Development and Validation of UV Spectrophotometric Method for the Determination of Aprepitant in Bulk and Capsule Formulation

In conjunction with other antiemetic medications, Aprepitant, an antiemetic and substance p/neurokinin 1 (NK1) receptor antagonist, is suggested for the prevention of acute and delayed nausea and vomiting related to initial and repeat rounds of highly emetogenic cancer chemotherapy. Human substance P/neurokinin 1 (nk1) receptors are selectively and highly affinitized by Aprepitant. The targets of current treatments for chemotherapy induced nausea and vomiting include serotonin (5-HT₃), dopamine, and corticosteroid receptors. Aprepitant has little to no affinity for these receptors (CINV). [1][2][3]

Figure A: Chemical Structure of Aprepitant

A review of the literature finds that some research has been done on the HPLC determination of Aprepitant in plasma and pharmaceutical formulations. To the best of our knowledge, no UV approach for analyzing Aprepitant in pharmaceutical formulations has been published in the literature [4][5][6][7]. The goal of this project is to develop and validate a rapid, simple, reliable, selective, sensitive, and low-cost UV Spectrophotometric method for determining Aprepitant in bulk drugs and commercial pharmaceutical formulations like capsules.

Drug Profile of Aprepitant [8]

Drug Name:  Aprepitant

Category: Anti-emetics

Chemical formula: C₂₃H₂₁F₇N₄0₃

IUPAC Name: 5-([(2R, 3S)-2-((R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy)-3-(4-               fluorophenyl)morpholino]methyl)-1H-1,2,4-triazol-3(2H)-one

Molecular Weight: 534.427g/mol

Melting point: 251-255ºC

Half life: 9-13 Hours

Mechanism of Action

Aprepitant is a highly specific antagonist of the G-protein coupled neurokinin-1 receptor [9][10][11]. Both the central and peripheral nervous systems have neurokinin-1 receptors. Substance P, a nociceptive neurotransmitter, is their major ligand. The central pattern generator, nucleus tractus solitarius (NTS), and region postrema are hypothesised to play essential roles in the vomiting reflex (Aprepitant). [12][13][14]

Since central neurokinin-1 receptors are located throughout these regions, aprepitant can be widely bound in these crucial areas. Aprepitant is anticipated to dramatically reduce the chance of the complicated vomiting reflex beginning by acting as a competitive antagonist in these locations. [15] Neurokinin-1 (NK-1) receptors can be found throughout the gastrointestinal system. The Aprepitant's binding to NK-1 receptors may decrease vagal afferent impulses, contributing to the antiemetic action. The proposed mechanism for the antipruritic action is antagonism of substance P, an endogenous NK1 receptor ligand.

Pharmacokinetics

Absorption: Aprepitant has a nonlinear pharmacokinetic profile. The Aprepitant's bioavailability declines with increasing dose, with the mean oral bioavailability being 67% for the 80 mg Aprepitant capsules and 59% for the 125 mg Aprepitant capsules. T max (time to peak plasma concentration) is about four hours. [16]

Distribution: Aprepitant has a high plasma protein binding (about 95%) and a high volume of distribution (Vss) of 70 L at steady state.

Metabolism: Aprepitant is metabolised in the liver via P450 CYP3A4 and CYP2C9, as well as CYP1A2 (minor) and CYP2C19 (minor). Main metabolites are N- and O-dealkylation products which are pharmacologically inactive. It is critical to understand that Aprepitant is both a dose-dependent inhibitor and inducer of CYP3A4. [17]

Excretion: Aprepitant metabolites are eliminated roughly 50% in urine and 50% in faeces.

ICH Guidelines (ICH Q2R1) for Analytical Procedure and Validation [18]

The analytical procedure refers to the manner of performing the analysis. It should detail the steps required to execute each analytical test. This includes, but is not limited to, the preparation of the sample, the reference standard, and the reagents, the use of the apparatus, the development of the calibration curve, the application of the formula for the calculation, and so on.

The subject of analytical procedure validation focuses on the four most frequent types of analytical procedures:

• Identification examinations
• Quantitative tests for impurity content - Limit tests for impurity control
• Quantitative analyses of the active moiety in drug substance or drug product samples, or other specified component(s) of the drug product

The analytical procedure's goal should be clearly known because it will govern the validation qualities that must be examined. The following are some typical validation factors to consider:

• Accuracy         
• Precision
• Repeatability
• Intermediate Precision
• Specificity
• Detection Limit
• Quantitation Limit
• Linearity
• Range

Moreover, revalidation may be required in the following situations:

• Modifications in the manufacture of the medicinal ingredient
• Changes in the finished product's composition
• Changes in the analytical procedure

EXPERIMENTAL

MATERIALS AND METHODS

Aprepitant was procured from Piramal Pharma Limited, Telangana. All the reagents were of analytical grade. The commercially available two marketed tablet brand containing Aprepitant, 125mg and 80mg in each tablet have been used for estimation.

Preparation of Standard stock solution

Weigh accurately and transfer about 80 mg of Aprepitant working standard into 100 ml volumetric standard flask. Add 50ml of Mobile phase. Sonicate for dissolve and makeup with the volume with mobile phase and mix.

Further take 5 ml of this solution in 50ml volumetric flask and make up to volume with mobile phase. Filter it through 0.45-micron syringe filter.

Preparation of sample solution:

Take equivalent to one capsule( i.e., 80 mg equivalent of Aprepitant) into 100 ml volumetric flask add 50ml of Mobile phase and keep the flask in sonicator for 30 minutes make up the volume up to mark with mobile phase and mix.

Further take 5 ml of this solution in 50ml volumetric flask and make up to volume with mobile phase. Filter it through 0.45-micron syringe filter.

Validation of method

The method was validated with respect to linearity and range, accuracy and limit of detection (LoD), limit of quantification (LoQ).

Linearity and range

The prepared aliquots (5-15μg/mL) were scanned for absorbance at λ_max Value 264nm. The absorbance range was found to be 0.1171-0.3513. These solution obeyed Beer-Lambert’s law in above concentration range with regression of 1.0000

Accuracy

The standard solution, Accuracy -80%, Accuracy -100% and Accuracy -120% solutions were injected. The amount found and amount added for Aprepitant mean recovery values were calculated and the results were summarized.

Limit of Detection (LoD) and Limit of Quantification (LoQ)

LoD (k =3.3) and LoQ (k =10) of the method was established according to ICH definition(C1 =ks/s, where C1 is LoD or LoQ, S is the mean standard deviation of blank determination, Sis the slope of standard curve and k is the constant related to confidence interval).LoD and LoQ of method reported.

RESULT AND DISCUSSION

ABSORBANCE MAXIMA

Figure B: UV Spectrum of Aprepitant in Sample solution

Discussion: The maximum absorbance obtained by 264 nm.

Figure C: Linearity curve for Aprepitant

Table1: Result for Linearity

Discussion: Five linear concentrations of Aprepitant were absorbed in a duplicate manner. Average absorbance were mentioned above and linearity equations obtained for Aprepitant y = 43.2401x0.0013. Correlation coefficient obtained R² =0.998 for the Aprepitant.

Table 2: Result for Accuracy of Aprepitant