Development And Validation for Simultaneous Estimation of Itraconazole and Terbinafine HCL By HPLC

Itraconazole (ITZ) is an antifungal medication that is a white to nearly white powder. Its molecular formula is C35H38Cl2N8O4, and its molecular weight is 706 g/mol. Its chemical formula is 4-[4-[4-[4-[[cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)- 1,3dioxolan-4-yl]methoxy] phenyl]piperazin-1-yl]phenyl]-2-[(1RS)- 1methylpropyl]-2,4-dihydro-3H-1,2,4 triazol-3- one] [1]. The TRB's structure was displayed in figure 1[2].

291.4 g/mol. The TRB's structure was displayed in figure 2 [3].

Table 1: Drug Profile of Itraconazole and Terbinafine

Chronic fungal infections can be treated with this medication for long-term maintenance due to its minimal toxicity profile of ITZ.[4] In the United States, itraconazole was authorised for medicinal use in 1992 after being patented in 1978[5]. It is included among the essential medications by the World Health Organisation [6]. Both itraconazole and terbinafine HCl are insoluble in water but readily soluble in acetonitrile, methanol, and dimethyl sulfoxide [7,8]. Antifungal illnesses like toenail onychomycosis are treated with a combination of itraconazole and terbinafine HCl, which inhibits fungal development by inhibiting covering [9]. According to the literature review, no reversed-phase high-performance liquid chromatography (RP-HPLC) technique has been documented for the estimate of terbinafine HCl with itraconazole in tablet form [10-14]. By preventing the creation of the sterol components of the fungal membrane, the azole antifungal medications work. They prevent lanosterol from being demethylated into ergosterols, the main sterol of fungal membranes, by inhibiting C-14 alpha demethylase. Fungal cell development is inhibited by this inhibitor because it alters the shape and function of membranes [15]. Compared to fluconazole, itraconazole had a wider range of activity. It has anti-aspergillus properties. Additionally, it has a license for use. in onchomycosis, histoplasmosis, sporotrichosis, and blastomycosis. It hardly penetrates the cerebral fluid and is more than 99 percent protein bound. As a result, it shouldn't be utilised for CNS injections or meningitis treatment [16]. Headache, nausea, diarrhoea, and abdominal pain are common side effects; more serious ones could include Heart failure, allergic reactions, Stevens-Johnson syndrome, and linear issues [17]. Terbinafine and itraconazole, a fixed-dose combination antiretroviral drug used to treat fungal infections, were first made available on the market in the combined dosage form known as "Itrogen-TR." It tends to accumulate in fatty tissues, skin, and nails due to its high lipophilicity One Terbinafine, like other allylamines, prevents the creation of ergosterol by blocking the enzyme fungal squalene monooxygenase, also known as squalene epioxidase, which is a component of the fungal cell wall manufacturing process. Through the suppression of the enzyme cytochrome P450 14?-demethylase, itraconazole is a highly selective inhibitor of fungal cytochrome P-450 sterol C-14 ?-demethylation. This enzyme is necessary for the formation of fungal cell walls and changes lanosterol into ergosterol. Toothers The According to a survey of the literature, there aren't many analytical techniques available for the simultaneous measurement of Terbinafine Itraconazole in biological samples, pharmaceutical dosage forms, and bulk. They are LC-MS/MS, HPLC, and UV Spectrophotometric techniques. There aren't many known analytical techniques for Terbinafine Itraconazole in pharmaceutical formulations and bulk. They are UPLC, HPLC, and UV spectrophotometric techniques. concurrent estimate Itraconazole Terbinafine of Therefore, an effort has been made to provide a straightforward, accurate, sensitive, dependable, and economical stability indicating RP-HPLC approach for concurrent measurement of itraconazole and terbinafine in pharmaceutical dosage form and bulk [18].

Solution preparation:-

For separation, a Phenomenex Luna C18 column (250 x 4.6mm, 5µ) was utilised. mobile phase made up of a combination of buffer and acetonitrile 7.8 gm of Delivered at a flow rate of 1.0 ml/min with detection at 210 nm, sodium dihydrogen orthophosphate and 1.8 gm of hexane sulfonic acid in a 50:50 v/v ratio in 1000 ml of water with pH adjusted to 4 were measured. After passing through a 0.45-inch nylon filter, the mobile phase was sonicated for fifteen minutes. The analysis was carried out at room temperature.

Standard stock solution preparation:-

25 mg of itraconazole and 62.5 mg of terbinafine were precisely weighed and then put to 50 ml volumetric flasks individually. Three-quarters of the diluents were added to the flask and gave it a 10-minute sonication. Diluents were added to the flask, which was marked "Standard stock solution." 500 µg/ml of itraconazole and 1250 µg/ml of terbinafine One millilitre of the stock solution was pipetted out of the aforesaid solution, transferred to a ten ml volumetric flask, and diluted. (50µg/ml of itraconazole and 125µg/ml of terbinafine).

Methodology for tablet analysis :-

After weighing 10 tablets and calculating their average weight, the weight of one tablet was placed into a 100 ml volumetric container. flask, followed by the addition of 25 ml of diluents and 25 minutes of sonication. The volume was then adjusted using the diluent and filtered using HPLC filters (2500 µg/ml of Terbinafine and 1000 µg/ml of itraconazole).

Validation Of Methods:-

Precision-

The method's reproducibility was confirmed by figuring out the percentage RSD of 8 replicate injections at 100% concentration (125 ?g/ml of Terbinafine and 50?g/ml of itraconazole) on the same day, and for intermediate precision, the percentage RSD was computed from multiple investigations conducted on various days.

Accuracy-

Terbinafine and itraconazole recovery percentage experiments at three distinct concentration levels (50, 100, and 150%) were used to assess accuracy. The amount recovered and the amount added were used to compute the recovery percentage. The fact that the recovery percentage was within the acceptable range shows how accurate the procedure was. (Admissibility criterion: recovery percentage in between 98 and 102).

Linearity-

Itraconazole and Terbinafine standard aliquots were used to create six working solutions with concentrations ranging from 23.5-78µg and /mL. 36.28-165.5µg/mL. Plotting calibration curves with observed peak areas against concentration was done, and then regression equations and the correlation coefficient on the curves for itraconazole and terbinafine were determined.

LOD & LOQ-

Using the formulas LOD = 3?/s and LOQ = 10 ?/s, the LOD and LOQ were determined using the calibration plot's slope(s) and the peak areas' standard deviation (SD).

Robustness-

By adjusting the chromatographic parameters, such as flow rate, mobile phase ratio, and temperature, the method's robustness was confirmed; nevertheless, no discernible changes were observed in the results, which all fell within the acceptable range as per ICH criteria. Samples were injected in duplicate while maintaining robustness conditions such as flow minus (0.7 ml/min), flow plus (0.9 ml/min), 65:35 mobile phase minus 55:45 mobile phase plus, temperature minus (22°C), and temperature plus (37°C). The criteria for system appropriateness was met. The percentage RSD was not exceeded.

Chromatogram of ITR & TER:-