UV Spectrophotometric Method Development and Validation for the Determination of Fluoxetine Hydrochloride in Bulk Drug and Commercial Formulation

Fluoxetine hydrochloride is a second-generation antidepressant drug that is classified as a selective serotonin reuptake inhibitor (SSRI). It regulates by growing the quantity of a biological chemical called serotonin in the brain. [1]. It was initially developed to treat depression, but since receiving FDA approval in 1987, it is now often used to treat depression and several other conditions. Major depressive disorder, bulimia and bipolar I’ are all treated with this SSRI. ‘N-methyl-3-phenyl-3-[4-(trifluoromethyl) phenoxy] propan-1-amine; hydrochloride’ is the IUPAC name of fluoxetine hydrochloride, which has the molecular formula ‘C₁₇H₁₉ClF₃NO’ with a molecular weight of ‘345.8 g/mol’. It is essentially a crystalline solid that ranges from white to off white. Fluoxetine hydrochloride is freely soluble in methanol and ethanol [2]. Fluoxetine causes prolonged amounts of 5-HT in particular areas of the brain by blocking the serotonin reuptake transporter at the presynaptic terminal. Though, because to its comparatively low affinity for 5-HT, ‘dopaminergic, adrenergic, cholinergic, muscarinic, and histamine receptors’, FLU has a far more satisfactory adverse effect profile than previously identified classes of antidepressants, such as tricyclic antidepressant [3].

Fig 1: Structure of Fluoxetine hydrochloride

MATERIALS AND METHODS

Materials

A gift sample of pure fluoxetine hydrochloride was received from institute. Loba Chemie Pvt. Ltd. was the supplier of ‘potassium dihydrogen phosphate, potassium chloride, sodium hydroxide, sodium chloride, and hydrochloric acid’ (HCl). Every additional chemical and reagent employed in the investigation was of analytical grade. Prodep (sun pharma) the marketed product of fluoxetine hydrochloride (label claimed 20 mg) was purchased from local market.

Equipment’s

UV-visible spectroscopy (Shimadzu 1900i) was used with ‘1 cm path length quartz cell’, analytical weighing balance, and ultrasonic bath sonicator were used in this study.

Preparation of buffer solution

For the preparation of 1000 ml 6.8 pH phosphate buffer take ‘28.80 gm disodium hydrogen phosphate and 11.5 gm of potassium dihydrogen phosphate’ dissolved into few amounts of distilled water. Then take the 1000ml volumetric flask and pour it into the volumetric flask and make up the volume upto 1000ml [4].

For the preparation of 1000ml 7.4 pH phosphate buffer take ‘2.38gm disodium hydrogen phosphate, 0.19gm potassium dihydrogen phosphate and 8gm sodium chloride’ and mixed into few quantities of distilled water. Then take 1000ml volumetric flask and pour the solution into volumetric flask and make up the volume upto 1000ml [4,5].

For the preparation of 1000ml 1.2 pH HCl buffer take ‘2gm sodium chloride in water and add 7.0 ml HCl’ with it. Then take 1000ml volumetric flask and pour the solution into it and adjust the volume upto 1000ml[5,6].

Preparation of standard solution

After weighing 10 mg of fluoxetine hydrochloride bulk drug, the sample's stock solution was made by dissolving it in 9:1(buffer: methanol) ratio of HCl and phosphate buffer solutions with a pH level of 1.2, 6.8, and 7.4, respectively and methanol. Each buffer solution's aliquot sample (10 mL) was placed in a volumetric flask, diluted to 10 mL to provide a concentration range of ‘2–20 mcg/ml’, and its corresponding wavelength was examined.

Preparation of sample solution

Triturate and crushed the ten tablets of fluoxetine hydrochloride in fine powder. 10mg equivalent tablet powder was weighed. Then the sample was prepared by dissolving the drug into solvent in 9:1 ratio (buffer: methanol). In this process prepared the sample in the concentration range of 2 to 20µg/ml. Filtered the sample by whatman filter paper and measure the absorbance of sample at 226nm.

Validation of method

By analyzing linearity, accuracy, limit of detection, and limit of quantification according with the International Council for Harmonization's guidelines, the side-by-side equation approach was verified [7].

Linearity

Buffer solution at pH 1.2, 6.8, and 7.4 was used to make up the volume after 1.6, 1.8, and 2.0 ml of solution were shifted from the stock solution to separate 10 ml of the volumetric flask. Fluoxetine hydrochloride concentrations ranged from 2 to 20 mcg/ml [8].

Precision

The precision takes into account how closely a set of results determined by examining several samples of a single analyte agree with one another. ‘Repeatability’ (system precision) and ‘intermediate’ (method precision) investigations confirmed the approach's accuracy. To determine the technique precision, the system precision investigation was conducted by replicating the absorbance of fluoxetine hydrochloride three times on the same day and on separate days [9].

LOD and LOQ

The lowest analyte concentration in a sample that may be identified but is typically not measured is known as the limit of detection, or LOD. Equations i & ii were used to compute LOD and LOQ [10].  LOD=3.3 ×IS

LOQ=10 ×IS

  Where, I is the standard deviation of intercept and S is mean value of slope.

Result And Discussion:

Major depressive disorder, bulimia, panic disorder, and bipolar I are all treated with fluoxetine hydrochloride, a selective serotonin reuptake inhibitor. According to reports, the UV Spectroscopy approach makes it feasible to estimate fluoxetine hydrochloride significantly. This method's accurate, precise, and selective approach to fluoxetine hydrochloride measurement has been successful.

The absorption spectra for fluoxetine hydrochloride showed a maximum absorbance at 226nm wavelength in different pH. So, the absorbance of fluoxetine hydrochloride in different pH was checked at 226nm of maximum wavelength. For the bulk drug, the linearity was found to be between 2 and 20 mcg/ml.

Fig 2: Determination of ?_max of fluoxetine hydrochloride

Quantification of unknown materials using an equipment such as a UV spectrophotometer. The absorbance of fluoxetine hydrochloride bulk in different concentration was determined in different pH. Calibration curve was performed three times and the mean values ± standard deviation was reported as shown in Table 1 & Table 2.

Table 1: Calibration data of fluoxetine hydrochloride bulk drug in different pH

Table 2: data of Fluoxetine hydrochloride marketed formulation

For the bulk drug and the marketed formulation, the linearity was found to be between 2 to 20 mcg/ml. Fig 2 & Fig 3 displays the calibration curves. The linear regression equations for bulk drug were found to be y = 00.0463x + 00.0602, y = 00.0417x +00.0749, y = 00.0408x + 00.0015 and for the marketed formulation found to be y = 00.0412x + 00.0825, y = 00.0386x + 00.0829, y = 00.0374x + 00.0816 in 1.2, 6.8 and 7.4 pH buffer respectively. “The correlation coefficient” (r²) value of the bulk drug was detected to be 0.999, 0.999, 0.998 and for marketed formulation was detected to be 0.9994, 0.9993, 0.999 in 1.2, 6.8 and 7.4 pH buffer solution respectively.

Fig 3: Calibration curve of bulk drug in different pH

Fig 4: Absorbance vs concentration curve of marketed formulation in different pH

According to Table 3 & Table 4, the relative standard deviation for 3 reproduces was less than 2% for both bulk drug & marketed formulation and system precision. At varying pH values, the LOD and LOQ results displayed in Table 5 were determined to be satisfactory.

Table 3: Systems precision data for bulk drug fluoxetine hydrochloride.

Table 4: Systems precision data for marketed drug fluoxetine hydrochloride.

Table 5: Detection and quantification data of fluoxetine hydrochloride