Analytical Method Development and Validation of UV-HPLC Method for The Estimation of Amoxapine

Amoxapine [2-Chloro-11-(piperazin-1-yl) dibenzo[b,f][1,4] oxazepane] is a tricyclic dibenzoxazepine derivative, has an antidepressant action. It is a sparse golden yellow crystalline powder, nearly insoluble in water, but soluble in organic solvents, such as chloroform and methanol^[1]. Amoxapine became notable due to the fact that it could treat depressive and psychotic symptoms making it unique to tricyclic antidepressants ^[2].

Figure 1: Structure of Amoxapine

The molecule has a tricyclic backbone and a piperazine ring, which helps it to be dibasic with pKa of about 8.7 and 3.9 at 25 °C^[3]. Amoxapine is a BCS class II and possesses a half-life of 8h. It has moderate oral bioavailability (36%) due to low water solubility, high first-pass effect, and high protein binding (90%)^[4]. In this study, we describe a simple and reproducible UV-HPLC method for the quantification of pure Amoxapine that is rapid, cost-effective, and suitable for routine analysis of pharmaceutical formulations and raw materials.

2. MATERIAL AND METHODS

2.1 Materials

Amoxapine was obtained as a gift sample from Almon Healthcare (Ahmedabad, India). Methanol (HPLC Grade) was procured from Rankem Chemicals (Thane, India). Double-distilled water was freshly prepared in the laboratory using standard distillation procedures. Disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride were purchased from Astron Chemicals (Ahmedabad, India).

A digital analytical balance was used (Shimadzu, Japan). pH measurements were performed using a pH meter (Lab India Instruments). Centrifugation was performed using a C-24 Plus centrifuge (Remi Instruments, Mumbai). UV absorbance measurements were conducted using a double beam UV-Visible spectrophotometer (Model: UV-1900, Shimadzu, Japan). High-Performance Liquid Chromatography (HPLC) analysis was performed using a Vanquish Core system from Thermo Fisher Scientific, Massachusetts, USA.

2.2 UV Spectrophotometric Method

2.2.1 Development of Spectrophotometric Method

2.2.1.1 Preparation of Stock Solution

Accurately weighed 10 mg Amoxapine was transferred to a 10 mL volumetric flask. Methanol (3–4 mL) was added to the above flask, the drug was dissolved properly and the final volume was made up to 10 mL with methanol to produce a stock solution of 1000 µg/mL.

2.2.1.2 Determination of λmax

1 mL from the stock solution (1000 µg/mL) was further diluted with 100 mL to produce 10 µg/mL. The spectrum of the resultant solution (10 µg/mL) was recorded in the range of 200-400 nm against methanol as a blank.

2.2.1.3 Calibration plot of Amoxapine in methanol

From the Stock solution, suitable dilutions were made to produce standard solutions of 4, 8, 12,16,20, and 24 µg/mL. The absorbance of the solutions was measured spectrophotometrically at 252 nm using methanol as a blank.

2.2.2 Validation of Spectrophotometric Method

Linearity and Range: The linear curve of the Amoxapine was obtained by plotting calibration curves with reference to standard drug solutions. Working range was determined as the lowermost/highest points of the calibration, which was determined by the analysis of the linear regression^[5].

Accuracy: The validation with the developed method will run using standard addition method at three concentrations, 80%, 100%, and 120%. Weighing known amounts of pure drug into pre-analysis sample bottles, we calculated percentage recovery as a measure of the proximity of obtained values to actual concentration.

Precision: Precision is the replicability of the procedure. This was assessed by analysis of three replicate samples of the same working solution. Intra-day and inter-day variations were used to prove the precision of the method.

Intra-day Precision: It was determined by analyzing the 8,12,16 µg/mL of Amoxapine for three different times in the same day.

Inter-day Precision: It was determined by measuring the 8,12,16 µg/mL of Amoxapine for three consecutive days.

LOD and LOQ: Detection limit and quantitation limit were achieved on the basis of standard deviation of y intercepts of calibration curves and the average slope of calibration curves.

LOD = 3.3 × Standard deviation of intercept/Slope

LOQ = 10 × Standard deviation of intercept/Slope

Ruggedness: Ruggedness refers to the reproducibility of test results under expected laboratory conditions, taking into account variations such as different instruments and different analysts^[6].

2.3 Chromatographic Method

2.3.1 Development of Chromatographic method

2.3.1.1 Chromatographic Conditions

The HPLC system consisted of UV-Visible Detector. The wavelength of detection set at 240nm. Separation was carried out on Endurus C18 Classic (100 × 4.6 mm, 3 µm) using 10 mM Ammonium acetate in channel A and Acetonitrile in channel B (1:1) as mobile phase with gradient elution at a flow rate of 1.5 mL/min. The mobile phase filtered through vacuum filtration and degassed with ultrasonicator prior to use. Chromatography was carried out at ambient temperature.

2.3.1.2 Standard & Sample Preparation:

Accurately weighed 10 mg of Amoxapine was transferred into a 10 mL volumetric flask and dissolved in 10 mM Ammonium acetate and Acetonitrile (1:1) to prepare a primary stock solution of 1000 µg/mL (1 mg/mL). From this stock, 1 mL was further diluted to 10 mL with 10 mM Ammonium acetate and Acetonitrile (1:1) to obtain a secondary stock solution of 100 µg/mL. Subsequent working standard concentrations (0.5, 1, 1.5, 2 and 2.5 µg/mL) required for calibration were also prepared by further diluting the secondary stock with 10 mM Ammonium acetate and Acetonitrile (1:1).

2.3.1.3 Injection sequence:

Separately injected (Prefiltered thorough syringe filter) 20 µL each of one diluent sample into the liquid chromatography, recorded the chromatograms and measured the responses for all peaks including the peaks of blank.

2.4 Validation of HPLC Method:^{[7, 8]}

Linearity: The linearity of the analytical method for Amoxapine was determined by studying standard calibration curves. The range of the analytical method was decided from the interval between the upper and lower level of the calibration curves.

Accuracy: Accuracy of the method was assessed by standard addition method at three different concentration levels i.e. 50%, 100%, 150%. Standard concentration of 0.5,1, and 1.5 µg/mL was added into 1 µg/mL of drug concentration. The % Recoveries was calculated by applying regression equation.

Intra-day Precision: It was determined by analysing the standard solutions of Amoxapine (0.5,1.0,1.5 µg/mL) and at three different time intervals on same day.

Inter-day Precision: It was determined by analysing the combined standard solution of Amoxapine (0.5,1.0,1.5 µg/mL) on three consecutive days. The results were reported in terms of % RSD.

LOD and LOQ: The limit of detection and the limit of quantification were determined as a function of the standard deviation of the intersections and the average slope of the calibration curves.

Robustness: Stand ard solution of Amoxapine (1.5 µg/mL)) were used and analysed at different flow rate (1.2,1.5,1.7 mL/min) and wavelength (242,238,240 nm).

Ruggedness: It was verified by two different analysts maintaining the same environmental and experimental conditions. An appropriate concentration of 1.5 µg/mL Amoxapine was tested and the concentration determined.

System Suitability: Amoxapine standard solution (1.5 µg/mL) was prepared and analysed. Chromatograms for different parameters such as tailing factor, resolution and theoretical plates were studied to see whether or not they meet the recommended limit.

3. RESULT AND DISCUSSION

3.1 UV-Visible Spectrophotometric Method

3.1.1 Linearity study

To assess the linearity of the method, standard solutions of Amoxapine, ranging from 4 to 24 µg/mL, were prepared from the stock solution.

Figure 2: UV spectra of the methanolic solution of Amoxapine showing maximum absorption at λmax = 252.00 nm.

Figure 3:  Calibration plot of Amoxapine in methanol

Table 1: Calibration data of Amoxapine in methanol

Table 2: Linear regression analysis by UV

3.1.2 Validation Parameters:

Method validation was performed according to ICH guidelines. The Accuracy of the method was determined at the 50%, 100% and 150% level by the standard addition method and the precision of the method was determined by the % RSD of intraday precision, interday precision ^{[9, 10]}.

Table 3: Result of Accuracy study

Table 4: Result of intra-day precision

Table 5: Result of inter-day precision

Table 6:  Result of robustness study

Table 7: Result of ruggedness study

Table 8: Specificity study

3.2.1 Selection of Analytical Wavelength:

Amoxapine standard solutions (10 µg/mL) were scanned in the UV region of 200-400 nm and spectra were recorded. Amoxapine were observed to show absorbance at 240 nm. So, the detection wavelength used was 240 nm.

3.2.2 Optimized Parameter (Gradient HPLC):

Figure 4: Chromatograph of Amoxapine by HPLC

3.2.3 Linearity Study:

Amoxapine was found to be linear in the concentration range of 0.5-2.5 µg/mL.

Figure 5: HPLC chromatograms of Amoxapine (0.5–2.5 µg/mL) showing retention at 4.162 min with concentration-dependent peak response.

Figure 6: Calibration curve of Amoxapine by HPLC

Table 8: Specificity study

Table 9: Result of calibration curve

Table 10: Summary of method validation parameter

3.2.4 Validation Parameters:

This method was validated according to the ICH guidelines. The percentage of recoveries of Amoxapine was found in the range of 99.97 to 100.4%. The precision of the method was determined by the % RSD. The LOD and LOQ of Amoxapine were found to be 0.048 and 0.146 µg /mL, respectively. For the robustness study, the effect of the change in wavelength and flow rate on the mean peak area, % RSD and % assay was studied. The percentage of RSD of each peak in all variables was found to be less than 2% ^{[11, 12]}.

Accuracy

Accuracy was studied by standard addition method and % recovery found was within acceptable limit.

Table 11: Result of Accuracy by HPLC

Mean % Recovery: 99.849%

Intra-day and inter-day precision assures the repeatability of test results. The % RSD found was below 2.

Table 12A: Result of intraday precision

Table 12B: Result of Interday precision

Robustness

Robustness was studied by different deliberate variations in the chromatographic conditions.

Table 13: Result of robustness study

Ruggedness

Ruggedness was studied by different analyst.

Table 14: Result of Ruggedness

Specificity

Excipients and impurities were not interacting with the standard drug; hence method is specific.

Table 15: Data for specificity study

Table 16: Result of system Suitability