Int J Pharm Pharm Sci, Vol 7, Issue 12, 30-35Original Article
ASSAY METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTITATIVE ESTIMATION OF DILOXANIDE FUROATE AND TINIDAZOLE IN TABLETS BY RP-HPLC
P. SWETHA, D. VIJAY KUMAR, B. RAKESH, A. ASHOK KUMAR*
Department of Pharmaceutical Analysis and Quality Assurance, Vijaya College of Pharmacy, Munaganur (Village), Hayathnagar (Mandal), Hyderabad 501511, India
Email: ashok576@gmail.com
Received: 04 Feb 2015 Revised and Accepted: 27 Oct 2015
ABSTRACT
Objective: To develop an accurate, precise and linear Reverse-Phase High-Performance Liquid Chromatographic (RP-HPLC) method for simultaneous quantitative estimation of Diloxanide furoate and Tinidazole in tablets and validate as per ICH guidelines.
Methods: The optimized method uses a reverse phase column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), a mobile phase of triethylammonium phosphate buffer (pH 2.3):acetonitrile in the proportion of 40:60 v/v, flow rate of 1.0 ml/min and a detection wavelength of 270 nm using a UV detector.
Results: The developed method resulted in Diloxanide furoate eluting at 4.07 min and Tinidazole at 2.52 min. Diloxanide furoate exhibited linearity in the range 31.25-93.75μg/ml, while Tinidazole exhibited linearity in the range 37.5-112.5μg/ml. The precision is exemplified by relative standard deviations of 0.90% for Diloxanide furoate and 0.68% for Tinidazole. Percentage Mean recoveries were found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Diloxanide furoate and Tinidazole were found to be 68.53µg/ml and 97.87µg/ml respectively, while the limit of quantitation (LOQ) for Diloxanide furoate and Tinidazole were found to be 207.677µg/ml and 296.6µg/ml respectively.
Conclusion:A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of Diloxanide furoate and Tinidazole in tablets and validated as per ICH guidelines. Hence, it can be used for the routine analysis of Diloxanide furoate and Tinidazole in tablets in various pharmaceutical industries.
Keywords: RP-HPLC, Diloxanide furoate, Tinidazole, Method development, Validation.
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
Tinidazole (fig. 1) chemically is 1-[2-(ethyl sulphonyl) ethyl]-2-methyl-5-nitro-1H-imidazole, (fig. 2). It is used as antiprotozoal agent. Tinidazole is a prodrug and the anti-protozoal action of tinidazole results from the reduction of nitro group of tinidazole in Trichomonas by a ferredoxin-mediated electron transport system. As a result of this reduction, a free nitro radical is generated and is believed to be responsible for the antiprotozoal activity. This toxic free radical covalently binds to DNA, resulting in DNA damage and leads to cell death [1]. It has a molecular formula of C8H13N3O4S and a molecular weight of 247.272 g/mol.
Fig. 1: Structure of Tinidazole
Diloxanide furoate (fig. 2)chemically is 4-(N-methyl-2, 2-dichloro-acetamido) phenyl-2-furoate having the molecular formula as C14H11Cl2NO4 and the molecular weight as 328.147 g/mol [2]. It is an effective drug for the treatment of asymptotic persons who are passing cysts of Entameba histolytica [3]. It acts principally in the bowel lumen and is used in the treatment of the intestinal amoebiasis. Diloxanide furoate has been used in the treatment of the asymptotic carriers of Entameba histolytica [3] and is excellent amoebicide for cyst passers [4, 5].
A detailed literature survey reveals that there exists literature on chromatographic methods for Tinidazole in combination with other drugs [6-12] and similarly Diloxanide furoate in combination with other drugs [13-17] in various matrices. While there are only two RP-HPLC assay methods reported for the simultaneous quantitative estimation of Diloxanide furoate and Tinidazole in pharmaceutical dosage forms using potassium dihydrogen orthophosphate as buffer (pH 5.0) and mixed phosphate buffer pH 6.5 [18-19].
Fig. 2: Structure of Diloxanide furoate
As there is no literature reported on working using triethyl-ammonium phosphate buffer as aqueous media along with acetonitrile as mobile phase, we here report a new and a rapid RP-HPLC validated method for the simultaneous quantitative estimation of Diloxanide furoate and Tinidazole in tablets using triethyl-ammonium phosphate buffer (pH 2.3) as per ICH guidelines.
MATERIALS AND METHODS
Chemicals and reagents
Analytically pure sample of Diloxanide furoate and Tinidazole with purities greater than 95% were obtained as gift samples from Chandra Labs, Hyderabad, India and tablet formulation [Metroquin] was procured from Medplus pharmacy, Hyderabad, India with labelled amount 250 mg and 300 mg of Diloxanide furoate and Tinidazole respectively. Acetonitrile (HPLC grade) was obtained from Sigma Aldrich (Hyderabad, India), water (HPLC grade), Triethylamine (AR grade), orthophosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, India), 0.22 and 0.45μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.
Instrument
HPLC analysis was performed on Shimadzu LC-20AD Prominence Liquid Chromatography comprising an LC-20AD pump, Shimadzu SPD-20A Prominence UV-VISIBLE detector and a reverse phase C18 column, Waters Symmetry (250 X 4.6 mm; 5μ). A manually operating Rheodyne injector with 20 μl sample loop was equipped with the HPLC system. The HPLC system was controlled with “Lab solutions lite” software. A double beam UV-visible spectrophotometer (Shimadzu, model UV-1800) having two matched quartz cells with 1 cm light path and loaded with UV probe software (version 2.41) was used for recording of spectra and measuring absorbance. An electronic analytical weighing balance (0.1 mg sensitivity, Shimadzu AY 220), digital pH meter (DELUX model 101), a sonicator (sonica, model 2200 MH).
Methods
Selection of wavelength
The suitable wavelength for the HPLC analysis was determined by recording UV spectrums in the range of 200-400 nm for individual drug solutions of Tinidazole and Diloxanide furoate. Suitable wavelength selected for simultaneous estimation is 270 nm (fig. 3-4).
Chromatographic conditions
The developed method uses a reverse phase C18 column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), a mobile phase of triethylammonium phosphate buffer (pH 2.3):acetonitrile in the proportion of 40:60 v/v, flow rate of 1.0 ml/min and a detection wavelength of 270 nm using a UV detector.
Buffer preparation
The buffer solution was prepared by adding 5 ml of triethylamine to 1000 ml of HPLC grade water and later pH was adjusted to 2.3 using 30% v/v of orthophosphoric acid in water. The buffer was filtered through 0.45µ filter to remove all fine particles.
Mobile phase preparation
The mobile phase was prepared by mixing buffer and acetonitrile in the ratio of 40:60 v/v and later it was sonicated for 10 min for the removal of air bubbles.
Diluent
Diluent used is the mobile phase itself
Preparation of standard solution
12 mg of Tinidazole and 10 mg of Diloxanide furoate were weighed accurately in 100 ml of volumetric flask and dissolved in 80 ml of mobile phase and volume was made up with mobile phase. From stock solution 75 µg/ml of Tinidazole and 62.5 µg/ml of Diloxanide furoate were prepared further by appropriate dilution. This is treated as working standards solution, 100% target concentration.
Preparation of sample solution
10 tablets were weighed and taken into a mortar, crushed and then uniformly mixed. Test stock solution of Tinidazole (750μg/ml) and Diloxanide furoate (625 μg/ml) were prepared by taking tablet powder equivalent to 75 mg of Tinidazole and 62.5 mg of Diloxanide furoate to 80 ml of mobile phase which is sonicated for a min and later made up to 100 ml with mobile phase. This solution was filtered using 0.22 micron syringe filter. 1 ml of the stock solution was pipetted out and made up to 10 ml to get working sample solution equivalent to a concentration of 75µg/ml for Tinidazole and 62.5µg/ml for Diloxanide furoate, concentrations equal to 100% target concentration.
RESULTS AND DISCUSSION
Method development
A Reverse phase HPLC method was developed keeping in mind the system suitability parameters i.e. resolution factor (Rs) between peaks, Tailing factor (T), the number of theoretical plates (N), runtime and the cost effectiveness.
Fig. 3: UV spectrum of standard Diloxanide furoate
Fig. 4: UV spectrum of standard tinidazole
The optimized method developed resulted in the elution of Tinidazole at 2.52 min and Diloxanide furoate at 4.07 min. fig. 5-8 represents chromatograms of the blank solution, standard solutions individually and mixture of standard solutions respectively. The total run time is 6 min. System suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N), peak resolution (Rs) and Tailing factor (T) were evaluated for six replicate injections of the standards at working concentration. The results given in table 1were within acceptable limits.
In order to test the applicability of the developed method to a commercial formulation, ‘Metroquin’ tablets were chromatographed at working concentration and it is shown in fig. 9. The sample peaks were identified by comparing relative retention times with the standard solutions (Fig. 5-8).
System suitability parameters were within the acceptance limits, ideal for the chromatograph ed sample. Integration of separated peak area was done and each drug concentration was determined by using the peak area concentration relationship obtained in the standardization step. The protocol affords reproducible quantification of the two drugs with error less than 10%, which is the standard level in any pharmaceutical quality control.
Fig. 5: Typical chromatogram of blank solution
Fig. 6: Typical chromatogram of Diloxanide furoate standard solution
Fig.7: Typical chromatogram of Tinidazole standard solution
Fig. 8: Typical chromatogram of mixture of standard solutions
Fig. 9: Typical chromatogram of sample solution
Table 1: System suitability studies results
Parameters |
Acceptance Limits |
Tinidazole |
Diloxanide furoate |
Retention time (min) |
- |
2.52 |
4.07 |
Resolution factor (Rs) |
Not less Than 2 |
9.027 |
|
Number Of Theoretical plates (N) |
Not less Than 2000 |
4076 |
7648 |
Tailing factor (T) |
Not More Than 2 |
1.919 |
1.884 |
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. HPLC method developed was validated according to International Conference on Harmonization (ICH) guidelines [20] for validation of analytical procedures. The method was validated for the parameters like linearity, accuracy, system precision, intra-day precision, limit of detection (LOD) and limit of quantitiation (LOQ).
Specificity
Fig. 5-9 for blank, individual and mixture of standards drug solutions and sample solution chromatogram reveal that the peaks obtained in the standards solution and sample solution at working concentrations are only because of the drugs as blank has no peak at the retention time of Tinidazole and Diloxanide furoate standards. Accordingly it can be concluded that, the method developed is said to be specific.
Precision
System precision
Six replicate injections of the mixture of standards solution at working concentration showed % RSD(Relative Standard Deviation) less than 2 concerning peak area for both the drugs, which indicates the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in table 2.
Method precision
Method precision was determined by performing assay of the sample under the test of repeatability (Intraday precision) at working concentrations.
Repeatability (Intraday precision)
Six consecutive injections of the sample from the same homogeneous mixture at working concentration showed % RSDless than 2 concerning % assay for both the drugs which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results (table 3).
Linearity
Standards solutions of Diloxanide furoate and Tinidazole at different concentrations were prepared. Calibration curves (fig. 10-11) were constructed by plotting the concentration level versus corresponding peak area for both the drugs.
The results show an excellent correlation between peak areas and concentration within the concentration range of 377.5-112.5µg/ml for Tinidazole and 31.25-93.75µg/ml for Diloxanide furoate (tables 4-5).The correlation coefficients were greater than 0.995 for both the drugs, which meet the method validation acceptance criteria and hence the method is said to be linear for both the drugs.
Accuracy
Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of both the drugs at three different levels (50-150%). At each level, three determinations were performed. Percent mean recovery is calculated as shown in table 7. The accepted limits of mean recovery are 98%-102% and all observed data were within the required range, which indicates good recovery values and hence the accuracy of the method developed.
Table 2: System precision results of Diloxanide furoate and Tinidazole
n |
Diloxanide furoate |
Tinidazole |
||
RT |
Peak area |
RT |
Peak area |
|
1 |
4.075 |
4407555 |
2.521 |
764673 |
2 |
4.070 |
4439757 |
2.519 |
755950 |
3 |
4.078 |
4399433 |
2.527 |
763716 |
4 |
4.070 |
4380246 |
2.520 |
756354 |
5 |
4.069 |
4429097 |
2.519 |
754293 |
Average |
4.072 |
4411217.6 |
2.521 |
758997 |
SD |
0.0039 |
23694.41 |
0.0033 |
4818.79 |
% RSD |
0.095 |
0.53 |
0.13 |
0.63 |
Table 3: Intraday precision results of Diloxanide furoate and Tinidazole
N |
Diloxanide furoate |
Tinidazole |
% Assay |
% Assay |
|
1 |
98.8 |
101.4 |
2 |
98.6 |
101.2 |
3 |
98.2 |
101.2 |
4 |
98.3 |
101.2 |
5 |
99.0 |
99.6 |
6 |
98.7 |
100.5 |
Average |
98.6 |
100.8 |
SD |
0.30 |
0.686 |
% RSD |
0.304 |
0.68 |
Table 4: Calibration data for Diloxanide furoate
% Level |
Concentration (µg/ml) |
Peak area |
50 |
31.25 |
2216411 |
75 |
46.87 |
3309655 |
100 |
62.5 |
4451303 |
125 |
78.12 |
5623684 |
150 |
93.75 |
6578781 |
Regression equation |
y=70647.99x+20608.41 |
|
Regression coefficient |
0.999 |
Table 5: Calibration data for Tinidazole
% Level |
Concentration (µg/ml) |
Peak area |
50 |
37.5 |
391612 |
75 |
56.25 |
584953 |
100 |
75 |
784818 |
125 |
93.75 |
990979 |
150 |
112.5 |
1159699 |
Regression equation |
Y=10358.29x+5536.2 |
|
Regression coefficient |
0.999 |
Fig. 10: Linearity graph of Diloxanide furoate
Fig. 11: Linearity graph of Tinidazole
Table 6: Results of Accuracy studies for Diloxanide furoate and Tinidazole
Level (%) |
Diloxanide furoate |
Tinidazole |
||
% Recovery |
% Mean |
% Recovery |
% Mean |
|
50 |
101.7 |
99.5 |
||
50 |
101.0 |
100.55 |
98.3 |
99.3 |
50 |
100.1 |
100.1 |
||
100 |
98.2 |
98.8 |
||
100 |
98.3 |
99.23 |
101.4 |
100.3 |
100 |
101.2 |
100.7 |
||
150 |
98.1 |
99.7 |
||
150 |
99.4 |
98.63 |
100.1 |
99.66 |
150 |
98.4 |
100.1 |
||
Table 3: Ruggeness results of Diloxanide furoate and Tinidazole.
N |
Diloxanide furoate |
Tinidazole |
% Assay |
% Assay |
|
1 |
100.6 |
98.4 |
2 |
96.1 |
99.9 |
3 |
98.6 |
100.5 |
4 |
98.2 |
98.8 |
5 |
98.3 |
101.4 |
6 |
100.5 |
100.7 |
Average |
98.4 |
99.9 |
SD |
1.435 |
1.157 |
% RSD |
1.16 |
1.157 |
Sensitivity
The sensitivity of measurement of Diloxanide furoate and Tinidazole by use of the proposed method was estimated in terms of the limit of quantitation (LOQ) and limit of detection (LOD). LOQ and LOD were calculated by the use of the equations LOD = 3.3s/S and LOQ = 10s/S where s is the standard deviation of response of calibration plots and S is the slope of the corresponding calibration plot. The limit of detection (LOD) for Diloxanide furoate and Tinidazole were found to be 68.53µg/ml and 97.87µg/ml respectively, while limit of quantitation (LOQ) for Diloxanide furoate and Tinidazole were found to be 207.677µg/ml and 296.6 µg/ml respectively.
Ruggedness
Ruggedness was evaluated by performing assay of the formulations by different analyst by injecting six consecutive injections of the sample at working concentration from the same homogeneous mixture of tablets. This study showed % RSDless than 2 concerning % assay for both the drugs which indicate that the method developed is rugged and hence can be understood that the method gives reproducible results irrespective of analyst (table 7).
CONCLUSION
A reverse phase HPLC isocratic method developed has been validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, limit of detection and limit of quantitation, for for simultaneous quantitative estimation of Diloxanide furoate and Tinidazole in Metroquin tablets. The developed method resulted in Diloxanide furoate eluting at 4.07 min and Tinidazole at 2.52 min. Diloxanide furoate exhibited linearity in the range 31.25-93.75μg/ml, while Tinidazole exhibited linearity in the range 37.5-112.5μg/ml. The precision is exemplified by relative standard deviations of 0.90% for Diloxanide furoate and 0.68% for Tinidazole. Percentage Mean recoveries were found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Diloxanide furoate and Tinidazole were found to be 68.53µg/ml and 97.87µg/ml respectively, while limit of quantitiation (LOQ) for Diloxanide furoate and Tinidazole were found to be 207.677µg/ml and 296.6µg/ml respectively.
ACKNOWLEDGEMENT
The authors would like to thank the management of Vijaya College of pharmacy, Hyderabad for providing the necessary facilities to carry out of this research work and also Chandra labs, Hyderabad for providing drugs in form of gift samples.
CONFLICT OF INTERESTS
Declared None
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