Analytical Method Development and Validation of Nitazoxanide by

RP-HPLC Method in API and Tablet Dosage Forms

 

Devshree Yashwantbhai Patel1, Javesh Kashinath Patil2, Harsha Vasudev Chaudhari3,

Ruchita M Kothari4

1Department of Pharmaceutical Quality Assurance,

P.S.G.V.P. Mandal’s College of Pharmacy, Shahada, Dist - Nandurbar, 425409, Maharashtra, India.

2Associate Professor, Department of Pharmaceutical Quality Assurance,

P.S.G.V.P. Mandal’s College of Pharmacy, Shahada, Dist. - Nandurbar, 425409, Maharashtra, India.

3Department of Pharmaceutical Quality Assurance,

P.S.G.V.P. Mandal’s College of Pharmacy, Shahada, Dist - Nandurbar, 425409, Maharashtra, India.

4Department of Pharmaceutical Quality Assurance,

P.S.G.V.P. Mandal’s College of Pharmacy, Shahada, Dist - Nandurbar, 425409, India.

*Corresponding Author E-mail: devshree744@gmail.com

 

ABSTRACT:

A Simple, precise and accurate RP-HPLC method was developed for determination of of Nitazoxanide in API and tablet dosage forms. The chromatographic separation was done on Cosmosil C18 Column (250mm x 4.6mm,5µm) using mobile phase MEOH+ 0.05% (OPA with TEA) Water (70+30% v/v) at flow rate 0.7ml/min and wavelength detection 340nm.The retention time for Nitazoxanide was found to be 4.333 min. Linearity was observed in the range10-50μg/ml (y=55.38x-37.01 and R˛ = 0.9976). The LOD and LOQ was found to be 0.4258 and 1.289 respectively. The Precision as indicated by repeatability study was having %RSD less than 2. The accuracy, intraday and interday were also found to be less than 2. Hence the proposed method is successfully developed and validated.

 

KEYWORDS: Nitazoxanide, RP-HPLC.

 

 

 

 

INTRODUCTION:

Nitazoxanide is a class of antiparasitic medications known as anthelmintics or anthelminthics eliminates internal parasites such as helminths and parasitic worms from the body by either killing or stunning them without seriously harming the host. Nitazoxanide Chemical name is [2-[(5-nitro-1,3-thiazol-2-yl) carbamoyl] phenyl] acetate. The Molecular weight is 307.28g/mol.

 

 

The Molecular formula is C12H9N3O5S.Nitazoxanide is a synthetic benzamide with antiprotozoal activity. Nitazoxanide exerts its antiprotozoal activity by interfering with the pyruvate ferredoxin/flavodoxin oxidoreductase-dependent   transfer reaction, which is essential to anaerobic energy metabolism. Nitazoxanide is reduced by the PFOR enzyme, which inhibits energy metabolism. However, interference with the PFOR enzyme-dependent electron transfer reaction may not be the only pathway by which nitazoxanide exhibits antiprotozoal activity. Nitazoxanide is active against G. lamblia and Cryptosporidium parvum.

 

In the literature review we found that there is method developed on this drug but in combination but not on single drug. So, we had developed and validated a RP-HPLC method on Nitazoxanide by pure and Tablet dosage form.

 

Fig. no. 1: Structure of Nitazoxanide

 

 

EXPERIMENTAL WORK:

Preparation of Stock Standard Solution:

Standard Solution Stock:

Accurately weigh and transfer 10mg Nitazoxanide working standard into 10ml volumetric flask as about diluent Methanol completely and make volume up to the mark with the same solvent to get 1000µg/ml standard (stock solution) and 15 min sonicate to dissolve it and the resulting stock solution 0.1ml was transferred to 10 ml volumetric flask and the volume was made up to the mark with mobile phase Methanol: Water (0.1% OPA with TEA) Water, prepared in (70ml MEOH: 30ml WATER v/v) solvent.

 

Optimized chromatography condition:

·       Analytical column: Cosmosil C18 Column (250mm x 4.6mm), 5µm partical size.

·       Mobile phase: MEOH+0.05% (OPA with TEA) Water (70+30% v/v)

·       Injection volume: 20µl

·       Flow rate: 0.7ml/min

·       Detection: 340nm

·       Run Time: 10min

 

METHOD DEVELOPMENT:3-4

Method development and optimization in liquid chromatography is still an attractive field of research for theoreticians. Complex mixtures or samples required systematic method development involving accurate modelling of the retention behaviour of the analyte. Among all, the liquid chromatographic methods, the reversed phase systems based on modified silica offers the highest probability of successful results. However, a large number of (system) variables (parameters) affect the selectivity and the resolution.

RESULTS AND DISCUSSION:

Analytical of Method Validation:5-8

Linearity:

From Nitazoxanide standard stock solution, different working standard solution (10-50μg/ml) were prepared in mobile phase 20 μl of sample solution was injected into the chromatographic system using mixed volume loop injector Chromatograms were recorded. The area for each concentration were recorded. (Table-1 and figure-2).

 

Table no 1. Linearity of Nitazoxanide

Sr. No.

For HPLC Method

For UV Method

Conc. μg/ml

Area Nitazoxanide

Conc. μg/ml

Area Nitazoxanide

1

10

567.94

5

0.195

2

20

1034.08

10

0.421

3

30

1583.81

15

0.61

4

40

2164.20

20

0.789

5

50

2771.86

25

0.989

 

 

Fig.no.2 Calibration curve of Nitazoxanide

 

Table no 2. Regression equation data for Nitazoxanide

Regression Equation Data Y=mx+c

Slope(m)

55.38x

0.0391 x

Intercept(c)

37.01

0.014

Correlation Coefficient

0.997

0.9984

 

Accuracy:

Recovery studies were performed to validate the accuracy of developed method. To pre analysed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analysed.

 

Accuracy 80%

 

Fig.no.3: Chromatogram of Accuracy 80%

Accuracy 100%                                                                        Accuracy 120%

 

Fig.no.4: Chromatogram of Accuracy 100%                                                  Fig.no.5: Chromatogram of Accuracy 120

 

Table no 3: Result of Recovery data for Nitazoxanide

Drug

Sr No

Level (%)

Amt. taken

(μg/m)

Amt. Added

(μg/ml)

Absorbance

Mean*±SD

Amt. recovered

Mean*±SD

%Recovery

Mean *± S.D.

 

NTZ

1

80%

10

8

18.02±0.06

8.02±0.06

100.28±0.71

2

100%

10

10

20.04±0.05

10.04±0.05

100.39±0.55

3

120%

10

12

21.97±0.11

11..97±0.11

99.78±0.98

*Mean of each 3 reading

 

Table no 4: Statistical Validation of Recovery Studies Nitazoxanide

Sr.no.

conc

Area 1

Area 2

Area 3

Mean

STDV

% RSD

1

80 %

0.198

0.2

0.199

0.199

0.001

0.502

2

100 %

0.354

0.354

0.357

0.355

0.001

0.478

3

120 %

0.386

0.386

0.384

0.385

0.001

0.286

 

Accuracy of method is ascertained by recovery studies performed at different levels of concentrations (80%, 100% and 120%). The % recovery was found to be within 99-100%

 

System suitability parameters: (Repeatability):

To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of Nitazoxanide system suitability parameters were studied.

 

Fig.no.6: Chromatogram of System suitability No- 1

 

Fig.no 7: Chromatogram of System suitability No- 2

Table no.5: Repeatability studies on Nitazoxanide

Sr. No.

Concentration of Nitazoxanide (mg/ml)

Peak area

Amount found (mg)

% Amount found

1

20

1080.35

20.19

100.96

2

20

1080.13

 

 

 

Mean

1080.24

 

 

 

SD

0.02

 

 

 

%RSD

0.11

 

 

 

Repeatability studies Nitazoxanide was found to be, The %RSD was less than 2, which shows high percentage amount found in between 99% to 100% indicates the analytical method that concluded.

 

Precision:

The method was established by analyzing various replicates standards of Nitazoxanide All the solution was analyzed thrice in order to record any intra-day & inter-day variation in the result that concluded.

 

Fig No .8: Chromatogram of Precision

Fig No.9: Chromatogram Intra-day precision.

 

Fig No.10: Chromatogram Inter-day precision

Table no.6: Result of Intraday and Inter day Precision for Nitazoxanide

Drug

Concn

(µg/ml)

Intraday Precision

Interday Precision

Mean*±SD

% Amt Found

%RSD

Mean*±SD

% Amt Found

%RSD

 

NTZ

20

1042.85±5.93

97.45

0.57

1088.98±1.90

101.65

0.17

30

1589.11±7.93

97.87

0.50

1596.66±3.24

98.30

0.20

40

2161.30±7.06

99.00

0.33

2160.55±2.21

99.20

0.10

*Mean of each 3 reading

 

Intraday and Inter day Precision for Nitazoxanide which shows the high precision %amount in between 97% to 101% indicates to analytical method that concluded

 

Robustness:

The Robustness of a method is its ability to remain unaffected by small deliberate changes in parameters. To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate, wavelength on retention time and tailing factor of drug peak was studied.

 

The mobile phase composition was changed in (±1 ml/min-1) proportion and the flow rate was varied by of optimized chromatographic condition. Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

 

Flow Rate Change 0.6ml

FigNo.11. Chromatogram of Flow rate change 0.6ml

 

 

Flow Rate Change 0.8 ml:

Fig No 12. Chromatogram of Flow rate change 0.8 ml

 

Mobile phase composition Change: 69ml MEOH + 0.05% (OPA with TEA) 31mlWater

Fig.no.13: Chromatogram of Mobile phase composition change 69ml MEOH + 0.05 %(OPA) 31mlWater

 

 

 

Mobile phase composition Change: 71ml MEOH+0.05%(OPA)29ml Water

 

Wavelength Change 339 nm

 

Fig.no.14: Chromatogram of Mobile phase composition change 71ml MEOH+0.05% (OPA with TEA)29 ml Water

Fig.no.15: Chromatogram of comp change wavelength change 339 nm

 

Wavelength Change 341 nm

 

Fig.no .16: Chromatogram of comp change wavelength change 341 nm

 

Table no.7: Result of Robustness Study of Nitazoxanide

Parameters

Conc.(µg/ml)

Amount of detected(mean±S.D.)

%RSD

MP composition(69ml+31ml)

Methanol + 0.05%(OPA)water

20

1003.23±15.78

1.66

MP composition(71ml+29ml)

Methanol + 0.05% (OPA)water

20

1016.86±14.36

0.10

Wavelength change 339 nm

20

1124.53±8.80

0.78

Wavelength Change 341 nm

20

971.51±3.55

0.37

Flow rate change(0.6ml)

20

921.92±15.29

1.66

Flow rate change(0.8ml)

20

1039.47±1.07

0.10

 

6. Limit Detection:

The LOD is the lowest limit that can be detected. Based on the S.D. deviation of the response and the slope The limit of detection (LOD) may be expressed as:

LOD = 3.3 (SD)/S

= 3.3 X 7.142 /55.386

= 0.4258

 

where, SD = Standard deviation of Y intercept

 S = Slope

 

The LOD of Nitazoxanide was found to be 0.4258 (μg/mL) analytical method that concluded.

 

Limit Quantification:

The LOQ is the lowest concentration that can be quantitatively measured. Based on the S.D. deviation of the response and the slope,

The quantitation limit (LOQ) may be expressed as:

LOQ = 10 (SD)/ S

=10 X 7.142 / 55.386

= 1.289

 

where, SD = Standard deviation Y intercept

S = Slope

 

The LOQ of Nitazoxanide was found to be 1.289 (μg/mL) analytical method that concluded.

 

Analysis of tablet formulation:

Procedure:

Weigh 20 Nitazoxanide tablets and calculated the average weigh 0.2037mg accurately weigh and transfer the sample equivalent to 20.37mg Nitazoxanide into 10 ml volumetric flask. Add about 10ml of diluent and sonicate to dissolve it completely and make volume up to the mark with diluents. Mix well and filter through 0.45µm filter. Further pipette 0.2ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents. (20µg/ml). The amounts of Nitazoxanide per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated two times with tablet formulation. Tablet Assay for % Label claim for %RSD Calculated.

 

Brand Name: - NOZOA (MICRO 100 mg)

Total weight of 20-tab Powder wt. = 4.076 Gms

Avg Powder Weight = 0.2037 Gms. /Tab

Eq. Wt. for 10 mg= 10 x 0.2037 /100   = 13.5    mg

Take 20.37 mgs in 10 ml MeOH i.e. =   1000 µg/ml tab solution

 

Fig.no.17: Chromatogram for Marketed Formulation

 

 

Table no. 8: Analysis of marketed formulation.

Sr. No

Amount present in mg

Area(I)

Amount found in mg

% Label claim

NTZ

NTZ

NTZ

NTZ

1

30

1545.48

29.29

97.63

2

30

1609.49

29.73

99.10

Mean

 

1597.49

29.51

98.37

SD

 

16.96

0.31

0.34

%RSD

 

1.06

1.05

0.33

Analysis of marketed formulation were also % Label Claim was found to be 97-99% Satisfactory are concluded.

 

Tablet Assay for % Label Claim

Table no. 9: Tablet for % Label claim

Sample

Label claimed

%Label claimed. ± SD

%RSD

Nitazoxanide

Ritomune =100mg

98.37±0.34

      0.33

 

Tablet Assay for % Label claim for were also was found to be 100% and %RSD   are less than 2 satisfactory result that concluded.

 

CONCLUSION:

Simple, rapid, accurate and precise RP-HPLC have been developed and validated for the routine analysis of Nitazoxanide in API and tablet dosage forms. Both methods are suitable for the simultaneous determination of Nitazoxanide in Single-component formulations without interference of each other. The developed methods are recommended for routine and quality control analysis of the investigated drugs in two component pharmaceutical preparations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also, the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms.

 

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Received on 07.08.2024      Revised on 14.10.2024

Accepted on 30.11.2024      Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):211-216.

DOI: 10.52711/2231-5675.2024.00038

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