INTRODUCTION:

Itraconazole, chemically known as 2-butan-2-yl-4-[4-[4-[4-[[(2R,4S)-2-(2,4-dichlorophenyl)-2- (1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-1,2,4-triazol-3-one belongs to a class of drugs known as azole antifungals used to treat systemic and superficial fungal infections like aspergillosis, blastomycosis, coccidioidomycosis, histoplasmosis and paracoccidioidomycosis.

ITZ inhibits ergosterol synthesis, which helps to maintain the cell membrane in fungi which leads to fungal membrane abnormalities that increase permeability and disrupt fungal cell membrane integrity^1-3. (Fig 1)

Fig. 1: Chemical structure of ITZ

Secnidazole, chemically known as 1-(2-methyl-5-nitroimidazol-1-yl) propan-2-ol is a nitroimidazole antibiotic used to treat bacterial vaginosis. It works by killing bacteria or preventing their growth by damaging their DNA. They are also effective in the treatment of amoebiasis, giardiasis, trichomoniasis and bacterial vaginosis^4-⁶. (Fig 2)

Fig. 2: Chemical structure of SEC

The combination of ITZ and SEC is used in treatment of Oral to Vaginal infections of diverse etiology. It is used to treat Vulvovaginitis, Tricomonas Vaginalis and bacterial vaginosis⁷.

On literature survey it was found that ITZ and SEC have been estimated individually as well as simultaneously in combination with other drugs. It was found that a chemometric analysis has been employed for their simultaneous estimation in combined dosage form⁸. However, no other methods have been developed and neither any method is available in pharmacopoeia. In the view of the need for a suitable method for routine analysis in combined formulations, attempts have been made to develop simple, precise & accurate analytical method for the simultaneous estimation of titled drugs.

EXPERIMENTAL:

Chemicals and Reagents:

ITZ was procured from Anvik Biotech, Sonipat, Haryana as a gift sample where as SEC pure drug was procured from Lincoln Pharmaceuticals, Gujarat, India. The combined formulation was obtained from the local market. All chemicals and reagents used were of analytical grade.

Instrumentation:

The present work was carried out by using Shimadzu model equipped with SPD-20AD UV detector, isocratic pump LC- 20 AD and loop injector. Chromatographic analysis was performed on Enable C18 column having 250mm x 4.6mm i.d. and 5µm particle size. All the drugs and chemicals were weighed on Shimadzu electronic balance.

Chromatographic conditions:

Enable C18 column (250 x 4.6mm i.d, 5μ) was used for separation. The mobile phase consists of methanol: PDP buffer (adjusted to pH 5.5 with triethylamine) in the ratio of 90:10v/v with a flow rate of 1.5ml/min with detection at wavelength 257nm. The Injection volume was 20μl and the analysis was performed at ambient temperature.

Preparation of mobile phase:

Preparation of buffer solution: 0.136g of potassium dihydrogen phosphate was dissolved in 100ml of HPLC grade water (0.01M).

The mobile phase was prepared by mixing methanol and 0.01M potassium dihydrogen phosphate having pH 5.5 in the ratio of 90:10v/v and sonicating for 15min followed by filtration through 0.45µm membrane filter.

Preparation of standard stock solution of ITZ and SEC:

Accurately weighed 100mg of ITZ and 100mg of SEC pure drug was transferred into separate 100ml volumetric flasks and was dissolved using methanol: PDP buffer and subjected to sonication for 15 min followed by filtration and diluted up to the mark with the same mobile phase (1000µg/ml). From these stock solutions further dilutions were made to obtain 10, 20, 30, 40 and 50µg/ml of ITZ and SEC.

Preparation of sample stock solution:

20 tablets were taken and the average weight of one tablet was found out. All the tablets were finely powdered and the amount equivalent to 100mg of ITZ was taken and transferred to 100ml volumetric flask (which also contains SEC). Dissolved it using methanol: PDP buffer and the mixture was sonicated for 15 min followed by filtration and diluted up to 100ml with methanol: PDP buffer (1000µg/ml). From this stock solution further dilutions were prepared to bring the drugs in their working concentration range.

Method validation:

The developed RP-HPLC method was validated for the following parameters as per ICH guidelines^9-11.

Specificity:

It refers to the analytical method ability to distinguish between the analyte and other impurities in the sample. The Retention time of ITZ was found to be 6.037 minutes while that of SEC was found to be 3.788 minutes. There was no interference observed for blank sample at the retention times of ITZ and SEC which confirms the specificity of the method. The chromatograms of blank, ITZ, SEC and drug mixture are shown in Fig. 3-6 respectively.

Fig. 3: Chromatogram of Blank at 257nm

Fig. 4: Chromatogram of ITZ at 257nm

Fig. 5: Chromatogram of SEC at 257nm

Fig. 6: Chromatogram of mixture of ITZ and SEC at 257nm

Linearity:

The linearity of the method was determined at five concentration levels ranging from 10-50µg/ml for both ITZ and SEC. The peak areas of each concentration were recorded and a calibration curve was constructed by plotting peak area versus concentration. The regression coefficients, slope and intercept were calculated from the curve. The calibration curve of both the drugs is shown in Fig. 7 and 8. The statistical data for the calibration curve of ITZ and SEC is given in the Table 1.

Fig. 7: Calibration curve for ITZ at 257 nm by RP-HPLC method

Fig. 8: Calibration curve for SEC at 257 nm by RP-HPLC method

Table 1: Statistical data of ITZ and SEC at 257 and 311nm by RP-HPLC method

SI. No	Concentration (µg/ml)		Area
SI. No	ITZ	SEC	ITZ	SEC
1	10	10	519824	199205
2	20	20	965936	308569
3	30	30	1387059	429556
4	40	40	1874823	574492
5	50	50	2354721	681395

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value and the value found. Recovery studies were carried out by adding 80%, 100%, 120% of the standard drug solution of ITZ and SEC to the known amount of sample solution by standard addition method (Table 2). The Statistical validation data for accuracy determination is shown in Table No 3. The assay result of formulation is shown in Table 4.

Precision:

The precision of an analytical procedure is the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

The intra-day precision was carried out by analyzing ITZ 40 μg/ml and SEC 10μg/ml at 3 different time points on the same day. The inter-day precision was carried out by analyzing ITZ 40μg/ml and SEC 10μg/ml at 3 different time points on 3 different days. The statistical validation data for both intra-day and inter-day precision has been given in the Table 5.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection and quantification of the drug was calculated with the standard deviation and slope.

LOD = 3.3*σ /S

LOQ = 10*σ /S

σ = Standard deviation

S = Slope of the calibration curve

Robustness:

Robustness of a method is a measure of its capacity to remain unaffected by small and deliberate changes that are made in method parameters. Robustness was performed by altering the flow rate (± 1ml/min) and wavelength (± 2 nm). It was observed that even with small changes in method conditions there was no marked change in the results which implies that the developed method was robust. The robustness results were within the accepted limits of ≤ 2 % RSD. The result is summarized in Table No. 6.

Ruggedness:

Ruggedness is the degree of reproducibility of test results obtained by the same samples under variety of test conditions within the specified conditions. The solution containing 40 µg/ml of ITZ and 20 µg/ml of SEC was injected three times by different analysts and the retention times and tailing factors were recorded. The results were summarized in Table No. 7.

RESULTS AND DISCUSSION:

It was discovered that the developed RP-HPLC method was accurate, precise and robust. The method was validated in accordance with ICH guidelines. The retention times were found to be 6.037 min and 3.788 min for ITZ and SEC respectively with a run time of 8 min which allows the analysis of a large number of samples in a short period of time. The linearity of this method was assessed by regression analysis of the standard calibration curve. The calibration curves were linear over the concentration of 10-50 µg/ml with correlation co-efficient = 0.999 and 0.998 for ITZ and SEC respectively and are depicted along with the summary of system suitability parameters and validation in Table No. 8. The intra-day and inter-day precision (%RSD) at different concentration levels was found to be less than 2%. The recovery studies were carried out which confirmed the accuracy of the proposed method. The calculated LOQ and LOD concentrations confirmed that the methods were sufficiently sensitive.

Table 2: Determination of accuracy of ITZ and SEC

Level of % recovery	Amount taken from formulation (µg/ml)		Amount of standard drug added(µg/ml)		Total amount recovered (µg/ml)		% Recovery
Level of % recovery	ITZ	SEC	ITZ	SEC	ITZ	SEC	ITZ	SEC
80%	9	12	7.2	9.6	16.19	21.59	99.93	99.95
	9	12	7.2	9.6	16.18	21.58	99.87	99.90
	9	12	7.2	9.6	16.10	21.57	99.38	99.86
100%	9	12	9	12	17.99	24.01	99.94	100.04
	9	12	9	12	17.95	23.99	99.72	99.95
	9	12	9	12	18.01	23.98	100.05	99.91
120%	9	12	10.8	14.4	19.72	26.39	99.59	99.96
	9	12	10.8	14.4	19.75	26.41	99.74	100.03
	9	12	10.8	14.4	19.82	26.38	100.10	99.92

Table 3: Statistical validation data for accuracy determination

Level of % Recovery	Mean*		Standard Deviation*		Co-efficient of Variation*		Standard Error*
Level of % Recovery	ITZ	SEC	ITZ	SEC	ITZ	SEC	ITZ	SEC
80%	99.98	100.01	0.02517	0.03512	0.000251	0.000351	0.01455	0.0203
100%	100.02	99.99	0.05132	0.04726	0.000513	0.000472	0.02966	0.02732
120%	99.99	99.98	0.04726	0.02517	0.000472	0.000251	0.02732	0.01455

*n = 3

Table 6: Robustness result for variations in flow rate and wavelengths

Method Parameter - Flow Rate (ml/min)
Flow rate (ml/min)	Level	Retention Time		Tailing factor
Flow rate (ml/min)	Level	ITZ	SEC	ITZ	SEC
1.4	-1	5.989	3.657	1.314	1.253
1.5	0	6.037	3.788	1.41	1.38
1.6	+1	6.102	3.801	1.510	1.417
Method parameter- Wavelength (nm)
Detection (nm)	Level	Retention time		Tailing factor
Detection (nm)	Level	ITZ	SEC	ITZ	SEC
255	-2	5.979	3.638	1.343	1.262
257	0	6.037	3.788	1.41	1.38
259	+2	6.12	3.813	1.565	1.409

Table 7: Ruggedness result for variations in analyst

Method Parameter	Retention Time		Tailing factor
Analysts	ITZ	SEC	ITZ	SEC
Analysts 01	6.037	3.788	1.41	1.380
Analysts 02	6.041	3.801	1.398	1.378

Table 8: Summary of validation and system suitability parameters of ITZ and SEC

Parameters	ITZ	SEC
Linearity range (µg/ml)	10-50	10-50
Retention time (min)	6.037	3.788
Tailing factor	1.41	1.38
Resolution factor	6.300
Theoretical plate	3164		2696

CONCLUSION:

The developed RP-HPLC method for the simultaneous estimation of ITZ and SEC in bulk and pharmaceutical formulation was found to be simple, precise and accurate. The devised technique was validated in accordance with ICH guidelines. The solvent used was methanol: potassium dihydrogen phosphate buffer (PDP) pH 5.5(90:10v/v) which was economical. The proposed method demonstrated high recoveries and the %RSD values were discovered to be less than 2%, confirming its accuracy and precision. Since this approach is straightforward, it may be used in laboratories that lack complex analytical equipments like LC-MS/MS or GC-MS/MS. Therefore, this approach may be used for the routine quality control of ITZ and SEC in bulk and formulations.

ACKNOWLEDGEMENT:

The authors are thankful to Anvik Biotech, Haryana and Lincoln Pharmaceuticals, Gujarat, India for providing the gift samples of Itraconazole and Secnidazole. The authors are also thankful to National College of Pharmacy, Shivamogga for providing necessary facilities to carry out research work.

REFERENCES:

1. https://www.drug bank.ca/drugs/Itraconazole.

2. Ahmed M, Sanjana N R, Shetty S A. Development and validation of UV Spectrophotometric methods for the simultaneous estimation of Itraconazole and Terbinafine hydrochloride in bulk drug and pharmaceutical formulations. Int J Pharm Ph Res. 2020; Vol. 18 (3): 486-495.

3. https://www.ncbi.nlm.nih.gov/Itraconazole.

4. https://www.drug bank.ca/drugs/Secnidazole.

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9. Jitta SR, Bhaskaran NA, Shirodkar RK. Development and Validation of RP-HPLC method for Quantification of Total, Free and Entrapped Ritonavir in Lipid Nanocarriers and Drug content of Film Coated Fixed Dose Formulation. Indian J Pharm Ed Res. 2022; 56(1): 240-246.

10. Bharti A, Kumbhare RM, Jeyaseelan C. HPLC Method Development and Validation for the Quantification of Related Impurities in Testosterone Cypionate Active Pharmaceutical Ingredient. Indian J Pharm Ed Res. 2022; 56(3s): s547-s558.

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Received on 23.08.2023 Modified on 29.02.2024

Asian J. Res. Pharm. Sci. 2024; 14(3):216-220.

DOI: 10.52711/2231-5659.2024.00035

Components	Mean*	Standard Deviation*	Co-efficient of Variation*	Standard Error*
ITZ	99.94	0.9511	0.0095	0.3882
SEC	99.9	0.7409	0.0074	0.30243

Components		Mean*	Standard Deviation*	Co-efficient of Variation*	Standard Error*
ITZ	Intra-day	100.03	0.208973	0.002089	0.08564
ITZ	Inter-day	99.68	0.42334	0.004247	0.09984
SEC	Intra-day	100.86	0.663325	0.006576	0.27185
SEC	Inter-day	100.02	0.90673	0.009065	0.21385