To Development and Validation RP-HPLC Method for Tinidazole in Bulk and Pharmaceutical Formulation.

Abstract

A simple, precise, accurate, and robust Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative estimation of Tinidazole in bulk and pharmaceutical dosage forms. Chromatographic separation was achieved using a mobile phase of Acetonitrile and Water (90:10 v/v), with the detection wavelength set at 270 nm. The method demonstrated excellent linearity in the concentration range of 5–30 µg/ml, with a correlation coefficient (r²) of 0.9996. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were found to be 2.5070 µg/ml and 7.5912 µg/ml respectively. Validation parameters such as system suitability, specificity, accuracy, precision (intra-day and inter-day), robustness, and sensitivity were conducted in accordance with ICH guidelines, confirming the reliability and reproducibility of the method. Recovery studies showed results within the acceptable range, indicating no interference from excipients. The proposed method is suitable for routine analysis of Tinidazole in both bulk drug and pharmaceutical formulations due to its simplicity, sensitivity, and cost-effectiveness.

Keywords

Introduction

High Performance Liquid Chromatography (HPLC) is a leading analytical technique in contemporary pharmaceutical sciences, utilized for both qualitative and quantitative analysis of various compounds. This method, a form of column chromatography, employs high-pressure pumps to propel a liquid mobile phase containing the sample mixture through a column packed with a solid stationary phase. The technique is based on the principle of differential partitioning of compounds between the mobile and stationary phases. As analytes traverse the column, their interaction with the stationary phase—determined by polarity and solubility—affects their retention time, thereby facilitating precise separation and identification. HPLC is highly versatile, capable of detecting compounds in trace amounts, down to parts per trillion, making it suitable for applications in pharmaceutical, environmental, forensic, and chemical research. Among the various types of HPLC, Reverse Phase HPLC (RP-HPLC) is the most widely employed technique due to its capacity to handle a broad range of polar and non-polar compounds. RP-HPLC is particularly favored for the analysis of pharmaceutical drugs due to its reliability, reproducibility, and precision. The current research endeavor focuses on the development and validation of a robust, simple, and accurate RP-HPLC method for the determination of Tinidazole in both bulk drug and pharmaceutical dosage forms.

Tinidazole is a synthetic nitroimidazole derivative recognized for its potent antiprotozoal and antibacterial activities. Its IUPAC name is 1-(2-ethylsulfonylethyl)-2-methyl-5-nitroimidazole, and it possesses a molecular formula of C?H??N?O?S with a molecular weight of 247.27 g/mol. Pharmacologically, Tinidazole is effective against protozoal infections caused by Trichomonas vaginalis, Giardia duodenalis, and Entamoeba histolytica. The drug functions as a prodrug, which, upon entering the microbial cell, undergoes reduction of its nitro group by a ferredoxin-mediated electron transport system. This reduction generates free nitro radicals, which are cytotoxic and interact with DNA to cause strand breakage and inhibition of nucleic acid synthesis, ultimately leading to cell death.

Fig. No. 1.1 Structure of Tinidazole

Tinidazole is rapidly and completely absorbed when administered orally under fasting conditions. When taken with food, there is a delay in time to maximum concentration (Tmax) by approximately two hours and a slight reduction in peak plasma concentration (Cmax) by about 10%. It exhibits a volume of distribution of approximately 50 liters and has plasma protein binding of approximately 12%. The drug is extensively metabolized in the liver, primarily through CYP3A4-mediated oxidation, hydroxylation, and conjugation reactions. Its major metabolite is the 2-hydroxymethyl derivative. The elimination half-life ranges between 12 to 14 hours, and the drug is excreted in urine (20–25% unchanged) and feces (approximately 12%). Given its clinical significance and widespread use in antiprotozoal therapy, the establishment of a validated, sensitive, and reproducible RP-HPLC method for Tinidazole is essential for ensuring quality control in pharmaceutical formulations. This method development will provide a reliable tool for routine analysis of Tinidazole in both bulk and finished dosage forms, fulfilling regulatory requirements and supporting consistent therapeutic efficacy.

Review of Literature

1 Method development and validation for the determination of tinidazole by reverse phase HPLC technique, Amit Kumar De, Ashok Kumar Bera and Biswajit Pal (2015) The aim of the current study is to develop a simple, specific, rapid and precise quantification technique for the estimation of tinidazole from tablet dosage form. Successful separation of the drug was carried out on a Cis column (particle size 5 µm, 250 mm length x 4 mm i.d.) using a mobile phase consisting of a 5.3 mM phosphate buffer solution and acetonitrile in the ratio of 60:40 (v/v). The detection wavelength is 318 nm. The method has been validated as per ICH (Q2) guidelines on the basis of accuracy, precession, linearity, sensitivity and robustness. The method is found to be linear with limit of detection and limit of quantitation 0.25µg/ml and 0.76µg/ml respectively. The average elution time is only 5.0 minutes with the analyte elution taking place at about 3.0 minutes making the method rapid and cost effective for routine analysis.

2. Titrimetric and Spectrophotometric Determination of Tinidazole Tablets L.O. Okunrobo (2007) A simple, sensitive, rapid, reproducible, economical and easily accessible method for the determination of tinidazole tablets is described. From the experiment carried out there is the need for recrystallisation of tinidazole tablet before it is analysized. Ultraviolet (UV) absorption analysis corresponds to the result obtained with non-aqueous titration. And the best solvent to be use for non-aqueous titration is acetic anhydride and crystal violet indicator can be used.

3. Method for Determination of Tinidazole using Direct UV-Visible Spectrophotometry and Differential Spectrophotometry in Pure and Tablet Dosage Forms, L Singh, S Nanda (2014)Three simple, rapid, selective, precise and accurate spectrophotometric methods for the determination of tinidazole in tablet formulation were developed. The first method was based on the direct absorbance measurements of tinidazole in 0.5 N NaOH with a max of 368.6 nm and linearity range of 20-150 µg/ml. The second method was based on direct measurement of absorbance at 279.2 nm for tinidazole in 0.5 N HCI whereby the linearity range was 50-150 µg/ml. The third method was based on the differential spectra between tinidazole solution in 0.5 N NaOH and 0.5 N HCI. The maxima recorded was 368.8 nm while the minima was 276 nm with a linearity range of 20-120 µg/ml. The methods were validated by determining accuracy, precision, limit of detection, limit of quantitation and performing recovery studies. The developed methods were successfully applied in the analysis of commercial samples of tinidazole and could therefore be used in the routine analysis of tinidazole formulations.

MATERIALS AND METHODS

Tinidazole from Lupin Pharmaceuticals, Inc., Methanol, Acetonitrile, Water, from Merck Lie Sciences Pvt. Ltd, Mumbai, Potassium dihydrogen phosphate from Research Fine Chem. Indu.

Preparation of mobile phase

70 ml of HPLC grade Acetonitrile was added to 30ml of Water i.e. in 90: 10 v/v proportions. The pH was adjusted to 6, 7 and 8 with Triethylamine and orthophosphoric acid. The solution was filtered through 0.45µ membrane filter and then sonicated in sonicator bath for 10 min.

Preparation of stock solutions of Tinidazole

Stock solution was prepared by dissolving 10 mg Tinidazole in water and then diluted with Water in 10 ml of volumetric flask to get concentration of 1000 µg/ml. From the resulting solution 0.1 ml was diluted to 10 ml with water to obtain concentration of 10 µg/ml of Tinidazole and labelled as standard stock Tinidazole.

Selection of detection wavelength

From the standard stock solution further dilutions were done using water and scanned over the range of 200-400 nm and the spectra were overlain. It was observed that drug showed considerable absorbance at 270 nm at 270 nm.

Results and Discussion

The proposed HPLC method was validated in terms of system sultability, specificity, precision, accuracy and robustness as per the International Conference on Harmonization (ICH) guidelines

1. Linearity:

Table 1.1: Linearity Result of Tinidazole

Sr. No.	Concentration (µg/ml)	Peak Area
		Tinidazole
1	5	329620
2	10	665231
3	15	998222
4	20	1342803
5	25	1635604
6	30	19983125

Table 1.2: Characteristic parameters of Tinidazole for the proposed HPLC method.

Parameter	Result
	Tinidazole
Calibration range (µg/ml)	05-30
Detection wavelength (nm)	270
Regression equation (y*)	y=65847x+6778.3
Slope (b)	65847
Intercept (a)	6778.3
Correlation coefficient(r2)	0.9996
Limit of Detection (µg/ml)	2.5070
Limit of Quantitation (µg/ml)	7.5912

2. System Suitability:

Table 1.3: System suitability studies of Tinidazole by HPLC method.

Sr. No.	Properties	Values
1	Retention time	4.1
2	Area	1342580
3	Asymmetry	1.08

3. Specificity:

Table 1.4: Specificity of Tinidazole by HPLC method

Concentration	API Area	Tablet Area
20	1342566	1368789
20	1354894	1379866
20	1355897	1389789
20	1356323	1330021
20	1354890	1398779
20	1394231	1381998
Mean	1359800	1374874
SD	17652.22	24159.46
RSD	1.30	1.76

Figure 1.2: Chromatogram of Blank

Figure 1.3: Chromatogram of Sample

Figure 1.4: Chromatogram of Standard

4. Sensitivity:

(The sensitivity of measurement of Tinidazole by use of the proposed method was estimated in terms of the limit of detection (LOD) and the limit of quantification (LOQ). The LOD and LOQ were calculated by the use of signal to noise ratio.

5. Precision:

Table 1.5: Intraday Precision of Tinidazole at 270

Table 1.6: Interday Precision of Tinidazole at 270

6. Accuracy:

Recovery studies by the standard addition method were performed with a view to justify the accuracy of the proposed method. (Previously analysed samples of Tinidazole (20 µg/ml) were spiked with 80, 100, and 120% extra Tinidazole standard and the mixtures were analysed by the proposed method.

Table 1.7: Accuracy of Tinidazole at 270 nm.

7. Robustness:

Table 1.8: Robustness of Tinidazole at 270 nm

Table 1.9: Robustness of Tinidazole at 270 nm

CONCLUSION

From literature review few analytical methods appeared for the determination of Tinidazole includes HPLC, HPTLC and UV-Visible spectrophotometric methods. In view of the above fact, some simple analytical methods were planned to develop with sensitivity, accuracy, precision and economical. In the present investigation RP-HPLC method for the quantitative estimation of Tinidazole in bulk drug and pharmaceutical formulations as per ICH guidelines has been developed. It was concluded that, the proposed RP-HPLC method applying developed for the Tinidazole in bulk and in its formulations was simple, selective, sensitive, Accurate, precise and rapid. The proposed method was sufficiently sensitive and reproducible for the Analysis of Tinidazole within a short analysis Time. The method was proved to be superior to most of the reported methods. The mobile phases was simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence these methods can easily be adopted as an alternative method to reported ones for the routine determination of Tinidazole depending upon the availability of chemicals and nature of other ingredients present in the sample.

The validation study shows that the developed method is accurate, rapid, precise, reproducible and inexpensive with acceptable correlation co-efficient, RSD (%) and standard deviations which make it versatile and valuable for simultaneous determination of Tinidazole in pharmaceutical dosage forms.

ACKNOWLEDGMENT

The authors sincerely acknowledge the support of the Department of Pharmaceutical Chemistry and the laboratory staff for providing the necessary facilities and guidance to carry out this research work. We also extend our gratitude to Lupin Pharmaceuticals for providing the Tinidazole sample used in this study.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest regarding the publication of this research work.

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