6. METHOD DEVELOPMENT OF DRUGS 6.1 …shodhganga.inflibnet.ac.in/bitstream/10603/50474/14/14...Table 6.1.1.1: List of the instruments used in the UV-Spectrophotometry Sr. No. Instruments

Page 126

6. METHOD DEVELOPMENT OF DRUGS

6.1 GLICLAZIDE

6.1.1 SPECTROPHOTOMETRIC METHOD DEVELOPMENT FOR THE

ESTIMATION OF GLICLAZIDE IN ITS SOLID DOSAGE FORM

6.1.1.1Instruments and Materials

6.1.1.1.1 Instruments

Table 6.1.1.1: List of the instruments used in the UV-Spectrophotometry

Sr. No. Instruments Make Model

1 UV 1)Shimadzu

2)Perkin Elmer

1)UV-Pharmaspec-1700

2)Lambda 19

2 Analytical balance Mettler Toledo AX205

3 Sonicator Compact Ultrasonic Branson 2510

4 pH meter Thermo Orion 420 A+

5 Milli-Q water source Thermofisher Scientific Branstead D3750

6 Deionised Water Plant Millipore Elix- 3 system

� UV- visible double beam spectrophotometer with matched quartz cells

(1cm).

1) Make: SHIMADZU

Model UV-Pharmaspec-1700

Scanning Range 185 – 3200 nm

Slit Width 2 nm

Photometric Accuracy ± 0.003 Å

Wavelength Accuracy ± 0.15 nm

Baseline Flatness ± 0.001 Å

Stray Light 0.04% max

Wavelength Display 0.1 nm increments

Page 127

Noise Level 0.02 s

2) Make: Perkin Elmer, USA,

Model: Lambda 19

� pH meter

Make: Thermo Orion

Model: 420 A+

� Analytical Balance

Model: AX205

Make: Mettler Toledo

Maximum capacity: 220g

� Sonicator

Model: Branson 2510

Compact Ultrasonic Cleaner

0-60 Sonic/minute

� Milli-Q water

Make: Thermofisher scientific

Model: Branstead D3750

Hollow fiber filter

Gamma Irradiated

Pore size: 0.2 µm

Max operating pressure: 50 psi

Page 128

� Deionised Water Plant

Make: Millipore

Model: Milli Q academic (water output 1.5L)

6.1.1.1.2 Materials

Gliclazide: Reddy’s Laboratories,Hyderabad, India.

Methanol (AR Grade) E.Merck Ltd., Mumbai, India

6.1.1.2 Marketed Formulation:

Azukon (Torrent),Gujarat.

Tablet 80mg

6.1.1.3 Development and Optimization of Spectrophotometric method

6.1.1.3.1 Selection of Solvent for Gliclazide.

Gliclazide is freely soluble in solvents like dichloromethane and methanol .

Gliclazide is practically insoluble in water.Initially spectrum of Gliclazide was

scanned in methanol. Owing to the solubility of Gliclazide in the methanol and also

there was no shift in absorbance maxima, methanol was selected as solvent.

6.1.1.3.2 Selection of analytical wavelength for Gliclazide.

The solution of Gliclazide was prepared in methanol at a concentration of 6 µg /ml. It

was scanned in the wavelength range of 200-400 nm. Maximum absorbance was

obtained at 228 nm. This analytical wavelength was selected for determination of

Gliclazide. The standard solutions of 2-10 µg/ml were prepared and spectrums of

these concentrations were obtained with n = 5. The overlain spectrum of Gliclazide at

different concentration was recorded.(Fig.no.6.1.1.1)

6.1.1.3.3Preparation of standard stock solution

Gliclazide standard stock solution (100µg/ml)

Page 129

Accurately weighed powder of Gliclazide (100 mg) was transferred to a separate 100

ml volumetric flask and dissolved and diluted to the mark with methanol. Further 10

ml was taken and diluted to 100 ml with same solvent to give a standard stock

solution containing 100µg/ml Gliclazide.

6.1.1.3.4 Calibration Curve for Gliclazide (2-10µg/ml)

Appropriate volumes of aliquots from standard stock solution was transferred to

different volumetric flasks of 10 ml capacity. The volumes were adjusted to the mark

with methanol to obtain concentration of 2, 4, 6, 8 and 10µg/ml. The curve of solution

against methanol as a blank was recorded. Absorbance was measured at 228nm

against methanol as a blank and the plot of absorbance vs. concentration was plotted.

The straight-line equation was determined.(Fig.no.6.1.1.2)

6.1.1.3.5 Sample preparation

Twenty Gliclazide tablets were weighed and powdered. The tablet powder equivalent

to 100 mg of Gliclazide transferred in to a 100 ml volumetric flask. Methanol (100

ml) was added to it and sonicated for 20 min. The solution was filtered through

whatman filter paper No. 41 and the volume was adjusted up to the mark with

methanol to produce sample solution containing Gliclazide 1000 µg/ml. 10 ml of

resulting solution was taken in 100 ml volumetric flask and volume made up to 100

ml to contain Gliclazide 100 µg/ml (Stock solution A).

From the Stock solution A, 0.6 ml was transferred to volumetric flask of 10 ml

capacity. Volume was made up to the mark with methanol to give a solution

containing 6µg/ml Gliclazide (Solution 1). This solution was used for the estimation

of Gliclazide.

6.1.1.3.6 Estimation of Gliclazide by UV Spectroscopic Method

The solution 1 was measured at 228 nm for quantification of Gliclazide. The amount

of Gliclazide present in the sample solution was determined. From the absorbance

obtained in the spectrum, the amount of drug was calculated.

6.1.1.4 Method Validation, Results and Discussion

Page 130

Various validation Parameters

LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 2-10µg/ml with respect to

the test concentration. A standard stock solution was prepared and further diluted to

attain concentration of about 2, 4, 6, 8 & 10 µg/ml of sample concentration. Each

standard preparation was recorded in six replicates. The mean absorbance at each

level was calculated and a graph of mean absorbance v/s concentration (%) was

plotted. The correlation co-efficient (r), y-intercept and slope of regression line were

calculated and recorded in Table: 6.1.1.3.

Table 6.1.1.2: Result of calibration readings at 228nm for Gliclazide

Concentration

(µg/ml)

Absorbance at 228nm

Mean ± S.D. (n=6)

% RSD

2 0.084 ± 0.001 1.683

4 0.164 ± 0.002 1.590

6 0.248 ± 0.004 1.506

8 0.334 ± 0.004 1.068

10 0.428 ± 0.006 1.333

Figure 6.1.1.1: Overlain Spectrum of Gliclazide in methanol at 228nm.

Page 131

Figure 6.1.1.2: Calibration Curve of Gliclazide at 228 nm.

Table 6.1.1.3: Statistical data for Gliclazide by Spectrophotometry method

Parameter Gliclazide

Linearity Range (µg/ml) 2-10

Slope 0.0428

Intercept 0.0056

Standard deviation of intercept 0.003

Acceptance criteria

The correlation coefficient value should not be less than 0.995 over the working

range.

Conclusion:

The correlation coefficient value was 0.9991 for Gliclazide.The absorbances obtained

were directly proportional to the concentration of analyte in the sample. The method

can, therefore be termed as linear in the range considered. Based on the linearity

results, the working range of the method could be established as 2-10µg/ml of the

working concentration.

ACCURACY

Procedure

Page 132

The accuracy of the analytical method for assay was established at three levels in

triplicate, viz. 50%, 100% and 150% of the test concentration. Standard was prepared

as per method and sample preparations were done by mixing known amount of

Gliclazide working standard with placebo. Amount found, % Recovery and mean

Recovery was calculated at each level and recorded in Table 6.1.1.4.

Table 6.1.1.4: Data derived from Accuracy Experiment

Level

%

Sets

Absorbance

Drug

Added

(µg/ml)

Amount

recovered

(µg/ml)

%Recovery

Mean %

Recovery

%

RSD

50 1 0.377 3 8.96 99.555

99.222

0.008 50 2 0.373 3 8.85 98.333

50 3 0.378 3 8.98 99.777

100 1 0.508 6 12.02 100.166

100.5

0.004 100 2 0.513 6 12.12 101.000

100 3 0.509 6 12.04 100.333

150 1 0.632 9 14.91 99.400

99.466

0.004 150 2 0.631 9 14.87 99.133

150 3 0.635 9 14.98 99.866

Acceptance Criteria

% Recovery (individual) and mean % Recovery at each level should be within 98.0-

102.0 with % RSD not be more than 2.0.

Conclusion:

The % Recovery at each level, mean % Recovery and % RSD met the established

acceptance criteria.Hence, the method can be termed accurate in the considered range.

PRECISION

METHOD PRECISION (REPEATABILITY)

Procedure

Page 133

Method precision was established by assaying five standard and sample preparations

under same conditions. Individual assay value, mean assay value and % RSD were

calculated and recorded in Table 6.1.1.5 and Table 6.1.1.6.

Table 6.1.1.5: Repeatability (Method precision) data for Gliclazide standard at

228nm

Conc. 2µg/ml 4µg/ml 6µg/ml 8µg/ml 10µg/ml

Absorbance 0.086 0.162 0.252 0.338 0.433

0.084 0.164 0.247 0.330 0.423

0.082 0.163 0.248 0.332 0.423

0.084 0.168 0.245 0.332 0.422

0.083 0.166 0.242 0.338 0.432

0.085 0.161 0.251 0.331 0.434

Mean 0.084 0.164 0.247 0.334 0.428

S.D 0.001 0.003 0.004 0.004 0.006

% RSD 1.683 1.590 1.506 1.068 1.334

Table 6.1.1.6: Repeatability of sample application data for Gliclazide

Concentration Gliclazide(6µg/ml)

Absorbance 0.248

0.253

0.244

0.248

0.252

0.252

Mean 0.249

S.D. 0.003

% RSD 1.383

Acceptance Criteria

Page 134

% RSD of six preparations for method precision should not be more than 2.0%.

Conclusion:

The result obtained were well within the acceptance criteria. The method could

therefore be termed as precise.

INTER-DAY AND INTRA-DAY PRECISION (INTERMEDIATE PRESICION)

Variation of results within the same day (Intra-day) and variation of results between

days (Inter-day) were analysed.

Intra-day precision was determined by analysing Gliclazide for six times in the same

day at 228nm.

Inter-day precision was determined by analysing the drug daily for three days at

228nm.

Table 6.1.1.7: Intra-day precision data for Gliclazide at 228nm

Conc. µg/ml Intraday (n=6) % RSD

4 0.164 ± 0.003 1.219

6 0.247 ± 0.002 0.809

8 0.333 ± 0.004 1.249

Table 6.1.1.8: Inter-day precision data for Gliclazide at 228nm

Conc. µg/ml Interday (n=6) % RSD

4 0.165 ± 0.003 1.519

6 0.248 ± 0.004 1.632

8 0.332 ± 0.005 1.388

Acceptance Criteria

RSD for six preparations should not be more than 2.0%.

Page 135

Overall % RSD of method precision and intermediate precision should not more than

2.0%.

Conclusion:

The results obtained were well within the acceptance criteria. The method can

therefore be termed as precise and rugged.

REPRODUCIBILITY

Procedure

The absorbance readings were measured at different laboratory using another

spectrophotometer by another analyst and the value obtained were evaluated to verify

their reproducibility.

Table 6.1.1.9: Determination of Reproducibility

Instrument 1 (n=6) Instrument 2 (n=6)

0.396 ± 0.0037

%RSD = 0.43

0.398 ± 0.0043

%RSD = 0.62

Acceptance Criteria

% RSD for six preparation for Reproducibility study should not be more than 2.0%

Conclusion

The results obtained were within the acceptance criteria. The method can therefore be

termed as Reproducible.

ROBUSTNESS

Procedure

The robustness of the method was established by making deliberate minor variations

in the wavelength. The effects of changes were recorded in Table 6.1.1.10.

Page 136

Table 6.1.1.10: Data derived from Robustness experiment

Parameter % RSD(n=6)

Normal Condition Changed Conditions

Wavelength (228nm) 0.5 0.32(-5nm) 0.2 (+5nm)

Acceptance criteria

The % RSD for six replicate injections for the analyte peak should not be more than

2.0%.

Conclusion

Results were within acceptance criteria. Hence, method can be termed as Robust.

SOLUTION STABILITY

Procedure

The standard and sample solutions were prepared as per method and initial

absorbance was noted down. The standard and sample preparations were re-analyzed

by examining at regular intervals for 24 hrs and recorded in Table 6.1.1.11.

Table 6.1.1.11: Solution stability study

Time Absorbanceof

standard(6µg/ml)

at 228nm

Absorbanceof

sample(6µg/ml)

at228nm

% Assay

standard Sample

9.00a.m 0.252 0.252 100.312 100.312

2.00p.m 0.253 0.252 100.70 100.312

6.00p.m 0.248 0.247 98.754 98.365

9.00a.m 0.247 0.247 98.364 98.364

Acceptance criteria

Page 137

The difference in the assay value obtained at different time interval should not be

more than 2.0 % from the initial value.

Conclusion

The solution stability was checked for the sample preparation and standard

preparation for 24 hours. The results obtained are well within the acceptance criteria.

Therefore, the standard preparation and sample preparation were stable in solution

form for 24 hours at room temperature.

SYSTEM SUITABILITY TEST

Procedure

System suitability was performed by taking absorbance of standard solution six times

and calculating its average and % RSD value. System suitability test was performed at

the start of study for each parameter.

Table 6.1.1.12: System Suitability Test

Standard Absorbance Average % RSD

1 0.246

0.248

1.354

2 0.252

3 0.251

4 0.248

5 0.244

6 0.252

Acceptance criteria

The % RSD should not be more than 2.0%.

Conclusion

The % RSD was complied with acceptance criteria. Therefore the system was suitable

for use.

Page 138

6.1.1.5 Summary of validation Parameters

Table 6.1.1.13: Summary of Validation Parameters of Spectrophotometry

Sr.No. Parameter Results

1 Linearity (µg/ml) 2-10

2 Recovery % 98.87-99.85

3 Repeatability (% RSD, n=6) 1.383

4 Precision (%CV)

Intra-day

Inter-day

0.809-1.249

1.388-1.632

5 LOD (µg/ml) 0.215

6 LOQ (µg/ml) 0.651

7 Specificity Specific

8 Robustness (% RSD) < 0.7

9 Solution Stability Suitable for 24 hrs.

6.1.1.6 Assay Results of Marketed Formulation

Table 6.1.1.14: Assay Results of Marketed Formulation

Set Amount(µg/ml) %Assay %RSD(n=3)

1 6 98.555 0.834

Page 139

6.1.2 RP-HPLC METHOD DEVELOPMENT FOR GLICLAZIDE IN ITS

SOLID DOSAGE FORM.


6.1.2.1.1Instruments

Table 6.1.2.1: List of the instruments used in RP-HPLC

Sr. No. Instrument Make Model

1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000




5 Milli-Q water source Thermofisher scientific Branstead D3750

6 Deionised Water Plant Millipore Elix-3 system

HPLC

Model LC20AT

Column C18 Hypersil BDS(thermomake)

Dimension 250×4.6mm; 5µ

Pump Isocratic system; Back pressure 5000psi

Flow rate: 1 ml/min

Injector Rhenodyne valve with 20µl fixed loop

Detector SPD20A

6.1.2.1.2 Materials:

Gliclazide: Reddy,s Laboratories,Hyderabad, India

Methanol (HPLC Grade) – E. Merck (India) Ltd., Mumbai

Page 140

Water (HPLC Grade) – Milli Q

Acetonitrile (HPLC Grade) – E. Merck (India) Ltd., Mumbai


Azucon (Aventis Pharma Ltd),Gujarat.

Tablet 30mg

6.1.2.3 Development and Optimization of HPLC method

6.1.2.3.1 Selection of Detection Wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper

selection of detection wavelength is the one that gives good response for the drugs

that are to be detected. In the present study drug solution of 10µg/ml was, therefore,

prepared in solvent mixtures of water and ACN (30:70). This drug solution was then

scanned in the UV region of 200-400 nm and the spectrum was recorded.(Fig.

no.6.1.2.1)

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

250

500

750

1000

1250

1500

mAU

224.8

8

256.4

9

204.1

3

Figure 6.1.2.1: UV spectrum of Gliclazide

6.1.2.3.2 Selection of chromatographic conditions

Proper selection of the HPLC condition depends upon the nature of the sample (ionic

or ionizable or neutral molecule), its molecular weight and solubility. The drug

selected for the present study is polar in nature and hence either reversed phase or ion-

exchange or ion-pair chromatography can be used. Reverse phase HPLC was selected

for initial separation because of its simplicity.

Page 141

To optimize the chromatographic conditions, the effect of chromatographic variables

such as mobile phase pH, flow rate and solvent ratio were studied. The resulting

chromatograms were recorded and the chromatographic parameters such as capacity

factor, asymmetric factor and column efficiency were calculated. The conditions that

gave the best resolution, symmetry and capacity factor were selected for analysis.

6.1.2.4 Estimation of Gliclazide by RP-HPLC method

6.1.2.4.1 Preparation of Mobile Phase

300 ml of water and 700 ml of Acetonitrile were mixed and filtered through 0.45µ

filter paper, sonicated for 10 minutes to degas and used as mobile phase. Mobile

phase was used as diluent.

6.1.2.4.2 Preparation of standard stock solution

Gliclazide Standard stock solution (100 ppm)

Accurately weighed Gliclazide standard (10 mg) was transferred to a 100 ml

volumetric flask and dissolved in about 10 ml diluent, then diluted to mark to obtain

standard stock solution (100µg/ml). An aliquot of the solution (5.0 ml) was

transferred to a 50.0 ml volumetric flask and diluted to mark with diluent to obtain

working standard solution (10µg/ml).

6.1.2.4.3 Calibration curve for Gliclazide(50-150µg/ml)

Appropriate volume of aliquots from standard Gliclazide stock solution was

transferred to different volumetric flasks of 10ml capacity. The volume was adjusted

to the mark with the diluent to obtain the concentration of 5, 7.5, 10, 12.5, 15µg/ml.

Calibration curve of each solution against the diluent was recorded at 256nm and the

graph of absorbance v/s concentration was plotted. The straight line equation was

determined from calibration curve.(Fig. no.6.2.1.6)

6.1.2.4.4 Sample Preparation

Page 142

Twenty Gliclazide tablets were taken and crushed to make powder. The accurately

weighed powder equivalent to 10 mg of Gliclazide was transferred into 100 ml

volumetric flask and dissolved in about 10 ml diluent by sonication for 25 minute and

then volume was made up to 100 ml with diluent to obtain the sample stock solution

(100µg/ml). An aliquot of the sample stock solution (5.0ml) was transferred to a 50ml

volumetric flask and diluted to mark with mobile phase to obtain working sample

solution (10ug/ml).

6.1.2.5 Estimation of Gliclazide in its solid dosage form

The prepared sample solution(20µl) was chromatographed for 10 minutes using

mobile phase.From the peak area obtained in the chromatogram, the amount of the

drug was calculated.


Figure 6.1.2.2: Chromatogram of Gliclazide using water:methanol (30:70)

Page 143

Figure 6.1.2.3: Chromatogram of Gliclazide using water:ACN (40:60)

Figure 6.1.2.4: Chromatogram of Gliclazide using water:ACN (35:65)

Figure 6.1.2.5: Chromatogram of Optimized chromatographic condition for

Gliclazide

Table 6.1.2.2: Optimized chromatographic conditions for Gliclazide

Sr.No. Parameter Conditions

1 Mobile Phase Water:Acetonitrile (30:70)

2 Pump mode Isocratic

3 Stationary Phase Hypersil BDS(250×4.6 mm id, 5µm particle size)

column

4 Flow rate(ml/min) 1

5 Volume of Injection 20µl

6 Detection 256nm

Page 144

wavelength

7 Run time (min) 8 min

8 Diluent Mobile Phase

Various Validation Parameters

LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 5 to 15µg/ml with respect to


attain concentration of about 5, 7.5, 10, 12.5, and 15µg/ml of sample concentration.

Each standard preparation was injected six replicates. The mean area at each level was

calculated and a graph of mean area v/s concentration (%) was plotted. The

correlation co-efficient (r), y-intercept and slope of regression line were calculated

and recorded in Table 6.1.2.4.

Table 6.1.2.3: Result of calibration readings for Gliclazide

Concentrations

(µg/ml)

Gliclazide

Area

Mean ± S.D. (n=6) CV

5 1678.147 ± 9.710 0.579

7.5 2505.323 ± 14.744 0.589

10 3354.926 ± 17.897 0.533

12.5 4172.391 ± 30.716 0.736

15 5039.921 ± 37.771 0.749

Page 145

Figure 6.1.2.6: Calibration curve of Gliclazide at 256nm

Table 6.1.2.4: Statistical data for Gliclazide by RP-HPLC

Parameter Gliclazide

Linear Range(µg/ml) 5-15

Slope 335.62

Intercept 6.1039

Standard deviation of Intercept 4.765

Acceptance criteria


range.

Conclusion:

The correlation coefficient value was found to be 1.0 for Gliclazide.

The areas obtained were directly proportional to the concentration of analyte in the

sample. The method can, therefore be termed as linear in the range considered. Based

on the linearity results, the working range of the method could be established as 5-

15µg/ml of working concentration.

ACCURACY

Procedure

Page 146

The accuracy of the analytical method for assay of Gliclazide was established at three

levels in triplicate,viz. 80%, 100%, and 120% of the test concentration. Standard was

prepared as per method and sample preparations were done by mixing known amount

of Gliclazide working standard with placebo. Amount found, % Recovery and Mean


Table 6.1.2.5: Data derived from Accuracy experiment

Level Set Area Amount

added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean

% Recovery

%

RSD

80 1 2668.316 8 7.968 99.607

99.998

0.401 80 2 2689.779 8 8.032 100.406

80 3 2678.317 8 7.998 99.979

100 1 3386.182 10 10.107 101.075

100.016

1.119 100 2 3311.417 10 9.884 98.847

100 3 3354.547 10 10.012 100.126

120 1 4088.231 12 12.199 101.661

100.987

1.012 120 2 4081.267 12 12.178 101.488

120 3 4013.803 12 11.977 99.813

Acceptance Criteria

% Recovery (individual) and mean % recovery at each level should be within 98.0-


Conclusion: The % Recovery at each level, mean % Recovery and % RSD met the

established acceptance criteria.Hence, the method can be termed accurate in the

considering range.

PRECISION


Page 147

Procedure

Method precision was established by taking five standard and sample preparations



Table 6.1.2.6: Determination of Method Precision (Repeatability)

Concentration 5(µg/ml) 7.5(µg/ml) 10(µg/ml) 12.5(µg/ml) 15(µg/ml)

Area 1675.665 2512.136 2343.779 2750.126 3406.432

1625.888 2528.324 2343.779 2750.654 3406.765

1667.363 2433.233 2332.15 2753.145 3408.412

1699.286 2491.164 2275.123 2751.89 3409.431

1662.482 2511.127 2241.345 2751.765 3442.876

1668.672 2521.264 2289.217 2750.635 3408.315

Mean 1666.559 2499.541 2304.232 2751.369 3413.705

S.D 23.790 34.81 17.808 29.93 36.17

%RSD 1.427 1.392 0.531 0.717 0.716

n=6 determinations


Concentration Gliclazide

10µg/ml

Area 3345.032

3237.263

3324.653

3321.889

3367.242

3268.565

Mean 3310.774

S.D. 48.711

% RSD 1.471

Page 148

Acceptance Criteria


Conclusion:

The results obtained were well within the acceptance criteria. The method could





Table 6.1.2.8: Precision data

Intraday Inter-day

Conc. 5(µg/ml) 10(µg/ml) 15(µg/ml) 5(µg/ml) 10(µg/ml) 15(µg/ml)

Area 1689.14 3385.17 5087.814 1658.985 3398.658 4987.074

1664.085 3331.447 4982.045 1697.579 3328.128 5108.023

1678.382 3348.206 5027.315 1679.087 3361.646 5047.417

Mean 1677.202 3354.941 5032.391 1678.550 3362.810 5047.504

S.D. 12.569 27.487 53.07 19.30259 35.279 60.474

% RSD 0.749 0.819 1.055 1.149 1.049 1.198

Acceptance Criteria



2.0%.

Conclusion:



Page 149

REPRODUCIBILITY

Procedure

The Area readings were measured at different laboratory using another HPLC

instrument by another analyst and the value obtained were evaluated to verify their

reproducibility.


Instrument 1

Area ± S.D.(n=6)

Instrument 2

Area ± S.D.(n=6)

3328.026 ± 42.306

%RSD = 1.271

3333.905 ± 7.739

%RSD = 0.232

Acceptance criteria

% RSD for six preparation for Reproducibility study should not be more than 2.0%.

Conclusion



ROBUSTNESS

Procedure


in the following method parameters.

1. Flow rate

2. Organic phase ratio

The effect of changes observed on system suitability parameters were recorded in

table 6.1.2.10.

Page 150

Table 6.1.2.10: Data derived from Robustness study of Gliclazide

Robust condition Area %RSD

M.P(Water:ACN)32:68 3348.239 ± 30.693 0.917

M.P(Water:ACN)28:72 3357.171± 33.571 0.999

Flow rate +0.2ml/min 3189.765±26.397 0.828

Flow rate -0.2ml/min 3527.925±19.510 0.553

RSD = Relative standard deviation of area of 6 standard preparations. For each robust

condition, one standard preparation was analysed 6 times.

Note

Theoretical plate and asymmetry values are from the first injection of the system

suitability set.

% RSD was calculated for the six replicate injections of standard preparation

Acceptance criteria

Number of Theoretical plates for the analyte peak should not be less than 2000.

Asymmetry value for the analyte peak should not be more than 2.0.

The % RSD for six replicate injection for the analyte peak should not be more than

2%

Conclusion

The system suitability parameter and absolute difference between area obtained from

normal condition and varied conditions were well within acceptance criteria, hence

method can be termed as robust.

SOLUTION STABILITY

Procedure

Page 151

The standard and sample solutions were prepared as per method and analysed at

regular intervals. Results were recorded in Table 6.1.2.11.


Time AreaofGLZ

standard(10µg/ml)

AreaofGLZ

Sample(10µg/ml)

% Assay

standard sample

9.00a.m 3363.294 3345.456 100.393 99.862

2.00p.m 3345.047 3324.678 99.849 99.243

6.00p.m 3330.811 3323.456 99.425 99.206

9.00a.m 3318.958 3312.298 99.072 98.874

Acceptance criteria

The difference in the assay value obtained at different time intervals should not be

more than 2.0% from the initial value.

Conclusion



Therefore, the standard and sample preparation were stable in solution form for 24

hours at room temperature.


Procedure



the start of study for each parameter. The value of system suitability results obtained

were recorded in Table 6.1.2.12.

Page 152

Table 6.1.2.12: System suitability test

System Suitability Parameter Gliclazide

Retention times (RT) 5.703

Theoritical plates (N) 7039

Tailing factor (AS) 1.405

% RSD (n=6) 0.56

Note

System suitability values are from the first injection of six replicates of standard.

% RSD is calculated from six replicate injections of standard.

Acceptance criteria

The % RSD should not be more than 2.0%

Conclusion

The system suitability parameters are well within acceptance criteria. Therefore the

system and chromatographic conditions are suitable for use.

6.1.2.7 Summary of validation parameters of RP-HPLC Method

Table 6.1.2.13: Summary of validation parameters of Gliclazide by RP-HPLC

method

Sr.No. Parameter Result

1. Linearity Range (μg/ml) 5– 15

2. Correlation co-efficient (r2) 1.0

3. Accuracy (%Recovery) (n=3) 99.998 to 100.987

4. Precision (%CV) 1.049-1.198

Page 153

Inter-day precision

Intra-day precision

0.749-1.055

5. Limit of detection (μg/ml) 0.046

6 Limit of quantitation (μg/ml) 0.142

7. Specificity Specific

8 Robustness(%RSD) <1.1

9 Solution stability Stable for 24hrs

6.1.2.8 Assay result of marketed formulation

Table 6.1.2.14: Assay result of marketed formulation


1 10 98.83 0.45

Page 154

6.1.3 HPTLC METHOD DEVELOPMENT FOR THE ESTIMATION OF

GLICLAZIDE IN ITS SOLID DOSAGE FORM



Table 6.1.3.1: List of the instruments used in HPTLC


1 HPTLC Camag Linomat 5

2 Analytical Balance Mettler Toledo AX205




6 Deionised water Plant Millipore Elix-3 system

High Performance Thin Layer Chromatography (HPTLC)

Camag Linomat 5

Semiautomatic application, band application by spray on technique (2 -500µl)

Camag twin trough glass chamber (10 × 10 and 20 × 10)

Camag TLC scanner 3

Scanning speed up to 100mm/s, Spectral range 190 – 800nm

Camag Reprostar 3 with digital camera

For 254nm, 366 nm and with light

Camag UV cabinet with dual wavelength UV lamp

Dual wavelength 254/ 366nm

Stationary Phase: silica gel G60 F254 coated on aluminum sheet

Hamilton 100µl HPTLC syringe

Page 155

Data Resolution: 100µm/step

6.1.3.1.2 Materials

GLICLAZIDE: Reddy,s Laboratories,Hyderabad, India

Ethyl Acetate (HPLC Grade) - E.Merck (India) Ltd., Mumbai.

Toluene (A.R. Grade) – E.Merck (India) Ltd., Mumbai.

Hexane (A.R. Grade) – E.Merck (India) Ltd., Mumbai

Methanol (A.R. Grade) – E.Merck (India) Ltd., Mumbai.


Azukon (Torrent), Gujarat

Tablet 80mg

6.1.3.3 Development and Optimization of HPTLC Method

6.1.3.3.1 Selection of detection wavelength

The sensitivity of HPTLC method that uses UV detection depends upon proper

selection of detection wavelength. An ideal wavelength is the one that gives good

response for the drugs that are to be detected.

In the present study drug solution of 1000µg/ml was prepared in Methanol and from

the stock solution further dilution was made with methanol to get a final solution of

500µg/ml. This drug solution was then scanned in the UV region of 200-400nm and

spectrum was recorded. Also the UV absorbing compound Gliclazide was determined

by densitometric scanning after chromatography development. The light intensity re-

emitted by chromatographic zones is usually lower than the sorbent layer around it.

Therefore, absorption spectra of compound determined directly on HPTLC plates was

almost similar to the one recorded when the substance in solution.


Page 156

Proper selection of chromatographic condition for HPTLC method depends upon the

nature of the sample (ionic, ionizable or neutral molecule), molecular weight and

solubility.


such as mobile phase composition and solvent ratio were studied. The resulting

developed plates were studied and recorded accordingly. The conditions that gave the

best result were selected for estimation.

Stationary phase: Precoated silica gel G60F254 Aluminum sheet, 10 × 10cm

(E.Merck, Germany), and thickness of layer 0.2mm. Plate was pre washed using

methanol and allowed to dry in oven at 500C for 15minutes and allow to come to

room temperature and used immediately.

Mobile phase: Methanol: Toluene: Glacial Acetic Acid (9:1:0.02)

Chamber saturation time: 20 minutes

Distance run: 70mm

Temperature: 270C

Wavelength: 232nm

Slit dimension: 6mm

Scanning speed: 100nm/sec

Spotting parameter:

Band width: 6mm

Spaced between bands: 11.6mm

Syringe capacity: 100µl

6.1.3.4 Estimation of Gliclazide by HPTLC method


Page 157

A mixture of Methanol: Toluene: (9:1v/v) previously prepared and then add 0.02 ml

of Glacial Acetic Acid and filtered through 0.45 µm filter paper in a flask was used as

a mobile phase.

6.1.3.4.2 Preparation of Standard stock solution (500µg/ml)

Gliclazide standard stock solution: (500µg/ml)

A 50 mg of standard Gliclazide accurately was weighed and transferred to a 100 ml

volumetric flask and dissolved in 50 ml Methanol. The flask was sonicated for 10 min.

The flask was shaken and volume was made up to the mark with Methanol to give a

solution containing 500µg/ml Gliclazide.

6.1.3. 4.3 Calibration curve for Gliclazide (500-3000ng/spot)

Appropriate volume of aliquot from standard Gliclazide stock solution was transferred

to same volumetric flasks of 10 ml capacity. Above solution containing 500ng/µl

Gliclazide Stock solution was filled in the syringe and under nitrogen stream by a

semiautomatic sample applicator; it was applied in form of band of each drug on a

single plate having concentration of 500 to 3000ng/spot of Gliclazide. Plate was

developed using Methanol: Toluene: Glacial Acetic Acid (9:1:0.02 v/v/v) at 25±1 ○C

and dried in air. Developed plates were subjected to densitometric measurements in

absorbance mode at wavelength 232 nm using TLC Scanner 3. Plot of peak area vs.

concentration for the Gliclazide was obtained. Spectra of drug were recorded in the

range of 200-400 nm and purity of chromatographic peak was checked by scanning

individual peak at 3 different positions (peak start, peak apex and peak end).


Twenty tablets and capsules were weighed and finely powdered. The powder

equivalent to 25 mg Gliclazide was accurately weighed and transferred to volumetric

flask of 50 ml capacity. 10 ml of methanol was transferred to volumetric flask and

sonicated for 10 min. The flask was shaken and volume was made up to the mark with

methanol. The above solution was filtered through whatman filter paper (0.45µ).

6.1.3.4.5 Estimation of Gliclazide in its solid dosage form

Page 158

8 µl of the prepared sample was applied on pre-washed TLC plate, developed in the

above mobile phase, dried in air and photometrically analyzed as described above

(Fig. 6.1.3.14). From the peak area obtained in the chromatogram, the amount of the



Figure 6.1.3.1: Chromatogram of Figure 6.1.3.2: Chromatogram of

Gliclazide std using mobile phase Gliclazide std using mobile phase

ACN: Ethyl Acetate (4:6) ACN: Toluene: Glacial Acetic Acid

(4:6:0.02)

Page 159


Gliclazide std using mobile phase Gliclazide std using mobile phase

ACN:Toluene: Glacial Acetic Acid Methanol: Toluene: Glacial Acetic Acid

(3:7:0.2) (9:1:0.02)

Figure 6.1.3.5: Chromatogram of Gliclazide sample using Methanol: Toluene:

Glacial Acetic Acid(9:1:0.02)

Page 160


Gliclazide std (500ng/spot) Gliclazide Std (1000ng/spot)


Gliclazide std (1500ng/spot) Gliclazide Std (2000ng/spot)


Gliclazide std (2500ng/spot) Gliclazide std (3000ng/spot)

Page 161

Figure 6.1.3.12: View of all tracks of Gliclazide at 232nm (500-3000ng/spot)

Figure 6.1.3.13: Spectrum of Gliclazide at 232nm (500-3000ng/spot)

Figure 6.1.3.14: Photograph of HPTLC Plates of Gliclazide

6.1.3.6 Various validation parameters

LINEARITY AND RANGE

Procedure

Page 162

Linearity was determined at six levels over the range of 500-3000ng/spot. Each

standard preparation was analysed. The area at each level was calculated and a graph

of mean area v/s concentration (%) was plotted. The correlation co-efficient (r), y-

intercept and slope of regression line were calculated and recorded in Table 6.1.3.3.

Table 6.1.3.2: Linearity study of Gliclazide at 232nm

Amount Sprayed(µL) Amount/Spot(ng) Area Rf-value

2 500 3226.7 0.62

4 1000 5311.5 0.60

6 1500 7000.3 0.62

8 2000 8238.3 0.62

10 2500 9823.8 0.62

12 3000 11445.6 0.63

Figure 6.1.3.15: Calibration curve of Gliclazide at 232nm

Table 6.1.3.3: Statistical data for Gliclazide by HPTLC method

Parameter Gliclazide (232nm)

Linearity Range (ng/spot) 500-3000

Correlation co-efficient 0.995

Slope (s) 3.1924

Intercept 1921.5


Acceptance criteria

The correlation coefficient value should not be less than 0.995 over the working range.

Page 163

Conclusion

The correlation coefficient value was found to be 0.995


sample. The method can, therefore be termed as linear in the specified range.

ACCURACY

Procedure




Gliclazide working standard with placebo. Amount found, % Recovery and mean%



Level

%

Set Area Amount

added(ng)

Amount

recovered

%

Recovery

Mean %

Recovery

%

RSD

50 1 5118.6 1000 1001.472 100.147

100.049

0.103 50 2 5109.4 1000 998.590 99.859

100 1 8231.8 2000 1976.663 98.877

98.846

0.21 100 2 8234.6 2000 1977.569 98.828

150 1 11443.2 3000 2982.615 99.421

99.464

0.05 150 2 11444.6 3000 2983.054 99.435

150 3 11454.3 3000 2986.0919 99.536

Acceptance Criteria



Conclusion:



Page 164

PRECISION


Procedure

Method precision was established by assaying six standard and sample preparations



Table 6.1.3.5: Repeatability (Method precision) data for Gliclazide standard

Conc. 500

ng/spot

1000

ng/spot

1500

ng/spot

2000

ng/spot

2500

ng/spot

3000

ng/spot

Area 3226.7 5311.5 7000.3 8238.3 9823.8 11445.6

3225.6 5311.8 7004.7 8238.4 9823.4 11444.8

3225.8 5314.2 7005.2 8239.4 9824.6 11446.6

3226.4 5314.2 7003.6 8234.8 9823.6 11445.6

3224.8 5311.6 7004.6 8236.5 9824.6 11445.6

3225.6 5214.8 7000.3 8238.2 9824.3 11446.8

Mean 3225.816 5313.016 7003.116 8237.606 9824.05 11445.833

S.D. 0.671 1.534 2.243 1.663 0.521 0.742

% RSD 0.021 0.028 0.032 0.020 0.005 0.006


Concentration Gliclazide(2000ng/spot)

Area 8234.5

8228.5

8233.4

8234.5

8228.8

8238.6

Mean 8233.05

S.D. 3.844

Page 165

% RSD 0.047

Acceptance Criteria


Conclusion:



INTER-DAY AND INTRA-DAY PRECISION (INTERMEDIATE PRECISION)



Intra-day precision was determined by analysing Gliclazide for six times in the same

day at 232nm.


232nm.


Intra-day Inter-day

Conc. 1000

(µg/ml)

2000

(µg/ml)

3000

(µg/ml)

1000

(µg/ml)

2000

(µg/ml)

3000

(µg/ml)

Area 5211 8234.3 11465.3 5112.4 8238.2 11456.3

5116.3 8231.6 11456.2 5116.4 8234.6 11446.7

5114.2 8226.6 11444.7 5118.4 8233.4 11456.4

Mean 5147.167 8230.833 11455.4 5115.733 8235.4 11453.133

S.D. 55.291 3.907 10.323 3.055 2.498 5.572

% RSD 1.074 0.047 0.090 0.059 0.030 0.049

Acceptance Criteria


Page 166


2.0%.

Conclusion:



REPRODUCIBILITY

Procedure

The absorbance readings were measured at different laboratory using another HPTLC

Instrument by another analyst and the value obtained were evaluated to verify their

reproducibility.


Instrument 1

Area ± S.D(n=6)

Instrument 2

Area ± S.D(n=6)

8233.05 ± 3.844

%RSD = 0.047

8234.35 ± 2.857

%RSD = 0.035


Conclusion



ROBUSTNESS

Procedure



Page 167


Parameter % RSD (n=6)



Acceptance criteria


2.0%.

Conclusion

Results were within acceptance criteria hence, method can be termed as Robust.

SOLUTION STABILITY

Procedure





Time Area % Assay

Standard Sample standard Sample

9.00a.m 8234.6 8241.3 98.877 98.982

2.00p.m 8233.8 8238.5 98.864 98.938

6.00p.m 8228.4 8234.4 98.779 98.874

9.00a.m 8218.4 8232.4 98.623 98.843

Acceptance criteria

Page 168



Conclusion






Procedure





Standard Area Average % RSD

1 8219.5

8232.683

0.081

2 8234.6

3 8237.7

4 8236.5

5 8234.6

6 8233.2

Acceptance criteria


Conclusion


for use.

Page 169


Table 6.1.3.12: Summary of Validation Parameter of HPTLC


1 Linearity (ng/spot) 500-3000

2 Recovery % 98.846-100.049


4 Precision (%CV)

Intra-day (n=6)

Inter-day (n=6)

0.030-0.059

0.047-1.074

5 LOD (ng/spot) 0.865

6 LOQ (ng/spot) 2.621


8 Selectivity Selective





Set Amount(ng) %Assay %RSD(n=3)

1 2000 99.12 0.52

Page 170

6.2 GLIBENCLAMIDE


ESTIMATION OF GLIBENCLAMIDE IN ITS SOLID DOSAGE FORM

6.2.1.1 Instruments and Materials




1 UV 1)Shimadzu

2)Perkin Elmer


2)Lambda 19




5 Milli-Q water source Thermofisher

Scientific

Branstead D3750


UV- visible double beam spectrophotometer with matched quartz cells (1cm).

1) Make: SHIMADZU



Slit Width 2 nm






Noise Level 0.002 Abs

2) Make: Perkin Elmer, USA, Model: Lambda 19

Page 171

� pH meter

Make: Thermo Orion

Model: 420 A+


Model: AX205



� Sonicator

Model: Branson 2510


0-60 Sonic/minute

� Milli-Q water



Hollow fiber filter

Gamma Irradiated

Pore size: 0.2 µm



Make: Millipore

Model: Elix-3 system (water output 3L)



Page 172

6.2.1.1.2 Materials

Glibenclamide: Reddy,s Laboratories,Hyderabad, India.

Sodium hydroxide (AR Grade) E.Merck (India) Ltd., Mumbai.


Gluconil (Bal Pharma),Karnataka.

Tablet 5mg


6.2.1.3.1 Selection of Solvent for Glibenclamide.

Glibenclamide is soluble in solvents like methanol, dichloromethane and ethanol.

Glibenclamide is practically insoluble in water. The spectrum of Glibenclamide was

scanned in methanol. Owing to the solubility of Glibenclamide in methanol and also

there was no shift in absorbance maxima , methanol was selected as solvent.

6.2.1.3.2 Selection of analytical wavelength for Glibenclamide.

The solution of Glibenclamide was prepared in methanol at a concentration of 100 µg

/ml. It was scanned in the wavelength range of 200-400 nm. Data were recorded at an

interval of 1 nm. Maximum absorbance was obtained at 300 nm. This analytical

wavelength was selected for determination of Glibenclamide. The standard solutions

of 10-100 µg/ml were prepared and spectrums of these concentrations were obtained

with n = 6. The overlain spectrum of Glibenclamide at different concentration was

recorded (Fig. no.6.2.1.1).


Glibenclamide standard stock solution (100µg/ml)

Accurately weighed powder of Glibenclamide (100 mg) was transferred to a separate

100 ml volumetric flask and dissolved and diluted to the mark with methanol. Further

10 ml was taken and diluted to 100 ml with same solvent to give a standard stock

solution containing 100µg/ml Glibenclamide.

6.2.1.3.4 Calibration Curve for Glibenclamide (10-100µg/ml)

Page 173



with methanol to obtain concentration of 10, 20, 40, 60, 80 and 100µg/ml. The curve

of solution against methanol as a blank was recorded. Absorbance was measured at

300nm against methanol as a blank and the graph of absorbance vs. concentration

was plotted. The straight-line equation was determined.(Fig.no.6.2.1.2)


Twenty Glibenclamide tablets were weighed and powdered. The tablet powder

equivalent to 100 mg of Glibenclamide transferred in to a 100 ml volumetric flask.

Methanol (100 ml) was added to it and sonicated for 20 min. The solution was filtered

through whatman filter paper No. 41 and the volume was adjusted up to the mark with

methanol to produce sample solution containing Glibenclamide 1000 µg/ml. 10 ml of


ml to contain Glibenclamide 100 µg/ml (Stock solution A).

From the Stock solution A, 2ml was transferred to volumetric flask of 10 ml capacity.

Volume was made up to the mark with methanol to give a solution containing

20µg/ml Glibenclamide (Solution 1). This solution was used for the estimation of

Glibenclamide.

Estimation of Glibenclamide by UV Spectroscopic Method

6.2.1.3.6 The solution 1 was measured at 300 nm for quantification of Glibenclamide.

The amount of Glibenclamide present in the sample solution was determined. From

the absorbance obtained in the spectrum, the amount of drug was calculated.



LINEARITY AND RANGE

Procedure

Linearity was determined at six levels over the range of 10-100µg/ml with respect to


attain concentration of about 10, 20, 40, 60, 80 & 100 µg/ml of sample concentration.

Page 174

Each standard preparation was recorded in six replicates. The mean absorbance at

each level was calculated and a graph of mean absorbance v/s concentration (%) was



Table 6.2.1.2: Result of calibration reading at 300nm for Glibenclamide

Concentration

(µg/ml)

Absorbance at 300nm

Mean ± S.D. (n=6)

% RSD

10 0.098 ± 0.001 1.117

20 0.145 ± 0.001 0.575

40 0.301 ± 0.001 0.271

60 0.433 ± 0.002 0.413

80 0.564 ± 0.001 0.207

100 0.713 ± 0.001 0.106

Figure 6.2.1.1: Overlain Spectrum of Glibenclamide (10 -30µg/ml) in methanol at

300nm.

Page 175

Figure 6.2.1.2: Calibration Curve of Glibenclamide at 300 nm.

Table 6.2.1.3: Statistical data for Glibenclamide by Spectrophotometry method

Parameter Glibenclamide


Slope 0.0069

Intercept 0.0209


Acceptance criteria


Conclusion:

The correlation coefficient value was 0.9988 for Glibenclamide.

The absorbances obtained were directly proportional to the concentration of analyte in

the sample. The method can, therefore be termed as linear in the range considered.

Based on the linearity results, the working range of the method could be established

as 10-100µg/ml of the working concentration.

ACCURACY

Procedure




y = 0.0069x + 0.0209R² = 0.9988

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60 80 100 120

ab

sorb

an

ce

concentration(µg/ml)

calibration curve of Glibenclamide

Series1

Linear (Series1)

Linear (Series1)

Page 176

Glibenclamide working standard with placebo. Amount found, % Recovery and mean



Level

%

Set

Absorbance

Amount

Added

(µg/ml)

Amount

recovered

(µg/ml)

%Recovery

Mean %

Recovery

%

RSD

50 1 0.642 30 90.06 100.067

99.8

0.257 50 2 0.641 30 89.8 99.778

50 3 0.639 30 89.6 99.556

100 1 0.851 60 120.3 100.250

100.639

0.599 100 2 0.859 60 121.6 101.333

100 3 0.852 60 120.4 100.333

150 1 1.057 90 150.12 100.08

99.796

0.415 150 2 1.056 90 149.98 99.987

150 3 1.048 90 148.98 99.320

Acceptance Criteria



Conclusion:


acceptance criteria. Hence, the method can be termed accurate in the considered range.

PRECISION


Procedure




Page 177

Table 6.2.1.5: Repeatability (Method precision) data for Glibenclamide standard

at 300nm

Concentration 10µg/ml 20µg/ml 40µg/ml 60µg/ml 80µg/ml 100µg/ml

Absorbance 0.099 0.145 0.302 0.431 0.565 0.713

0.098 0.146 0.302 0.432 0.565 0.712

0.098 0.146 0.301 0.434 0.564 0.713

0.099 0.146 0.301 0.433 0.564 0.712

0.098 0.144 0.302 0.432 0.562 0.713

0.096 0.146 0.300 0.436 0.563 0.714

Mean 0.098 0.146 0.301 0.433 0.564 0.713

S.D. 0.001 0.001 0.001 0.002 0.001 0.001

% RSD 1.118 0.575 0.271 0.413 0.207 0.106

Table 6.2.1.6: Repeatability data for Glibenclamide

Concentration Glibenclamide(60µg/ml)

Absorbance 0.439

0.434

0.432

0.433

0.436

0.438

Mean 0.435

S.D. 0.003

% RSD 0.644

Acceptance Criteria


Conclusion:



Page 178




Intra-day precision was determined by analysing Glibenclamide for six times in the

same day at 300nm.


300nm.

Table 6.2.1.7: Intra-day precision data for Glibenclamide at 300nm

Conc. (µg/ml) Intra-day (n=6) % RSD

40 0.304 ± 0.002 0.657

60 0.434 ± 0.003 0.740

80 0.566 ± 0.003 0.568

Table 6.2.1.8: Inter-day precision data for Glibenclamide at 300nm

Conc. (µg/ml) Inter-day (n=6) % RSD

40 0.307 ± 0.003 1.048

60 0.439 ± 0.003 0.695

80 0.570 ± 0.002 0.304

Acceptance Criteria



2.0%.

Conclusion:

The results obtained were well within the acceptance criteria. The method can there

fore be termed as precise and rugged.

REPRODUCIBILITY

Page 179

Procedure

The absorbance readings of solutions (60µg/ml) were measured at different laboratory

using another spectrophotometer by another analyst and the value obtained were

evaluated to verify their reproducibility.


Instrument 1(n=6) Instrument 2(n=6)

0.432 ± 0.002

%RSD = 0.23

0.431 ± 0.003

%RSD = 0.64

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure







Acceptance criteria


2.0%.

Page 180

Conclusion


SOLUTION STABILITY

Procedure





Time Absorbance of

standard(60µg/ml)

at 300nm

Absorbance of

sample(60µg/ml)

at 300nm

% Assay

standard sample

9.00a.m 0.435 0.435 100.024 100.024

2.00p.m 0.434 0.434 99.783 99.782

6.00p.m 0.433 0.432 99.541 99.299

9.00a.m 0.433 0.431 99.541 99.058

Acceptance criteria



Conclusion






Procedure

Page 181





Standard Absorbance Average % RSD

1 0.439

0.435

0.644

2 0.434

3 0.432

4 0.433

5 0.436

6 0.438

Acceptance criteria


Conclusion


for use.


Table 6.2.1.13 Summary of Validation Parameter of Spectrophotometry



2 Recovery % 98.87-99.85


4 Precision (CV)

Intra-day (n=6)

Inter-day (n=6)

0.568-0.740

0.304-1.048

5 LOD (µg/ml) 1.017

6 LOQ (µg/ml) 3.081

Page 182







1 60 101 0.42

Page 183

6.2.2 RP-HPLC METHOD DEVELOPMENT FOR GLIBENCLAMIDE IN

ITS SOLID DOSAGE FORM.





1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000






HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A


Glibenclamide: Reddy,s Laboratories,Hyderabad, India


Page 184



Potassium dihydrogen phosphate (AR Grade) – Rankem(India) Ltd., Mumbai

Triethyl amine (AR Grade)- Finar

Orthophosphoric acid (AR Grade)- Rankem(India) Ltd., Mumbai


Gluconil (Bal Pharma),Karnataka.

Tablet 5mg


6.2.2.3.1 Selection of Detection Wavelength




prepared in solvent mixtures of Buffer (pH 4.0) and ACN (30:70). This drug solution

was then scanned in the UV region of 200-400 nm and the spectrum was

recorded.(Fig. no.6.2.2.1)

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

100

200

300

400

500

600

700

800

mAU

246.0

4

220.5

7

279.3

9

Figure 6.2.2.1: UV spectrum of Glibenclamide

6.2.3 Selection of chromatographic conditions

Page 185











6.2.2.4 Estimation of Glibenclamide by RP-HPLC method

6.2.2.4.1 Preparation of 0.05M buffer (pH 4.0)

Accurately weighed 6.8gm of potassium dihydrogen phosphate was dissolved in 800

ml of water and then volume made up to 1000ml with water and adjusted pH of the

solution with orthophosphoric acid (5%) .


300 ml of buffer (pH 4.0) and 700 ml of Acetonitrile were mixed and filtered through

0.45µ filter paper, sonicated for 10 minutes to degas and used as mobile phase.

Mobile phase was used as diluent.


Glibenclamide Standard stock solution (300 ppm)

Accurately weighed Glibenclamide standard (30 mg) was transferred to a 100 ml





Page 186

6.2.2.4.4 Calibration curve for Glibenclamide(50-150µg/ml)

Appropriate volume of aliquots from standard Glibenclamide stock solution was


to the mark with the diluent to obtain the concentration of 15, 22.5, 30, 37.5 and

45µg/ml. Calibration curve of each solution against the diluent was recorded at

279nm and the graph of absorbance v/s concentration was plotted. The straight line

equation was determined from calibration curve.


Twenty Glibenclamide tablets were taken and crushed to make powder. The

accurately weighed powder equivalent to 3 mg of Glibenclamide was transferred into

100 ml volumetric flask and dissolved in about 10 ml diluent by sonication for 25

minute and then volume was made up to 100 ml with diluent to obtain sample stock

solution (300µg/ml). An aliquot of the solution (5.0ml) was transferred to a 50ml


solution (30ug/ml).

6.2.2.4.6 Estimation of Glibenclamide in its solid dosage form

The prepared sample solution was chromatographed for 10 minutes using mobile

phase.From the peak area obtained in the chromatogram, the amount of the drug was

calculated.


Page 187

Figure 6.2.2.2: Chromatogram of Glibenclamide using water:methanol(40:60)

Figure 6.2.2.3: Chromatogram of Glibenclamide using water:methanol (30:70)

Figure 6.2.2.4: Chromatogram of Glibenclamide using water: ACN (40:60)

Figure 6.2.2.5: Chromatogram of Glibenclamide using buffer: ACN (35:65)

Page 188


Glibenclamide

Table 6.2.2.2: Optimized chromatographic conditions for Glibenclamide


1 Mobile Phase 0.05M Potassium dihydrogen

phosphate(pH 4.0 adjusted with ortho

phosphoric acid):Acetonitrile (30:70)


3 Stationary Phase Hypersil BDS(250×4.6 mm id, 5µm

particle size) column


5 Volume of

Injection (µl)

20µl

6 Detection

wavelength(nm)

279nm




Page 189

LINEARITY AND RANGE

Procedure



attain concentration of about 15, 22.5, 30, 37.5 & 45 µg/ml of sample concentration.

Each standard preparation was recorded in six replicates. The mean absorbance at

each level was calculated and a graph of mean absorbance v/s concentration (%) was



Table 6.2.2.3: Result of calibration readings for Glibenclamide

Concentrations

(µg/ml)

Glibenclamide

Area


15 2130.054 ± 15.587 0.732

22.5 3191.779 ± 16.120 0.505

30.5 4268.011 ± 14.678 0.344

37.5 5316.284 ± 81.207 1.528

45 6384.372 ± 33.974 0.532

Figure 6.2.2.7: Calibration curve of Glibenclamide at 279nm

Page 190

Table 6.2.2.4: Statistical data for Glibenclamide by RP-HPLC

Parameters Glibenclamide


Slope 141.78

Intercept 4.844


Acceptance criteria


range.

Conclusion:

The correlation coefficient value were found to be 1.000 for Glibenclamide.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Glibenclamide was established at

three levels in triplicate,viz. 80%, 100%, and 120% of the test concentration. Standard

was prepared as per method and sample preparations were done by mixing known

amount of Glibenclamide working standard with placebo. Amount found, % Recovery

and Mean Recovery was calculated at each level and recorded in Table 6.2.2.5.

Page 191


Level

(%)

Sets Area Amount

added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean

% Recovery

% RSD

80 1 3415.776 24 24.057 100.241

100.639

0.431 80 2 3445.094 24 24.264 101.103

80 3 3427.08 24 24.153 100.574

100 1 4341.82 30 30.589 101.965

100.888

1.057 100 2 4251.843 30 29.955 99.849

100 3 4294.479 30 30.266 100.852

120 1 5167.294 36 36.412 101.144

100.911

0.253 120 2 5157.541 36 36.343 100.953

120 3 5141.451 36 36.229 100.637

Acceptance Criteria


102.0 with % RSD not be more than 2.0



considering range.

PRECISION


Procedure




Page 192


Concentration 15(µg/ml) 22.5(µg/ml) 30(µg/ml) 37.5(µg/ml) 45(µg/ml)

Area 2137.125 3190.839 4260.932 5162.347 6380.073

2113.626 3213.111 4269.42 5396.799 6418.33

2156.362 3168.406 4295.005 5305.933 6329.038

2120.022 3206.68 4269.42 5359.348 6392.806

2132.848 3184.409 4252.395 5337.933 6367.321

2120.342 3187.23 4260.896 5335.346 6418.664

Mean 2130.054 3191.779 4268.011 5316.284 6384.372

S.D. 15.587 16.120 14.678 81.207 33.974

%RSD 0.732 0.505 0.344 0.532 0.532

n=6 determinations

Table 6.2.2.7: Repeatability of sample application data for Glibenclamide

Concentration Glibenclamide(30µg/ml)

Area 4260.456

4268.564

4269.456

4268.327

4260.642

4236.486

Mean 4260.655

S.D. 12.514

% RSD 0.294

Acceptance Criteria


Page 193

Conclusion:






Table 6.2.2.8 Precision data

Intra-day Inter-day

Conc.(µg/ml) 50 100 150 50 100 150

Area 2152.094 4316.328 6443.941 2156.362 4303.512 6488.787

2122.171 4226.813 6335.433 2117.895 4226.785 6341.816

2132.848 4252.395 6367.321 2128.569 4277.93 6392.806

Mean 2135.704 4265.178 6382.231 2134.275 4269.409 6407.803

S.D. 15.16 46.106 55.76 19.858 39.066 74.62

% RSD 0.710 1.081 0.874 0.930 0.915 1.165

Acceptance Criteria



2.0%.

Conclusion:



Page 194

REPRODUCIBILITY

Procedure



reproducibility.


Instrument 1

Area ± S.D.(n=6)

Instrument 2

Area ± S.D.(n=6)

4270.882 ± 36.853

%RSD = 0.863

4258.246 ± 36.565

%RSD = 0.859

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure




2. Flow rate

3. pH of buffer


table 6.2.2.10.

Page 195

Table 6.2.2.10: Data derived from Robustness study of Glibenclamide


M.P(buffer:ACN)77:23 4248.115 ± 27.9104 0.657

M.P(buffer:ACN)73:27 4277.895 ± 41.9323 0.980

Flow rate 1.2ml/min 4033.565 ± 19.9010 0.493

Flow rate 0.8ml/min 4463 ± 39.253 0.879

pH of buffer 4.2 4236.833 ± 44.5702 1.052

pH of buffer 3.8 4260.871 ± 43.0777 1.011



Note


suitability set.

% RSD was calculated for the six replicate injections of standard preparation.

Acceptance criteria




2%.

Conclusion


normal condition and varied condition were well within acceptance criteria, hence


Page 196

SOLUTION STABILITY

Procedure




Time Areaof

GLBstandard(30µg/ml)

Area of GLB

sample(30µg/ml)

% Assay

standard sample

9.00a.m 4226.245 4316.328 100.928 101.365

2.00p.m 4224.456 4316.321 100.903 101.365

6.00p.m 4216.675 4309.998 100.575 101.216

9.00a.m 4212.658 4310.221 99.793 101.222

Acceptance criteria



Conclusion






Procedure



Page 197



Table 6.2.2.12 System suitability test

System Suitability Parameter Glibenclamide




% RSD (n=6) 0.2

Note



Acceptance criteria


Conclusion



Page 198


Table 6.2.2.13: Summary of validation parameters of Glibenclamide by RP-

HPLC method


1. Linearity Range (μg/ml) 15 –45

2. Correlation co efficient (r2) 1.0


4. Precision (%CV)

Inter-day precision

Intra-day precision

0.915-1.165

0.710-1.080






6.2.2.6 Assay results of marketed formulation

Table 6.2.2.14: Assay results of marketed formulation


1 30 99.863 0.862

Page 199

6.2.3 HPTLC METHOD DEVELOPMENT FOR THE ESTIMATION OF

GLIBENCLAMIDE IN ITS SOLID DOSAGE FORM



Table 6.2.3.1 List of the instruments used in HPTLC


1 HPTLC Camag Linomat 5

2 Analytical Balance Mettler Toledo AX205




6 Deionised water Plant Millipore Elix-3 system

High Performance Thin Layer Chromatography (HPTLC)

Camag Linomat 5

Semiautomatic application, band application by spray on technique (2 -500µl)

Camag twin trough glass chamber (10 × 10 and 20 × 10)

Camag TLC scanner 3

Scanning speed up to 100mm/s, Spectral range 190 – 800nm

Camag Reprostar 3 with digital camera

For 254nm, 366 nm and with light

Camag UV cabinet with dual wavelength UV lamp

Dual wavelength 254/ 366nm

Stationary Phase: silica gel G60 F254 coated on aluminum sheet

Hamilton 100µl HPTLC syringe

Page 200

Data Resolution: 100µm/step

6.2.3.1.2 Materials

GLIBENCLAMIDE: Reddy,s Laboratories,Hyderabad, India

Ethyl Acetate (HPLC Grade) - E.Merck (India) Ltd., Mumbai.

Toluene (A.R. Grade) – E.Merck (India) Ltd., Mumbai.

Hexane (A.R. Grade) – E.Merck (India) Ltd., Mumbai

Methanol (A.R. Grade) – E.Merck (India) Ltd., Mumbai.


Gluconil(Bal Pharma),Karnataka

Tablet 5mg

6.2.3.3 Development and Optimization of HPTLC Method

6.2.3.3.1 Selection of detection wavelength

The sensitivity of HPTLC method that uses UV detection depends upon proper

selection of detection wavelength. An ideal wavelength is the one that gives good

response for the drugs that are to be detected.

In the present study drug solution of 1000µg/ml was prepared in Methanol and from

the stock solution further dilution was made with methanol to get a final solution of

500µg/ml. This drug solution was then scanned in the UV region of 200-400nm and

spectrum was recorded. Also the UV absorbing compound Glibenclamide was

determined by densitometric scanning after chromatography development. The light

intensity re-emitted by chromatographic zones is usually lower than the sorbent layer

around it. Therefore, absorption spectra of compound determined directly on HPTLC

plates was almost similar to the one recorded when the substance in solution.


Page 201

Proper selection of chromatographic condition for HPTLC method depends upon the

nature of the sample (ionic, ionizable or neutral molecule), molecular weight and

solubility.


such as mobile phase composition and solvent ratio were studied. The resulting

developed plates were studied and recorded accordingly. The conditions that gave the

best result were selected for estimation.

Stationary phase: Precoated silica gel G60F254 Aluminum sheet, 10 × 10cm

(E.Merck, Germany), and thickness of layer 0.2mm. Plate was pre washed using

methanol and allowed to dry in oven at 500C for 15minutes and allow to come to

room temperature and used immediately.

Mobile phase: Toluene: Ethyl Acetate: Hexane: Glacial Acetic Acid (6:2:2:0.1)

Chamber saturation time: 20 minutes

Distance run: 70mm

Temperature: 270C

Wavelength: 230nm

Slit dimension: 6mm

Scanning speed: 100nm/sec

Spotting parameter:

Band width: 6mm

Spaced between bands: 11.6mm

Syringe capacity: 100µl

6.2.3.4 Estimation of Glibenclamide by HPTLC method


Page 202

A mixture of Toluene: Hexane: Ethyl Acetate: Glacial Acetic Acid (6:2:2:0.1 v/v/v/v)

previously prepared and then add 0.1 ml of Glacial Acetic Acid and filtered through

0.45 µm filter paper in a flask was used as a mobile phase.

6.2.3.4.2 Preparation of Standard stock solution (500µg/ml)

Glibenclamide standard stock solution: (500µg/ml)

A 50 mg of standard Glibenclamide accurately was weighed and transferred to a 100

ml volumetric flask and dissolved in 50 ml Methanol. The flask was sonicated for 10

min. The flask was shaken and volume was made up to the mark with Methanol to

give a solution containing 500µg/ml Glibenclamide.

6.2.3.4.3 Calibration curve for Glibenclamide (500-1000ng/spot)

Appropriate volume of aliquot from standard Glibenclamide stock solution was

transferred to same volumetric flasks of 10 ml capacity. Above solution containing

500ng/µl Glibenclamide. Stock solution was filled in the syringe and under nitrogen

stream by a semiautomatic sample applicator; it was applied in form of band of each

drug on a single plate having concentration of 500 to 3000ng/spot of Glibenclamide.

Plate was developed using Toluene: Hexane: Ethyl Acetate: Glacial Acetic Acid

(6:2:2:0.1 v/v/v/v) at 25±1 ○C and dried in air. Developed plates were subjected to

densitometric measurements in absorbance mode at wavelength 230 nm using TLC

Scanner 3. Plot of peak area vs. concentration for the Glibenclamide was obtained.

Spectra of drug were recorded in the range of 200-400 nm and purity of

chromatographic peak was checked by scanning individual peak at 3 different

positions (peak start, peak apex and peak end).


Twenty tablets and capsules were weighed and finely powdered. The powder

equivalent to 25 mg Glibenclamide was accurately weighed and transferred to

volumetric flask of 50 ml capacity. 10 ml of methanol was transferred to volumetric

flask and sonicated for 10 min. The flask was shaken and volume was made up to the

mark with methanol. The above solution was filtered through whatman filter paper

(0.45µ).

6.2.3.5 Estimation of Glibenclamide in its solid dosage form

Page 203

8 µl of the prepared sample was applied on pre-washed TLC plate, developed in the

above mobile phase, dried in air and photometrically analyzed as described above .

(Fig.no.6.2.3.13).From the peak area obtained in the chromatogram, the amount of the




Glibenclamide std using mobile Glibenclamide std using mobile

Phase (methanol: Hexane:GAA) Phase (methanol: Toluene: Hexane:GAA)

3:7:0.1 3:2:5:0.1

Figure 6.2.3.3: Chromatogram of Figure 6.2.3.4 Chromatogram of

Glibenclamide std using optimised Glibenclamide sample using

mobile phase optimised mobile phase

Page 204


Glibenclamide std (500ng/spot) Glibenclamide std (1000ng/spot)


Glibenclamide std (1500ng/spot) Glibenclamide std (2000ng/spot)

Figure6.2.3.9:Chromatogramof Figure6.2.3.10Chromatogramof

Glibenclamide std(2500ng/spot) Glibenclamide std(3000ng/spot)

Page 205

Figure 6.2.3.11: View of all tracks of Glibenclamide

Figure 6.2.3.12: Spectrum of Glibenclamide at 230nm (500-3000ng/spot)

Figure 6.2.3.13: Photograph of HPTLC Plates of Glibenclamide

Various validation parameters

Page 206

LINEARITY AND RANGE

Procedure

Linearity was determined at six levels over the range of 500-3000ng/spot. Each

standard preparation was analysed. The area at each level was calculated and a graph

of mean area v/s concentration (%) was plotted. The correlation co-efficient (r), y-

intercept and slope of regression line were calculated and recorded in Table 6.2.3.2.

Table 6.2.3.2: Linearity study of Glibenclamide at 230nm

Amount Sprayed

(µL)

Amount/Spot

(ng)

Area Rf-value

2 500 8995.2 0.47

4 1000 11258.5 0.44

6 1500 13634.5 0.42

8 2000 15513.3 0.42

10 2500 17193 0.41

12 3000 19713.1 0.42

Figure 6.2.3.14: Calibration curve of Glibenclamide at 230nm

Page 207

Table 6.2.3.3: Statistical data for Glibenclamide by HPTLC method

Parameter Glibenclamide (230nm)

Linearity Range (ng/spot) 500-3000

Correlation co-efficient 0.9968

Slope (s) 4.1897

Intercept 7053.5


Acceptance criteria


Conclusion

The correlation coefficient value was found to be 0.9968


sample. The method can, therefore be termed as linear in the specified range.

ACCURACY

Procedure




Glibenclamide working standard with placebo. Amount found, % Recovery and

mean% Recovery was calculated at each level and recorded in Table 6.2.3.4.

Page 208


Level

%

Set Area Amount

added(ng)

Amount

recovered(ng)

%

Recovery

Mean %

Recovery

%

RSD

50 1 11258.2 1000 1003.58 100.358

100.243

0.039 50 2 11252.5 1000 1002.22 100.222

50 3 11249.4 1000 1001.48 100.148

100 1 15511.2 2000 2018.689 100.934

100.813

0.111 100 2 15481.2 2000 2011.528 100.576

100 3 15510.6 2000 2018.545 100.927

150 1 19713.3 3000 3021.648 100.722

100.750

0.018 150 2 19720.5 3000 3023.367 100.779

150 3 19716.9 3000 3022.508 100.750

Acceptance Criteria



Conclusion:



PRECISION


Procedure




Page 209

Table 6.2.3.5: Repeatability (Method precision) data for GLB standard at 230nm.

Conc. 500

ng/spot

1000

ng/spot

1500

ng/spot

2000

ng/spot

2500

ng/spot

3000

ng/spot

Area 8995.2 11258.5 13634.5 15513.3 17193 19713.1

8986.3 11263.4 13643.2 15521.2 17193.4 19721.1

8984.6 11262.4 13638.6 15516.4 17187.4 19714.6

8993.2 11253.6 13634.5 15521.6 17192.7 19718.1

8994.8 11253.5 13643.2 15519.6 17189.4 19716.4

8983.3 11254.5 13641.2 15513.4 17194 19721.6

Mean 8989.567 11257.65 13639.2 15517.58 17191.65 19717.48

S.D. 5.420947 4.469 4.013 3.756 2.632 3.438

% RSD 0.060303 0.039 0.029 0.024 0.015 0.017

Table 6.2.3.6: Repeatability of sample application data for Glibenclamide

Concentration Glibenclamide

2000ng/spot

Absorbance 15515.03

15516.3

15521.6

15516.2

15518.4

15526.4

Mean 15518.99

S.D. 4.307

% RSD 0.028

Acceptance Criteria


Conclusion:

Page 210



INTER-DAY AND INTRA-DAY PRECISION (INTERMEDIATE PRES-

CISION)



Intra-day precision was determined by analysing Glibenclamide for six times in the

same day at 230nm.


230nm.


Intra-day Inter-day

Conc.( µg/ml) 1000 2000 3000 1000 2000 3000

Area 11258 15509.8 19703.5 11249 15520.1 19721.4

11256.4 15555.3 19716.9 11258.4 15521.3 19720.5

11253.7 15512.5 19712.4 11249.4 15518.4 19714.4

Mean 11256.03 15525.87 19710.93 11252.27 15519.93 19718.77

S.D 2.173 25.526 6.819 5.315 1.457 3.808

% RSD 0.019 0.164 0.035 0.047 0.009 0.019

Acceptance Criteria



2.0%.

Conclusion:



Page 211

REPRODUCIBILITY

Procedure

The absorbance readings were measured at different laboratory using another HPTLC

Instrument by another analyst and the value obtained were evaluated to verify their

reproducibility.


Instrument 1

Area ± S.D(n=6)

Instrument 2

Area ± S.D(n=6)

15522.9 ± 16.494

%RSD = 0.106

15509.32 ± 14.339

%RSD = 0.092

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure




Parameter % RSD (n=6)



Acceptance criteria


2.0%.

Page 212

Conclusion


SOLUTION STABILITY

Procedure





Time Area % Assay

Standard Sample standard Sample

9.00a.m 15521 15534.4 101.0514 101.2113

2.00p.m 15516.4 15522.5 100.9965 101.0693

6.00p.m 15511.8 15518 100.9416 101.0156

9.00a.m 15505.4 15516.4 100.8652 100.9965

Acceptance criteria



Conclusion






Procedure

Page 213





Standard Area Average % RSD

1 15509.8

15516.23

0.029

2 15515.3

3 15512.5

4 15520.1

5 15521.3

6 15518.4

Acceptance criteria


Conclusion


for use.

Page 214


Table 6.2.3.12: Summary of Validation Parameters of HPTLC


1 Linearity (ng/spot) 500-3000

2 Recovery % 100.243-100.813


4 Precision (%CV)

Intra-day

Inter-day

0.019-0.164

0.009-0.047

5 LOD (ng/spot) 3.111

6 LOQ (ng/spot) 9.428


8 Selectivity Selective





Set Amount(ng) %Assay %RSD(n=3)

1 2000 100.14 0.61

Page 215


ESTIMATION OF GLIPIZIDE IN ITS SOLID DOSAGE FORM





1 UV 1)Shimadzu

2)Perkin Elmer


2)Lambda 19




5 Milli-Q water Thermofisher

Scientific

Branstead D3750


UV- Visible double beam spectrophotometer with matched quartz cells (1cm).

1. Make: SHIMADZU



Slit Width 2 nm






Noise Level 0.02 S

2. Make: Perkin Elmer, USA, Model: Lambda 19

� pH meter

Page 216

Make: Thermo Orion

Model: 420 A+


Model: AX205


Maximum capacity: 22

� Sonicator

Model: Branson 2510


0-60 Sonic/minute

� Milli-Q water



Hollow fiber filter

Gamma Irradiated

Pore size: 0.2 µm



Make: Millipore




6.3.1.1.2 Materials

Page 217

Glipizide: Reddy, s Laboratories, Hyderabad, India.

Sodium hydroxide (AR Grade) E.Merck Ltd., Mumbai,India.


Glez(Aristo Pharmaceuticals Ltd.),Maharashtra.

Tablet 5mg


6.3.1.3.1 Selection of Solvent

Glipizide is insoluble in solvents like water and practically insoluble in ethanol. But it

dissolves in dilute solution of alkali hydroxides. Initially spectrum of Glipizide was

scanned in 0.1M NaOH. Owing to the solubility of Glipizide in the 0.1M NaOH and

also there was no shift in absorbance maxima of Glipizide, 0.1M NaOH was selected

as solvent.

6.3.1.3.2 Selection of analytical wavelength for Glipizide.

The solution of Glipizide was prepared in 0.1 N NaOH at a concentration of 20 µg /ml.

It was scanned in the wavelength range of 200-400 nm. Maximum absorbance was

obtained at 276.0 nm. This analytical wavelength was selected for determination of

Glipizide. The standard solutions of 10-30 µg/ml were prepared and spectrums of

these concentrations were obtained with n = 5. The overlain spectrum of Glipizide at

different concentration was recorded.(Fig.no.6.3.1.1)


Glipizide standard stock solution (100µg/ml)

Accurately weighed powder of Glipizide (100 mg) was transferred to a separate 100

ml volumetric flask and dissolved and diluted to the mark with 0.1M NaOH. Further

10 ml was taken and diluted to 100 ml with same solvent to give standard stock

solution containing 100µg/ml Glipizide.

6.3.1.3.4 Calibration Curve for Glipizide (10-30µg/ml)

Page 218



with 0.1M NaOH to obtain concentration of 10, 15, 20, 25 and 30µg/ml. The curve of

solution against the 0.1M NaOH as a blank was recorded. Absorbance was measured

at 276nm against 0.1M NaOH as a blank and the plot of absorbance vs. concentration

was plotted. The straight-line equation was determined.(Fig.no.6.3.1.2)


Twenty Glipizide tablets were weighed and powdered. The tablet powder equivalent

to 100 mg of Glipizide transferred in to a 100 ml volumetric flask. 0.1M NaOH (100

ml) was added to it and sonicated for 20 min. The solution was filtered through

whatman filter paper No. 41 and the volume was adjusted up to the mark with 0.1M

NaOH to produce sample solution containing Glipizide 1000 µg/ml. 10 ml of


ml to contain Glipizide 100 µg/ml (Stock solution A).

From the Stock solution A, 2 ml was transferred to volumetric flask of 10 ml capacity.

Volume was made up to the mark with 0.1M NaOH to give a solution containing

20µg/ml.Glipizide (Solution 1). This solution was used for the estimation of Glipizide.

6.3.1.3.6 Estimation of Glipizide by UV Spectroscopic Method

The solution 1 was measured at 276 nm for quantification of Glipizide. The amount of

Glipizide present in the sample solution was determined. From the absorbance

obtained in the spectrum, the amounts of drug was calculated.



LINEARITY AND RANGE

Procedure



attain concentration of about 10,15,20,25 & 30 µg/ml of glipizide. Each standard

preparation was recorded in six replicates. The mean absorbance at each level was

Page 219

calculated and a graph of mean absorbance v/s concentration was plotted. The

correlation co-efficient (r), y-intercept, slope of regression line were calculated and

recorded in Table: 6.3.1.3.

Table 6.3.1.2: Result of calibration reading at 276nm for Glipizide

Concentration

(µg/ml)

Absorbance at 276nm

Mean ± S.D. (n=6)

% RSD

10 0.266 ± 0.003 1.089

15 0.396 ± 0.002 0.462

20 0.532 ± 0.002 0.440

25 0.681 ± 0.002 0.327

30 0.832 ± 0.004 0.521

Figure 6.3.1.1: Overlain Spectrum of Glipizide

Page 220

Figure 6.3.1.2: Calibration Curve of Glipizide at 276 nm.

Table 6.3.1.3: Statistical data for Glipizide by Spectrophotometry method

Parameter Glipizide (at 276nm)


Slope 0.0283

Intercept 0.0248


Acceptance criteria


Conclusion:

The correlation coefficient value was 0.999 for Glipizide.





ACCURACY

Procedure




Page 221

Glipizide working standard with placebo. Amount found, % Recovery and Mean



Level

%

Sets Absorbance Amount

Added

(µg/ml)

Amount

recovered

(µg/ml)

%

Recovery

Mean%

Recovery

%

RSD

50 1 0.820 10 29.86 99.533

98.955

0.506 50 2 0.813 10 29.6 98.666

50 3 0.813 10 29.6 98.666

100 1 1.082 20 39.48 98.7

98.633

0.409 100 2 1.096 20 39.6 99.0

100 3 1.083 20 39.28 98.2

150 1 1.361 30 49.09 98.68

98.366

0.255 150 2 1.372 30 49.34 98.46

150 3 1.368 30 49.23 98.44

Acceptance Criteria



Conclusion:



PRECISION


Procedure




Page 222

Table 6.3.1.5: Repeatability (Method precision) data for Glipizide standard at

276nm


Absorbance 0.266 0.398 0.530 0.683 0.830

0.264 0.396 0.536 0.68 0.840

0.264 0.399 0.533 0.682 0.832

0.269 0.394 0.534 0.684 0.829

0.263 0.396 0.532 0.678 0.833

0.270 0.398 0.53 0.680 0.828

Mean 0.266 0.396 0.533 0.681 0.832

S.D. 0.003 0.002 0.002 0.002 0.004

% RSD 1.089 0.462 0.440 0.327 0.521

Table 6.3.1.6: Repeatability of data for Glipizide

Concentration Glipizide

20µg/ml

Absorbance 0.525

0.548

0.548

0.534

0.542

0.546

Mean 0.541

S.D. 0.009

% RSD 0.925

Acceptance Criteria


Conclusion:

Page 223






Intra-day precision was determined by analysing Glipizide for six times in the same

day at 276nm.


276nm.

Table 6.3.1.7: Intra-day precision data for Glipizide at 276nm

Conc. (µg/ml) Intra-day (n=6) % RSD

15 0.386 ± 0.003 0.761

20 0.549 ± 0.009 1.589

25 0.685 ± 0.003 0.365

Table 6.3.1.8: Inter-day precision data for Glipizide at 276nm

Conc. (µg/ml) Inter-day (n=6) % RSD

15 0.381 ± 0.004 1.103

20 0.546 ± 0.008 1.399

25 0.686 ± 0.004 0.573

Acceptance Criteria


Overall % RSD of method precision and intermediate precision should not be more

than 2.0%.

Page 224

Conclusion:

The results obtained were well within the acceptance criteria. The method can there

fore be termed as precise and rugged.

REPRODUCIBILITY

Procedure

The absorbance readings of solutions(20µg/ml) were measured at different laboratory

using another spectrophotometer by another analyst and the value obtained were

evaluated to verify their reproducibility.


Instrument 1 (n=6) Instrument 2 (n=6)

0.538 ± 0.013

%RSD = 1.29

0.544 ± 0.007

%RSD = 0.733

Acceptance criteria


Conclusion


termed as reproducible.

ROBUSTNESS

Procedure


in the wavelength and the effects of changes were recorded in Table 6.3.10.

Page 225





Acceptance criteria


2.0%.

Conclusion


SOLUTION STABILITY

Procedure





Time Absorbance of

standard(20µg/ml)

at 276nm

Absorbance of

sample(20µg/ml)

at 276nm

% Assay

Standard sample

9.00a.m 0.543 0.544 100.318 100.494

2.00p.m 0.542 0.544 100.141 100.494

6.00p.m 0.541 0.543 99.964 100.318

9.00a.m 0.541 0.542 99.964 100.141

Acceptance criteria

Page 226



Conclusion






Procedure

System suitability was performed by taking absorbance of standard solution(20µg/ml)

six times and calculating its average and % RSD value. System suitability test was

performed at the start of study for each parameter.

Table 6.3.1.12 System Suitability Test

Sr. no. Absorbance Average % RSD

1 0.533

0.544

0.73

2 0.546

3 0.542

4 0.542

5 0.555

6 0.548

Acceptance criteria


Conclusion


for use.


Page 227

Table 6.3.1.13: Summary of Validation Parameter of Spectrophotometry



2 Recovery % 98.366-98.955


4 Precision (%CV)

Intra-day

Inter-day

0.365-1.589

0.573-1.399

5 LOD (µg/ml) 2.895

6 LOQ (µg/ml) 8.775







1 20 100.09 1.366

Page 228

6.3.2 RP-HPLC METHOD DEVELOPMENT FOR GLIPIZIDE IN ITS SOLID

DOSAGE FORMS





1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000






HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A


Glipizide: Reddy,s Laboratories,Hyderabad, India.


Page 229



Potassium dihydrogen phosphate (AR Grade)-Rankem(India) Ltd., Mumbai

Triethyl amine (AR Grade) -Finar



Amaryl (Aventis Pharma Ltd),Maharashtra.

Tablet 5mg


6.3.2.3.1 Detection of Wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper selection of

detection wavelength is the one that gives good response for the drugs that are to be

detected. In the present study drug solution of 100µg/ml was, therefore, prepared in solvent

mixtures of Buffer (pH 7.5) : ACN (40:60) and 0.1%TEA. This drug solution was then

scanned in the UV region of 200-400 nm and the spectrum was recorded.(Fig.no.6.3.2.1)

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

0

100

200

300

400

500

600

700mAU

231.9

0

208.1

9

242.1

8

Figure 6.3.2.1: UV spectrum of Glipizide


Proper selection of the HPLC condition depends upon the nature of the sample (ionic or

ionizable or neutral molecule), its molecular weight and solubility. The drug selected for the

Page 230

present study is polar in nature and hence either reversed phase or ion-exchange or ion-pair

chromatography can be used. Reverse phase HPLC was selected for initial separation

because of its simplicity.

To optimize the chromatographic conditions, the effect of chromatographic variables such

as mobile phase pH, flow rate and solvent ratio were studied. The resulting chromatograms

were recorded and the chromatographic parameters such as capacity factor, asymmetric

factor and column efficiency were calculated. The conditions that gave the best resolution,

symmetry and capacity factor were selected for analysis.

6.3.2.4 Estimation of Glipizide by RP-HPLC method


300 ml of water and 700 ml of methanol were mixed and 0.1%TEA was added and adjusted

pH 7.5 of the solution with orthophosphoric acid (1%) and filtered through 0.45µ filter

paper, sonicated for 10 minutes to degas and used as mobile phase. Mobile phase was used

as diluent.


Glipizide Standard stock solution (1000 ppm)

Accurately weighed Glipizide standard (100 mg) was transferred to a 100 ml volumetric

flask and dissolved in about 10 ml diluent, then diluted to mark to obtain standard stock

solution (1000µg/ml). An aliquot of the solution (5.0 ml) was transferred to a 50.0 ml

volumetric flask and diluted to mark with diluents to obtain working standard solution

(100µg/ml).

6.3.2.4.3 Calibration curve for Glipizide(50-150µg/ml)

Appropriate volume of aliquots from standard Glipizide stock solution was transferred to

different volumetric flasks of 10ml capacity. The volume was adjusted to the mark with the

diluent to obtain the concentration of 50, 75, 100, 125 and 150µg/ml.Calibration curve of

each solution against the diluent was recorded at 232nm and the graph of absorbance v/s

Page 231

concentration was plotted. The straight line equation was determined from calibration

curve.(Fig.no.6.3.2.6)


Twenty Glipizide tablets were taken and crushed to make powder. The accurately weighed

powder equivalent to 100 mg of Glipizide was transferred into 100 ml volumetric flask and

dissolved in about 10 ml diluent by sonication for 25 minute and then volume was made up

to 100 ml with diluent to obtain sample stock solution (1000µg/ml). An aliquot of the

sample stock solution (5.0ml) was transferred to a 50ml volumetric flask and diluted to

mark with mobile phase to obtain working sample solution (100ug/ml).

6.3.2.4.6 Estimation of Glipizide in its solid dosage form

The prepared sample solution(20µl) was chromatographed for 10 minutes using mobile

phase.From the peak area obtained in the chromatogram, the amount of the drug was

calculated.


Figure 6.3.2.2: Chromatogram of Glipizide using water:ACN (50:50)

Page 232

Figure 6.3.2.3: Chromatogram of Glipizide using water:methanol (40:60)

Figure 6.3.2.4: Chromatogram of Glipizide using water:methanol:TEA (pH 7.5with

phosphoric acid) (40:60:0.1)

Figure 6.3.2.5: Chromatogram of Optimized chromatographic condition for Glipizide

Page 233

Table 6.3.2.2: Optimized chromatographic conditions for Glipizide


1 Mobile Phase Water:Methanol:TEA(30:70:0.1)(pH

7.5 adjusted withorthophosphoric acid

1%)





5 Volume of Injection (µl) 20µl

6 Detection wavelength

(nm)

242nm




LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 7.5 to 22.5µg/ml with respect to

the test concentration. A standard stock solution was prepared and further diluted to attain

concentration of about 7.5, 11.25, 15, 18.75 and 22.5µg/ml of sample concentration. Each

standard preparation was injected six replicates. The mean area at each level was calculated

and a graph of mean area v/s concentration (%) was plotted. The correlation co-efficient (r),

y-intercept and slope of regression line were calculated and recorded in Table 6.3.2.4.

Page 234

Table 6.3.2.3: Result of calibration readings for Glipizide

Concentrations

(µg/ml)

Glipizide

Area


7.5 1613.076 ± 8.646 0.536

11.25 2424.717 ± 14.969 0.617

15 3234.34 ± 22.641 0.700

18.75 4056.142 ± 30.159 0.744

22.5 4840.861 ± 37.919 0.783

Figure 6.3.2.6: Calibration curve of Glipizide at 242nm

Table 6.3.2.4: Statistical data for Glipizide by RP-HPLC

Parameters Glipizide

Linear Range(µg/ml) 7.5-22.5

Slope 215.65

Intercept 0.9707


Acceptance criteria


Page 235

Conclusion:

The correlation coefficient value was found to be 1.0 for Glipizide.

The areas obtained were directly proportional to the concentration of analyte in the sample.

The method can, therefore be termed as linear in the range considered. Based on the

linearity results, the working range of the method could be established as 7.5-22.5µg/ml of

working concentration.

ACCURACY

Procedure

The accuracy of the analytical method for assay of Glipizide was established at three levels

in triplicate,viz. 80%, 100%, and 120% of the test concentration. Standard was prepared as

per method and sample preparations were done by mixing known amount of Glipizide

working standard with placebo. Amount found, % Recovery and Mean Recovery was

calculated at each level and recorded in Table 6.3.2.5.

Table 6.3.2.5:Data derived from Accuracy experiment

Level

%

Sets Area Amount

Added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean

% recovery

% RSD

80 1 2577.615 0.25 11.957 99.644

100.068

0.483 80 2 2602.188 0.25 12.071 100.594

80 3 2585.986 0.25 11.996 99.967

100 1 3206.614 0.375 14.874 99.160

100.161

1.053 100 2 3274.58 0.375 15.189 101.261

100 3 3235.774 0.375 15.009 100.062

120 1 3940.953 0.5 18.279 101.552

100.333

1.055 120 2 3873.066 0.5 17.964 99.803

120 3 3866.916 0.5 17.936 99.644

Page 236

Acceptance Criteria

% Recovery (individual) and mean % Recovery at each level should be within 98.0-102.0

with % RSD not be more than 2.0.


established acceptance criteria.Hence, the method can be termed accurate in the considering

range.

PRECISION


Procedure

Method precision was established by taking five standard and sample preparations under

same conditions. Individual assay value, mean assay value and % RSD were calculated and

recorded in Table 6.3.2.6 and Table 6.3.2.7.

Table6.3.2.6: Determination of Method Precision (Repeatability)

Conc. 50(µg/ml) 75(µg/ml) 100(µg/ml) 125(µg/ml) 150(µg/ml)

Area 1611.678 2427.954 3229.775 4054.002 4835.944

1605.212 2408.565 3258.923 4009.451 4879.497

1629.429 2447.419 3199.245 4102.854 4778.173

1614.852 2432.835 3236.263 4062.111 4845.601

1608.425 2423.099 3223.365 4045.905 4826.267

1608.86 2408.432 3258.468 4062.526 4879.686

Mean 1613.076 2424.717 3234.34 4056.142 4840.861

S.D. 8.646 14.969 22.641 30.159 37.919

%RSD 0.536 0.617 0.700 0.744 0.783

n=6 determinations

Page 237

Table 6.3.2.7: Repeatability of sample application data for Glipizide

Concentration Glipizide

100µg/ml

Area 3224.238

3258.865

3266.245

3234.642

3223.468

3260.424

Mean 3244.647

S.D. 19.405

% RSD 0.598

Acceptance Criteria


Conclusion:

The results obtained were well within the acceptance criteria. The method could therefore be

termed as precise.


Variation of results within the same day (Intra-day) and variation of results between days

(Inter-day) were analysed.

Page 238


Intra-day Inter-day

Conc.(µg/ml) 50 100 150 50 100 150

Area 1622.951 3255.685 4884.346 1634.248 3200.769 4816.604

1600.385 3199.245 4773.33 1592.315 3265.359 4918.494

1608.425 3223.365 4826.267 1618.106 3249.163 4845.601

Mean 1610.587 3226.098 4827.981 1614.89 3238.43 4860.233

S.D. 11.437 28.319 55.528 21.151 33.606 52.497

% RSD 0.710 0.878 1.150 1.309 1.038 1.080

Acceptance Criteria


Overall % RSD of method precision and intermediate precision should not more than 2.0%.

Conclusion:

The results obtained were well within the acceptance criteria. The method can therefore be

termed as precise and rugged.

REPRODUCIBILITY

Procedure

The Area readings were measured at different laboratory using another HPLC instrument by

another analyst and the value obtained were evaluated to verify their reproducibility.


Instrument 1

Area ± S.D (n=6)

Instrument 2

Area ± S.D (n=6)

3240.25 ± 20.947

%RSD = 0.646

3230.385± 17.301

%RSD = 0.535

Page 239

Acceptance criteria

% RSD for six preparation for Reproducibility study should not be more than 2.0%.

Conclusion



ROBUSTNESS

Procedure

The robustness of the method was established by making deliberate minor variations in the

following method parameters.


2. Flow rate

3. pH of buffer

The effect of changes observed on system suitability parameters were recorded in Table

6.3.2.10.

Table 6.3.2.10: Data derived from Robustness study of Glipizide


M.P.(water:MeOH:TEA)28:72:0.01 3220.693± 23.550 0.731

M.P.( water:MeOH:TEA)32:68:0.01 3231.509 ± 39.726 1.229

Flow rate 1.2ml/min 3054 ± 22.759 0.754

Flow rate 0.8ml/min 3383.897 ± 23.608 0.698

pH of buffer 7.7 3213.701 ± 33.887 1.054

pH of buffer 7.3 3225.537±35.518 1.101

Page 240



Note

Theoretical plate and asymmetry values are from the first injection of the system suitability

set.


Acceptance criteria



The % RSD for six replicate injection for the analyte peak should not be more than 2%.

Conclusion

The system suitability parameter and absolute difference between area obtained from normal

condition and varied conditions were well within acceptance criteria, hence method can be

termed as robust.

SOLUTION STABILITY

Procedure

The standard and sample solutions were prepared as per method and analysed at regular

intervals. Results were recorded in Table 6.3.2.11.

Page 241


Time Areaof GLP(standard)

(100µg/ml)

Area of GLM (Sample)

(100µg/ml)

% Assay

standard sample

9.00a.m 3251.585 3250.246 100.550 100.509

2.00p.m 3234.858 3223.242 100.033 99.674

6.00p.m 3231.923 3220.326 99.942 99.584

9.00a.m 3216.029 3210.988 99.451 99.295

Acceptance criteria

The difference in the assay value obtained at different time intervals should not be more

than 2.0% from the initial value.

Conclusion

The solution stability was checked for the sample preparation and standard preparation for

12 hours. The results obtained are well within the acceptance criteria. Therefore, the

standard and sample preparations were stable in solution form for 12 hours at room

temperature.


Procedure

System suitability was performed by taking absorbance of standard solution six times and

calculating its average and % RSD value. System suitability test was performed at the start

of study for each parameter. The value of system suitability results obtained were recorded

in Table 6.3.2.12.

Page 242


System Suitability Parameter Glipizide




% RSD (n=6) 0.36

Note



Acceptance criteria


Conclusion

The system suitability parameters are well within acceptance criteria. Therefore the system

and chromatographic conditions are suitable for use.

Page 243


Table 6.3.2.13: Summary of validation parameters of Glipizide by RP-HPLC method


1. Linearity Range (μg/ml) 7.5– 22.5

2. Correlation co efficient (r2) 1

3. Accuracy(%Recovery) (n=3) 100.068 to100.333

4.

Precision (%CV)

Inter-day precision

Intra-day precision

1.08-1.309

0.710-1.150





9 Solution stability Stablefor24hrs

6.3.2.7 Assay result of marketed formulation

Table 6.3.2.14: Assay result of marketed formulation


1 15 99.84 0.62

Page 244

6.4 GLIMEPIRIDE


ESTIMATION OF GLIMEPIRIDE IN ITS SOLID DOSAGE FORM



Table: 6.4.1.1 List of the instruments used in the UV-Spectrophotometry


1 UV 1)Shimadzu

2)Perkin Elmer


2)Lambda 19





Scientific

Branstead D3750


� UV- visible double beam spectrophotometer with matched quartz cells

(1cm).

1) Make: SHIMADZU



Slit Width 2 nm






Noise Level 0.002 Abs

Page 245

2)Make: Perkin Elmer, USA,

Model: Lambda 19

� pH meter

Make: Thermo Orion

Model: 420 A+


Model: AX205



� Sonicator

Model: Branson 2510


0-60 Sonic/minute

� Milli-Q water



Hollow fiber filter

Gamma Irradiated

Pore size: 0.2 µm



Page 246

Make: Millipore




6.4.1.1.2Materials

Glimepiride: Hetero Labs Ltd, Andhra Pradesh, India

Methanol (AR Grade) E.Merck Ltd., Mumbai, India



Tablet 2mg


6.4.1.3.1 Selection of Solvent for Glimepiride.

Glimepiride is freely soluble in solvents like dichloromethane and methanol .

Glimepiride is practically insoluble in water. The spectrum of Glimepiride was

scanned in methanol. Owing to the solubility of Glimepiride in the methanol and also

there was no shift in absorbance maxima, methanol was selected as solvent.

6.4.1.3.2 Selection of analytical wavelength for Glimepiride.

The solution of Glimepiride was prepared in methanol at a concentration of 6 µg /ml.

It was scanned in the wavelength range of 200-400 nm. Maximum absorbance was

obtained at 228 nm. This analytical wavelength was selected for determination of

Glimepiride. The standard solutions of 10-18µg/ml were prepared and spectrums of

these concentrations were obtained with n = 5. The overlain spectrum of Glimepiride

at different concentration was recorded.(Fig.no.6.4.1.1)


Glimepiride standard stock solution (100µg/ml)

Page 247

Accurately weighed powder of Glimepiride (100 mg) was transferred to a separate

100 ml volumetric flask and dissolved and diluted to the mark with methanol. Further

10 ml was taken and diluted to 100 ml with same solvent to give a standard stock

solution containing 100µg/ml Glimepiride.

6.4.1.3.3 Calibration Curve for Glimepiride (10-18µg/ml)



with methanol to obtain concentration of 10, 12, 14, 16 and 18µg/ml. The curve of

solution against methanol as a blank was recorded. Absorbance was measured at

228nm against methanol as a blank and the plot of absorbance vs. concentration was

plotted. The straight-line equation was determined.(Fig.no.6.4.1.2)


Twenty Glimepiride tablets were weighed and powdered. The tablet powder

equivalent to 20 mg of Glimepiride transferred in to a 100 ml volumetric flask.

Methanol (100 ml) was added to it and sonicated for 20 min. The solution was filtered

through whatman filter paper No. 41 and the volume was adjusted up to the mark with

methanol to produce sample solution containing Glimepiride 200 µg/ml. 10 ml of


ml to contain Glimepiride 20 µg/ml (Stock solution A).

From the Stock solution A, 7 ml was transferred to volumetric flask of 10 ml capacity.

Volume was made up to the mark with methanol to give a solution containing

14µg/ml Glimepiride (Solution 1). This solution was used for the estimation of

Glimepiride.

6.4.1.3.4 Estimation of Glimepiride by UV Spectroscopic Method

The solution 1 was measured at 228 nm for quantification of Glimepiride. The amount

of Glimepiride present in the sample solution was determined. From the absorbance

obtained in the spectrum, the amount of drug was calculated.

Page 248

6.4.1.3.6 Method Validation, Results and Discussion


LINEARITY AND RANGE

Procedure



attain concentration of about 10, 12, 14, 16 & 18 µg/ml of sample concentration. Each

standard preparation was recorded in six replicates. The mean absorbance at each

level was calculated and a graph of mean absorbance v/s concentration (%) was



Table 6.4.1.2: Result of calibration readings at 228nm for Glimepiride

Concentration

(µg/ml)

Absorbance at 228nm

Mean ± S.D. (n=6)

% RSD

10 0.457 ± 0.008 1.654

12 0.538 ± 0.009 1.807

14 0.617 ± 0.009 1.422

16 0.694 ± 0.001 0.204

18 0.786 ± 0.009 1.235

Page 249

Figure 6.4.1.1: Overlain Spectrum of Glimepiride

Figure 6.4.1.2: Calibration Curve of Glimepiride at 228 nm.

Table 6.4.1.3: Statistical data for Glimepiride by Spectrophotometry method

Parameter Glimepiride (at 228nm)


Slope 0.0407

Intercept 0.0396


Acceptance criteria

Page 250


range.

Conclusion:

The correlation coefficient value was 0.9972 for Glimepiride.





ACCURACY

Procedure




Glimepiride working standard with placebo. Amount found, % Recovery and mean

Recovery was calculated at each level and recorded in Table 6.6.

Table 6.4.1.4 Data derived from Accuracy Experiment

Level

%

Sets

Absorbance

Drug

Added

(µg/ml)

Amount

recovered

(µg/ml)

%Recovery

Mean %

Recovery

%

RSD

50 1 0.332 7 7.01 100.105

100.221

0.459 50 2 0.331 7 6.982 99.756

50 3 0.334 7 7.056 100.803

100 1 0.621 14 14.073 100.524

100.408

0.336 100 2 0.618 14 14 100.000

100 3 0.622 14 14.098 101.362

150 1 0.914 21 21.237 101.129

101.362

0.218 150 2 0.916 21 21.286 101.362

150 3 0.918 21 21.335 101.595

Page 251

Acceptance Criteria



Conclusion:


acceptance criteria. Hence, the method can be termed accurate in the considered

range.

PRECISION


Procedure




Table 6.4.1.5: Repeatability (Method precision) data for Glimepiride standard at

228nm


Absorbance 0.466 0.548 0.618 0.695 0.797

0.444 0.536 0.600 0.696 0.771

0.456 0.528 0.620 0.694 0.782

0.458 0.543 0.624 0.692 0.783

0.454 0.548 0.623 0.694 0.796

0.462 0.526 0.616 0.693 0.786

Mean 0.457 0.538 0.617 0.694 0.786

S.D 0.008 0.013 0.004 0.001 0.009

% RSD 1.654 1.59 1.506 0.203 1.23

Page 252

Table 6.4.1.6: Repeatability of sample application data for Glimepiride

Concentration Glimepiride

14µg/ml

Absorbance 0.618

0.624

0.622

0.618

0.634

0.632

Mean 0.625

S.D. 0.007

% RSD 1.103

Acceptance Criteria


Conclusion:






Intra-day precision was determined by analysing Glimepiride for six times in the same

day at 228nm.


228nm.

Page 253

Table 6.4.1.7: Intra-day precision data for Glimepiride at 228nm

Concentration

(µg/ml)

Intra-day

(n=6)

% RSD

12 0.543 ± 0.005 0.927

14 0.647 ± 0.06 0.861

16 0.694 ± 0.005 0.711

Table 6.4.1.8: Inter-day precision data for Glimepiride at 228nm

Concentration

(µg/ml)

Inter-day

(n=6)

% RSD

12 0.544 ± 0.002 0.367

14 0.624 ± 0.007 1.136

16 0.693 ± 0.005 0.727

Acceptance Criteria



2.0%.

Conclusion:



REPRODUCIBILITY

Procedure

The absorbance readings were measured at different laboratory using another

spectrophotometer by another analyst and the value obtained were evaluated to verify

their reproducibility.

Page 254


Instrument 1

(n=6)

Instrument 2

(n=6)

0.648 ± 0.006

%RSD = 0.58

0.642 ± 0.004

%RSD = 0.42

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure






Wavelength (228nm) 0.246 0.24(-5nm)0.46(+5nm)

Acceptance criteria


2.0%.

Conclusion

Results were within acceptance criteria. Hence, method can be termed as Robust.

Page 255

SOLUTION STABILITY

Procedure





Time Absorbanceof

standard(14µg/ml) at

228nm

Absorbanceof

sample(14µg/ml) at

228nm

% Assay

standard Sample

9.00a.m 0.628 0.626 101.746 101.397

2.00p.m 0.624 0.628 101.048 101.746

6.00p.m 0.614 0.622 99.301 100.699

9.00a.m 0.614 0.618 99.301 100

Acceptance criteria



Conclusion






Procedure

Page 256





Sr. no. Absorbance Average % RSD

1 0.616

0.619

0.705

2 0.614

3 0.618

4 0.622

5 0.626

6 0.621

Acceptance criteria


Conclusion


for use.

6.4.1.3.7 Summary of validation Parameters

Table 6.4.1.13: Summary of Validation Parameter of Spectrophotometry



2 Recovery % 100.221-100.407


4 Precision (CV)

Intra-day (n=6)

Inter-day (n=6)

0.711-0.927

0.368-1.136

5 LOD (µg/ml) 0.727

Page 257

6 LOQ (µg/ml) 2.202




6.4.1.3.8 Assay Results of Marketed Formulation



1 14 100.264 0.533

Page 259

6.4.2 RP-HPLC METHOD DEVELOPMENT FOR GLIMEPIRIDE IN ITS

SOLID DOSAGE FORM.





1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000





scientific

Branstead D3750


HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A


Glimepiride: Hetero Labs Ltd, Andhra Pradesh, India



Page 260






Tablet 1mg

Tablet 2mg


Selection of Detection Wavelength


selection of detection wavelength.An ideal wavelength is the one that gives good

response for the drugs that are to be detected. In the present study drug solution of

100µg/ml was, therefore, prepared in solvent mixtures of Buffer (pH 4.5) and ACN

(40:60). This drug solution was then scanned in the UV region of 200-400 nm and the

spectrum was recorded.(Fig. no.6.4.2.1)

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

0

250

500

750

1000

1250

1500

mAU

339.9

0

308.8

2

232.6

1

336.8

8

Figure 6.4.2.1: UV spectrum of Glimepiride

6.4.2.4 Selection of chromatographic conditions






Page 261


such as mobile phase pH, flow rate, and solvent ratio were studied. The resulting




6.4.2.5 Estimation of Glimepiride by RP-HPLC method

6.4.2.5.1 Preparation of 0.02M buffer (pH 4.5)

Accurately weighed 2.72gm of potassium dihydrogen phosphate was dissolved in

1000 ml of water and adjusted pH of the solution with orthophosphoric acid (5%) .


400 ml of buffer(pH 4.5) and 600 ml of Acetonitrile were mixed and filtered through




Glimepiride Standard stock solution (1000µg/ml)

Accurately weighed Glimepiride standard (100 mg) was transferred to a 100 ml


standard stock solution (1000µg/ml). An aliquot of the standard stock solution (5.0 ml)

was transferred to a 50.0 ml volumetric flask and diluted to mark with diluent to

obtain working standard solution (100µg/ml).

6.4.2.5.4 Calibration curve for Glimepiride(50-150µg/ml)

Appropriate volume of aliquots from standard Glimepiride stock solution was


to the mark with the diluent to obtain the concentration of 50, 75, 100, 125, 150µg/ml.

Calibration curve of each solution against the diluent was recorded at 232nm and the


determined from calibration curve.(Fig.no.6.4.2.6)

Page 262


Twenty Glimepiride tablets were taken and crushed to make powder. The accurately

weighed powder equivalent to 100 mg of Glimepiride was transferred into 100 ml


then volume was made up to 100 ml with diluent to obtain sample stock solution

(1000µg/ml). An aliquot of the sample stock solution (5.0ml) was transferred to a

50ml volumetric flask and diluted to mark with mobile phase to obtain working

sample solution (100ug/ml).

6.4.2.5.6 Estimation of Glimepiride in its solid dosage form





Figure 6.4.2.2: Chromatogram of Glimepiride using phosphate buffer:ACN

(50:50)

Figure 6.4.2.3: Chromatogram of Glimepiride using water:ACN (50:50)

Page 263

Figure 6.4.2.4: Chromatogram of Glimepiride using phosphate buffer: ACN (pH

5.0) (50:50)


Glimepiride

Table 6.4.2.2: Optimized chromatographic conditions for Glimepiride



phosphate(pH 4.5 adjusted with ortho

phosphoric acid):Acetonitrile (40:60)




4 Flow rate(ml/min) 1.0

5 Volume of Injection (µl) 20

6 Detection wavelength (nm) 232



Page 264


LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 50 to 150µg/ml with respect

to the test concentration. A standard stock solution was prepared and further diluted to

attain concentration of about 50, 75, 100, 125, and 150µg/ml of sample concentration.




and recorded in Table 6.4.2.4.

Table 6.4.2.3: Result of calibration readings for Glimepiride

Concentrations

(µg/ml)

Glimepiride

Area


50 1157.742 ± 21.011 1.815

75 1644.859 ± 2.189 0.133

100 2304.232 ± 42.272 1.835

125 2751.369 ± 1.112 0.040

150 3413.705 ± 14.334 0.419

Figure 6.4.2.6: Calibration curve of Glimepiride at 232nm

Page 265

Table 6.4.2.4: Statistical data for Glimepiride by RP-HPLC

Parameters Glimepiride


Slope 22.474

Intercept 7.0071


Acceptance criteria


Conclusion:

The correlation coefficient value was found to be 0.9965 for Glimepiride.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Glimepiride was established at



amount of Glimepiride working standard with placebo. Amount found, % Recovery

and Mean Recovery was calculated at each level and recorded in Table 6.4.2.5.

Page 266


Level

%

Sets Area Amount

added

(µg/ml)

Amount

recovered

(µg/ml)

%

Recovery

Mean%

Recovery

% RSD

80 1 1818.205 80 80.591 100.738

100.737

0.673 80 2 1830.402 80 81.134 101.417

80 3 1805.923 80 80.044 100.055

100 1 2258.525 100 100.183 100.183

100.083

1.056 100 2 2278.916 100 101.091 101.091

100 3 2231.402 100 98.976 98.976

120 1 2713.723 120 120.437 100.365

100.395

1.055 120 2 2743.559 120 121.765 101.471

120 3 2686.303 120 119.217 99.348

Acceptance Criteria





considering range.

PRECISION


Procedure




Page 267


Concentration 50(µg/ml) 75(µg/ml) 100(µg/ml) 125(µg/ml) 150(µg/ml)

Area 1149.568 1643.245 2343.779 2750.126 3406.432

1154.234 1645.432 2343.779 2750.654 3406.765

1178.231 1648.234 2332.15 2753.145 3408.412

1188.069 1642.567 2275.123 2751.89 3409.431

1141.57 1646.321 2241.345 2751.765 3442.876

1134.781 1643.355 2289.217 2750.635 3408.315

Mean 1157.742 1644.859 2304.232 2751.369 3413.705

S.D. 21.011 2.189 42.272 1.111 14.334

%RSD 1.815 0.133 1.835 0.040 0.419

n=6 determinations

Table 6.4.2.7: Repeatability of sample application data for Glimepiride

Concentration Glimepiride

100µg/ml

Area 2343.779

2322.564

2241.654

2254.876

2241.654

2249.298

Mean 2275.638

S.D. 45.34297

% RSD 1.99

Acceptance Criteria


Conclusion:



Page 268





Intraday Inter-day


Area 1137.046 2301.82 3445.822 1132.512 2273.218 3367.413

1149.568 2332.217 3476.444 1146.236 2295.922 3399.439

1122.304 2274.203 3406.232 1118.377 2242.535 3314.261

Mean 1136.306 2302.746 3442.832 1132.375 2270.558 3360.371

S.D. 13.647 29.018 35.201 13.930 26.792 43.02

%RSD 1.201 1.260 1.022 1.230 1.180 1.280

Acceptance Criteria



2.0%.

Conclusion:



REPRODUCIBILITY

Procedure



reproducibility.

Page 269


Instrument 1

Area ± S.D (n=6)

Instrument 2

Area ± S.D (n=6)

2272.565 ± 43.154

%RSD = 0.48

2264.396 ± 42.997

%RSD = 0.76

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure



1.Organic phase ratio

2.Flow rate

3.pH of buffer


table 6.4.2.10.

Table 6.4.2.10: Data derived from Robustness study of Glimepiride


M.P(buffer:ACN)42:58 2242.495 ± 32.692 1.458

M.P(buffer:ACN)38:62 2262.694 ± 23.075 1.019

Flow rate +0.2ml/min 1814.994±19.604 1.080

Flow rate -0.2ml/min 2720.231±36.579 1.345

Page 270

pH of buffer 4.7 2266.576±36.344 1.604

pH of buffer 4.3 2272.292±27.828 1.225



Note


suitability set.


Acceptance criteria




2%.

Conclusion




SOLUTION STABILITY

Procedure



Page 271


Time Area of

GLMstandard

(100µg/ml)

Area of GLM

Sample

(100µg/ml)

% Assay

standard sample

9.00a.m 2244.292 2234.058 99.549 99.095

2.00p.m 2234.670 2229.902 99.122 98.909

6.00p.m 2233.785 2231.836 99.082 98.995

9.00a.m 2230.893 2228.88 98.954 98.864

Acceptance criteria



Conclusion






Procedure





Page 272


SystemSuitability Parameter Glimepiride




% RSD (n=6) 0.2%

Note



Acceptance criteria


Conclusion



Page 273

6.4.3 Summary of validation parameters of RP-HPLC Method

Table 6.4.2.13: Summary of validation parameters of Glimepiride by RP-HPLC

method


1. Linearity Range (μg/ml) 50 – 150

2. Correlation coefficient (r2) 0.9965

3. Accuracy (%Recovery) (n=3) 100.083 to100.737

4.

Precision (%CV)

Inter-day precision

Intra-day precision

1.18-1.28

1.02-1.26






6.4.4 Assay results of marketed formulation

Table 6.4.2.14: Assay results of marketed formulation

Set Amount(µg/ml) %Assay %RSD (n=3)

1 100 99.76 0.65

2 100 98.11 0.84

Page 273

6.5 RP-HPLC METHOD DEVELOPMENT FOR METFORMIN

HYDROCHLORIDE IN ITS SOLID DOSAGE FORM.

6.5.1 Instruments and Materials

6.5.1.1 Instruments

Table 6.5.1: List of the instruments used in RP-HPLC


1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000





scientific

Branstead D3750


HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A

6.5.1.2 Materials:

Metformin HCl: Indica Laboratories Ltd, Gujarat, India




Page 274

6.5.2 Marketed Formulation:

Forson (Unison Pharmaceuticals Ltd),Gujarat.

Tablet 500mg

6.5.3 Development and Optimization of HPLC method

6.5.3.1 Selection of Detection Wavelength




prepared in solvent mixtures of water and ACN (25:75). This drug solution was then

scanned in the UV region of 200-400 nm and the spectrum was recorded.(Fig.

no.6.5.1)

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

250

500

750

1000

1250

1500

1750

2000mAU

242.9

9

Figure 6.5.1: Spectrum of Metformin HCl

6.5.4 Selection of chromatographic conditions











Page 275

6.5.4 Estimation of Metformin HCl by RP-HPLC method

6.5.4.1 Preparation of Mobile Phase

250 ml of water and 750 ml of Acetonitrile were mixed and filtered through 0.45µ

filter paper, sonicated for 10 minutes to degas and used as mobile phase. Mobile

phase was used as diluent.

6.5.4.2 Preparation of standard stock solution

Metformin HCl Standard stock solution (100 ppm)

Accurately weighed Metformin HCl standard (10 mg) was transferred to a 100 ml





6.5.4.3 Calibration curve for Metformin HCl(5-15µg/ml)

Appropriate volume of aliquots from standard Metformin HCl stock solution was


to the mark with the diluent to obtain the concentration of 5, 7.5, 10, 12.5 and

15µg/ml.Calibration curve of each solution against the diluent was recorded at 242nm

and the graph of absorbance v/s concentration was plotted. The straight line equation

was determined from calibration curve(Fig.no.6.5.7).

6.5.4.4 Sample Preparation

Twenty Metformin HCl tablets were taken and crushed to make powder. The

accurately weighed powder equivalent to 10 mg of Metformin HCl was transferred

into 100 ml volumetric flask and dissolved in about 10 ml diluent by sonication for 25

minute and then volume was made up to 100 ml with diluent to obtain sample stock

solution (100µg/ml). An aliquot of the solution (5.0ml) was transferred to a 50ml


solution (10µg/ml).

Page 276

6.5.4.5 Estimation of Metformin HCl in its solid dosage form




6.5.5 Method Validation, Results and Discussion

Figure 6.5.2: Chromatogram of Metformin HCl using water:methanol (60:40)


Page 277


Figure 6.5.5: Chromatogram of Metformin HCl using water:ACN (40:60)

Figure 6.5.6: Chromatogram of Optimized chromatographic condition for

Metformin HCl

Page 278

Table 6.5.2: Optimized chromatographic conditions for Metformin HCl


1 Mobile Phase Water:Acetonitrile (25:75)





5 Volume of

Injection (µl)

20µl

6 Detection

wavelength (nm)

242nm




LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 5 to 15µg/ml with respect to






and recorded in Table 6.5.3.

Page 279

Table 6.5.3: Result of calibration readings for Metformin HCl

Concentrations

(µg/ml)

Metformin HCl

Area


5 525.326 ± 3.445 0.656

7.5 785.295 ± 4.632 0.589

10 1049.081 ± 6.525 0.622

12.5 1318.545 ± 8.115 0.615

15 1573.412 ± 14.259 0.906

Figure 6.5.7: Calibration curve of Metformin HCl at 242nm

Table 6.5.4: Statistical data for Metformin HCl by RP-HPLC

Parameters Metformin HCl


Slope 105.18

Intercept 1.4386


Acceptance criteria


range.

Page 280

Conclusion:

The correlation coefficient value were found to be 0.9999 for Metformin HCl.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Metformin HCl was established at



amount of Metformin HCl working standard with placebo. Amount found, %

Recovery and mean recovery was calculated at each level and recorded in Table 6.5.5.

Table 6.5.5: Data derived from Accuracy experiment

Level

%

Sets Area Amount

added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean

%

recovery

% RSD

80 1 841.601 8 8.015 100.190

100.566

0.387 80 2 848.139 8 8.077 100.967

80 3 844.541 8 8.043 100.539

100 1 1040.74 10 9.909 99.085

100.192

1.159 100 2 1065.078 10 10.139 101.399

100 3 1051.328 10 10.009 100.092

120 1 1256.56 12 11.960 99.670

100.709

1.006 120 2 1282.099 12 12.203 101.694

120 3 1270.343 12 12.085 100.762

Acceptance Criteria



Page 281

Conclusion: The % Recovery at each level, Mean % Recovery and % RSD met the


considered range.

PRECISION


Procedure



calculated and recorded in Table 6.5.6 and Table 6.5.7.

Table 6.5.6: Determination of Method Precision (Repeatability)

Conc. 5(µg/ml) 7.5(µg/ml) 10(µg/ml) 12.5(µg/ml) 15(µg/ml)

Area 524.234 785.487 1049.884 1318.848 1588.353

526.757 793.356 1058.366 1305.708 1588.353

520.342 782.107 1038.337 1330.8 1563.115

525.512 787.058 1052.975 1321.476 1577.334

523.119 780.241 1047.782 1316.203 1571.053

526.452 780.162 1048.12 1318.675 1552.345

Mean 524.403 784.735 1049.244 1318.618 1573.425

S.D. 2.414 5.065 6.631 8.113 14.263

%RSD 0.460 0.645 0.632 0.615 0.906

n=6 determinations

Page 282

Table 6.5.7: Repeatability of sample application data for Metformin HCl

Concentration Metformin

HCl(100µg/ml)

Area 1045.032

1037.263

1023.654

1021.432

1037.21

1038.985

Mean 1033.929

S.D. 9.300

% RSD 0.899

Acceptance Criteria


Conclusion:






Page 283

Table 6.5.8:Precision data

Intra-day Inter-day

concentration 5(µg/ml) 10(µg/ml) 12(µg/ml) 5(µg/ml) 10(µg/ml) 12(µg/ml)

Area 529.231 1042.566 1588.366 517.636 1042.566 1561.573

516.022 1055.1 1557.221 524.831 1057.688 1592.846

523.459 1047.782 1571.053 525.551 1051.975 1577.254

Mean 522.904 1048.482 1572.213 522.672 1050.743 1577.224

S.D. 6.621 6.296 15.604 4.376 7.635 15.636

% RSD 1.266 0.601 0.993 0.837 0.726 0.991

Acceptance Criteria



2.0%.

Conclusion:



REPRODUCIBILITY

Procedure



reproducibility.

Table 6.5.9: Determination of Reproducibility

Instrument 1

Area ± S.D (n=6)

Instrument 2

Area ± S.D (n=6)

1045.226 ± 2.252 1040.426± 9.017

Page 284

%RSD = 0.215 %RSD = 0.867

Acceptance criteria

% RSD for six preparations for Reproducibility study should not be more than 2.0%.

Conclusion



ROBUSTNESS

Procedure


in the following method parameters


2. Flow rate


Table 6.5.10.

Table 6.5.10: Data derived from Robustness study of Metformin HCl


M.P(water:ACN)27:73 1046.204 ± 10.807 1.033

M.P(water:ACN)23:77 1050.075 ± 12.431 1.184

Flow rate 1.2ml/min 991.85 ± 11.502 1.159

Flow rate 0.8ml/min 1097.312 ± 2.493 0.227



Note


suitability set.


Page 285

Acceptance criteria




2%.

Conclusion




SOLUTION STABILITY

Procedure


regular intervals. Results were recorded in Table 6.5.11.

Table 6.5.11: Solution stability study

Time Areaof

METstandard(10µg/ml)

Areaof

METsample(10µg/ml)

% Assay

standard sample

9.00a.m 1048.114 1045.456 99.786 99.534

2.00p.m 1039.669 1024.672 98.983 97.557

6.00p.m 1032.136 1023.456 98.267 97.442

9.00a.m 1029.931 1022.124 98.057 97.315

Acceptance criteria



Page 286

Conclusion



Therefore, the standard and sample preparation were stable in solution form for 24



Procedure




were recorded in Table 6.5.12.

Table 6.5.12: System suitability test

System Suitability Parameter Metformin HCl


Theoritical plates (N) 3040.565


% RSD (n=6) 0.43

Note



Acceptance criteria


Conclusion



Page 287


Table 6.5.13: Summary of validation parameters of Metformin HCl by RP-

HPLC method


1. Linearity Range (μg/ml) 5– 15



4. Precision (%CV)

Inter-day precision

Intra-day precision

0.726-0.991

0.601-1.266






6.5.6 Assay result of marketed formulation

Table 6.5.14: Assay result of marketed formulation


1 10 99.98 0.56

Page 288

6.6 RP-HPLC METHOD DEVELOPMENT FOR REPAGLINIDE IN ITS

SOLID DOSAGE FORM.


6.6.1.1 Instruments



1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000





scientific

Branstead D3750


HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A

6.6.1.2 Materials:

Repaglinide: Restech Pharmaceutical Ltd, Gujarat, India

Page 289




Potassium dihydrogen phosphate (AR Grade)-Rankem(India) Ltd., Mumbai

Triethyl amine (AR Grade)- Finar



Eurepa(Torrent),Gujarat

Tablet 2mg






prepared in solvent mixtures of water: methanol:TEA(15:85:0.02). This drug solution


recorded.(Fig.no.6.6.1)

190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 nm

250

500

750

1000

1250

1500

1750

2000mAU

217.5

3

246.2

2

205.5

1

Figure 6.6.1: UV spectrum of Repaglinide

Page 290












6.6.4 Estimation of Repaglinide by RP-HPLC method


150 ml of water and 850 ml of methanol were mixed and 0.2ml of TEA was added

and then filtered through 0.45µ filter paper, sonicated for 10 minutes to degas and

used as mobile phase.Mobile phase was used as diluent.


Repaglinide Standard stock solution (200 ppm)

Accurately weighed Repaglinide standard (20 mg) was transferred to a 100 ml





6.6.4.3 Calibration curve for Repaglinide(10-30µg/ml)

Appropriate volume of aliquots from standard Repaglinide stock solution was


Page 291

to the mark with the diluent to obtain the concentration of 10, 15, 20, 25 and 30µg/ml.

Calibration curve of each solution against the diluent was recorded at 246 nm and the


determined from calibration curve.(Fig.no.6.6.9)


Twenty Repaglinide tablets were taken and crushed to make powder. The accurately

weighed powder equivalent to 20 mg of Repaglinide was transferred into 100 ml


then volume was made up to 100 ml with diluent to obtain sample stock solution



solution (20ug/ml).

6.6.4.5 Estimation of Repaglinide in its solid dosage form





Figure 6.6.2: Chromatogram of Repaglinide using water:methanol (50:50)

Page 292

Figure 6.6.3: Chromatogram of Repaglinide using water:methanol (35:65)

Figure 6.6.4: Chromatogram of Repaglinide using water: methanol (15:85)

Figure 6.6.5: Chromatogram of Repaglinide using buffer:methanol (35:65)

Page 293

Figure 6.6.6: Chromatogram of Repaglinide using water:methanol:TEA

(40:60:0.02)

Figure6.6.7:ChromatogramofRepaglinideusing water:methanol:TEA(30:70:0.02)

Figure 6.6.8: Optimised Chromatographic condition for Repaglinide

Page 294

Table 6.6.2: Optimized chromatographic conditions for Repaglinide


1 Mobile Phase Water:methanol:TEA(15:85:0.02)




4 Flowrate(ml/min) 1

5 Volume of

Injection (µl)

20µl

6 Detection

wavelength (nm)

246nm


8 Diluent Mobile phase


LINEARITY AND RANGE

Procedure

Linearity was determined at five levels over the range of 10 to 30 µg/ml with respect


attain concentration of about 10, 15, 20, 25, and 30 µg/ml of sample concentration.





Page 295

Table 6.6.3: Result of calibration readings for Repaglinide

Concentrations

(µg/ml)

Repaglinide

Area


10 956.030 ± 06.511 0.681

15 1431.707 ± 8.276 0.578

20 1911.232 ± 12.666 0.663

25 2417.354 ± 38.840 1.607

30 2865.931 ± 16.008 0.559

Figure 6.6.9: Calibration curve of Repaglinide

Table 6.6.4: Statistical data for Repaglinide by RP-HPLC

Parameters Repaglinide


Slope 96.109

Intercept 5.729


Acceptance criteria


range.

Page 296

Conclusion:

The correlation coefficient value were found to be 0.9997 for Repaglinide.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Repaglinide was established at



amount of Repaglinide working standard with placebo. Amount found, % Recovery

and mean Recovery was calculated at each level and recorded in Table 6.6.5.


Level

%

Sets Area Amount

Added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean

% Recovery

% RSD

80 1 1517.278 16 15.847 99.042

100.087

1.060 80 2 1549.786 16 16.185 101.155

80 3 1532.994 16 16.010 100.064

100 1 1936.806 20 20.212 101.059

100.038

1.556 100 2 1893.154 20 19.758 98.788

100 3 1921.564 20 20.053 100.266

120 1 2300.446 24 23.995 99.981

100.085

0.327 120 2 2311.281 24 24.108 100.451

120 3 2296.823 24 24.020 99.824

Page 297

Acceptance Criteria




established acceptance criteria. Hence, the method can be termed accurate in the

considered range.

PRECISION


Procedure





Concentration 10(µg/ml) 15(µg/ml) 20(µg/ml) 25(µg/ml) 30(µg/ml)

Area 961.632 1432.551 1922.432 2400.633 2863.772

964.342 1427.742 1891.465 2424.692 2892.321

956.371 1442.234 1931.593 2386.154 2843.012

956.211 1426.324 1912.223 2410.191 2869.462

953.234 1427.814 1922.724 2381.886 2751.765

952.128 1427.674 1911.724 2491.451 2750.635

Mean 957.319 1430.723 1915.360 2415.834 2863.369

S.D. 4.768 6.030 13.869 40.231 16.810

%RSD 0.498 0.422 0.724 1.665 0.587

n=6 determinations

Page 298

Table 6.6.7: Repeatability of sample application data for Repaglinide

Concentration Repaglinide

20µg/ml

Area 1845.032

1877.263

1873.654

1921.432

1937.21

1908.985

Mean 1893.929

Std. Dev. 34.456

% RSD 1.819

Acceptance Criteria


Conclusion:






Page 299

Table 6.6.8: Precision data

Intra-day Inter-day


Area 964.527 1921.554 2889.537 966.426 1931.033 2840.861

947.351 1896.597 2843.716 944.491 1898.498 2895.277

953.049 1908.111 2857.974 956.921 1915.724 2869.462

Mean 954.975 1908.754 2863.742 955.946 1915.085 2868.533

S.D. 8.748 12.491 23.459 10.999 16.276 27.219

% RSD 0.916 0.654 0.819 1.151 0.849 0.949

Acceptance Criteria



2.0%.

Conclusion:



REPRODUCIBILITY

Procedure


instrument by another analyst .

Page 300


Instrument 1

Area ± S.D(n=6)

Instrument 2

Area ± S.D(n=6)

1940.870 ± 5.128

%RSD = 0.264

1940.426 ± 9.018

%RSD = 0.465

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure




2. Flow rate

The effect of changes observed on system suitability parameters were recorded in Table 7.14.

Page 301

Table 6.6.10: Data derived from Robustness study of Repaglinide


M.P.(water:methanol:TEA)

17:83:0.02

1907.486 ± 16.502 0.865

M.P.(water:methanol:TEA)

13:87:0.02

1911.975 ± 21.005 1.099

Flow rate +0.2ml/min 1811.949 ± 17.708 0.977

Flow rate -0.2ml/min 2002.151 ± 16.480 0.823



Note


suitability set.


Acceptance criteria




2%.

Conclusion


normal condition and varied condition were well within acceptance criteria, hence


Page 302

SOLUTION STABILITY

Procedure




Time Area of REPA

standard(20µg/ml)

Area of REPA

sample(20µg/ml)

% Assay

standard sample

9.00a.m 1919.979 1915.456 100.183 99.948

2.00p.m 1916.381 1914.672 99.996 99.907

6.00p.m 1915.435 1913.386 99.947 99.841

9.00a.m 1913.625 1912.654 99.853 99.802

Acceptance criteria



Conclusion






Procedure

System suitability was performed by taking absorbance of standard solution five times


Page 303



Table 6.6.12: System suitability test parameter

System Suitability Parameter Repaglinide




% RSD (n=5) 0.32

Note

System suitability values are from the first injection of five replicates of standard.

% RSD is calculated from five replicate injections of standard.

Acceptance criteria


Conclusion



Page 304


Table 6.6.13: Summary of validation parameters of Repaglinide by RP-HPLC

method

Sr.No. Parameters Results

1 Linearity 10-30µg/ml



4.

Precision (%CV)

Inter-day precision

Intra-day precision

0.849-1.151

0.654-0.916









1 20 99.76 0.65

Page 305

6.7 RP-HPLC METHOD DEVELOPMENT FOR SAXAGLIPTIN

HYDROCHLORIDE IN ITS SOLID DOSAGE FORM.


6.7.1.1 Instruments



1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 200





scientific

Branstead D3750


HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A

6.7.1.2 Materials:

Saxagliptin HCl: Triveni Chemicals, Gujarat, India





Page 306

Orthophosphoric acid (AR Grade)- Rankem(India) Ltd.,Mumbai

6.7.2 Marketed Preparation:

Onglyza(Astra Zeneca),Karnataka.

Tablet 5mg









Figure 6.7.1: UV spectrum of Saxagliptin HCl












Page 307

6.7.4 Estimation of Saxagliptin HCl by RP-HPLC method

6.7.4.1 Preparation of 0.02M buffer (pH 4.5)


800ml of water and then made up the volume to 1000 ml with water and adjusted pH

of the solution with orthophosphoric acid (5%).






Saxagliptin HCl Standard stock solution (200 ppm)

Accurately weighed Saxagliptin HCl standard (20 mg) was transferred to a 100 ml





6.7.4.4 Calibration curve for Saxagliptin HCl(50-150µg/ml)

Appropriate volume of aliquots from standard Saxagliptin HCl stock solution was


to the mark with the diluent to obtain the concentration of 50, 75, 100, 125,

150µg/ml.Calibration curve of each solution against the diluent was recorded at

220nm and the graph of absorbance v/s concentration was plotted. The straight line

equation was determined from calibration curve.(Fig.no.2.7.11)


Twenty Saxagliptin HCl tablets were taken and crushed to make powder. The

accurately weighed powder equivalent to 20 mg of Saxagliptin HCl was transferred

into 100 ml volumetric flask and dissolved in about 10 ml diluent by sonication for 25

minute and then volume was made up to 10 ml with diluents to obtain sample stock

solution (200µg/ml).An aliquot of the sample stock solution (5.0 ml) was transferred

to a 10.0 ml volumetric flask and diluted to mark with diluents to obtain working

sample solution (100µg/ml).

Page 308

2.7.4.6 Estimation of Saxagliptin HCl in its solid dosage form





Figure 6.7.2: Chromatogram of Saxagliptin HCl using water:methanol(50:50)

Figure 6.7.3: Chromatogram of Saxagliptin HCl using water:methanol (30:70)

Figure 6.7.4: Chromatogram of Saxagliptin HCl using water:methanol: ACN

(30:40:30)

Page 309

Figure 6.7.5: Chromatogram of Saxagliptin HCl using water:ACN (50:50)

Figure 6.7.6: Chromatogram of Saxagliptin HCl using buffer:ACN (55:45)



Page 310


Figure 6.7.10: Optimized chromatographic condition for Saxagliptin HCl

Table 6.7.2: Optimized chromatographic conditions for Saxagliptin HCl



phosphate(pH 4.5 adjusted with

ortho phosphoricacid):Acetonitrile

(70:30)


3 Stationary Phase Hypersil BDS(250×4.6 mm id,

5µm particle size) column


5 Volume of Injection

(µl)

20µl

6 Detection

wavelength(nm)

220nm



Page 311


LINEARITY AND RANGE

Procedure








Table 6.7.3: Result of calibration readings for Saxagliptin HCl

Concentrations

(µg/ml)

Saxagliptin HCl

Area


50 2328.276 ± 16.715 0.718

75 3504.704 ± 30.100 0.859

100 4676.066 ± 42.059 0.899

125 5799.668 ± 110.392 1.903

150 6971.990 ± 63.646 0.913

Figure 6.7.11: Calibration curve of Saxagliptin HCl

Page 312

Table 6.7.4: Statistical data for Saxagliptin HCl by RP-HPLC

Parameter Saxagliptin HCl


Slope 46.342

Intercept 22.24


Acceptance criteria


range.

Conclusion:

The correlation coefficient value was found to be 0.9999 for Saxagliptin HCl.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Saxagliptin HCl was established at



amount of Saxagliptin HCl working standard with placebo. Amount found, %

Recovery and mean recovery was calculated at each level and recorded in Table 6.7.5.

Page 313


Acceptance Criteria





considered range.

PRECISION


Procedure




Level

%

Sets Area Amount

Added

(µg/ml)

Amount

Recovered

(ug/ml)

%

Recovery

Mean%

Recovery

% RSD

80 1 3733.157 80 80.076 100.096

99.815

1.271 80 2 3764.007 80 80.742 100.928

80 3 3671.041 80 78.736 98.420

100 1 4718.009 100 101.329 101.329

100.719

1.221 100 2 4623.83 100 99.296 99.296

100 3 4727.463 100 101.533 101.533

120 1 5599.811 120 120.356 100.297

99.991

0.834 120 2 5618.454 120 120.759 100.633

120 3 5530.105 120 118.853 99.044

Page 314



Area 2332.327 3576.234 4684.365 5627.595 6986.015

2329.125 3489.876 4708.679 5775.149 6916.162

2342.876 3486.643 4678.234 5932.966 7062.969

2346.23 3459.864 4685.684 5788.304 7010.441

2349.124 3528.648 4768.886 5812.707 6898.330

2321.426 3586.866 4624.422 5876.529 6951.025

Mean 2336.851 3521.355 4691.711 5802.208 6970.823

S.D. 10.890 51.640 46.997 104.064 61.524

%RSD 0.466 1.466 1.00 1.794 0.882

n=6 determinations

Table 6.7.7: Repeatability of sample application data for Saxagliptin HCl

Concentration Saxagliptin

HCl(100µg/ml)

Area 4645.053

4737.453

4614.447

4764.889

4668.865

4766.342

Mean 4699.508

S.D. 65.301

% RSD 1.389

Acceptance Criteria


Conclusion:

Page 315







Intra-day Inter-day


Area 2321.697 4648.078 6958.072 2326.355 4629.409 6972.055

2300.753 4601.491 6888.369 2300.77 4657.388 6916.21

2352.016 4718.078 7048.846 2359.018 4704.051 7048.911

Mean 2324.822 4655.882 6965.095 2328.714 4663.616 6979.058

S.D. 25.773 58.684 80.469 29.196 37.709 66.627

% RSD 1.108 1.260 1.155 1.253 0.809 0.955

Acceptance Criteria



2.0%.

Conclusion:



REPRODUCIBILITY

Procedure


instrument by another analyst and the values obtained were evaluated to verify their

reproducibility.

Page 316


Instrument 1

Area ± S.D (n=6)

Instrument 2

Area ± S.D (n=6)

4673.52 ± 59.007

%RSD = 1.262

4748.851 ± 50.865

%RSD = 1.071

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure


in the following method parameters


2. Flow rate

3. pH of buffer


Table 6.7.10.

Table 6.7.10: Data derived from Robustness study of Saxagliptin HCl


M.P.(buffer:ACN)72:28 4662.065 ± 49.141 1.054

M.P.(buffer:ACN)68:32 4657.443 ± 56.568 1.215

Flow rate 1.2ml/min 4427.708±33.431 0.755

Flow rate 0.8ml/min 4912.598±40.452 0.823

pH of buffer 4.7 4662.129±70.691 1.516

pH of buffer 4.3 4641.892±62.897 1.355

Page 317



Note


suitability set.


Acceptance criteria




2%.

Conclusion




SOLUTION STABILITY

Procedure




Time Area of SAX

standard(100µg/ml)

Area of SAX

sample(100µg/ml)

% Assay

standard sample

9.00a.m 4719.168 4757.388 101.023 101.841

2.00p.m 4678.004 4615.429 98.802 100.142

6.00p.m 4759.118 4713.377 100.898 101.877

9.00a.m 4637.559 4612.234 98.733 99.275

Page 318

Acceptance criteria



Conclusion



Therefore, the standard and sample preparations were stable in solution form for 24



Procedure






System Suitability Parameter Saxagliptin HCl




% RSD (n=6) 0.3%

Note



Acceptance criteria


Conclusion

Page 319




Table 6.7.13: Summary of validation parameters of Saxagliptin HCl by RP-

HPLC method





4.

Precision (%CV)

Inter-day precision

Intra-day precision

0.809-1.254

1.108-1.260









1 100 99.81 0.34

Page 320

6.8 RP-HPLC METHOD DEVELOPMENT FOR VILDAGLIPTIN IN ITS

SOLID DOSAGE FORM.

6.8.1Instruments and Materials

6.8.1.1Instruments



1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series2000






HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A

6.8.1.2 Materials:

Vildagliptin: Apollo Life Sciences Pvt Ltd,Mumbai, India




Potassium dihydrogen phosphate (AR Grade), Rankem(India) Ltd.

Orthophosphoric acid (AR Grade), Rankem(India) Ltd.

Page 321


Jalra (USV Ltd),Maharashtra.

Tablet 50mg

6.8.3Development and Optimization of HPLC method








Figure 6.8.1: UV spectrum of Vildagliptin












200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

250

500

750

1000

1250

1500

1750

2000

215.1

Page 322

6.8.4 Estimation of Vildagliptin by RP-HPLC method



800ml of water and then volume adjusted to 1000 ml with water and adjusted pH of

the solution with orthophosphoric acid (5%).






Vildagliptin Standard stock solution (200 ppm)

Accurately weighed Vildagliptin standard (20 mg) was transferred to a 100 ml





6.8.4.4 Calibration curve for Vildagliptin(10-30µg/ml)

Appropriate volume of aliquots from standard Vildagliptin stock solution was


to the mark with the diluent to obtain the concentration of 10, 15, 20, 25 and



was determined from calibration curve.(Fig. no.6.8.10)


Twenty Vildagliptin tablets were taken and crushed to make powder. The accurately

weighed powder equivalent to 20 mg of Vildagliptin was transferred into 100 ml

volumetric flask and dissolved in about 10 ml diluent by sonication for 25 minutes

and then volume was made up to 100 ml with diluent to obtain sample stock solution



solution (20ug/ml).

Page 323

6.8.5 Estimation of Vildagliptin in its solid dosage form





Figure 6.8.2: Chromatogram of Vildagliptin using water:methanol(50:50)

Figure6.8.3:Chromatogram of Vildagliptin using water:methanol:ACN

(30:40:30)

Figure 6.8.4: Chromatogram of Vildagliptin using water: ACN (50:50)

Page 324

Figure 6.8.5: Chromatogram of Vildagliptin using buffer:ACN (50:50)




Page 325

Figure 6.8.9: Chromatogram of optimized chromatographic conditions for

Vildagliptin

Table 6.8.2: Optimised chromatographic conditions for Vildagliptin


1 Mobile Phase 0.02M Potassium dihydrogen phosphate(pH 4.5

adjusted with ortho phosphoric acid):Acetonitrile

(75:25)


3 Stationary Phase Hypersil BDS(250×4.6 mm id, 5µm particle

size) column


5 Volume of

Injection (µl)

20µl

6 Detection

wavelength(nm)

215nm




LINEARITY AND RANGE

Procedure



Page 326






Table 6.8.3: Result of calibration readings for Vildagliptin

Concentrations

(µg/ml)

Vildagliptin

Area


10 3089.257 ± 21.2012 0.686

15 4550.931 ± 79.8116 1.753

20 6177.331 ± 60.19537 0.974

25 7748.548 ± 49.42296 0.638

30 9222.697 ± 96.08078 1.042

Figure 6.8.10: Calibration curve of Vildagliptin at 215nm

Table 6.8.4: Statistical data for Vildagliptin by RP-HPLC

Parameters Vildagliptin


Slope 309.29

Intercept 28.046


Page 327

Acceptance criteria


range.

Conclusion:

The correlation coefficient value was found to be 0.9997 for Vildagliptin.





ACCURACY

Procedure

The accuracy of the analytical method for assay of Vildagliptin was established at

three levels in triplicate,viz. 80%, 100%, and 120% of the test concentration.Amount

found, % Recovery and mean % Recovery was calculated at each level and recorded

in Table6.8.5.

Table 6.8.5: Data derived from accuracy experiment

Level

%

Sets Area Amount

Added

(µg/ml)

Amount

Recovered

(µg/ml)

%

Recovery

Mean%

Recovery

% RSD

80 1 4941.642 16 15.886 100.425

100.492

0.702 80 2 4981.198 16 16.014 101.225

80 3 4911.978 16 15.791 99.826

100 1 6251.683 20 20.122 101.519

100.721

1.211 100 2 6239.457 20 20.083 101.321

100 3 6115.864 20 19.683 99.323

120 1 7413.461 24 23.879 100.249

100.432

0.379 120 2 7467.639 24 24.054 100.979

120 3 7426.989 24 23.922 100.432

Page 328

Acceptance Criteria





considered range.

PRECISION


Procedure






Area 3095.324 4658.685 6186.226 7765.453 9246.876

3076.423 4620.76 6166.487 7741.598 9304.326

3086.45 4623.226 6187.568 7768.885 9068.899

3096.56 4488.453 6223.965 7741.806 9138.342

3088.529 4481.642 6260.864 7772.685 9137.223

3076.242 4623.864 6166.346 7756.983 9336.926

Mean 3086.59 4582.772 6198.576 7757.902 9205.432

S.D. 8.832 77.012 37.058 13.579 106.379

%RSD 0.286 1.680 0.598 0.175 1.156

n=6 determinations

Table 6.7.7: Repeatability of sample application data for Vildagliptin

Concentration Vildagliptin(20µg/ml)

Area 6179.805

6167.342

6059.456

Page 329

6224.864

6260.678

6260.876

Mean 6192.17

S.D. 75.980

% RSD 1.227

Acceptance Criteria


Conclusion:



INTERDAY AND INTRADAY PRECISION (INTERMEDIATE PRESICION)




Intra-day Inter-day


Area 3076.098 6223.045 9304.347 3096.208 6179.804 9248.771

3114.815 6087.601 9332.153 3086.908 6167.497 9230.228

3056.031 6260.264 9137.978 3105.483 6145.78 9082.71

Mean 3082.315 6190.303 9258.159 3096.199 6164.360 9187.236

S.D. 29.881 90.868 105.004 9.287 17.227 90.996

% RSD 0.969 1.467 1.134 0.299 0.279 0.990

Acceptance Criteria



2.0%.

Page 330

Conclusion:



REPRODUCIBILITY

Procedure



reproducibility.


Instrument 1

Area ± S.D.(n=6)

Instrument 2

Area ± S.D.(n=6)

6171.885 ± 51.2508

%RSD = 0.83

6191.571 ± 57.947

%RSD = 0.94

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure




2. Flow rate

3. pH of buffer


Table 6.8.10.

Table 6.8.10: Data derived from Robustness study of Vildagliptin


M.P.(buffer:ACN)77:23 6179.862 ± 77.427 1.253

M.P.(buffer:ACN)73:27 6173.681 ± 81.040 1.31

Page 331

Flow rate 1.2ml/min 5879.385±55.712 0.947

Flow rate 0.8ml/min 6515.741±65.971 1.012

pH of buffer 4.7 6188.081±65.457 1.058

pH of buffer 4.3 6203.504±85.363 1.376



Note


suitability set.


Acceptance criteria




2%.

Conclusion




SOLUTION STABILITY

Procedure




Time Area of VIL

standard(20µg/ml)

Area of VIL

Sample(20µg/ml)

% Assay

standard sample

9.00a.m 6243.133 6210.486 101.380 100.852

2.00p.m 6190.181 6223.242 100.524 101.059

6.00p.m 6160.223 6122.428 99.685 99.429

9.00a.m 6155.223 6120.988 99.429 99.406

Page 332

Acceptance criteria



Conclusion






Procedure





Table 6.8.12: System suitability test parameter

System Suitability Parameter Vildagliptin




% RSD (n=6) 0.13

Note



Acceptance criteria


Conclusion



Page 333

2.8.7 Summary of validation parameters of Vildagliptin

Table 6.8.13: Summary of validation parameters of Vildagliptin by RP-HPLC

method


1. LinearityRange (μg/ml) 10 – 30

2. Correlation coefficient (r2) 0.9997

3. Accuracy (%Recovery) 100.492 to 100.721

4. Precision (%CV)

Inter-day precision

Intra-day precision

0.299-0.990

0.969-1.467


6 Limit of quantitation (μg/ml) 2.042l







1 20 99.76 0.46

Page 334

6.9 RP-HPLC METHOD DEVELOPMENT FOR SITAGLIPTIN PHOSPHATE

IN ITS SOLID DOSAGE FORM.

6.9.1Instruments and Materials

6.9.1.1 Instruments



1 HPLC Shimadzu

Perkin Elmer

LC20AT

Series 2000





scientific

Branstead D3750


HPLC

Model LC20AT




Flow rate: 1 ml/min


Detector SPD20A

6.9.1.2 Materials:

Sitagliptin phosphate: Triveni Pharmaceutical Ltd, Gujarat, India

Page 335





Triethyl amine (AR Grade) Finar



Januvia (MSD Pharma),Puducherry.

Tablet 50mg






prepared in solvent mixtures of Buffer (pH 4.5):ACN (50:50) . This drug solution was

then scanned in the UV region of 200-400 nm and the spectrum was


200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

250

500

750

1000

1250

1500

1750

2000

2250

2500

2750

mAU

242.0

1

208.5

7

263.6

7

Figure 6.9.1: UV spectrum of Sitagliptin phosphate

Page 336






for initial separation because of its simplicity .






6.9.4 Estimation of Sitagliptin phosphate by RP-HPLC method



800ml of water and then made up volume to 1000 ml with water and adjusted pH of

the solution with orthophosphoric acid (5%) .





Sitagliptin phosphate Standard stock solution (1000 ppm)

Accurately weighed Sitagliptin phosphate standard (100 mg) was transferred to a 100

ml volumetric flask and dissolved in about 10 ml methanol, then diluted to mark to

obtain standard stock solution (1000µg/ml). An aliquot of the solution (5.0 ml) was



Page 337

6.9.4.4 Calibration curve for Sitagliptin phosphate(25-75µg/ml)

Appropriate volume of aliquots from standard Sitagliptin phosphate stock solution

was transferred to different volumetric flasks of 10ml capacity. The volume was

adjusted to the mark with the diluent to obtain the concentration of 25, 37.5, 50, 62.5,



was determined from calibration curve.(Fig.no.2.9.8)


Twenty Sitagliptin phosphate tablets were taken and crushed to make powder. The

accurately weighed powder equivalent to 100 mg of Sitagliptin phosphate was

transferred into 100 ml volumetric flask and dissolved in about 10 ml diluent by

sonication for 25 minute and then volume was made up to 100 ml with diluent to

obtain sample stock solution (1000µg/ml). An aliquot of the sample stock solution

(5.0ml) was transferred to a 50ml volumetric flask and diluted to mark with mobile

phase to obtain working standard solution (100ug/ml).

6.9.4.6 Estimation of Sitagliptin phosphate in its solid dosage form





Page 338

Figure 6.9.2: Chromatogram of Sitagliptin phosphate using water:methanol

(30:70)

Figure 6.9.3: Chromatogram of Sitagliptin phosphate using

water:methanol:ACN (40:30:30)

Figure 6.9.4: Chromatogram of Sitagliptin phosphate using phosphate buffer:

ACN (pH 4.0) (50:50)


ACN (pH 5.0) (50:50)

Page 339


ACN (pH 5.5) (50:50)

Figure 6.9.7: Chromatogram of Optimized chromatographic condition for

Sitagliptin phosphate

Table 2.9.2: Optimized chromatographic conditions for Sitagliptin phosphate


1 Mobile Phase 0.02M Potassium dihydrogen phosphate(pH

4.5 adjusted with ortho phosphoricacid)

:Acetonitrile (50:50).


3 Stationary Phase Hypersil BDS(250×4.6 mm id, 5µm particle

size) column


5 Volume of Injection

(µl)

20µl

Page 340

6 Detection

wavelength(nm)

263nm

7 Run time (min) 10min

8 Diluent Methanol


LINEARITY AND RANGE

Procedure








Table 6.9.3: Result of calibration readings for Sitagliptin phosphate

Concentrations

(µg/ml)

Sitagliptin

phosphate

Area


25 1634.931 ± 8.376 0.512

37.5 2401.632 ± 13.781 0.574

50 3172.877 ± 15.183 0.478

62.5 3958.7 ± 28.931 0.731

75 4711.326 ± 37.681 0.799

Page 341

y = 61.679x + 91.95

R2 = 1

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 10 20 30 40 50 60 70 80

Series1

Linear (Series1)

Figure 6.9.8: Calibration curve of Sitagliptin phosphate

Table 6.9.4: Statistical data for Sitagliptin phosphate by RP-HPLC

Parameters Sitagliptin phosphate


Slope 61.67

Intercept 91.95


Acceptance criteria


range.

Conclusion:

The correlation coefficient value was found to be1.0 for Sitagliptin phosphate.





ACCURACY

Procedure

Page 342

The accuracy of the analytical method for assay of Sitagliptin phosphate was

established at three levels in triplicate,viz. 80%, 100%, and 120% of the test

concentration. Standard was prepared as per method and sample preparations were

done by mixing known amount of Sitagliptin phosphate working standard with

placebo. Amount found, % Recovery and mean % Recovery was calculated at each

level and recorded in Table 6.9.5.


Level

%

Sets Area Amount

Added

(µg/ml)

Amount

Recovered

(µg/ml)

% Recovery Mean

% Recovery

% RSD

80 1 2592.609 40 40.543 101.358

99.172

1.841 80 2 2510.161 40 39.206 98.016

80 3 2513.286 40 39.669 98.143

100 1 3194.033 50 50.294 100.588

100.241

0.293 100 2 3179.031 50 50.051 100.102

100 3 3176.921 50 50.017 100.033

120 1 3775.633 60 59.723 99.539

100.230

1.508 120 2 3866.862 60 61.203 102.004

120 3 3761.146 60 59.488 99.148

Acceptance Criteria



Conclusion: The % recovery at each level, mean % Recovery and % RSD met the


considered range.

PRECISION


Page 343

Procedure





Conc. 25(µg/ml) 37.5(µg/ml) 50(µg/ml) 62.5(µg/ml) 75(µg/ml)

Area 1633.493 2397.652 3178.383 3964.641 4715.961

1630.497 2380.866 3191.049 3988.337 4687.681

1630.247 2416.804 3168.823 3940.929 4753.631

1642.545 2407.233 3165.597 3917.141 4758.55

1647.466 2392.821 3184.682 3948.825 4678.55

1625.34 2414.414 3148.727 3992.327 4673.583

Mean 1634.931 2401.632 3172.877 3958.7 4711.326

S.D. 8.375 13.781 15.182 28.930 37.680

%RSD 0.512 0.574 0.478 0.731 0.799

n=6 determinations

Table 6.9.7: Repeatability of sample application data for Sitagliptin phosphate

Concentration Sitagliptin

phosphate(50µg/ml)

Area 3178.383

3183.242

3148.327

3184.648

3189.654

3188.432

Mean 3178.781

S.D. 15.448

% RSD 0.486

Page 344

Acceptance Criteria


Conclusion:







Intra-day Inter-day


Area 1624.568 3245.234 4356.238 1632.514 3273.543 4367.413

1649.342 3232.234 4364.986 1662.463 3237.824 4399.268

1682.468 3268.687 4318.683 1618.326 3242.432 4354.684

Mean 1652.126 3248.718 4346.636 1637.768 3251.266 4373.788

S.D. 29.050 18.475 24.599 22.533 19.429 22.966

% RSD 1.758 0.569 0.566 1.376 0.598 0.525

Acceptance Criteria



2.0%.

Conclusion:



Page 345

REPRODUCIBILITY

Procedure



reproducibility.


Instrument 1

Area ± S.D (n=6)

Instrument 2

Area ± S.D (n=6)

3238.552 ± 22.135

%RSD = 0.683

2799.918± 28.814

%RSD = 1.03

Acceptance criteria


Conclusion



ROBUSTNESS

Procedure




2. Flow rate

3. pH of buffer

Page 346


table 6.9.10.

Table 2.9.10: Data derived from Robustness study of Sitagliptin phosphate

Robust condition Area ± S.D %RSD

M.P.(buffer:ACN:TEA)

52:48:0.1

2738.102 ± 6.938 0.253

M.P.(buffer:ACN:TEA)

48:52:0.1

3005.201 ± 18.037 0.600

Flow rate 1.2ml/min 2800.654 ± 9.574 0.342

Flow rate 0.8ml/min 2974.911 ± 7.649 0.257

pH of buffer 4.7 2881.026±11.469 0.398

pH of buffer 4.3 2900.96±11.972 0.413



Note


suitability set.


Acceptance criteria




2%.

Page 347

Conclusion




SOLUTION STABILITY

Procedure




Time Area of SITA

standard(50µg/ml)

Area of SITA

sample(50µg/ml)

% Assay

standard sample

9.00a.m 3200.982 3210.486 100.8133 101.1215

2.00p.m 3186.863 3222.258 100.3555 101.5032

6.00p.m 3142.34 3212.964 98.91179 101.2018

9.00a.m 3140.233 3200.543 98.84346 100.7991

Acceptance criteria



Conclusion




hours.

Page 348


Procedure






System Suitability Parameter Sitagliptin phosphate




% RSD (n=6) 0.23

Note



Acceptance criteria


Conclusion



Page 349


Table 6.9.13: Summary of validation parameters of Sitagliptin phosphate by RP-

HPLC method

Sr.No. Parameters Result


2. Correlation co efficient (r2) 1

3. Accuracy (%Recovery) 99.172 to 100.241

4. Precision (%CV)

Inter-day precision

Intra-day precision

0.525-1.376

0.566-1.758









1 50 99.76 0.34

Documents

6. METHOD DEVELOPMENT OF DRUGS 6.1 …shodhganga.inflibnet.ac.in/bitstream/10603/50474/14/14...Table 6.1.1.1: List of the instruments used in the UV-Spectrophotometry Sr. No. Instruments