Chapter 3 Materials & Methods 03. MATERIALS &...

Chapter 3 Materials & Methods

Suresh Gyan Vihar University, Jaipur 57

03. MATERIALS & METHODS

3.1 INSTRUMENTATION

A. Shimadzu UV- 1700

The present work has been done on Shimadzu UV- 1700 series

spectrophotometer. It has a double beam-double detector configuration. In this

configuration, the sample beam and the reference beam enter different detectors

respectively. So it is necessary to use two detectors with similar characteristics.

The advantage of this configuration is that it is not necessary to enter two beams

always in the same detector, as in case of single detector configuration, so the

large space is allowed in the sample compartment. The hardware specifications

and software specifications of the instrument are given below:

Figure 3.1: UV Spectrophotometer (Shimadzu 1700)

a. Hardware specifications 97

Table-3.1: Hardware Specification of Shimadzu UV-1700

Measurements Wavelength Range : 190-1100 nm

Wavelength Display : 0.1nm units

Wavelength Scanning Speeds

GOTO WL command

: approx, 6000nm/min



Very Fast (sampling pitch;2.0nm)

Fast (sampling pitch;1.0nm)

Medium (sampling pitch; 1.2nm

Slow (sampling pitch: 1.0nm)

Very slow (sampling pitch:1.0nm)

Very slow (sampling pitch;0.1nm)

: approx, 3000nm/min

: approx 2000nm/min

: approx 260nm/min

: approx, 190nm/min

: approx,100nm/min

: approx. 10nm/min

Light source switching: Automatic switching with wavelength range.

Photometric Range: Absorbance :- 0.5 to 3.0 Abs

Transmittance: 0-300%

Baseline Correction: Automatic correction using computer memory

Light Source: 20W halogen lamp, Deuterium lamp

Monochromator: Uses aberration- correcting concave blazed holographic

grating.

Detector: Silicon photodiode

Sample compartment: Interior dimensions 110 x 230 x 105 (mm) (W x D x H )

(Partial depth 155mm)

Weight: 17 kg

Operating temperature/Humidity:

Temperature range: 15 to 350 C

Humidity range : 35 to 80% (150 C to blow 30

0 C), 35 to 70% (30

0 C to 35

0 C)

Turning on power & initialization: Initialized in approximately 4 minutes.

Screen Display and Sheet keys:

The modes & setting in the various screens can be selected using the numeric keys

0 through 9 or the function keys F1 through F4.

b. Software Specifications

Table-3.2: Software Specificaton of Shimadzu UV-1700

Specifications Salient Features

1. Photometric 1. Fixed Wavelength measurement

2. Quantitation by K-factor method

3. Save/ Load table data



2. Spectrum 1.Measurement mode : Absorbance, transmittance,

and energy (E)

2.Wavelength range : 190 nm to 110 nm

(Min scan range: 10nm )

3. Scan speeds: Very fast, Fast, medium, Slow, Very slow

4. Vertical axis recording range:

ABC: - 3.99 (Min range: - 01 to 0.01 T %)

E: - 399 to 399 (min range: - 1 to 1)

5. Scan repetitions: 1 to 99 times.

6. Recording method: Overlay/Sequential selectable

7. Spectrums Data Processing Functions

* Peak/Valley detection (possible up to 20 pcs.)

* The four rules of arithmetic (constant, between data)

* Derivative processing (1 to 4 order)

* Smoothing processing

* Area Calculation

* Point-pick processing

* Reduce/Expand (zoom)

* Display date with cursor

* Save/Load date function

3.Quantitation 1. Measurement method:

One- wavelength quantitation, Two-wavelength/

Three-wavelength quantitation, Quantitation by

Derivative (1 to 4 order) calculation

2. Functions regarding calibration curve

Automatically calculate concentration by K-factor method

Automatically calculate concentration by one point calibration

curve method.

Multi-point calibration curve (1 to 3 order Regression

calibration curve)

1 to 3 order regression calibration curve



Number of standard samples (2 to 10)

Select pass-though-origin conditions

Calibration curve preparation by repeat measurement (12 to

9 times) and taking the average measurement value

Display of calibration curve formula

Display of relative coefficient of calibration curve

3. Measurement: Quantitation by repeat measurement (1 to 9

times) & taking the average measurement value

4. Save/Load table data function

5. Automatic data print function: Output measurement result

to printer by every measurement.

4. Kinetics

1. One-wavelength kinetics

Measurement time; 1 to 6500 sec (min)

2. Two-wavelength kinetics

3. Rate-measurement

4. Save/Load curve date function

5. Output of curve data.

5. Time scan 1. Measurements mode: Absorbance (ABS) ,

Transmittance (%T), and Energy (E)

2. Measurement time. 1 to 6500sec/ min

3. One cell measurement

Waveform date processing function

Sample temperature control

4. Save/ Load of curve date function

5. Output of curve data

6. Waveform print: Printing of wave from in A5 size is available

by a printer supporting the control command ESC/P

6. Multi-

Component

Analysis

1. Up to 8 components

2. Pure and mixed samples of each constituent component can be

used as standard samples.

3. Measurements parameters and standard sample data can be



stored.

4. Measurements parameters and standard sample data can be

stored.

5. Equally spaced wavelengths or random wavelengths can be

selected for measurements wavelengths.

6. Quantitation can be done by calling up spectra.

7. Multi-

wavelength

measurement

1. Number of measurement wavelength: Max. 8 wavelengths.

2. Photometric mode: Absorbance (ABS),

3. Wavelength calculation: Calculation using photometric value

is possible.

Ratio and difference between the photometric value of two

wavelengths, three wavelength calculation.

4 data calculation : (K1 x A1 + K2 x A2 + K3 x A3+ K4 x

A4x )x K5

4 data calculation: K5 x (K1 x A1 + K2 x A2) (K3 x A3 + K4

x A4).

4. Sample selection by wavelength: It is possible to select

sample for each wavelength for one time measurement.

8.Optional

Program Pack

It helps to display the measurement parameters & screen of the

optional program.

9. Utilities

mode

This is the mode for setting the instrument’s operating

parameters, such as the light source switching wavelength,

printer setup or the number of data columns displayed.

B. FT-IR (BRUKER ALFA)

In the Present work, chemical identification of

drug molecule done by FT-IR for that BRUKR

ALFA model is used. It has a two module one is

traditional KBr module and another one is ATR

module.

Figure 3.2: Bruker AlFA FT-IR



a. KBr Module (Universal sampling module)

The universal sampling module enables you to analyze all kind of samples: solids,

liquids and gases. This transmission sample compartment with its 2x3" standard

sample holder can hold a variety of gas cells and liquid cells. Solid samples can

be investigated in a standard pellet holder or using a magnetic film holder.

Samples with a reflective surface are analyzed with a dedicated accessory for

reflection measurements.

b. Eco-ATR

ATR is an easy to use FT-IR sampling method that is ideal for both solids and

liquids and does not require any sample preparation. The Eco ATR is a single

reflection ATR sampling module with a very

attractive cost/performance ratio. It is equipped with a versatile high throughput

ZnSe ATR crystal for the analysis of powders, solids, pastes and liquids.

C. Weighing Balance: CITIZEN CX 26590

Brand Name: Citizen

Model No.: CX 265

Capacity: 60/20 g.

Readability: 0.01/0.1 mg

Temp Drift: +/- 2 ppm

Pan Size: 85 mm

Calibration Type: Internal.

Figure 3.2: Weighing Balance: Citizen CX 265

D. Sonicator91

Company Name: PCI Analytics Pvt. Ltd.

Technical Specification:

Operating frequency 33 3 KHz

Input voltage range of 170V AC - 270V AC,

50 Hz, 1Ph

Micro controller based timer with range

0 to 30/99 minutes Figure 3.3: Sonicator

Thermostatic controlled temperature controller



3.2 CHEMICALS

A. Drugs

The all drugs for this research work were obtained as gift sample from the

different companies listed below:

Table-3.3: List of Poorly Water-Soluble Drugs and their Supplier

Sr.

No.

Drugs Supplied By

1. AMLO Amlodipine Besylate Matrix Laboratories, Mumbai

2. OLM Olmesartan Medoxamil GSK, Mumbai and Hetero Drugs

Ltd., Baddi,

3. HCZ Hydrochlorothiazide Matrix Laboratories, Mumbai &

Hetero Drugs Ltd.,Baddi,

4. TSM Torasemide Macleodes Ltd, Mumbai,

5. PD Pramipexole

Dihyrochloride

Sun Pharmaceuticals Ind. Ltd

6. FZ Furazolidone GSK Ltd. Mumbai.

7. LOM Lomefloxacin Hcl Intas Pharma Ltd. Mumbai

8. CP Citalopram Hydrobromide Nicolas Piramal Lab., Mumbai,

9. ZIP Ziprasidone HCl Torrent Pharmaceuticals Ltd

10. ENT Entacapone Sun Pharmaceuticals Ind. Ltd

11. MCM Meloxicam Intas Laboratories Pvt Ltd

12. LER Lercanidipine HCl Glenmark Pharm. Ltd. Nashik,

13. KET Ketoconazole IPCA Laboratories,

14. EPS Eprosartan maleate Dishman Pharma & Chem Ltd.,

15. LEVO Levofloxacin Intas Laboratories Pvt Ltd

16. OZ Ornidazole Swan pharmaceutical, Indore

17. MTR Metronidazole Swan pharmaceutical, Indore

18. OFL Ofloxacin Swan pharmaceutical, Indore

19. MTO Metoprolol succinate Unichem Laboratories Ltd.

20. TEL Telmisartan Unichem Laboratories Ltd.

Mumbai



B. Solvent and Reagent

Table-3.4: List of Hydrotropic Agent Used

Sr. No. Hydrotropic Agent Company

1. Ammonium Acetate Merck Chemical Division

2. Ascorbic Acid Merck Chemical Division

3. Citric acid Merck Chemical Division

4. Ibuprofen sodium Merck Chemical Division

5. Niacinamide Merck Chemical Division

6. Nicotinamide Merck Chemical Division

7. Phenol Merck Chemical Division

8. Resocrinol Merck Chemical Division

9. Sodium acetate Merck Chemical Division

10. Sodium Ascorbate Merck Chemical Division

11. Sodium Benzoate Merck Chemical Division

12. Sodium citrate Merck Chemical Division

13. Sodium salicylate Merck Chemical Division

14. Sodium Tartrate Merck Chemical Division

15. Urea Merck Chemical Division

C. Marketed Formulation

Table-3.5: List of Market Formulation Used

Sr.

No

.

Drugs Brand Label claim/

Strength Manufacture

A Amlodipine Besylate

Amdepin 10 mg Cadila Pharma.

Amlopres 10 mg Cipla Ltd

Amlovas 10 mg Macleods

B Olmesartan

Medoxamil

Olmecip 10 mg Cipla Ltd.

Pinom 20 20 mg Lupin lab.Ltd.

C Hydrochlorothiazide Aquazide 25 mg Sun Pharma Ind. Ltd.,

Klorzide 25 mg Zydus Ltd

D Torasemide Dyamide 20 mg Macleodes Ltd

Dytor 20 mg Cipla Ltd



E Furazolidone Furoxon 100 mg GSk Pharma Ltd

Lomocyp 100 mg Cyper Pharma

F Lomefloxacin HCL Lomitas 400 mg Intas Lab. Pvt Ltd

Lomeflox 400 mg IPCA Lab. Ltd.

G Pramipexole

Dihyrochloride

Pramipex 0.5 mg Sun Pharma. Ind.Ltd.

Parpex 0.5 mg Zydus Cadila

Healthcare Ltd

H Citalopram

Hydrobromide

Cetopam 10 mg Sun Pharma. Ind.Ltd.,

Citara 10 mg Intas Pharma Ltd.

I Ziprasidone

Hydrochloride

Azona * 80mg Torrent Pharma. Ltd.

Zipsydon 80 mg Sun Rise International

Labs Ltd.

J Entacapone Entacom 200mg Intas Pharma Ltd.

K Meloxicam M -Cam 7.5mg Unichem Lab. Ltd.

Movac 7.5mg Alkem Lab. Ltd

L Lercanidipine HCL Landip 10mg Micro Labs Ltd

Lerka 10mg Piramal Healthcare

M Ketoconazole Fungicide 200mg Torrent Pharma. Ltd.

Ketozole 200mg Ranbaxy (Rexcel div.)

N

Eprosartan mesylate

and

Hydrochlorothiazide

Teveten®

HCT,

EPS-600 mg

HCZ- 25mg Solvay Pharmaceuticals

O

Olmesartan

medoxamil and

Hydrochlorothiazide-

Olmetor-

H,

OLM-20mg,

HCZ-12.5mg Hetero Drugs Ltd.

P

Levofloxacin

Hemihydrates and

Ornidazole

Levoflox

–OZ,

LEVO-250

mg, OZ-500mg Cipla Limited

Q Metronidazole &

Furazolidone Metrofur

MTR-200mg

FZ-100mg Western Remedies

R Metronidazole and

Ofloxacin

Oflaswift

– m

MET-100 mg

OFL-50 mg Swift Medicare pvt.ltd.

S

Olmesartan

Medoxamil

Metoprolol Succinate

Olsar-M OLM- 20 mg

MTO-50 mg Unichem laboratories

T Metoprolol Succinate

and Telmisartan

Telsar

Beta

MTO-50 mg,

TEL-40 mg Unichem laboratories

*Available in capsule dosage form; rest of the formulation are in tablet dosage

form



Method- 3A

Spectrophotometric Method Development and Validation for Quantitative

Estimation of Amlodipine Besylate in Bulk Drug and Their Dosage Forms by

Using Hydrotropic Agent

Apparatus

As per the section 3.1

Reagents and Standards


Theme

Preliminary solubility studies

Selection of hydrotropic agent.

Establishment of stability profile of drugs in hydrotropic solution.

Wavelength selection for linearity study.

Linearity range and calibration graph.

Method development.

Tablet analysis

Validation of developed method.

3A.1. Preliminary Solubility Studies

Solubility of amlodipine besylate was determined at 28±1 °C. An definite amount

of drug was placed to screw capped 30 ml glass vials containing different aqueous

systems viz. distilled water, buffer (pH 8.2) and 2 M sodium acetate solution and

other hydrotropic agent for 8 hrs.. After 8 hrs all solution were filtered through

whatman filter paper No. 41. The filtrates were diluted suitably and analyzed

spectrophotometrically against water. Enhancement of solubility of drug was

more than 75 fold.

3A.2. Selection of Hydrotropic Agent

AMLO was scanned in various hydrotropic agents in the spectrum mode over the

UV range (200-400) and 2M sodium acetate was found to be most appropriate

because:

AMLO is soluble in it (75 fold enhancement of solubility)

AMLO is stable in it (as shown in 3A.3)



AMLO exhibit good spectral characteristics in it.

Sodium acetate solution has no interference with the λmax of AMLO

365nm (Figure 4A.1)

3A.3 Establishment of Stability Profile

Stability of AMLO was observed by dissolving in 2 M sodium acetate solution

used as hydrotropic agent. Solution of AMLO was prepared in the conc. of 150

g/ml and scanned under time scan for 30 min. Spectra of drug under time scan

shows that drug are stable in hydrotropic solution.

3A. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solution (Stock-A)

Standard stock solutions were prepared by weighed accurately 100 mg of the

AMLO which was transferred in to 100 ml volumetric flask containing 10 ml of

2M sodium acetate solution and the flask was sonicated for about 10 min to

solubilize the drug and the volume was made up to the mark with hydrotropic

agent to get a concentration of 1000 µg/ml (Stock-A).

Preparation of Working Standard Solution

The Aliquots ranging from 50-250 μg/m1 were prepared from stock solution

(1000 μg/ml) and absorbance was noted at 365 nm against distilled water as

blank. Calibration curve was plotted between concentrations versus absorbance

(Figure 4A.2). Result of linearity data (Table 4A.1) and their optical

characteristics has reported in Table 4A. 2.

3A.5 Analysis of Tablet Formulation

Three marketed formulation Amdepin (Cadila pharma ltd), Amlopres (Cipla Ltd)

and Amlovas (Macleods) were selected for tablet analysis. Twenty tablets of each

formulation were weighed and ground to a fine powder. An accurately weighed

powder sample equivalent to 10 mg of AMLO was transferred to a 10 ml of

volumetric flask containing 10 ml of 2 M sodium acetate solution. The flask was

shaken for about 10 min to solubilize the drug. The solution was filtered through

whatman filter paper No. 41. The filtrate was diluted appropriately with distilled

water and analyzed on UV spectrophotometer against water as blank. Drug

content of tablet formulation were calculated using calibration curve and value are



reported in Table 4A.3 and Table 4A.4.

3A.6 Validation of Method

a. Linearity

Linearity of AMLO was established by response ratios of drug. Response ratio of

drug was calculated by dividing the absorbance with respective concentration

Table 4A. 5. Then a graph was plotted between concentration and response ratio

Figure 4A.3.

b. Accuracy

Recovery studies were performed by adding the definite amount of drug using

pre-analyzed tablet solution. These studies were performed in two ways: one by

adding fixed amount of pure drug solution to the final dilution while varying the

concentration of tablet sample solution in the final dilution and second, by

varying the amount of drug solution added to the final dilution keeping the

concentration of sample solution in the final dilution constant and the calculate

the recovery in both the cases and result of recovery studies are presented in

Table 4A. 6. Recovery analysis was repeated at 6 replicate of 4 concentrations

levels.

c. Precision

Precision of the methods was studied at three level as at repeatability,

intermediate precision (Day to Day and analyst to analyst) and reproducibility.

Repeatability was performed by analyzing same 6 concentrations of drug for 5

times. Day to Day was performed by analyzing 6 different concentration of the

drug for three days in a week.

Reproducibility was performed by analyzing 6 different concentrations for 5 times

in different labs. The results are reported in Table 3A. 7.



Method- 3B

Application of Hydrotropic Solubilization Phenomenon for Quantitative

Analysis of Olmesartan Medoxamil in Tablet

Apparatus




Theme






Method development.

Tablet analysis


3B.1. Preliminary Solubility Studies

Solubility of OLM was determined at 28±1°C. A definite amount of drug was

added to screw capped 30 ml glass vials containing different aqueous systems viz.

distilled water, 8 M urea and 2 M sodium acetate solution and mixture of 2 M

sodium acetate and 8 M Urea (50:50% V/V) solution for 8 hrs. After 8 hrs all

solutions were filtered through whatman filter paper No. 41. The filtrates were

diluted suitably and analyzed spectrophotometrically against water. There was

more than 44 fold solubility enhanced in mixed hydrotropic solution as compare

with distilled water.

3B.2. Selection of Hydrotropic Agent

OLM was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and mixture of 2 M sodium acetate and 8 M Urea (50:50% V/V)

solution were found to be most appropriate because:

OLM is soluble in it (44 fold enhancement of solubility)

OLM is stable in hydrotropic agent (as shown in 3B. 3)



OLM exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of OLM

257nm Figure 4B.1.

3B.3 Establishment of Stability Profile

Stability of OLM was observed by dissolving in mixture of 2 M sodium acetate

and 8 M urea (50:50% V/V) solution used as hydrotropic agent. Solution of OLM

was prepared in the conc. of 30 g/ml and scanned under time scan for 30 min.

Spectra of drug under time scan shows that drug are stable in hydrotropic

solution.

3B. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the OLM was transferred in to 100 mL volumetric

flask containing 80 ml of mixture of 2 M sodium acetate and 8 M urea (50:50%

V/V) solution and the flask were sonicated for about 10 min to solubilize the drug

and the volume was made up to the mark with mixed hydrotropic agent to get a

concentration of 1000 µg/ml (Stock-A).


The standard solution (1000 µg/ml) was further diluted with distilled water to

obtain 10, 20, 30, 40 and 50 µg /ml solution and absorbance were noted at 257 nm

against distilled water as blank. Calibration curve was plotted between

concentrations versus absorbance Figure 4B.2. Observation of linearity data has

been reported in the Table 4B. 1. The Result of their optical characteristics shown

in Table 4B.2.

3B.5 Analysis of tablet formulation

Two Marketed formulation Olmecip-10 (Cipla Ltd.) and Pinom 20 (Lupin

laboratories Ltd.) were selected for tablet analysis. Twenty tablets of each

formulation were weighed and ground to a fine powder. An accurately weighed

powder sample equivalent to 100 mg of OLM was transferred to 80 ml of

volumetric flask containing mixed hydrotropic solution. The flask was sonicated

for about 10 min to solubilize the drug and the volume was made up to mark. The

solution was filtered through whatman filter paper No. 41. The filtrate was diluted



appropriately with distilled water and was analyzed on UV spectrophotometer

against distilled water as blank. Drug content of tablet formulation were

calculated using calibration curve Figure 4B.2 and value are reported in Table

4B.3. The statistical evaluation of tablet analysis has reported in Table 4B.4.

3B.6 Validation of method

a. Linearity

Linearity of OLM was established by response ratios of drug. Response ratio of


Table 4B. 5. Then a graph was plotted between concentration and response ratio

Figure 4B. 3.

b. Accuracy

To evaluate the recovery studies, to pre-analyzed tablet solution, a definite

amount of drug was added and then its recovery was studied. These studies were

performed, in pre-analyzed tablet solution ranging from 10-50 μg/ml, bulk drug

samples 10μg/ml was added as spiked concentrations and drug contents were

determined by the proposed analytical method. Result of recovery studies are

presented in Table 4B. 6. Recovery analysis was repeated at 6 replicate of 5

concentrations levels.

c. Precision







in different labs. The results are reported in Table 4B. 7.



Method-3C

Novel Spectrophotometric Quantitative Estimation of Hydrochlorothiazide

in Bulk Drug and their Dosage Forms by Using Hydrotropic Agent

Apparatus




Theme






Method development.

Tablet analysis


3C.1. Preliminary Solubility Studies

Solubility of HCZ was determined in distilled water and hydrotropic solution of 2

M sodium acetate and 8 M Urea separately at 28±1°. A defined amount of drug

was placed to screw capped 30 ml glass vials containing distilled water and

hydrotropic agents for 8 hrs. After 8 hrs both solution were filtered through

whatman filter paper No. 41. The filtrates were diluted suitably and analyzed

spectrophotometrically against water. There was more than 55 and 70 fold

solubility enhanced in 2 M sodium acetate and 8 M Urea solution respectively, as

compare with distilled water.

3C.2. Selection of Hydrotropic Agent

HCZ was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and 2 M sodium acetate and 8 M Urea were found to be most

appropriate because:

HCZ is soluble in it (55 and 72 fold enhancement of solubility)

HCZ is stable in both hydrotropic agent (as shown in 3C. 3)



HCZ exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of HCZ

272nm (Figure 4C.1 and 4C. 2)

3C.3 Establishment of Stability Profile

Stability of HCZ was observed by dissolving in 2 M sodium acetate and 8 M Urea

solution used as hydrotropic agent. Solution of HCZ was prepared in the conc. of

20 g/ml and scanned under time scan for 30 min. Spectra of drug under time

scan shows that drug are stable in hydrotropic solution.

3C. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the HCZ was transferred in to 100 ml volumetric

flask containing 50 ml of 2 M sodium acetate and 8 M urea solution separately

and the flask were sonicated for about 10 min to solubilize the drug and the

volume was made up to the mark with hydrotropic agent to get a concentration of

1000 µg/ml (Stock-A).



obtain 10, 20, 30, 40 and 50 µg /ml solution from sodium acetate. Likewise the

dilution ranging from 5-25µg/ml was prepared from stock solution containing

urea and absorbance was noted at 272 nm against distilled water as blank.

Calibration curve was plotted between concentrations versus absorbance; Figure

4C.3 and Figure 4C. 4. Observation of linearity data has been reported in the

Table 4C.1 and Table 4C.2. The Result of their optical characteristics is shown

in Table 4C.3.

3C.5 Analysis of Tablet Formulation

Two marketed formulation Aquazide (Sun Pharma) and Klorzide (Zydus Ltd)

were selected for tablet analysis. Twenty tablets of each formulation were

weighed and ground to a fine powder. An accurately weighed powder sample

equivalent to 25 mg of HCT was transferred to two different 100 ml of volumetric

flask containing 100 ml of 2 M sodium acetate and 8 m urea solution. The flask

was sonicated for about 10 min to solubilize the drug. The solution was filtered



through whatman filter paper No 41. The filtrate was diluted appropriately with

distilled water and was analyzed on UV spectrophotometer against distilled water

as blank. Drug content of tablet formulation were calculated using calibration

curve and value are reported in Table 4C.4 and Table 4C. 5. The statistical

evaluation of tablet analysis has reported in Table 4C.6.

3C.6 Validation of Method

a. Linearity

Linearity of HCZ was established by response ratios of drug. Response ratio of


Table 4C. 7. Then a graph was plotted between concentration and response ratio

Figure 4C.5 and Figure 4C.6.

b. Accuracy



performed in by adding fixed amount of pure drug solution to the final dilution

while varying the concentration of tablet sample solution in the final dilution and

result of recovery studies are presented in Table 4C. 8. Recovery analysis was

repeated at 6 replicate of 5 concentrations levels.

c. Precision







in different labs. The results are reported in Table 4C. 9.



Method-3D

Novel Spectrophotometric Quantitative Estimation of Torasemide in Tablets

Using Mixed Hydrotropic Agent

Apparatus




Theme






Method development.

Tablet analysis


3D.1. Preliminary Solubility Studies

Solubility of TSM was determined at 28±1°C. A definite amount of drug was

added to screw capped 30 ml glass vials containing different aqueous systems viz.



solution were filtered through whatman filter paper No. 41. The filtrates were




3D.2. Selection of Hydrotropic Agent

TSM was scanned in hydrotropic agent in the spectrum mode over the UV range



TSM is soluble in it (86 fold enhancement of solubility)

TSM is stable in hydrotropic agent (as shown in 3D. 3)



TSM exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of TSM

288nm (Figure 4D.1)

3D.3 Establishment of Stability Profile

Stability of TSM was observed by dissolving in mixture of 2 M sodium acetate

and 8 M Urea (50:50% V/V) solution used as hydrotropic agent. Solution of TSM

was prepared in the conc. of 30 g/ml and scanned under time scan for 30 min.


solution.

3D. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the TSM was transferred in to 100 ml volumetric

flask containing 80 ml of mixture of 2 M sodium acetate and 8 M Urea (50:50%






obtain 10, 20, 30, 40 and 50 µg /ml solution and absorbance was noted at 288 nm

against distilled water as blank. Calibration curve was plotted between

concentrations versus absorbance; Figure 4D.2. Observation of linearity data has

been reported in the Table 4D.1. The Result of their optical characteristics is

shown in Table 4D.2.

3D.5 Analysis of Tablet Formulation

Two marketed formulation Dyamide (Macleodes Ltd) and Dytor (Cipla Ltd) were

selected for tablet analysis. Twenty tablets of each formulation were weighed and

ground to a fine powder. An accurately weighed powder sample equivalent to 20

mg of TSM was transferred to 100 ml of volumetric flask containing mixed

hydrotropic solution. The flask was sonicated for about 10 min to solubilize the

drug and the volume was made up to mark. The solution was filtered through

whatman filter paper No. 41. The filtrate was diluted appropriately with distilled



water and was analyzed on UV spectrophotometer against distilled water as blank.

Drug content of tablet formulations were calculated using calibration curve and

values are reported in Table 4D.3. The statistical evaluation of tablet analysis has

reported in Table 4D.4.

3D.6 Validation of Method

a. Linearity

Linearity of TSM was established by response ratios of drug. Response ratio of

drug calculated by dividing the absorbance with respective concentration Table

4D. 5. Then a graph was plotted between concentration and response ratio Figure

4D.3

b. Accuracy



performed, in pre-analyzed tablet solution ranging from 10-50 μg /ml, bulk drug

samples 10 μg /ml was added as spiked concentrations and drug contents were

determined by the proposed analytical method. Results of recovery studies are

presented in Table 4D. 6. Recovery analysis was repeated at 6 replicate of 5


c. Precision






Reproducibility was performed by analyzing 6 different for 5 times in different

labs. The results are reported in Table 4D. 7.



Method-3E

Novel UV-Spectrophotometric Method for Quantitative Estimation of

Furazolidone using Mixed Hydrotropic Agent

Apparatus




Theme






Method development.

Tablet analysis


3E.1. Preliminary Solubility Studies

Solubility of FZ was determined at 28±1°C. A definite amount of drug was added

to screw capped 30 ml glass vials containing different aqueous systems viz.

distilled water, mixed hydrotropy using 2 M sodium acetate, 8 M urea, 2 M

niacinamide and 2 M sodium benzoate solution (25:25:25:25% V/V) as

hydrotropic agent for 8 hrs. After 8 hrs all solution were filtered through whatman

filter paper No. 41. The filtrates were diluted suitably and analyzed

spectrophotometrically against water. There was more than 32 fold solubility

enhanced in mixed hydrotropic solution as compare with distilled water.

3E.2. Selection of Hydrotropic Agent

FZ was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and mixture of 2 M sodium acetate, 8 M urea, 2 M niacinamide and 2

M sodium benzoate solution (25:25:25:25% V/V) as hydrotropic agent were

found to be most appropriate because:

FZ is soluble in it (32 fold enhancement of solubility)



FZ is stable in hydrotropic agent (as shown in 3E. 3)

FZ exhibit good spectral characteristics in it.

Sodium acetate, urea, niacinamide and sodium benzoate has no

interference with the λmax of FZ 360nm (Figure 4E.1)

3E.3 Establishment of Stability Profile

Stability of FZ was observed by dissolving in mixture of 2 M sodium acetate, 8 M

urea, 2 M niacinamide and 2 M sodium benzoate solution (25:25:25:25% V/V) as

hydrotropic agent. Solution of FZ was prepared in the conc. of 30 g/ml and

scanned under time scan for 30 min. Spectra of drug under time scan shows that

drug are stable in hydrotropic solution.

3E. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the FZ was transferred in to 100 ml volumetric

flask containing 80 ml of mixture of 2 M sodium acetate, 8 M urea, 2 M

niacinamide and 2 M sodium benzoate solution (25:25:25:25% V/V) as

hydrotropic agent and the flask were sonicated for about 10 min to solubilize the

drug and the volume was made up to the mark with mixed hydrotropic agent to

get a concentration of 1000 µg/ml (Stock-A).




against distilled water as blank. Spectra of FZ is shown in Figure 4E.1


4E.2. Observations of linearity data has been reported in the Table 4E. 1 and The

Result of their optical characteristics are shown in Table 4E. 2

3E.5 Analysis of Tablet Formulation

Two marketed formulation Furoxon (Glaxo Smithkline Pharmaceuticals Ltd.) and

Lomocyp (Cyper Pharma) were selected for tablet analysis containing 100 mg FZ.

Twenty tablets were accurately weighed, ground to fine powder. An accurately

weighed quantity of powder equivalent to 100 mg of FZ was transferred into 100

ml volumetric flask containing 80 ml of mixed hydrotropic solution. The flask



was sonicated for about 20 min to solublize the drug. Volume was adjusted to

mark with hydrotropic agent and filtered through whatman filter paper no. 41.

Absorbance of samples solutions were analyzed on UV spectrophotometer at 360

nm against R.O. water as blank. Drug content of tablet formulations were

calculated using calibration curve and values are reported in Table 4E.3. The

statistical evaluation of tablet analysis has reported in Table 4E.4.

3E.6 Validation of Method

a. Linearity

Linearity of FZ was established by response ratios of drug. Response ratio of drug

was calculated by dividing the absorbance with respective concentration Table

4E. 5. Then a graph was plotted between concentration and response ratio Figure

4E.3.

b. Accuracy



performed, in pre-analyzed tablet solution ranging from 10-50 μg /ml, bulk drug

samples 10μg/ml was added as spiked concentrations and drug contents were

determined by the proposed analytical method. Result of recovery studies are

presented in Table 4E.6. Recovery analysis was repeated at 5 replicate of 5


c. Precision





drug for three days in a week. Reproducibility was performed by analyzing same

concentration of drugs for five times in different lab. The results are shown in

Table 4E. 7.

d. LOD and LOQ

LOD and LOQ of the proposed method were calculated by using the standard

deviation method and results are shown in the Table 4E. 8.



Method-3F

Eco Friendly Spectrophotometric Method for Quantitative Estimation of

Lomefloxacin Using Hydrotropic Approach

Apparatus




Theme






Method development.

Tablet analysis


3F.1. Preliminary Solubility Studies

A definite amount of drug was added to a screw capped 25 ml of volumetric flask

containing different aqueous systems viz. distilled water, buffer of pH 6.4, buffer

of pH 8.2, and 8M urea solution. The volumetric flasks were shaken mechanically

for 12 hrs at 25±1°C in a mechanical shaker. These solutions were allowed to

equilibrate for next 24 hrs and then centrifuged for 5 min at 2000 rpm. The

supernatant liquid was taken for appropriate dilution after filtered through

whatman filter paper #41 and analyzed spectrophotometrically against

corresponding RO water as blank. After analysis, it was found that the

enhancement in the solubility of LOM was to be more than and 43 folds in 8M

urea solution as compared to solubility studies in other solvents.

3F.2. Selection of Hydrotropic Agent

LOM was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and 8 M urea as hydrotropic agent were found to be most appropriate

because:



LOM is soluble in it (43 fold enhancement of solubility)

LOM is stable in hydrotropic agent (as shown in 3F. 3)

LOM exhibit good spectral characteristics in it.

Urea solution has no interference with the λmax of LOM 281nm (Figure

4F.1)

3F.3 Establishment of Stability Profile

Stability of LOM was observed by dissolving in 8 M urea as hydrotropic agent.

Solution of LOM was prepared in the conc. of 15 g/ml and scanned under time

scan for 30 min. Spectra of drug under time scan shows that drug are stable in

hydrotropic solution.

3F. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the LOM was transferred in to 100 ml volumetric

flask containing 80 ml of 8 M urea as hydrotropic agent and the flask were

sonicated for about 10 min to solubilize the drug and the volume was made up to

the mark with hydrotropic agent to get a concentration of 1000 µg/ml (Stock-A).



obtain 5, 10, 15, 20 and 25µg /ml solution and absorbance were noted at 281 nm

against distilled water as blank. A spectrum of LOM is shown in Figure 4F.1.


4F.2. Observation of linearity data has been reported in the Table 4F. 1 The

Result of their optical characteristics is shown in Table 4F. 2.

3F.5 Analysis of Tablet Formulation

Two marketed formulation Lomitas (Intas Laboratories Pvt. Ltd.) and Lomeflox -

400 (IPCA Laboratories Ltd.) were selected for tablet analysis i.e. containing 400

mg LOM. Twenty tablets were accurately weighed, average weight determined

and ground to fine powder. An accurately weighed quantity of powder equivalent

to 100 mg of LOM was transferred into 100 ml volumetric flask containing 80 ml

of hydrotropic solution. The flask was sonicated for about 20 min to solublize the

drug. Volume was adjusted to mark with hydrotropic agent and filtered through



whatman filter paper no. 41. The resulting solution was further diluted to get

concentration of 5, 10, 15, 20 and 25 μg/ml of LOM. Absorbances of sample

solutions were analyzed on UV spectrophotometer at 281 nm against R.O. water

as blank. Drug content of tablet formulations were calculated using calibration

curve and values are reported in Table 4F.3. The statistical evaluation of tablet

analysis has reported in Table 4F.4.

3F.6 Validation of Method

a. Linearity

Linearity of LOM was established by response ratios of drug. Response ratio of


Table 4F. 5. Then a graph was plotted between concentration and response ratio

(Figure 4F.3).

b. Accuracy

To check the degree of accuracy of the method, recovery studies were performed

in triplicate by standard addition method at 80%, 100% and 120%. In pre-

analyzed tablet solution, a definite amount of drug was added and then its

recovery was studied. These studies were performed in by adding fixed amount of

pure drug solution to the final dilution while varying the concentration of tablet

sample solution in the final dilution. The percentage recovery and percentage

relative standard deviation of the recovery were calculated and reported in Table

4F. 6.

c. Precision






Reproducibility was performed by analyzing same concentration of drugs for five

times in different lab. The results are reported in Table 4F. 7.



Method-3G

Ecofriendly Spectrophotometric Method Development and Their Validation

for Quantitative Estimation of Pramipexole Dihyrochloride Using Mixed

Hydrotropic Agent

Apparatus




Theme






Method development.

Tablet analysis


3G.1. Preliminary Solubility Studies

Solubility of PD was determined at 25±1°C. A definite amount of drug was added

to screw capped 30 ml glass vials containing different aqueous systems viz.



solution were filtered through whatman filter paper No. 41. The filtrates were




3G.2. Selection of Hydrotropic Agent

PD was scanned in hydrotropic agent in the spectrum mode over the UV range



PD is soluble in it (46 fold enhancement of solubility)



PD is stable in hydrotropic agent (as shown in 3G. 3)

PD exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of PD

262nm (Figure 4G.1)

3G.3 Establishment of Stability Profile

Stability of PD was observed by dissolving in mixture of 2 M sodium acetate and

8 M urea (50:50% V/V) solution used as hydrotropic agent. Solution of PD was

prepared in the conc. of 45 g/ml and scanned under time scan for 30 min.


solution.

3G. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the PD was transferred in to 100 ml volumetric

flask containing 80 ml of mixture of 2 M sodium acetate and 8 M Urea (50:50%







against distilled water as blank. A Spectra of PD is shown in Figure 4G.1.


4G.2. Observation of linearity data has been reported in the Table 4G. 1 The

Result of their optical characteristics has shown in Table 4G.2.

3G.5 Analysis of Tablet Formulation

Two marketed formulation Pramipex (Sun Pharmaceuticals Ind. Ltd.), Parpex

(Zydus Cadila Healthcare Ltd.) were selected for tablet analysis. Twenty tablets of

PD were weighed and ground to a fine powder. An accurately weighed powder

sample equivalent to 10 mg of PD was transferred to 10 ml of volumetric flask

containing 8 ml of mixed hydrotropic solution, containing 8 M urea and 2 M

sodium acetate solution (50:50%v/v). The flask was warm and sonicate for about



30 min to solublized the drug. The solution was filtered through whatman filter

paper No 41. The filtrate was diluted appropriately with distilled water and was

analyzed on UV spectrophotometer against distilled water as blank. Drug content

of tablet formulation were calculated using calibration curve and values are

reported in Table 4G.3. The result of statistical evaluation of tablet analysis is

reported in Table 4G.4.

3G.6 Validation of Method

a. Linearity

Linearity of PD was established by response ratios of drug. Response ratio of drug

calculated by dividing the absorbance with respective concentration Table 4G. 5.

Then a graph was plotted between concentration and response ratio Figure 4G.3.

b. Accuracy



performed by taking different conc. ranging from 15-75µg/ml and analyze them.

Bulk drug samples ranging from 15-75 µg/ml were added as spiked concentrations

and drug contents were determined by the proposed analytical method. Result of

recovery studies from tablet are reported in Table 4G. 6. Recovery analysis was

repeated at 3 replicate of 3 concentrations levels.

c. Precision







in different labs. The results are reported in Table 4G. 7.



Method-3H

Economical Spectrophotometric Method for Quantitative Estimation of

Citalopram Hydrobromide Using Hydrotropic Solubilization Technique

Apparatus




Theme






Method development.

Tablet analysis


3H.1. Preliminary Solubility Studies


containing different aqueous systems viz. distilled water, buffer of pH 6.4, buffer

of pH 8.2, and 8M urea solution. The volumetric flasks were shaken mechanically

for 12 hrs at 25±1°C in a mechanical shaker. These solutions were allowed to

equilibrate for next 24 hrs and then centrifuged for 5 min at 2000 rpm. The


whatman filter paper no. 41 and analyzed spectrophotometrically against

corresponding solvent blank. After analysis, it was found that the enhancement in

the solubility of CP was to be more than 70 folds in 8M urea solution as compared

to solubility studies in other solvents.

3H.2. Selection of Hydrotropic Agent

CP was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and 8 M urea as hydrotropic agent was found to be most appropriate

because:



CP is soluble in it (70 fold enhancement of solubility)

CP is stable in hydrotropic agent (as shown in 3H. 3)

CP exhibit good spectral characteristics in it.

Urea solution has no interference with the λmax of CP 238nm (Figure

4H.1)

3H.3 Establishment of Stability Profile

Stability of CP was observed by dissolving in 8 M urea as hydrotropic agent.

Solution of CP was prepared in the conc. of 15 g/ml and scanned under time



3H. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the CP was transferred in to 100 ml volumetric

flask containing 80 ml of 8 M urea as hydrotropic agent and the flask was






against distilled water as blank. A spectrum of CP is shown in Figure 4H.1.

Calibration curve was plotted between concentrations versus absorbance;

Figure4H.2. Observation of linearity data has been reported in the Table 4H. 1

The Result of their optical characteristics shown in Table 4H. 2.

3H.5 Analysis of Tablet Formulation

Two marketed formulation Cetopam-10 mg (Sun Pharma) and Citara (Intas

Pharmaceuticals Ltd.) were selected for tablet analysis, i.e. containing 10 mg CP.

Twenty tablets were accurately weighed, average weight determined and ground

to fine powder. An accurately weighed quantity of powder equivalent to 10 mg of

CP was transferred into 10 ml volumetric flask containing 8 ml of hydrotropic

solution. The flask was sonicated for about 20 min to solublize the drug; volume

was adjusted to mark with hydrotropic agent and filtered through whatman filter



paper no. 41. The resulting solution was further diluted to get appropriate

concentration CP. Absorbance of sample solutions were analyzed on UV

spectrophotometer at 238 nm against R.O. water as blank. Drug content of tablet

formulations were calculated using calibration curve. The statistical evaluation of

tablet analysis has been reported in Table4H. 3. and Table4H.4.

3H.6 Validation of Method

a. Linearity

Linearity of CP was established by response ratios of drug. Response ratio of drug

was calculated by dividing the absorbance with respective concentration

Table4H. 5. Then a graph was plotted between concentration and response ratio

Figure 4H. 3.

b. Accuracy


in triplicate by standard addition method at 80%, 100% and 120%. In preanalyzed

tablet solution, a definite amount of drug was added and then its recovery was

studied. These studies were performed in by adding fixed amount of pure drug

solution to the final dilution while varying the concentration of tablet sample

solution in the final dilution. The percentage recovery and percentage relative

standard deviation of the recovery were calculated and reported in Table4H. 6.

c. Precision







times in different lab. The results are reported in Table4H. 7.



Method-3I

Novel Spectrophotometric Method for Estimation of Ziprasidone

Hydrochloride Monohydrate Using Hydrotropic Solubilization Technique

Apparatus




Theme






Method development.

Tablet analysis


3I.1. Preliminary Solubility Studies


containing different aqueous systems viz distilled water, different hydrotropic

agent and 2M Citric acid. The volumetric flasks were shaken mechanically for 12

hrs at 25±1°C in a mechanical shaker. These solutions were allowed to equilibrate

for next 24 hrs and then centrifuged for 5 min at 2000 rpm. The supernatant liquid

was taken for appropriate dilution after filtration through whatman filter paper no.

41 and analyzed spectrophotometrically against corresponding solvent blank.

After analysis, it was found that the enhancement in the solubility of ZIP was to

be more than 56 folds in 2M citric acid solution as compared to solubility studies

in other solvents.

3I.2. Selection of Hydrotropic Agent

ZIP was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and 2 M citric acid as hydrotropic agent were found to be most




ZIP is soluble in it (56 fold enhancement of solubility)

ZIP is stable in hydrotropic agent (as shown in 3I. 3)

ZIP exhibit good spectral characteristics in it.

Citric acid solution has no interference with the λmax of ZIP 314 nm.

3I.3 Establishment of Stability Profile

Stability of ZIP was observed by dissolving in 2 M citric acid as hydrotropic

agent. Solution of ZIP was prepared in the conc. of 60 g/ml and scanned under

time scan for 30 min. Spectra of drug under time scan shows that drug are stable

in hydrotropic solution.

3I. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the ZIP was transferred in to 100 ml volumetric

flask containing 80 ml of 2 M citric acid as hydrotropic agent and the flask was





obtain 20, 40, 60, 80 and 100µg /ml solution and absorbance were noted at 314nm

against distilled water as blank. A spectrum of ZIP is shown in Figure4I.1.

Calibration curve was plotted between concentrations versus absorbance;

Figure4I.2. Observation of linearity data has been reported in the Table 4I.1. The

Result of their optical characteristics has been reported in Table 4I. 2.

3I.5 Analysis of Tablet & Capsule Formulation

Two marketed formulation Azona capsule (Torrent Pharmaceuticals Ltd.),

Zipsydon tablet (Sun Rise International Labs Ltd.) were selected for analysis, i.e.

containing 80 mg ZIP. For tablet, twenty tablets were accurately weighed, average

weight determined and ground to fine powder and for capsule uncapped twenty

capsules and average weight determined. Accurately weighed quantity of powder

equivalent to 80 mg of ZIP was transferred into 100 ml volumetric flask and

dissolve in 80 ml of hydrotropic solution. The flask was sonicated for about 20

min to solublize the drug and filtered through whatman filter paper no. 41. The



resulting solution was further diluted. Absorbances of sample solutions were

analyzed on UV spectrophotometer at 314nm against R.O. water as blank. Drug

content of tablet and capsule formulation were calculated using calibration curve.

The statistical evaluation of tablet analysis has been reported in Table4I. 3. and

Table4I.4.

3I.6 Validation of Method

a. Linearity

Linearity of ZIP was established by response ratios of drug. Response ratio of


Table4I. 5. Then a graph was plotted between concentration and response ratio

Figure4I. 3.

b. Accuracy




recovery was studied. The percentage recovery and percentage relative standard

deviation of the recovery were calculated and reported in Table4I. 6.

c. Precision







times in different lab. The results has been reported in Table4I. 7.



Method-3J

A Novel Approach Using Hydrotropic Solubilization Technique for

Quantitative Estimation of Entacapone

Apparatus




Theme






Method development.

Tablet analysis


3J.1. Preliminary Solubility Studies


containing different aqueous systems viz. distilled water, different combination of

hydrotropic agent. The volumetric flasks were shaken mechanically for 12 hrs at

25±1°C in a mechanical shaker. These solutions were allowed to equilibrate for

next 24 hrs and then centrifuged for 5 min at 2000 rpm. The supernatant liquid

was taken for appropriate dilution after filtration through whatman filter paper

#41 and analyzed spectrophotometrically against corresponding solvent blank.

After analysis, it was found that the enhancement in the solubility of ENT was to

be more than 67 folds in 8 M Urea as compared to solubility studies in other

solvents.

3J.2. Selection of Hydrotropic Agent

ENT was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and 8 M Urea as hydrotropic agent were found to be most appropriate

because:



ENT is soluble in it (67 fold enhancement of solubility)

ENT is stable in hydrotropic agent (as shown in 3J. 3)

ENT exhibit good spectral characteristics in it.

Urea solution has no interference with the λmax of ENT i.e 378nm.

3J.3 Establishment of Stability Profile

Stability of ENT was observed by dissolving in 8 M Urea as hydrotropic agent.

Solution of ENT was prepared in the conc. of 12 g/ml and scanned under time



3J. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the ENT was transferred in to 100 ml volumetric

flask containing 80 ml of hydrotropic agent and the flask was sonicated for about

10 min to solubilize the drug and the volume was made up to the mark with mixed

hydrotropic agent to get a concentration of 1000 µg/ml (Stock-A).




against distilled water as blank. Spectra of ENT is shown in Figure 4J.1,

Calibration curve was plotted between concentrations versus absorbance

Figure4J.2. Observation of linearity data has reported in the Table 4J. 1 The

Result of their optical characteristics has been reported in Table 4J. 2.

3J.5 Analysis of Tablet Formulation

Marketed formulation Entacom (Intas Pharmaceuticals) was selected for tablet

analysis, i. e containing 200 mg ENT. Twenty tablets were accurately weighed,

average weight determined and ground to fine powder. An accurately weighed

quantity of powder equivalent to 100 mg of ENT was transferred into 100 ml

volumetric flask containing 80 ml of hydrotropic solution. The flask was

sonicated for about 20 min to solublize the drug; volume was adjusted to mark

with hydrotropic agent and filtered through whatman filter paper no. 41. The

Absorbance of sample solutions was analyzed on UV spectrophotometer at 378



nm against R.O. water as blank. Drug content of tablet formulation were

calculated using calibration curve and values are reported in Table4J. 3.

3J.6 Validation of method

a. Linearity

Linearity of ENT was established by response ratios of drug. Response ratio of


Table4J. 4. Then a graph was plotted between concentration and response ratio

Figure4J. 3.

b. Accuracy





deviation of the recovery were calculated and reported in Table4J. 5.

c. Precision







times in different lab. The results are reported in Table4J. 6.



Method-3K

Quantitative Estimation of Meloxicam: A Novel Approach Using

Hydrotropic Solubilization Technique

Apparatus




Theme






Method development.

Tablet analysis


3K.1. Preliminary Solubility Studies






was taken for appropriate dilution after filtration through whatman filter paper

#41 and analyzed spectrophotometrically against corresponding solvent blank.

After analysis, it was found that the enhancement in the solubility of MCM was to

be more than 32 folds in mixture of 8% phenol and 25% sodium benzoate solution

as compared to solubility studies in other solvents.

3K.2. Selection of Hydrotropic Agent

MCM was scanned in hydrotropic agent in the spectrum mode over the UV range

(200-400) and mixture of 8% phenol and 25% sodium benzoate solution as

hydrotropic agent were found to be most appropriate because:



MCM is soluble in it (32 fold enhancement of solubility)

MCM is stable in hydrotropic agent (as shown in 3K. 3)

MCM exhibit good spectral characteristics in it.

Phenol and sodium benzoate solution has no interference with the λmax of

MCM i.e. 362nm

3K.3 Establishment of Stability Profile

Stability of MCM was observed by dissolving in mixture of phenol and sodium

benzoate solution (8%:25%W/W) as hydrotropic agent. Solution of MCM was

prepared in the conc. of 45 g/ml and scanned under time scan for 30 min.


solution.

3K. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the MCM was transferred in to 100 ml volumetric


10 min to solubilize the drug and the volume was made up to the mark with mixed





against distilled water as blank. Spectra of MCM is shown in Figure 4K.1,

Calibration curve was plotted between concentrations versus absorbance Figure

4K.2. Observation of linearity data has been reported in the Table 4K.1. The

Result of their optical characteristics has shown in Table 4K. 2.

3K.5 Analysis of Tablet Formulation

Two marketed formulation M –Cam (Unichem Laboratories Ltd.), Movac (Alkem

Laboratories Ltd) were selected for tablet analysis, i.e. containing 7.5 mg MCM.

Twenty tablets were accurately weighed, average weight determined and ground

to fine powder. An accurately weighed quantity of powder equivalent to 100 mg

of MCM was transferred into 100 ml volumetric flask containing 80 ml of

hydrotropic solution. The flask was sonicated for about 20 min to solublize the



drug; volume was adjusted to mark with hydrotropic agent and filtered through

whatman filter paper no. 41. The Absorbance of sample solutions was analyzed on

UV spectrophotometer at 362 nm against R.O. water as blank. Drug content of

tablet formulation were calculated using calibration curve. The statistical

evaluation of tablet analysis is reported in Table4K.3 and Table 4K.4.

3K.6 Validation of Method

a. Linearity

Linearity of MCM was established by response ratios of drug. Response ratio of


Table 4K. 5. Then a graph was plotted between concentration and response ratio

Figure 4K. 3.

b. Accuracy





deviation of the recovery were calculated and reported in Table4K. 6.

c. Precision







times in different lab. The results are reported in Table4K. 7.



Method-3L

Quantitative Estimation of Lercanidipine Hydrochloride: A Novel Approach

Using Hydrotropic Solubilization Technique

Apparatus




Theme






Method development.

Tablet analysis


3L.1. Preliminary Solubility Studies






was taken for appropriate dilution after filtered through Whatman filter paper #41

and analyzed spectrophotometrically against corresponding solvent blank. After

analysis, it was found that the enhancement in the solubility of LER was to be

more than 61 folds in 2 M citric acid as compared to solubility studies in other

solvents.

3L.2. Selection of Hydrotropic Agent

LER was scanned in hydrotropic agent in the spectrum mode over the UV range





LER is soluble in it (61 fold enhancement of solubility)

LER is stable in hydrotropic agent (as shown in 3L. 3)

LER exhibit good spectral characteristics in it.

Citric acid solution has no interference with the λmax of LER i.e. 363 nm.

3L.3 Establishment of Stability Profile

Stability of LER was observed by dissolving in 2 M citric acid as hydrotropic

agent. Solution of LER was prepared in the conc. of 100 g/ml and scanned under



3L.4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the LER was transferred in to 100 ml volumetric


10 min to solubilize the drug and the volume was made up to the mark with




obtain 50, 100, 150, 200 and 250µg /ml solution and absorbance were noted at

363 nm against distilled water as blank. A spectrum of LER is shown in Figure

4L. 1, Calibration curve was plotted between concentrations versus absorbance

Figure 4L.2. Observation of linearity data has been reported in the Table 4L. 1

The Result of their optical characteristics has shown in Table 4L. 2

3L.5 Analysis of Tablet Formulation

Two marketed formulation Landip - 10 (Micro Labs Ltd), Lerka-10mg (Piramal

Healthcare) were selected for tablet analysis, i. e containing 10 mg LER. Twenty

tablets were accurately weighed, average weight determined and ground to fine

powder. An accurately weighed quantity of powder equivalent to 10 mg of LER

was transferred into 10 ml volumetric flask containing 8 ml of hydrotropic

solution. The flask was sonicated for about 20 min to solublize the drug; volume

was adjusted to mark with hydrotropic agent and filtered through whatman filter

paper no. 41. The Absorbance of sample solutions was analyzed on UV



spectrophotometer at 363 nm against R.O. water as blank. Drug content of tablet

formulations were calculated using calibration curve. The statistical evaluation of

tablet analysis has been reported in Table4L.3 and Table4L.4.

4L.6 Validation of Method

a. Linearity

Linearity of LER was established by response ratios of drug. Response ratio of


4L. 5. Then a graph was plotted between concentration and response ratio Figure

4L. 3.

b. Accuracy





deviation of the recovery were calculated and reported in Table4L. 6.

c. Precision







times in different lab. The results are shown in Table4L. 7.



Method-3M

Quantitative Estimation of Ketoconazole: A Novel Approach Using

Hydrotropic Solubilization Technique

Apparatus




Theme






Method development.

Tablet analysis


3M.1. Preliminary Solubility Studies






was taken for appropriate dilution after filtered through Whatman filter paper #41

and analyzed spectrophotometrically against corresponding solvent blank. After

analysis, it was found that the enhancement in the solubility of KET was to be

more than 39 folds in 2 M citric acid as compared to solubility studies in other

solvents.

3M. 2. Selection of Hydrotropic Agent

KET was scanned in hydrotropic agent in the spectrum mode over the UV range





KET is soluble in it (39 fold enhancement of solubility)

KET is stable in hydrotropic agent (as shown in 3M. 3)

KET exhibit good spectral characteristics in it.

Citric acid solution has no interference with the λmax of KET i.e 268 nm.

3M.3 Establishment of Stability Profile

Stability of KET was observed by dissolving in 2 M citric acid as hydrotropic

agent. Solution of KET was prepared in the conc. of 100 g/ml and scanned under



3M. 4 Linearity Range and Calibration Graph


Accurately weighed 100 mg of the KET was transferred in to 100 ml volumetric


10 min to solubilize the drug and the volume was made up to the mark with




obtain 100, 200, 300, 400 and 500µg /ml solution and absorbance were noted at

268 nm against distilled water as blank. Spectra of KET is shown in Figure 4M.

1, Calibration curve was plotted between concentrations versus absorbance

Figure 4M.2. Observation of linearity data has been reported in the Table 4M. 1

The Result of their optical characteristics has shown in Table 4M. 2

3M.5 Analysis of Tablet Formulation

Two marketed formulation Fungicide (Torrent Pharmaceuticals), Ketozole

(200mg) (Ranbaxy (Rexcel Division)) were selected for tablet analysis, i.e.

containing 200 mg KET. Twenty tablets were accurately weighed, average weight

determined and ground to fine powder. An accurately weighed quantity of powder

equivalent to 200 mg of KET was transferred into 100 ml volumetric flask

containing 80 ml of hydrotropic solution. The flask was sonicated for about 20

min to solublize the drug and volume was adjusted to mark with R.O. water and

filtered through Whatman filter paper no. 41. The Absorbance of samples



solutions were analyzed on UV spectrophotometer at 268 nm against R.O. water

as blank. Drug content of tablet formulations were calculated using calibration

curve and the statistical evaluation of tablet analysis reported in Table 4M.3 and

Table 4M.4.

3M.6 Validation of Method

a. Linearity

Linearity of KET was established by response ratios of drug. Response ratio of


4M. 5. Then a graph was plotted between concentration and response ratio Figure

4M.3.

b. Accuracy


in triplicate by standard addition method at 80%, 100% and 120%. In preanalyzed

tablet solution, a definite amount of drug was added and then its recovery was

studied. The percentage recovery and percentage relative standard deviation of the

recovery were calculated and reported in Table 4M.6.

c. Precision







times in different lab. The results are shown in Table 4M. 7.



Method-3N

Mixed Hydrotropy Solubilization Approach for Quantitative Estimation of

Eprosartan Mesylate and Hydrochlorothiazide by UV Spectrophotometer

Apparatus




Theme






Study of overlay spectra of drugs and selection of method.

Method development.

Tablet analysis


3N.1. Preliminary Solubility Studies

Solubility of both drugs was determined at 25±1°C. A definite amount of drugs

were added to two screw capped 25 ml of volumetric flask containing different

aqueous systems viz distilled water, buffer of pH 6.4, buffer of pH 8.2, Different

combination of hydrotropic agent and 2M sodium acetate and 8M urea solution.

The volumetric flasks were shaken mechanically for 12 h at 25±1°C in a

mechanical shaker. These solutions were allowed to equilibrate for next 24 h and

then centrifuged for 5 min at 2000 rpm. The supernatant liquid was taken for

appropriate dilution after filtered through whatman filter paper #41 and analyzed

spectrophotometrically against corresponding solvent blank. After analysis, it was

found that the enhancement in the solubility of EPS and HCZ was found to be

more than 56 and 74 folds respectively in mixture of 2M sodium acetate and 8M

urea solution (1:1) as compared to solubility studies in other solvents.



3N.2. Selection of Hydrotropic Agent

EPS and HCZ was scanned in hydrotropic agent in the spectrum mode over the

UV range (200-400) and mixture of 2 M sodium acetate and 8 M urea (50:50%

V/V) solution were found to be most appropriate because:

EPS and HCZ is soluble in it (56 and 74 fold enhancement of solubility)

EPS and HCZ is stable in hydrotropic agent (as shown in 3N. 3)

EPS and HCZ, both exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of EPS

and HCZ i.e. 267.5 and 271.5 nm respectively (Figure 4N.1).

3N.3 Establishment of Stability Profile

Stability of EPS and HCZ was observed by dissolving in mixed hydrotropic

agent. Solution of EPS and HCZ was prepared in the conc. of 45 g/ml and 15

g/ml respectively and scanned under time scan for 30 min. Spectra of drug under

time scan shows that drug are stable in hydrotropic solution.

3N. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of EPS and HCZ

Standard stock solutions were prepared by dissolving separately 100 mg of each

drug in mixed hydrotropic solution and the flask was sonicated for about 10 min

to solubilize the drug.

Preparation of Working Standard Solution for calibration curve

The standard solution (1000 µg/ml) was further diluted in ranging from 15-75

µg/ml for EPS and 5-25 µg/ml for HCZ. Calibration curve was plotted between

concentrations versus absorbance Figure 4N.2, Figure 4N.3. Linearity data of

both drugs has been reported in the Table 4N. 1 The results of their optical

characteristics are shown in Table 4N.2.

3N.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 267.5 nm and 271.5 nm for EPS

and HCZ respectively and isobestic point at 277 nm Figure 4N.1. Due to

difference in absorbance maxima and having no interference with each other so

both drug can be simultaneously estimated by simultaneous equation method

(Method A) and Q-analysis method (Method B)



Vierordt’s simultaneous equation method (Method A)

The wavelength 267.5 nm (λmax of EPS) and 271.5 nm (λmax of HCZ) was

selected. The absorbencies of EPS and HCZ were measured at 267.5 nm and

271.5 nm. This method of analysis is based on the absorption of drugs X and Y at

the wavelength maxima of the other. The quantification analysis of EPS and HCZ

in a binary mixture was performed by using Eqn-1 and Eqn-2.

CX = A2ay1- A1ay2 / ax2ay1- ax1ay2 ……..Eqn.1

CY = A1ax2- A2ax1 / ax2ay1-ax1ay2 ……...Eqn.2

Where CX and CY are the concentrations of EPS and HCZ respectively in the

diluted sample, ax1 and ax2 are absorptivities of EPS at λ1 and λ2, ay1 and ay2 are

absorptivities of HCZ at λ1 and λ2 respectively Table 4N.3. A1 and A2 are the

absorbances of samples at the 267.5 and 271.5 nm respectively.

Q-analysis method (Method B)

In this method absorbances of both the drugs were calculated at two selected

wavelengths; among which λ1 is the wavelength of isoabsorptive point of both

drugs and λ2 is the λmax of either drug among both drugs. From the overlain

spectra wavelength 277 nm (isoabsorption point) and 271.5 (λmax of HCZ) were

selected for study. The absorbencies at 277 nm and 271.5 nm for EPS were

obtained and similarly for HCZ absorbencies are measured at 277 nm and 271.5

nm. The concentrations of the individual components were calculated by using

the following equations;

CX = Qm-Qy/Qx-Qy)×A1 /ax1 …….. Eqn.3,

CY = Qm-Qx/Qy-Qx)×A1 /ay1 ……..Eqn.4

Where, Qm = A2 /A1, A1 is absorbance of sample at isoabsorptive point, A2 is

absorbance of sample at λmax of one of the two components. ax1 and ax2 represent

absorptivities of EPS at λ1 and λ2 and ay1 and ay2 denote absorptivities of HCZ at

λ1 and λ2 respectively Table 4N.3; CX and CY be the concentration of EPS and

HCZ respectively



3N.6 Analysis of Tablet Formulation

Twenty marketed tablets of EPS and HCZ (TEVETEN® HCT, Solvay

Pharmaceuticals) were weighed and ground to a fine powder; amount equal to 60

mg of EPS was taken in 10 ml volumetric flask. The HCZ present in this amount

of tablet powder was 2.5 mg. Then 8 ml of sodium acetate and urea solution was

added and the flask was sonicated for about 10 min to solubilize the drug

present in tablet powder and the volume was made up to the mark with

hydrotropic solution. After sonication filtration was done through whatman

filter paper No. 41. Filtrate was collected and further diluted with RO water to

get the final concentrations of both drugs in the working range. The absorbance of

final dilutions was observed at selected wavelengths and the concentrations were

obtained from simultaneous equation method and absorbance ratio method. The

result of tablet evaluation has reported in Table 4N.4.

3N.7 Validation of Method

a. Linearity

Linearity of EPS and HCZ was established by response ratios of drug. Response

ratio of drug was calculated by dividing the absorbance with respective

concentration Table 4N. 5. Then a graph was plotted between concentration and

response ratio Figure 4N.4, Figure 4N.5.

b. Accuracy

The accuracy of the proposed methods was assessed by recovery studies at three

different levels i.e. 80%, 100% and 120%. The recovery studies were carried out

by adding known amount of standard solution of EPS and HCZ to preanalysed

tablet solutions. The resulting solutions were then re-analysed by proposed

methods. Total amount of drug found and percentage recovery was calculated.

Result of recovery studies are reported in Table 4N. 6.

c. Precision

Precision of the methods was studied at three levels as at repeatability,

intermediate precision (Day to Day and analyst to analyst) and reproducibility

Table 4N. 7.



Method-3O

Economic Spectrophotometric Methods for Quantitative Estimation of

Olmesartan Medoxamil and Hydrochlorothiazide Using Mixed Hydrotropy

Apparatus




Theme







Method development.

Tablet analysis


3O.1. Preliminary Solubility Studies



aqueous systems viz. distilled water, buffer of pH 6.4, buffer of pH 8.2, Different

combination of hydrotropic agent and 2M sodium acetate and 8M urea solution.

The volumetric flasks were shaken mechanically for 12 h at 25±1°C in a





found that the enhancement in the solubility of OLM and HCZ was found to be





3O.2. Selection of Hydrotropic Agent

OLM and HCZ was scanned in hydrotropic agent in the spectrum mode over the



OLM and HCZ is soluble in it (58 and 74 fold enhancement of solubility)

OLM and HCZ is stable in hydrotropic agent (as shown in 3O. 3)

OLM and HCZ, both exhibit good spectral characteristics in it.


and HCZ i. e 250 nm and 271.5 nm respectively (Figure 4O.1.).

3O.3 Establishment of Stability Profile

Stability of OLM and HCZ was observed by dissolving in mixed hydrotropic

agent. Solution of OLM and HCZ was scanned under time scan for 30 min.


solution.

3O. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of OLM and HCZ



to solubilize the drug (Stock-A).


The standard solution (1000 µg/ml) was further diluted in different dilutions were

prepared ranging from 6-30 µg/ml for OLM and 5-25 µg/ml for HCZ. Calibration

curve was plotted between concentrations versus absorbance Figure 4O.2, Figure

4O.3. Linearity data of both drugs has been reported in the Table 4O. 1. The

Result of their optical characteristics shown in Table 4O.2.

3O.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 250 nm and 271.5 nm for OLM

and HCZ respectively and isobestic point at 261.2 nm Figure 4O.1. Due to







The wavelength 250 nm (λmax of OLM) and 271.5 nm (λmax of HCZ) was selected.

The absorbencies of OLM and HCZ were measured at 250 nm and 271.5 nm. This

method of analysis is based on the absorption of drugs X and Y at the wavelength

maxima of the other. The quantification analysis of OLM and HCZ in a binary

mixture was performed by using Eqn-1 and Eqn-2.



Where CX and CY are the concentrations of OLM and HCZ respectively in the

diluted sample, ax1 and ax2 are absorptivities of OLM at λ1 and λ2, ay1 and ay2 are

absorptivities of HCZ at λ1 and λ2 respectively Table 4O.3. A1 and A2 are the

absorbances of samples at the 250 and 271.5 nm respectively.





spectra wavelength 261.2 nm (isoabsorption point) and 271.5 (λmax of HCZ) were

selected for study. The absorbencies at 261.2 nm and 271.5 nm for OLM were

obtained and similarly for HCZ absorbencies are measured at 261.2 nm and 271.5

nm. The concentrations of the individual components were calculated by using the

following equations;





absorptivities of OLM at λ1 and λ2 and ay1 and ay2 denote absorptivities of HCZ

at λ1 and λ2 respectively Table 4O.3; CX and CY be the concentration of OLM and

HCZ respectively.

3O.6 Analysis of Tablet Formulation

Twenty marketed tablets of OLM and HCZ (Olmetor-H, Hetero Drugs Ltd.)

were weighed and ground to a fine powder; amount equal to 20 mg of OLM was



taken in 100 ml volumetric flask. The HCZ present in this amount of tablet

powder was 12.5 mg. Then 80 ml of mixed hydrotropic solution was added and

the flask was sonicated for about 10 min. T he volume was made up to the mark

with hydrotropic solution. Filter the solution through whatman filter paper No.

41. Filtrate was diluted with RO water to get the final concentrations of both

drugs in the working range. The absorbance of final dilutions was observed at

selected wavelengths and the concentrations were obtained from simultaneous

equation method and absorbance ratio method. The result of tablet evaluation has

been reported in Table 4O.4.

3O.7 Validation of Method

a. Linearity

Linearity of OLM and HCZ was established by response ratios of drug. Response

ratios of both drugs were calculated by dividing the absorbance with respective

concentration Table 4O. 5. Then a graph was plotted between concentration and

response ratio Figure 4O.4, Figure 4O.5.

b. Accuracy



by adding known amount of standard solution of OLM and HCZ to preanalysed



Result of recovery studies are reported in Table 4O. 6.

c. Precision


intermediate precision (Day to Day and analyst to analyst) and reproducibility,

Table 4O. 7.

d. Robustness:

For the robustness of the analytical method we changed the ratio of hydrotropic

solution. Instead the 50:50 ratios of sodium acetate and urea 60:40 sodium

acetate and urea were used as solvent Table 4O. 7.



Method-3P

Quantitative Estimation of Levofloxacin and Ornidazole by UV

Spectrophotometer: A Mixed Hydrotropy Solubilization Method

Apparatus




Theme







Method development.

Tablet analysis


3P.1. Preliminary Solubility Studies

Solubility of both drugs was determined at 25±1°C. A definite amount of drug

was added to two screw capped 25 ml of volumetric flask containing different

aqueous systems viz different combination of hydrotropic agent and 2M sodium

acetate and 8M urea solution. The volumetric flasks were shaken mechanically

for 12 h at 25±1°C in a mechanical shaker. These solutions were allowed to

equilibrate for next 24 h and then centrifuged for 5 min at 2000 rpm. The


whatman filter paper #41 and analyzed spectrophotometrically against

corresponding solvent blank. After analysis, it was found that the enhancement in

the solubility of LEVO and OZ was found to be more than 49 and 45 folds

respectively in mixture of 2M sodium acetate and 8M urea solution (1:1) as

compared to solubility studies in other solvents.



3P.2. Selection of Hydrotropic Agent

LEVO and OZ was scanned in hydrotropic agent in the spectrum mode over the



LEVO and OZ is soluble in it (49 and 45 fold enhancement of solubility)

LEVO and OZ is stable in hydrotropic agent (as shown in 3P. 3)

LEVO and OZ, both exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of

LEVO and OZ i. e 287 nm and 320 nm respectively (Figure 4P.1).

3P.3 Establishment of Stability Profile

Stability of LEVO and OZ was observed by dissolving in mixed hydrotropic

agent. Solution of LEVO and OZ was scanned under time scan for 30 min.


solution.

3P. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of LEVO and OZ





The standard solution (1000 µg/ml) was further diluted in ranging from 5-

25µg/ml for LEVO and 5-25 µg/ml for OZ. Calibration curve was plotted between

concentrations versus absorbance Figure 4P.2, Figure 4P.3. Linearity data of both

drugs has been reported in the Table 4P. 1. The Result of their optical

characteristics has shown in Table 4P.2.

3P.5 Study of Overlay Spectra of Drugs and Selection of Methods

The spectra exhibit major absorbance maxima at 287 nm and 320 nm for LEVO

and OZ respectively and isobestic point at 301 nm Figure 4P.1. Due to difference

in absorbance maxima and having no interference with each other so both drug

can be simultaneously estimated by simultaneous equation method (Method A)

and Q-analysis method (Method B)




The wavelength 287 nm (λmax of LEVO) and 320 nm (λmax of OZ) was selected.

The absorbencies of LEVO and OZ were measured at 287 nm and 320 nm. This


maxima of the other. The quantification analysis of LEVO and OZ in a binary




Where CX and CY are the concentrations of LEVO and OZ respectively in the

diluted sample, ax1 and ax2 are absorptivities of LEVO at λ1 and λ2, ay1 and ay2

are absorptivities of OZ at λ1 and λ2 respectively Table 4P.3. A1 and A2 are the

absorbances of samples at the 287 and 320 nm respectively.





spectra wavelength 301 nm (isoabsorption point) and 320 (λmax of OZ) were

selected for study. The absorbencies at 301 nm and 320 nm for LEVO were

obtained and similarly for OZ absorbencies are measured at 301 nm and 320 nm.

The concentrations of the individual components were calculated by using the

following equations;



where Qm = A2 /A1 , A1 is absorbance of sample at isoabsorptive point, A2 is


absorptivities of LEVO at λ1 and λ2 and ay1 and ay2 denote absorptivities of OZ at

λ1 and λ2 respectively Table 4P.3.; CX and CY be the concentration of LEVO and

OZ respectively.

3P.6 Analysis of Tablet Formulation

Twenty marketed tablets of LEVO and OZ (Levoflox –OZ, Cipla Limited) were

weighed and ground to a fine powder; amount equal to 250 mg of LEVO was



taken in 100 ml volumetric flask. The OZ present in this amount of tablet powder

was 500 mg. Then 80 ml of sodium acetate and urea solution was added and the

flask was sonicated for about 10 min to solubilize the drug present in tablet

powder and the volume was made up to the mark with hydrotropic solution.

After sonication filtration was done through whatman filter paper No. 41.

Filtrate was collected and further diluted with RO water to get the final

concentrations of both drugs in the working range. The absorbances of final

dilutions were observed at selected wavelengths and the concentrations were


statistical evaluation of tablet analysis has reported in Table 4P.4.

3P.7 Validation of Method

a. Linearity

Linearity of LEVO and OZ was established by response ratios of drug. Response

ratio of both drugs was calculated by dividing the absorbance with respective

concentration Table 4P. 5. Then a graph was plotted between concentration and

response ratio Figure 4P.4, Figure 4P.5.

b. Accuracy



by adding known amount of standard solution of LEVO and OZ to preanalysed



Result of recovery studies are reported in Table 4P. 6.

c. Precision



Table 3P. 7.

d. Robustness:



acetate and urea were used as solvent.



Method-3Q

Novel UV Spectrophotometer Methods for Quantitative Estimation of

Metronidazole and Furazolidone Using Mixed Hydrotropy Solubilization

Apparatus




Theme







Method development.

Tablet analysis


3Q.1. Preliminary Solubility Studies



aqueous systems viz. distilled water and different combination of hydrotropic

agent. The volumetric flasks were shaken mechanically for 12 h at 25±1°C in a

mechanical shaker. These solutions were allowed to equilibrate for next 24 hr. and




found that the enhancement in the solubility of MTR and FZ was found to be





3Q.2. Selection of Hydrotropic Agent

MTR and FZ was scanned in hydrotropic agent in the spectrum mode over the



MTR and FZ is soluble in it (36 and 28 fold enhancement of solubility)

MTR and FZ is stable in hydrotropic agent (as shown in 3Q. 3)

MTR and FZ, both exhibit good spectral characteristics in it.

Sodium acetate and urea solution has no interference with the λmax of MTR

and FZ, 319 and 364 nm respectively Figure 4Q.1.

3Q.3 Establishment of Stability Profile

Stability of MTR and FZ was observed by dissolving in mixture of 2 M sodium

acetate and 8 M urea (50:50% V/V) solution used as hydrotropic agent. Solution

of MTR and FZ was scanned under time scan for 30 min. Spectra of drug under

time scan shows that drug are stable in hydrotropic solution.

3Q. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of MTR and FZ





The standard solution (1000 µg/ml) was further diluted in different dilutions were

prepared ranging from 10-50 µg/ml for MTR and 5-25 µg/ml for FZ. Calibration

curve was plotted between concentrations versus absorbance Figure 4Q.2, Figure

4Q.3. Linearity data of both drugs has been reported in the Table 4Q. 1. The

Result of their optical characteristics has shown in Table 4Q.2.

3Q.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 319 nm and 364 nm for MTR

and FZ respectively and isobestic point at 339.2 nm Figure 4Q.1. Due to







The wavelength 319 nm (λmax of MTR) and 364 nm (λmax of FZ) was selected.

The absorbencies of MTR and FZ were measured at 319 nm and 364 nm. This


maxima of the other. The quantification analysis of MTR and FZ in a binary




Where CX and CY are the concentrations of MTR and FZ respectively in the

diluted sample, ax1 and ax2 are absorptivities of MTR at λ1 and λ2, ay1 and ay2 are

absorptivities of FZ at λ1 and λ2 respectively Table 4Q.3. A1 and A2 are the






spectra wavelength 339.2 nm (isoabsorption point) and 364 (λmax of FZ) were

selected for study. The absorbencies at 339.2 nm and 364 nm for MTR were

obtained and similarly for FZ absorbencies are measured at 339.2 nm and 364





Where, Qm = A2 /A1 , A1 is absorbance of sample at isoabsorptive point, A2 is


absorptivities of MTR at λ1 and λ2 and ay1 and ay2 denote absorptivities of FZ at

λ1 and λ2 respectively Table 4Q.3; CX and CY be the concentration of MTR and

FZ respectively

3Q.6 Analysis of Tablet Formulation

Twenty marketed tablets of MTR and FZ, Metrofur (Western Remedies.) were

weighed and ground to a fine powder; amount equal to 200 mg of MTR was



taken in 10 ml volumetric flask. The FZ present in this amount of tablet powder

was 100 mg. Then 80 ml of sodium acetate and urea solution was added and the

flask was sonicated for about 10 min to solubilize the drug present in tablet

powder and the volume was made up to the mark with hydrotropic solution.

After sonication filtration was done through whatman filter paper No. 41.

Filtrate was collected and further diluted with RO water to get the final




result of statistical evaluation of tablet analysis has reported in Table 4Q.4.

3Q.7 Validation of Method

a. Linearity

Linearity of MTR and FZ was established by response ratios of drug. Response

ratio of both drugs was calculated by dividing the absorbance with respective

concentration Table 4Q. 5. Then a graph was plotted between concentration and

response ratio Figure 4Q.4, Figure 4Q.5.

b. Accuracy



by adding known amount of standard solution of MTR and FZ to preanalysed



Result of recovery studies are reported in Table 4Q. 6.

c. Precision



Table 4Q. 7.

d. Robustness



acetate and urea were used as solvent Table 4Q. 7.



Method-3R

Novel Economic Method for Quantitation of Metronidazole and Ofloxacin

Using UV Spectrophotometer

Apparatus




Theme







Method development.

Tablet analysis


3R.1. Preliminary Solubility Studies




agent. The volumetric flasks were shaken mechanically for 12 hr. at 25±1°C in a





found that the enhancement in the solubility of MTR and OFL was found to be

more than 33 and 35 folds respectively in mixture of sodium benzoate: phenol

(25%:8%) as compared to solubility studies in other solvents.



3R.2. Selection of Hydrotropic Agent

MTR and OFL was scanned in hydrotropic agent in the spectrum mode over the

UV range (200-400) and mixture of sodium benzoate: phenol (25%:8%) solution

were found to be most appropriate because:

MTR and OFL is soluble in it (33 and 35 folds enhancement of solubility)

MTR and OFL is stable in hydrotropic agent (as shown in 3R. 3)

MTR and OFL, both exhibit good spectral characteristics in it.

Sodium Benzoate and Phenol solution has no interference with the λmax of

MTR and OFL, 319 nm and 330 nm respectively Figure 4R.1.

3R.3 Establishment of Stability Profile

Stability of MTR and OFL was observed by dissolving in mixture of sodium

benzoate: phenol (25%:8%) solution used as hydrotropic agent. Solution of MTR

and OFL was scanned under time scan for 30 min. Spectra of drug under time


3R. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of MTR and OFL






µg/ml for MTR and 10-50 µg/ml for OFL. Calibration curve was plotted between

concentrations versus absorbance Figure 4R.2 and Figure 4R.3. Observation of

linearity data of both drugs has reported in the Table 4R. 1. The Result of their

optical characteristics has shown in Table 4R.2.

3R.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 319 nm and 330 nm for MTR

and OFL respectively and isobestic point at 324.5 nm Figure 4R.1. Due to



(Method A) and Q-analysis method (Method B).




The wavelength 319 nm (λmax of MTR) and 330 nm (λmax of OFL) was selected.

The absorbencies of MTR and OFL were measured at 319 nm and 330 nm. This


maxima of the other. The quantification analysis of MTR and OFL in a binary




Where CX and CY are the concentrations of MTR and OFL respectively in the

diluted sample, ax1 and ax2 are absorptivities of MTR at λ1 and λ2, ay1 and ay2 are

absorptivities of OFL at λ1 and λ2 respectively Table 4R.3. A1 and A2 are the






spectra wavelength 324.5 nm (isoabsorption point) and 330 (λmax of OFL) were

selected for study. The absorbencies at 324.5 nm and 330 nm for MTR were

obtained and similarly for OFL absorbencies are measured at 324.5 nm and 330







absorptivities of MTR at λ1 and λ2 and ay1 and ay2 denote absorptivities of OFL

at λ1 and λ2 respectively Table 4R.3; CX and CY be the concentration of MTR

and OFL respectively

3R.6 Analysis of Tablet Formulation

Twenty marketed tablets of MTR and OFL Oflaswift – m (Swift Medicare

pvt.ltd.) were weighed and ground to a fine powder; amount equal to 100 mg of



MTR was taken in 10 ml volumetric flask. The OFL present in this amount of

tablet powder was 50 mg. Then 80 ml of hydrotropic agent solution was added

and the flask was sonicated for about 10 min to solubilize the drug present in

tablet powder and the volume was made up to the mark with hydrotropic

solution. After sonication filtration was done through whatman filter paper No.

41. Filtrate was collected and further diluted with hydrotropic agent to get the

final concentrations of both drugs in the working range. The absorbances of final



result and statistical evaluation of tablet analysis was reported in Table 4R.4.

3R.7 Validation of Method

a. Linearity

Linearity of MTR and OFL was established by response ratios of drug. Response

ratio of both drug calculated by dividing the absorbance with respective

concentration Table 4R. 5. Then a graph was plotted between concentration and

response ratio Figure 4R.4, Figure 4R.5.

b. Accuracy



by adding known amount of standard solution of MTR and OFL to preanalysed



Result of recovery studies are reported in Table 4R. 6.

c. Precision



Table 4R. 7.

d. Robustness


solution. Instead the 25%:8% ratios of sodium benzoate: phenol, (26%:7%) sodium

benzoate and phenol were used as solvent Table 4R. 7.



Method-3S

Ecofriendly UV Spectrophotometer Method for Quantitation of Olmesartan

Medoxamil and Metoprolol Succinate Using Hydrotropic Agent

Apparatus




Theme







Method development.

Tablet analysis


3S.1. Preliminary Solubility Studies









found that the enhancement in the solubility of OLM and MTO was found to be

more than 44 and 36 folds respectively in mixture of 2 M sodium acetate and urea

(50%:50%) as compared to solubility studies in other solvents.



3S.2. Selection of Hydrotropic Agent

OLM and MTO was scanned in hydrotropic agent in the spectrum mode over the

UV range (200-400) and mixture of 2 M sodium acetate and urea (50%:50%)


OLM and MTO is soluble in it (44 & 36 folds enhancement of solubility)

OLM and MTO is stable in hydrotropic agent (as shown in 3S. 3)

OLM and MTO, both exhibit good spectral characteristics in it.


and MTO, 257 nm and 274 nm respectively Figure 4S.1.

3S.3 Establishment of Stability Profile

Stability of OLM and MTO was observed by dissolving in mixture of 2 M sodium

acetate and urea (50%:50%) solution used as hydrotropic agent. Solution of OLM

and MTO was scanned under time scan for 30 min. Spectra of drug under time


3S. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of OLM and MTO





The standard solution (1000 µg/ml) was further diluted in anging from 10-50

µg/ml for OLM and 50-250 µg/ml for MTO. Calibration curve was plotted

between concentrations versus absorbance Figure 4S.2, Figure 4S.3. Observation

of linearity data of both drugs has reported in the Table 4S. 1 The Result of their

optical characteristics has shown in Table 4S.2.

3S.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 257 nm and 274 nm for OLM

and MTO respectively and isobestic point at 266.8 nm Figure 4S.1. Due to



(Method A) and Q-analysis method (Method B).




The wavelength 257 nm (λmax of OLM) and 274 nm (λmax of MTO) was selected.

The absorbencies of OLM and MTO were measured at 257 nm and 274 nm. This


maxima of the other. The quantification analysis of OLM and MTO in a binary




Where CX and CY are the concentrations of OLM and MTO respectively in the

diluted sample, ax1 and ax2 are absorptivities of OLM at λ1 and λ2, ay1 and ay2 are

absorptivities of MTO at λ1 and λ2 respectively Table 4S.3. A1 and A2 are the






spectra wavelength 266.8 nm (isoabsorption point) and 274 (λmax of MTO) were

selected for study. The absorbencies at 266.8 nm and 274 nm for OLM were

obtained and similarly for MTO absorbencies are measured at 266.8 nm and 274







absorptivities of OLM at λ1 and λ2 and ay1 and ay2 denote absorptivities of MTO

at λ1 and λ2 respectively Table 4S.3; CX and CY be the concentration of OLM and

MTO respectively

3S.6 Analysis of Tablet Formulation

Twenty marketed tablets of OLM and MTO Olsar-M (Unichem laboratories)

were weighed and ground to a fine powder; amount equal to 20 mg of OLM was



taken in 10 ml volumetric flask. The MTO present in this amount of tablet powder

was 50 mg. Then 80 ml of hydrotropic agent solution was added and the flask

was sonicated for about 10 min to solubilize the drug present in tablet powder

and the volume was made up to the mark with hydrotropic solution. After

sonication filtration was done through whatman filter paper No. 41. Filtrate

was collected and further diluted with hydrotropic agent to get the final




result and statistical evaluation of tablet analysis has reported in Table 4S.4.

3S.7 Validation of Method

a. Linearity

Linearity of OLM and MTO was established by response ratios of drug. Response


concentration Table 4S. 5. Then a graph was plotted between concentration and

response ratio Figure 4S.4, Figure 4S.5.

b. Accuracy



by adding known amount of standard solution of OLM and MTO to preanalysed



Result of recovery studies are reported in Table 4S. 6.

c. Precision



Table 4S. 7.

d. Robustness



acetate and urea were used as solvent Table 4S. 7.



Method-3T

Novel Economic Method for Quantitation of Metoprolol Succinate and

Telmisartan Using UV Spectrophotometer

Apparatus




Theme







Method development.

Tablet analysis


3T.1. Preliminary solubility studies









found that the enhancement in the solubility of MTO and TEL was found to be

more than 41 and 52 folds respectively in 2 M Citric Acid as compared to

solubility studies in other solvents.



3T.2. Selection of Hydrotropic Agent

MTO and TEL was scanned in hydrotropic agent in the spectrum mode over the

UV range (200-400) and 2M Citric acid solution were found to be most


MTO and TEL is soluble in it (41and 52 folds enhancement of solubility)

MTO and TEL is stable in hydrotropic agent (as shown in 3T. 3)

MTO and TEL, both exhibit good spectral characteristics in it.

Citric acid solution has no interference with the λmax of MTO and TEL, 274

nm and 291 nm respectively Figure 4T.1.

3T.3 Establishment of Stability Profile

Stability of MTO and TEL was observed by dissolving in 2 M Citric Acid

solution. Solution of MTO and TEL was prepared in the conc. of 30 g/ml and

150 g/ml respectively and scanned under time scan for 30 min. Spectra of drug

under time scan shows that drug are stable in hydrotropic solution.

3T. 4 Linearity Range and Calibration Graph

Preparation of Standard Stock Solutions of MTO and TEL



to solubilize the drugs (Stock-A).



µg/ml for MTO and 10-50 µg/ml for TEL. Calibration curve was plotted between

conc. versus absorbance Figure 4T.2, Figure 4T.3. Observations of linearity data

of both drugs has been reported in the Table 4T. 1. The Result of their optical

characteristics has shown in Table 4T.2.

3T.5 Study of Overlay Spectra of Drugs and Selection of Method

The spectra exhibit major absorbance maxima at 274 nm and 291 nm for MTO

and TEL respectively and isobestic point at 281.2 nm. Due to difference in

absorbance maxima and having no interference with each other so both drug can

be simultaneously estimated by simultaneous equation method (Method A) and

Q-analysis method (Method B).




The wavelength 274 nm (λmax of MTO) and 291 nm (λmax of TEL) was selected.

The absorbencies of MTO and TEL were measured at 274 nm and 291 nm. This


maxima of the other. The quantification analysis of MTO and TEL in a binary




Where CX and CY are the concentrations of MTO and TEL respectively in the

diluted sample, ax1 and ax2 are absorptivities of MTO at λ1 and λ2, ay1 and ay2 are

absorptivities of TEL at λ1 and λ2 respectively Table 4T.3. A1 and A2 are the






spectra wavelength 281.2 nm (isoabsorption point) and 291 (λmax of TEL) were

selected for study. The absorbencies at 281.2 nm and 291 nm for MTO were

obtained and similarly for TEL absorbencies are measured at 281.2 nm and 291

nm. The concentrationS of the individual components were calculated by using






absorptivities of MTO at λ1 and λ2 and ay1 and ay2 denote absorptivities of TEL

at λ1 and λ2 respectively Table 4T.3; CX and CY be the concentration of MTO

and TEL respectively

3T.5 Analysis of Tablet Formulation

Twenty marketed tablets of MTO and TEL, Telsar Beta (Unichem laboratories)

were weighed and ground to a fine powder; amount equal to 50 mg of MTO was



taken in 10 ml volumetric flask. The TEL present in this amount of tablet powder

was 40 mg. Then 80 ml of hydrotropic agent solution was added and the flask

was sonicated for about 10 min to solubilize the drug present in tablet powder

and the volume was made up to the mark with hydrotropic solution. After

sonication filtration was done through whatman filter paper No. 41. Filtrate

was collected and further diluted with hydrotropic agent to get the final




result and statistical evaluation of tablet analysis has reported in Table 4T.4.

3T.6 Validation of Method

a. Linearity

Linearity of MTO and TEL was established by response ratios of drug. Response


concentration Table 4T. 5. Then a graph was plotted between concentration and

response ratio Figure 4T.4, Figure 4T.5.

b. Accuracy



by adding known amount of standard solution of MTO and TEL to preanalysed



Result of recovery studies are reported in Table 4T. 6.

c. Precision



Table 4T. 7.

Chapter 3 Materials & Methods 03. MATERIALS &...

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