Analytical Method Development And Validation

For The Simultaneous Estimation Of Neomycin

And Clotrimazole By Rp- Hplc Method

J.Harika1 , P. Sowjanya2 , Dr.Gampa Vijaya Kumar3

1,2,3
Department of Pharmaceutical Analysis, KGR Institute of Technology and Mangement, Rampally, Keesara, Rangareddy,
Telangana, India
*Corresponding Author
Dr. Gampa Vijaya Kumar ,
Ph.D, Professor,&Principal,
KGR Instiute Of Management And Technology
Email id :[email protected]
DOI: 10.47750/pnr.2022.13.S09.961

High performance liquid chromatography is at present one of the most sophisticated tool of the analysis. The estimation of
Neomycin and Clotrimazole was done by RP-HPLC. The Phosphate buffer was pH 3.0 and the mobile phase was optimized with
consists of Methanol: Phosphate buffer mixed in the ratio of 70:30 % v/ v. Inertsil C 18 column C18 (4.6 x 150mm, 5m) or
equivalent chemically bonded to porous silica particles was used as stationary phase. The detection was carried out using UV
detector at 260 nm. The solutions were chromatographed at a constant flow rate of 0.8 ml/min. the linearity range of Neomycin
and Clotrimazole were found to be from 100-500 g/ml of Neomycin and 1-5g/ml of Clotrimazole. Linear regression coefficient
was not more than 0.999.The values of % RSD are less than 2% indicating accuracy and precision of the method. The percentage
recovery varies from 98-102% of Neomycin and Clotrimazole. LOD and LOQ were found to be within limit.The results obtained
on the validation parameters met ICH and USP requirements .it inferred the method found to be simple, accurate, precise and linear.
The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.

KEY WORDS : Methanol: Phosphate buffer, Inertsil C18 column, Neomycin and Clotrimazole.

INTRODUCTION

Clotrimazole IUPAC name 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole It is soluble in DMSO (25 mM),

chloroform (50 mg/mL), DMF, ethyl acetate, ethanol, It is an anti-fungal agent, 14-alpha demethylase inhibitor
and anti-infective agent.

Journal of Pharmaceutical Negative Results ¦ Volume 13 ¦ Special Issue 9 ¦ 2022 8211

 Clotrimazole

Neomycin sulphate IUPAC name (2R, 3S, 4R, 5R, 6R)-5-amino-2-(aminomethyl)-6-{[(1R, 2R, 3S, 4R, 6S)-
4, 6-diamino-2-{[(2S, 3R, 4S, 5R)-4-{[(2R, 3R, 4R, 5S, 6S)-3 amino-6-(aminomethyl)-4, 5- dihydroxyoxan-2-
yl]oxy}-3-hydroxy 5 (hydroxymethyl)oxolan-2-yl]oxy}-3-hydroxycyclohexyl]oxy}oxane-3, 4-diol.

Neomycin

MATERIALS AND METHOD

Instrumentation
System Alliance Waters 2690 separation module, Pump Analytical HPLC isocratic pump, Detector Photo diode array
detector, Software Empower 2 software, Column Agilent (250×4.6mm, 5µ) C-18 RP-column, Sonicator Analytical
Technologies Limited- Ultrasonic cleaner. U.V double beam spectrophotometer LABINDIA, UV 3000+pH meter,
Weighing machine

Chemicals
Neomycin and Clotrimazole Potassium dihydrogen orthophosphate, Water and Methanol for HPLC, Acetonitrile for
HPLC, Ortho phosphoric Acid.
METHOD DEVELOPMENT

Trial-5 (Optimised)

Chromatographic conditions

Column : Inertsil C18 (4.6 x 250mm, 5m)

Buffer pH : 3.0.
Mobile phase : 30% buffer 70% Methanol
Flow rate : 1.0ml per min
Wavelength : 260 nm
Temperature : ambient.
Run time : 10min.

Fig.No.1. Chromatogram showing trial-5 injection

Sample solution preparation

Journal of Pharmaceutical Negative Results ¦ Volume 13 ¦ Special Issue 9 ¦ 2022 8212

 Accurately weigh 10 tablets crush in mortor and pestle and transfer equivalent to 10 mg of Neomycin and
Clotrimazole (marketed formulation) sample into a 10mL clean dry volumetric flask add about 7mL of Diluent and
sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further
pipette 3 ml of Neomycin e and Clotrimazole of the above stock solution into a 10ml volumetric flask and dilute up
to the mark with diluent.

METHOD VALIDATION
• System Suitability
• Linearity
• Specificity
• Precision
• Intermediate Precision
• Accuracy
• Limit of Detection and Limit of Quantification
• Robustness

RESULTS AND DISCUSSION

System suitability

Table 1: Results of system suitability parameters for Neomycin and Clotrimazole

S.No Name Retention Area (µV Height USP USP USP plate
time(min) sec) (µV) resolution tailing count

1 Neomycin 2.5 124505 213642 1.2 4673.4

2 Clotrimazole 3.9 1308495 154566 60 1.3 6090.3

Precision

Table 2: Results of method precession for Neomycin

Injection Area

1302729
Injection-1

1302947
Injection-2

1303236
Injection-3
1303977
Injection-4
1309759
Injection-5

Average 1304529.8

Standard Deviation 2961.1

%RSD 0.2

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Table 3: Results of method precession for Clotrimazole

Injection Area

123149
Injection-1

123766
Injection-2

124271
Injection-3
124691
Injection-4
124956
Injection-5

Average 124162.7

Standard Deviation 725.6

0.6
%RSD

Intermediate Precession (Ruggedness)

Table 4: Results of Intermediate precision for Neomycin

Injection Area

1300148
Injection-1

1304520
Injection-2

1305937
Injection-3
1306476
Injection-4
130871
Injection-5

Average 1305070.2

Standard Deviation 3061.8

%RSD 0.2

Table 5: Results of Intermediate precision for Clotrimazole

Injection Area

122487
Injection-1

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 122626
Injection-2

122632
Injection-3
122702
Injection-4
122962
Injection-5

Average 122681.8

Standard Deviation 174.8

%RSD 0.1
Accuracy

Table-6 Accuracy (recovery) data for Neomycin

%Concentration Amount Amount
Mean
(at specification Area Added Found % Recovery
Recovery
Level) (mg) (mg)

50% 656659.5 5.0 5.036 100.7%

100% 1304258 10.0 10.003 100.0% 99.84%

150% 1854608 14.4 14.224 98.780%

Table-7 Accuracy (recovery) data for Clotrimazole

%Concentration Amount Amount

Mean
(at specification Area Added Found % Recovery
Recovery
Level) (mg) (mg)

50% 65800 5.3 5.34 100.8%

100% 124353 10 10.10 100.01% 100.51%

150% 177940 14.2 14.45 99.68%

Linearity

Table-8 Area of different concentration of Neomycin

S.No. Linearity Level Concentration Area

668934
1 I 100ppm

Journal of Pharmaceutical Negative Results ¦ Volume 13 ¦ Special Issue 9 ¦ 2022 8215

 956781
2 II 200ppm

1313873
3 III 300ppm

4 IV 400ppm 1563458
1867084
5 V 500ppm

Correlation Coefficient 0.999

Table-9 Area of different concentration of Clotrimazole

S.No Linearity Level Concentration Area

66510
1 I 1ppm

94701
2 II 2ppm

124802
3 III 3ppm

152731
4 IV 4ppm

179732
5 V 5ppm

Correlation Coefficient 0.999

Figure 2 Calibration graph for Neomycin

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 Figure 3 Calibration graph for Clotrimazole

Table-10 Analytical performance parameters of Neomycin and Clotrimazole

Parameters Neomycin Naloxone
Slope (m) 66574 12529
Intercept (c) 53592 50245
Correlation coefficient (R2) 0.999 0.999

LIMIT OF DETECTION

Table-11 Results of LOD

Drug name Baseline noise(µV) Signal obtained (µV) S/N ratio
Neomycin 52 152 2.9

Clotrimazole 52 156 3

LIMIT OF QUANTIFICATION

Table no-12 Results of LOQ

Drug name Baseline noise(µV) Signal obtained (µV) S/N ratio

Neomycin 52 522 10.03

Clotrimazole 52 524 10.1

Robustness

Table-13 Flow Rate (ml/min) data for Neomycin

System Suitability Results

Flow Rate (ml/min) USP Plate Count USP Tailing

S. No
1 0.6 5339.9 1.4
2 0.8 4673.4 1.3
3 1.0 5216.0 1.4

Table-14 flow rate (ml/min) data for Clotrimazole

System Suitability Results

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 Flow Rate (ml/min)
USP Plate Count USP Tailing
S. No
1 0.8 7063.3 1.3
2 1.0 6090.3 1.2
3 1.2 6998.0 1.3

Table -15 Change in Organic Composition in the Mobile Phase for Neomycin
Change in Organic System Suitability Results
S.No Composition in the
USP Plate Count USP Tailing
Mobile Phase

1 10% less 4508.4 1.3

2 *Actual 4673.4 1.4
3 10% more 4318.1 1.3

Table -16 Change in Organic Composition in the Mobile Phase for Clotrimazole
Change in Organic System Suitability Results
S.No Composition in the
USP Plate Count USP Tailing
Mobile Phase
1.2
1 10% less 6387.7

*Actual 6090.3 1.2

2

10% more 6232.5 1.2

3

SUMMARY AND CONCLUSION

The proposed analytical technique of RP-HPLC is simple, accurate and precise method for the simultaneous estimation
of Neomycin and Clotrimazole in pharmaceutical dosage forms has been developed. The method was validated as per
ICH guidelines. Statistical analysis proves that method is repeatable, sensitive for the analysis of Neomycin and
Clotrimazole in pharmceutical dosage forms.

BIBLIOGRAPHY
1. Dr. Abdul Rahaman Shaik, Development and Validation of New Analytical Method for the Estimation of Beclomethasone Dipropionate,
Clotrimazole and Neomycin Sulphate in Bulk and Pharmaceutical Dosage Forms April 2020Indo Global Journal of Pharmaceutical
Sciences 8(3):88-91
2. M. Sudhakar Rp-Hplc Method Development And Validation For The Simultaneous Estimation Of Clindamycin Phosphate And
Clotrimazole In Pharmaceutical Dosage Forms Int J Pharm Pharm Sci, Vol 7, Issue 1, 247-251
3. Joshi, S., Analytical Method Development and Validation for Simultaneous Estimation of Clotrimazole and Tinidazole by RP-HPLC
International Journal for Pharmaceutical Research Scholars (IJPRS) 2015
4. Mohammad Marouf Novel Rp-Tlc Densitometric Method For The Simultaneous Determination Of Ciprofloxacin, Chlorocresol,
Clotrimazole, And Fluocinolone Acetonidein Cream Dosage Form, Amir Alhaj Sakur, J. Global Trends Pharm Sci, 2020; 12 (2): 9151
- 9157

Journal of Pharmaceutical Negative Results ¦ Volume 13 ¦ Special Issue 9 ¦ 2022 8218