

111

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118

APPENDIX-I

RESEARCH PAPERS PUBLISHED

RESEARCH PAPERS PUBLISHED Dr C.M. BHASKAR REDDY M.SC; M.Ed; M.Phil; MBA; Ph.D PROFESSOR OF CHEMISTRY DEPT OF HUMANITIES &SCIENCES CHAITANYA BHARATI INSTITUTE OF TECHNOLOGY VIDYA NAGAR; PALLAVOLU PRODDATUR-516360 AP ; INDIA MOBILE : 9966476194 E-MAIL [email protected]

List of Research papers Published 1. U.V. visible spectrophotometric estimation of Anti biotic drugs by C.M. Bhaskar Reddy , Dr. G.V. Subba Reddy, and Dr N. Ananda kumar Reddy AJPTR 2016, Volume 6, Issue 6 2. U.V. visible spectrophotometric estimation of Emtricitabine by C.M. Bhaskar Reddy and Dr N. Ananda kumar Reddy ,IRJP 2016, 7(12) 3. U.V. visible spectrophotometric estimation of Ganciclovir by C.M. Bhaskar Reddy and Dr N. Ananda kumar Reddy ,IRJP 2016, 7(12) 4. U.V. visible spectrophotometric estimation of Anti retroviral drugs by C.M. Bhaskar Reddy , Dr N. Ananda kumar Reddy and Dr L.Maheswar Reddy AJPHR 2017, 5, (1)

119

5. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Spectrophotometric estimation of Fluconazole in pure drug and pharmaceutical formulation” . International Journal of Scientific & Engineering Research, Vol. No.3(9),Year 2012,Pages (pp) 1-6. 6. C.M.

Bhaskar Reddy, G.V.Subba Reddy,N.Ananda kumar Reddy “Development and validation of UV Spectrophotometric method for determination trifluoperazine hydrochloride in bulk drug and pharmaceutical dosage form” . International Journal of Scientific and Research publications, Vol. No.2(8),Year 2012,Pages (pp) 1-5.

7. C.M. Bhaskar Reddy and G.V.Subba Reddy, “A validated UV Spectrophotometric determination of an antiviral drug Zanamvir from tablet formulations” . Journal of chemical and pharmaceutical Research, Vol. No.4(7),Year 2012,Pages (pp)3624-3627. 8. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Development ,validation and application of UV Spectrophotometric method for the determination of Roxithromycin in bulk drug and pharmaceutical dosage form” . Journal of chemical and pharmaceutical Research, Vol. No.4(7),Year 2012,Pages (pp)3684-3687.

9. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Spectrophotometric studies of Vinblastin in bulk drug and and pharmaceutical formulations” . Journal of chemical and pharmaceutical Research Vol. No.4(7),Year 2012,Pages (pp) 3703-3707. 10. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Study and improvement of Spectrophotometric and HPLC methods for the determination of Nitazoxanide in pharmaceutical preparations” . Journal of chemical and pharmaceutical Research, Vol. No.4(7),Year 2012,Pages (pp) 3708-3714.

11. C.M. Bhaskar Reddy and G.V.Subba Reddy, “UV Spectrophotometric method for estimation of Efavirenz in bulk drug and tablet dosage form” . International Journal of Pharmaceutical sciences and Research, Vol. No.3(12),Year 2012,Pages (pp) 5033-5037.

12. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Spectrophotometric estimation of Flucloaxacillin in pure drug and pharmaceutical dosage formulation” .IOSR Journal of Pharmacy and Biological sciences, Vol. No.2(2),Year 2012,Pages (pp) 46-48.

13. C.M. Bhaskar Reddy and G.V.Subba Reddy, “Development ,validation of UV Spectrophotometric methods for the estimation of Ceftriaxone in bulk and tablet dosage form”. International Journal of Chemtech Research, Vol. No.5(1),Year 2012,Pages (pp) 472-477.

120

14. C.M. Bhaskar Reddy,A.Sai Tejaswini ,C. Dhanya,G.Lakshmi Lavanya ,G. Sai

Aparna , “Comphrehensive Microbial and Chemical Analysis of Potable Water During The Year 2014-15 Around Villages Of CBIT-VBIT Campus” . International Journal of Green and Herbal Chemistry, Vol. No.4(2),Year 2015,Pages (pp)284-292.

15. C.M.BhaskarReddy,U.Sulochana,N.Bhargavi,M.Sreedevi, P.Madhavi , “Comphrehensive analysis of drinking water around CBIT-VBIT campus during the academic year 2014-15” . International Journal of Advanced and Innovative Research, Vol. No.4(4),Year 2015,Pages (pp) 659-661. 16. C.M. Bhaskar Reddy,C.Chandrika,K.Tejaswini,R.N.S.Praveena,U.Anusha , “Analysis of Ground Water during the year 2014-15 areas around CBIT-VBIT campus” . International Journal of Scientific & Engineering Research, Vol. No.4(5),Year 2015,Pages (pp)216-220.

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