INTRODUCTION:

Nitazoxanide, also known by the brand name Alinia, is a synthetic nitrothiazolyl-salicylamide derivative and an anti-protozoal agent. It was approved for the treatment of epidemiological diseases caused by Cryptosporidium Phiramum and Giałdia Lambia in 1 year and older patients. After oral administration it is active metabolite, tizo carnide, hydrolyzed, which is 99% protein adherence. Peak densities are observed after 1-4 hours administration. It is excreted in urine, bile and stool.¹

Structure:

Chemical formula: C₁₂H₉N₃O₅S

Molecular weight: 307.283g/mol

Category: Antiparastitic agent

Ofloxacin²

A synthetic fluoroquinolone (fluoroquinolones) antibacterial agent that inhibits the supercoiling activity of bacterial DNA gyrase, halting DNA replication²

Synonyms: 8-Fluoro-3-methyl-9-(4-methyl-piperazin-1-yl)-6-oxo-2,3- dihydro-6H-1-oxa-3a-aza-phenalene-5-carboxylic acid.²

Structure:

Categories:

Anti-Bacterial Agents, Anti-Infective Agents, Anti-Infective Agents, Urinary, Antibacterial for Systemic Use

Chemical Formula: C₁₈H₂₀FN₃O₄

IUPAC Name: 7- fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-4-oxa-1- azatricyclo [7.3.1.0⁵,¹³]trideca-5(13),6,8,11-tetraene-11-carboxylic acid

MATERIALS AND METHODS:

The various materials and equipment’s used for the present study are summarized as follows.

Table 1: Instruments used

SL. No	Instrument	Model
1	HPLC	Hitachi LaChrome, 1575 model
2	UV/VIS spectrophotometer	Elico India, UV1800
3	pH meter	Adwa – AD 1020
4	Weighing machine	Afcoset ER-200A
5	Pipettes and Burettes	Borosil
6	Beakers	Borosil

Table 2: Chemicals used

SL. No	Chemical	Brand
1	Nitazoxanide	Supplied by Pharmatrain
2	Ofloxacin	Supplied by Pharmatrain
3	KH₂PO₄	Sd fine-Chem ltd; Mumbai
4	Water for HPLC	Sd fine-Chem ltd; Mumbai
5	Acetonitrile for HPLC	Loba Chem; Mumbai.
6	Methanol for HPLC	Loba Chem; Mumbai.

Table 3: Optimized Chromatographic Conditions

Column	Develosil ODS HG-5 RP C18, 5m, 15cmx4.6mm i.d.
Mobile phase	ACN: Methanol: Citric Acid (pH2.5 OPA)50:45:5
Flow rate	1ml per min
Wavelength	296nm
Injection volume	20ml
Run time	10 min.

Preparation of mobile phase:^3,4,6,7

Mobile phase was prepared by taking Acetonitrile: Methanol: Citric acid (0.02M, pH 2.5) (50:45:5v/v). The mobile phase is filtered by 0.45μm layer filter and has been formed in the ultrasonic bath. Mobile phase is pumped through the column at 1.0ml/min flow rate.

Preparation Sample solutions:^3,4,6,7

Accurately weighed and transferred 25mg of Ofloxacin and 25mg of Nitazoxanide working standard 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 0.6ml of the above stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

Standard Solution Preparation:^{3,4,6 and 7}

Accurately weighed 25mg of Ofloxacin and 25mg of Nitazoxanide were transferred to 25ml volumetric flask. Approximately 5ml added HPLC grade methanol and sonicated to dissolve. The volume is designed to identify with the same solvent. Then the mixture is diluted with 100ml with 3ml solution. The resulting solution was filtered by a 0.45μm filter and insulted under the ultrasonic bath for use before. Many of the standard work solutions from the above standard solution are made by serial dilution technique.

RESULTS AND DISCUSSION

LINEARITY:^5,6

Linearity range was found to be 0-50µg/ml for Nitazoxanide and 0-50µg/ml for Ofloxacin and Calibration Curves are shown below.

Fig 1: Calibration graph for Nitrazoxanide and Ofloxacin

Table 4: Linearity data of concentration of Nitrazoxanide and Ofloxacin

S. No	Nitrazoxanide		Nitrazoxanide and Ofloxacin
S. No	Concentration (µg/ml)	Area	Concentration (µg/ml)	Area
1	10	424838	10	1228747
2	20	904737	20	2638031
3	30	1302869	30	3983572
4	40	1746831	40	5249436
5	50	2250813	50	6979310

PRECISION:^5,6

Precision of the method was carried out for both sample solutions as described under experimental work. The corresponding chromatograms and results are shown below.

Fig 2: Chromatogram of Nitrazoxanide and Ofloxacin showing precision

Table 5: Results of Precision for Nitrazoxanide and Ofloxacin

Injec. No.	Nitrazoxanide		Ofloxacin
Injec. No.	Retention time (min)	Peak area	Retention time (min)	Peak area
1	9.93	3983572	2.02	1302869
2	9.93	3985214	2.02	1302586
3	9.93	3990228	2.02	1318521
4	9.92	3985261	2.01	1302569
5	9.92	3996512	2.02	1302896
Mean		3988157		1305888
SD		5295.407		7063.605
%RSD		0.9		0.9

Limit of Detection for Nitrazoxanide and Ofloxacin:^5,6

The lowest concentration of the sample was prepared with respect to the baseline noise and measured the signal to noise ratio.

Table 6: Results of LOD

Drug name	Result
Nitrazoxanide	0.06 µg/ml
Ofloxacin	0.02 µg/ml

Signal to noise ratio shall be 3 for LOD solution

The result obtained is within the limit.

Limit of Quantification for Nitrazoxanide and Ofloxacin: ^5and6

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio.

Table 6: Results of LOQ

Drug name	Result
Nitrazoxanide	1.2 µg/ml
Ofloxacin	0.04 µg/ml

Signal to noise ratio shall be 10 for LOQ solution

The result obtained is within the limit.

CONCLUSION:

A sensitive and selective stability indicting RP-HPLC method has been developed and validated for the analysis of Ofloxacin and Nitazoxanide. Based on peak purity results, obtained from the analysis of samples using described method, it can be concluded that the absence of co-eluting peak along with the main peak of Ofloxacin and Nitazoxanide indicated that the developed method is specific for the estimation of Ofloxacin and Nitazoxanide. Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility.

REFERENCES:

1. www.fda.gov/cder/drug/infopage/rosuvastatin/default. html

2. https://pubchem.ncbi.nlm.nih.gov/compound/ofloxacin

3. ICH, Text on Validation of Analytical Procedures, ICH – Q2A, International Conference on Harmonisation, IFPMA, Geneva, 1995; 2-3: A–1 to A–3.

4. ICH, Validation of Analytical Procedures Methodology, ICH – Q2B, International Conference on Harmonisation, 1996; p1-3.

5. ICH Guidelines, Q2 (R1) Validation of Analytical Procedures Text and Methodology, 2005; p1-6.

6. Gadapa Nirupa1 and M Ravi RP-HPLC Analytical process Development and Validation for Simultaneous analysis of two Drugs, Nitazoxanide, Ofloxacin and its Pharmaceutical Dosage Forms International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN: 0974-4290 Vol.4, No.2, pp775-783.

7. Prashant D. Ghode, Sunil P. Pawar, Stability Novel HPLC technique development and validation for the coincident estimation of Azithromycin and Ofloxacin in bulk and its dosage forms.

Received on 10.12.2020 Revised on 15.02.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):84-86.

DOI: 10.52711/2231-5675.2021.00016