Identification & Assay Methods for Aripiprazole

Sonal Shinde

Merck, Life Science Application Centre (GAC), Mumbai, India

Section Overview

• Introduction
• Experimental
• Results
• Related Products

Introduction

Figure 1. Chemical structure of Aripiprazole

Aripiprazole is an atypical antipsychotic and a partial dopamine agonist. It is primarily used in the treatment of schizophrenia, bipolar disorder, major depressive disorder, tic disorders, and irritability associated with autism. Aripiprazole was first approved by the U.S. Food and Drug Administration in November 2002 for schizophrenia and by the European Medicines Agency in June 2004 for acute manic and mixed episodes associated with bipolar disorder.

Common commercial brand names:
Abilify and Aripiprex

Aripiprazole was developed by Otsuka in Japan. In the United States, Otsuka America markets it jointly with Bristol-Myers Squibb. In 2010, sales were $4.6 billion globally; the patent expired in 2015.

In this compilation, we have used the USP 40–NF 35 experimental conditions for Aripiprazole in the following areas:

• Identification — FTIR
• Assay — HPLC (gradient method)
• Related Substances — HPLC (gradient method)

The HPLC methods are gradient methods, thus they are nonscalable.

The same chromatographic conditions are used for methods in both the assay and related substances, and a full validation protocol can be found using these USP Reference Standards: USP Aripiprazole RS and USP Aripiprazole Related Compound F RS.

Experimental

Identification and Assay

Definition

Aripiprazole contains not less than (NLT) 98.0% and not more than (NMT) 102.0% of aripiprazole (C₂₃H₂₇Cl₂N₃O₂), calculated on the dried basis.

Identification—FTIR <197K>

1. Infrared absorption
2. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

Assay Solutions and Analysis

• System suitability solution: 1 µg/mL each of USP Aripiprazole and USP Aripiprazole Related Compound F in Diluent
• Standard solution: 0.1 mg/mL of USP Aripiprazole in Diluent
• Sample solution: 0.1 mg/mL of aripiprazole in Diluent
• System Suitability
• Samples: System suitability solution and Standard solution
  Note: The relative retention times (RRT) for aripiprazole and aripiprazole related compound F are 1.0 and 1.1, respectively.

Assay—HPLC (gradient method)

Procedure (protect the solutions from light):

• Diluent: Acetonitrile, methanol, water, and acetic acid (30:10:60:1)
• Solution A: Acetonitrile and 0.05% trifluoroacetic acid (10:90)
• Solution B: Acetonitrile and 0.05% trifluoroacetic acid (90:10)
• Gradient: (Table 1)
  • Detector: UV 254 nm
  • Column: 4.6 mm × 10 cm (3 µm) packing L1 (e.g., Purospher^® STAR RP-18 endcapped)
  • Flow rate: 1.2 mL/min
  • Injection volume: 20 µL

Note: The gradient was established on an HPLC system with a dwell volume of approximately 650 µL.

Table 1. This is a gradient method and can therefore not be changed

Time (min)	Solution A (%)	Solution B (%)
0	80	20
2	80	20
10	65	35
20	10	90
25	10	90
26	80	20
35	80	20

Suitability Requirements

• Resolution: NLT 2.0 between aripiprazole and aripiprazole related compound F, System suitability solution
  
• Tailing factor: NMT 1.5 for aripiprazole, System suitability solution
  
• Relative standard deviation: NMT 1.0%, Standard solution
  

Analysis

• Samples: Standard solution and Sample solution
• Calculate the percentage of aripiprazole (C₂₃H₂₇Cl₂N₃O₂) in the portion of the sample taken.
• Result = (rU/rS) × (CS/CU) × 100
  rU = peak area from the Sample solution
  rS = peak area from the Standard solution
  CS = concentration of USP Aripiprazole in the Standard solution (mg/mL)
  CU = concentration of aripiprazole in the Sample solution (mg/mL)
• Acceptance criteria: 98.0%–102.0% on the dried basis

Impurities Analysis

• Inorganic Impurities
  1. Residue on Ignition—USP General Chapter 281: NMT 0.1%
  2. Heavy Metals, Method II—USP General Chapter 231¹: NMT 10 ppm
• Organic Impurities
  (Protect the solutions from light.) Diluent, Solution A, Solution B, Mobile phase, System suitability solution, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Analysis

• Samples: Sample solution
  Calculate the percentage of each impurity in the portion of aripiprazole taken.
• Result = (ri/rU) × (1/F) × 100
  ri = peak response of each impurity from the Sample solution
  rU = peak response of aripiprazole from the Sample solution
  F = relative response factor (Table 2)
  USP Reference Standards
• • USP Aripiprazole
  • USP Aripiprazole Related Compound F
    

¹Chapter valid until Jan. 1, 2018.

Table 2. Related substances (organic impurities)

Name	Relative Retention Time (RRT)	Relative Response Factor (RRF)	Acceptance Criteria (NMT, %)
Aripiprazole related compound G2	0.9	0.72	0.10
Aripiprazole	1.0	—	—
Aripiprazole related compound F3,4	1.1	1.0	—
Aripiprazole 4,4-dimer5	1.3	1.0	0.10
Any other impurity	—	—	0.10
Total impurities	—	—	0.50

²7-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butoxy}quinolin-2(1H)-one
³4-(2,3-Dichlorophenyl)-1-[4-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yloxy)butyl]piperazin 1-oxide
⁴For system suitability and identification purposes only
⁵(7,7′-((((ethane-1,1-diylbis(2,3-dichloro-4,1-phenylene))bis(piperazine-4,1-diyl))bis(butane-4,1-diyl))bis(oxy))bis(3,4-dihydroquinolin-2(1H)-one)

Results

Identification Data

Infrared Absorption

The reference <197K> in a monograph signifies that the substance under examination is mixed intimately with potassium bromide. We recommend potassium bromide for IR spectroscopy—Uvasol^® (1.04907).

Figure 2. IR spectrum of aripiprazole USP Reference Standard

Assay and Related Substances Data

Table 3.Chromatographic conditions

HPLC Parameters
Column:	Purospher® STAR RP-18 endcapped, 3 μm, 100 x 4.6 mm (1.50469)
Mobile phase:	[A] Acetonitrile and 0.05% trifluoroacetic acid (10:90 v/v); [B] Acetonitrile and 0.05% trifluoroacetic acid (90:10 v/v)
Gradient:	See Table 4
Flow rate:	1.2 mL/min
Pressure drop:	95–170 Bar (1377–2465 psi)
Column Temp:	40 ˚C
Detection:	UV, 254 nm; 10 μL cell
Injection:	20 µL
Diluent:	Acetonitrile, methanol, water, and acetic acid (30:10:60:1 v/v)
Sample:	1 μg/mL (1 ppm) each of aripiprazole and aripiprazole related compound F in diluent

Table 4. HPLC Gradient conditions used

Time (min)	A (%)	B (%)
0.01	80	20
2	80	20
10	65	35
20	10	90
25	10	90
26	80	20
35	80	20

Suitability Requirements

• Resolution: NLT 2.0 between aripiprazole and aripiprazole related compound F
  
• Tailing Factor: NMT 1.5 for aripiprazole
  
• Relative Retention Time (RRT):
      1.0 for aripiprazole and
      1.1 for aripiprazole related compound F

Figure 3. HPLC-UV chromatogram aripiprazole and aripiprazole related compound F

Table 5. Chromatographic data (System Suitability Solution)

Compound	Retention Time (min)	RRT	Tailing Factor	Resolution
Aripiprazole	12.0	1.0	1.4	—
Aripiprazole related compound F	12.5	1.05	1.3	2.8

Figure 4. HPLC-UV chromatogram (left) sample solution containing aripiprazole and aripiprazole related compound F in diluent, (right) aripiprazole standard solution

Table 6. Chromatographic data (100 ppm Standard Solution)

Compound	Retention Time (min)	Tailing Factor	Theoretical plates
Aripiprazole	12.0	1.4	16,118

Assay and Related Substances Validation Data

Table 7. Specificity determined by injection of System suitability solution and determination of the retention time and relative retention time for aripiprazole and aripiprazole related compound F.

A. Specificity
Compound	Retention time (min)	RRT	Tailing factor	Resolution
Aripiprazole	12.0	1.0	1.4	—
Aripiprazole related compound F	12.5	1.05	1.3	2.8

Table 8. Repeatability determined by injecting five samples with a standard solution containing 100.0 ppm USP Aripiprazole RS and 1.0 ppm of USP Aripiprazole Related Compound F RS

B. Repeatability
Standard Solution	Aripiprazole (Area Units)	Aripiprazole Related Compound F (Area Units)
1	60,114	6,047,450
2	60,363	6,080,108
3	60,308	6,086,256
4	60,174	6,081,386
5	60,316	6,089,267
Mean	60,255	6,076,893
STDEV	105.6	16,868.6
RSD (%)	0.18	0.28

Linearity, Limit of Detection (LOD), and Limit of Quantitation (LOQ)

Determined by injecting seven concentration levels from 0.1 to 1.5 ppm of USP Aripiprazole Related Compound F and nine concentration levels ranging from 1.0 to 150.0 ppm of aripiprazole.

Table 9. Sensitivity for aprazole related compound F

LOD (ppb)	LOQ (ppb)
7.6	23

Table 10. Sensitivity for aprazole

LOD (ppm)	LOQ (ppm)
0.57	1.7

Table 11.Calibration data aripiprazole related compound F

(ppm)	Area Units
0.1	6,020
0.25	15,062
0.5	29,604
0.8	48,154
1.0	60,630
1.2	73,335
1.5	92,098

Table 12.Calibration data Aripiprazole

(ppm)	Area Units
1.0	63,167
5.0	292,127
10.0	586,674
25.0	1,587,549
50.0	3,013,178
80.0	4,878,836
100.0	6,076,893
120.0	7,401,807
150.0	9,241,987
R2	0.9999

D. Linearity, LOD, LOQ (cont.)

Determined by injecting seven concentration levels from 0.1 to 1.5 ppm of USP Aripiprazole Related Compound F and nine concentration levels ranging from 1.0 to 150.0 ppm of aripiprazole.

Figure 5. Calibration curves (left) aripiprazole and (right) aripiprazole related compound F

Table 13. LOQ accuracy determined by injecting 10 Standard solutions at LOQ level of USP Aripiprazole RS.

Injection	Area units	SD Area units	RSD (%)
N=10	151,949	±424.9	0.28

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