HPLC Quantification of Aprepitant in Bulk and Capsules

Aprepitant (APT) is (SP) / neurokinin -1 (NK1) receptor antagonist chemically as 5-[(2R,3S)-2-[(1R)-1-(3,5-bis(trifluoromethyl)phenyl) ethoxy]-3- (4-fluorophenyl)-4 morpholinylmethyl-1,2dihydro-3H-1,2,4-triazol-3-one (fig. 1). It is a white to off-white crystalline solid, with a molecular weight of 534.43 and empirical formula of C₂₃H₂₁F₇N₄O₃. APT is a selective high affinity antagonist of human substance P/neurokinin 1 (NK1) receptors and it has little or no affinity for serotonin (5-HT3), dopamine, and corticosteroid receptors. A large number of drugs are available for prevention of Postoperative nausea and vomiting¹ of which 5-HTs receptor antagonists have occupied an important position because of their better efficacy and side effect profile with a disadvantage that it prevents only acute emesis.

A newer class of drugs namely neurokinin receptor antagonists provides an additional advantage of preventing both acute and delayed emesis. Various NK1 receptor antagonists include APT, GR-205171, CP-122721 and CJ-11974, of which APT has been approved for Postoperative nausea and vomiting and treatment of nausea in cancer chemotherapy, APT has been shown in animal models to inhibit emesis induced by cytotoxic chemotherapeutic agents, such as cisplatin, via central actions. Animal and human Positron Emission Tomography (PET) studies with APT have shown that it crosses the blood brain barrier and occupies brain NK1 receptors² and also showed that APT augments the antiemetic activity of the 5 HT3 receptor antagonist ondansetron and the corticosteroid dexamethasone and inhibits both the acute and delayed phases of cisplatin-induced emesis it has been recently demonstrated that substance P (SP) and neurokinin -1 (NK1) receptor antagonists induce cell proliferation and cell inhibition in human melanoma cells.

Literature review reveals that very few analytical methods has been established for the estimation of APT and its metabolite in human plasma^3,4,and other methods^5-9

Figure 1: Chemical structure of Aprepitant

Only one method was reported for the determination of APT in presence of its degradation products in oral liquid formulation in the literature. The objective of this work was to develop a new, simple, economic, rapid, precise and accurate HPLC method for quantitative analysis of Aprepitant and to validate the method in accor dance with ICH guidelines Q2(R1)¹⁰and apply the method to capsule formulation.

MATERIALS AND  METHODS

Aprepitant pure drug (API) was procured from Glenmark Pharmaceuticals Ltd.

1. Instrument Used

• Electronic Weighing Balance : Sartorius TE 214 S
• Ultrasonicator : RC Systems MU 1700
• UV–Visible Spectrophotometer : Shimadzu 1900i (LabSolutions software)
• Digital pH Meter : Labman
• Vacuum Pump : Servewell Instruments Pvt. Ltd.
• Membrane Filter : Supor 200, 0.45 µm (Pall India Pvt. Ltd)
• Hot Air Oven

2. HPLC System

• Liquid Chromatograph : Shimadzu LC-10AT
• Detector : UV–Visible Detector (Shimadzu SPD-10A)
• Analytical Column : BRISA LC2 C18 (250 mm × 4.6 mm, 5 µm)
• Data Processor : LC Solution Software, Version 2.1.4.93
• Injector : Rheodyne 7725i (Fixed loop of 50 µL)
• Syringe : Hamilton, 20 µL

3. Chemicals and Reagent

• Acetonitrile (HPLC grade)
• HPLC Grade Water
• Potassium Dihydrogen Orthophosphate Buffer (20 mM, HPLC grade)
• Aprepitant (Standard API)
• Orthophosphoric Acid (1% v/v, AR grade)
• Aprepitant Capsules

EXPERIMENTAL METHODOLOGY                                                                                    PREPARATION OF PHOSPHATE BUFFER

20mM of Phosphate buffer was prepared by dissolving 680.4 mg of potassium dihydrogen orthophosphate in 250 mL of water adjust the pH to 3.0 with 1% of Phosphoric acid. The working mobile phase was prepared in the ratio of 85:15 (Acetonitrile: Phosphate buffer) filtered, degassed and sonicated for 10 min.

PREPARATION OF STANDARD APREPITANT SOLUTION

Accurately weighed 10mg of Aprepitant standard was transferred into 10mL volumetric flask, 3-5mL of Mobile phase was added and sonicated for 5 min to dissolve it completely and the volume was made up to 10ml the volume with mobile phase to get 500µg/mL of standard Aprepitant solution and labelled as STD Stock.

PREPARATION OF SAMPLE APREPITANT SOLUTION

Aprepitant 1 capsule (Aprecap 80) were accurately weighed and their average weight was calculated. Accurately weighed a quantity of powder containing 114mg of Aprepitant and transferred to 10ml volumetric flask, solubilized in 10ml of mobile phase and sonicated for 15mins. After sonication the volume was made up to the 10ml mark with the mobile phase to obtain final concentration of 500µg/mL of Aprepitant and was labelled as ‘SMP STOCK’ and filtered through Whatman Filter paper (#41).

From ‘SMP STOCK’ 5mL was transferred to a 10mL volumetric flask and volume was made up to the mark with mobile phase to obtain a concentration of 500µg/mL of Aprepitant and labelled as ‘FINAL SMP’.

BLANK, FINAL STD STOCK and FINAL SMP solutions were filtered through 0.45µm nylon membrane filter and 20µL was injected into the HPLC system under standardized chromatographic conditions to get a stable baseline and to observe for peak of Aprepitant and any extra peak for 15 mins. The chromatograms obtained is represented.

SELECTION OF ANALYTICAL WAVELENGTH:

Using a Shimadzu 1900i UV-Visible Spectrophotometer, a standard solution of Aprepitant (500 µg/mL) made in a mobile phase of Acetonitrile and 20 mM Phosphate Buffer (pH 3.0) in a 85:15 (v/v) ratio was scanned in the UV range of 200–400 nm. At 264 nm, the highest absorbance (λmax) was recorded.

Fig 2: UV Spectra of Aprepitant(500µg/ml)

HPLC CHROMATOGRAPHIC CONDITIONS:

The stationary phase in reversed-phase chromatography was a Phenomenex BDS C18 column  (250 mm× 4.6 mm, 5 µm). The mobile phase was made up of 85:15 (v/v) Acetonitrile and phosphate buffer (pH 3.0), filtered through a 0.2 µm nylon membrane, the mobile phase was filtered and sonicated to remove any dissolved gases. 1.0 mL/min was the fixed flow rate. Twenty minutes was set as run duration, and 20 µL of the sample was injected once the column had been equilibrated with the mobile phase, after a stable baseline. A wavelength of 264nm was used for the analyte's detection. The chromatogram is presented in Fig 3.

Fig3: Chromatogram for Aprepitant (500µg/ml) in MP containing Acetonitrile: Phosphate buffer 20mM pH 3.0, (85:15) at 264nm

VALIDATION OF RP-HPLC METHOD:

RP-HPLC method developed for determination of Aprepitant was Validated as per ICH guidelines Q2 (R1) for various parameters. Results obtained are presented below.

SPECIFICITY

Specificity was performed to determine that there is no interference of excipients with peak of   Aprepitant in standard and sample solutions.

Fig 4: Overlain Chromatogram for specificity studies of Aprepitant (Standard and Sample)

LINEARITY AND RANGE

The linearity of an analytical procedure specifies the results which are directly proportional to the concentration of analyte in the sample. The linearity and range were determined from coefficient of correlation (R2) obtained by plotting AUC vs. CONCENTRATION at 264nm. The results obtained and calibration graph prepared is presented below.

Table 1: Data for concentration and peak area for the linearity studies of Apripetant

Fig5: Calibration graph for linear  ity and range for Aprepitant

Table 2: Linear Regression Analysis Data for Calibration Curve of Apripetant

LOD and LOQ

The lowest amount that can be detected and quantified was calculated from the respective calibration curve.

The LOD and LOQ were calculated using

LOD = 3.3 × Standard Deviation of Y-intercept

               Average slope of six calibration curves

LOQ = 10 × Standard Deviation of Y-intercept

               Average slope of six calibration curves

Table 3: Data for LOD and LOQ Studies for Aprepitant

ACCURACY

Accuracy studies were performed at three different levels (80%, 100% and 120%) and the % Recovery of Aprepitant were calculated and presented below.

Table 4: Percentage Recovery data for accuracy studies at three different levels

Fig 6: Overlain Chromatogram for Accuracy studies of Aprepitant at three different level

PRECISION

The precision of an analytical method was studied by performing intra-day, inter-day precision, repeatability and reproducibility studies.

Intra- Day and Inter- Day Precision

Intra-Day and Inter- Day Precision was performed to determine whether the developed method gives consistent results at different time intervals on the same day and for three consecutive days. The results obtained have been tabulated below.

Table5: Data for Intra-day and Inter-day precision studies for Aprepitant

REPEATABILITY

The test for repeatability was performed to check whether the developed method gives consistent results with same solution on the same day and same time. The chromatogram and data obtained are presented below.

Table 6: Data for Repeatability studies of Aprepitant

Fig7: Overlain Chromatogram for Repeatability studies of Aprepitant

REPRODUCIBILITY

Reproducibility was performed to ensure that the method is precise when different analysts performed the same analysis with the same method. The results obtained are presented below

Table 7: Data for reproducibility studies of Aprepitant

ROBUSTNESS

Robustness was evaluated on the basis of system suitability parameters to assess whether the system suitability parameters change with small but deliberate variations. The system suitability parameters for standard solution of Aprepitant were studied with variation in flow rate and organic phase ratio.

1. CHANGE IN FLOW RATE

Small but deliberate changes in flow rate (±3) were tested and the chromatogram and the data  obtained is presented below.

Table 8: Data for variation in Flow rate for Aprepitant

2. CHANGE IN WAVELENGTH

Small but deliberate changes in wavelength (±3nm) was tested and the chromatogram and the dat  obtained are presented below:

Table 9: Data for variation in wavelength for Aprepitant

SYSTEM SUITABILITY PARAMETERS

Various system suitability parameters such as Retention time (RT), theoretical plates (TP), tailing factor (TF) and area under curve (AUC) were determined from the data obtained. The results are tabulated and presented below:

Table 10: Results of System Suitability parameters for Aprepitant