1. INTRODUCTION:

Pharmaceutical analysis may be defined as the application of analytical procedures used to determine the purity, safety and quality of drugs and chemicals. The term “pharmaceutical analysis” is otherwise called quantitative pharmaceutical chemistry.

Pharmaceutical analysis includes both qualitative and quantitative analysis of drugs and pharmaceutical substances starts from bulk drugs to the finished dosage forms. In the modern practice of medicine, the analytical methods are used in the analysis of chemical constituents found in human body whose altered concentrations during disease states serve as diagnostic aids and also used to analyse the medical agents and their metabolites found in biological system.^1,2

The term “quality” as applied to a drug product has been defined as the sum of all factors, which contribute directly or indirectly to the safety, effectiveness and reliability of the product. These properties are built into drug products through research and during process by procedures collectively referred to as “quality control”. ³

1.1 High performance liquid chromatography (HPLC):

The term ‘chromatography’ covers those processes aimed at the separation of the various species of a mixture on the basis of their distribution characteristics between a stationary and a mobile phase. The principle of separation in normal phase mode and reverse phase mode is adsorption. When a mixture of components is introduced in to a column, they travel according to their relative affinities towards stationary phase. The component which has more affinity towards the adsorbent travels slower. The component which has less affinity towards stationary phase travels faster. Since no two components have the same affinity towards the stationary phase, the components are separated. HPLC is an analytical technique for resolution of solutes, in which separation is made by differential migration in a porous medium and migration is caused by flow of solvent. Chromatography is one of the widely used physiochemical methods of separation of inorganic and organic substances related in their composition and properties. Chromatography is characterized by two important features,

A. A very large interface area, and

B. A dynamic way of operation, which ensures a more effective separation of substances as compared to other methods.^4,5

1.2. Validation of analytical methods as per ICH guidelines⁶

Types of analytical procedures to be validated

The objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated. Typical validation characteristics which should be considered are listed below,

a. Accuracy

b. Precision

c. Repeatability

d. Intermediate precision

e. Specificity

f. Detection limit

g. Quantitation limit

h. Linearity

i. Range^7,8,9.

Furthermore revalidation may be necessary in the following circumstances:

· Changes in the synthesis of the drug substance.

· Changes in the composition of the finished product.

· Changes in the analytical procedure.^10,11.

1.3. QbD Approach:

As per US FDA QbD is defined as “Systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”. In a pharmaceutical QbD approach to product development, it helps to identify characteristics which are critical to quality from the patient’s perspective, convert them into the drug product critical quality attributes (CQAs), and establishes the relationship between formulation or manufacturing variables and CQAs to deliver a drug product with such CQAs to the patient in consistent manner. QbD consists of the following elements:

1. A quality target product profile (QTPP).

2. Critical material attributes (CMAs).

3. Critical process parameters (CPPs) and a thorough understanding of scale-up principles, linking CMAs and CPPs to CQAs.

4. A control strategy which consists specifications for the drug substance(s), excipient(s), and drug product as well as controls for each and every step of the manufacturing process.

5. Process capability and continual improvement.^12.

1.4 Force Degradation:

It is a procedure where the natural degradation rate of a product or material is amplified by the application of an additional external stress. Forced degradation studies are aimed to recognize reactions which might be fall to degrade the subjected chemical entity. It is typically performed before final formulation; forced degradation observes external stresses to rapidly shade material solidities. Longer term storage tests are preferably used to amount similar properties when final formulations are involved because of the stringent FDA regulations.

1.5 Drug Profile of Aripiprazole

Figure no. 1: Chemical structure of Aripiprazole

2. MATERIAL AND METHODS:

2.1 Chemicals, reagents and Instruments:

In method development and validation of preservatives following chemicals and reagents were used. Aripiprazole API was provided by R.S.I.T.C. Jalgaon. HPLC grade TEA and Orthophosphoric acid was provided by Avantor Performance material India Ltd. Thane, Maharashtra. HPLC grade methanol and water was provided by Merck Specialities Pvt. Ltd. Shiv Sager Estate ‘A’ Worli, Mumbai.

HPLC instrument (Agilent 1100 with auto sampler) (Chemstation software), UV-Spectrophotometer (Analytical Technologies Limited), Column(C₁₈) (Agilent C₁₈) (100mmX 4.6mm, 5µm)pH meter (VSI pH meter) (VSI 1-B), Balance (WENSAR™ High Resolution Balance), and Sonicator (Ultrasonic electronic instrument) were used.

Marketed formulation used was Apiz mouth dissolving tablet by Reliance life science Pvt. Ltd. The analysis of the drug was carried out on Agilent (S.K.) Gradient System UV Detector. Equipped with Reverse Phase (Agilent) C18 column (4.6mm x 100mm; 2.5µm), a SP930Dpump, a 20µl injection loop and UV730D (DAD) Absorbance detector and running Chemstation software.

3. EXPERIMENTAL WORK:

3.1 Analysis by UV spectrophotometer:

I) Solubility studies:

This finds that the Aripiprazole is soluble in Methanol, DMSO etc. Methanol was selected for further studies.

II) Aripiprazole standard stock solution: (Stock I)

An accurately weighed quantity, 10mg of Aripiprazole (ARP) was dissolved in methanol in a 10ml volumetric flask and volume made up to 100ml to produce a solution of 1000ug/ml.

III) Preparation of Stock Standard Solution :( Stock II) [Aripiprazole]

Accurately weight and transfer 10mg Aripiprazole working standard into 10ml volumetric flask as about diluent methanol completely and make volume up to the mark with the same solvent to get 1000µg/ml standard (stock solution) and 15 min sonicated to dissolve it and remove the unwanted gas, further an aliquots portion of Aripiprazole stock solution in ratio of 1 were mixed in volumetric flask in 10 ml and volume was adjusted up to mark with mobile phase from the resulting solution 0.05 ml was transferred to 10 ml volumetric flask and the volume was made up to the mark with MEOH :Water (0.1% OPA), prepared in (80ml MEOH : 20 ml Water (0.1% OPA))solvent .

IV) Determination of wavelength and linearity study:

Wavelength was selected from the spectrum of Aripiprazole obtained from UV spectrophotometer. A concentration of 10μg/ml solution of each drug is prepared in methanol and scanned in range from 400-200nm using above solvent as blank. The maximum absorbance i.e. λmax was found at 254 nm.

3.2 HPLC Chromatography:

3.2.1 Preparation of std. Aripiprazole solution: (Stock I)

An accurately weighed quantity, 10mg of Aripiprazole (ARP) was dissolved in methanol in a 10ml volumetric flask and volume made up to 10.0ml to produce a solution of 1000ug/ml. From the freshly prepared standard stock solution (1000ug/ml), 0.1ml stock solution was pipetted out in 10ml of volumetric flask and volume was made up to 10ml with mobile phase to get final concentration of 10ug/ml.

3.2.2 Preparation of std. Aripiprazole solution :(Stock III)

From the freshly prepared standard stock solution (1000ug/ml), 0.1ml stock solution was pipetted out in 10 ml of volumetric flask and volume was made up to 10ml with mobile phase to get final concentration 10ug/ml.

3.2.3 Selection of mobile phase:

Each mobile phase was vacuum degassed and filtered through 0.45µ membrane filter. The mobile phase was allowed to equilibrate until OPAby baseline was obtained. The standard solution containing mixture of Aripiprazole was run with different individual solvents as well as combinations of solvents were tried to get a good separation and stable peak. From the various mobile phases tried, mobile phase containing MEOH and Water (0.1% OPA) was selected since it gave sharp, well resolved peaks with symmetry within the limits and significant reproducible retention time for Aripiprazole.

3.3 Standard Marketed Formulation Solution:

To determine the content of Aripiprazole in marketed tablets (label claim 15mg of Aripiprazole), 20 tablets powder weighed 1.56 gm and average weight of powder was 0.78gm. Tablets were triturated and powder equivalent to weigh in 78mg the drug was extracted from the tablet powder with 10 ml MEOH.

To ensure complete extraction it was sonicated for 15 min. 0.3mL of supernatant was then diluted up to 10mL with mobile phase. The resulting solution was injected in HPLC and drug peak area was noted. (Fig No: 2).

Regression equation was generated using peak areas of standard solutions. Using the regression equation and peak area of the sample the amount of Aripiprazole in the sample was calculated. The amount of Aripiprazole Per tablet was obtained from the regression equation of the calibration curve as described in analysis of Tablet formulation are shown in (Table No.5).

4. RESULTS AND DISCUSSION:

4.1 Mobile phase optimization:

After the selection of suitable mobile phase, it was optimized for its reproducibility, sensitivity and accuracy. The optimized parameters for selected method are as below.

The final chromatographic conditions selected were as follow:

DAD Detector Agilent (S.K) Gradient System

· Analytical column : (Agilent) C18 column (4.6mm x 100mm)

· Injection volume : 20µl

· Flow rate : 0.7 ml/min

· Mobile phase : MEOH +0.1OPA(80+20% v/v)

· Detection : 254 nm

· Run Time : 15 min

Then after Taguchi screening method was used for preliminary screening for optimized batch

4.2 Design of Experiments:

Design of Experiments:

Taguchi screening method was used to perform Preliminary Screening and Optimization Data Analysis Preliminary experiments to identify the critical factors and to set their levels (maximum and minimum) for the experimental design. In this step various parameters were investigated as follows: selection of a chromatographic Column (C8 and C18), column temperature, mobile phase(ratio of ACN: MEOH :buffer), concentration of the buffer if present In the mobile phase, buffer pH, injection volume, mode of flow (isocratic/gradient) as well as determining the ideal flow rate. Based on the results obtained from the Taguchi screening, multiple linear regression analysis (MLRA) was applied for the studied Design using Design Expert® software version 9.0.02 to fit the full second-order polynomial equations with added Interaction terms. The method chosen to optimize separation of Aripiprazole with the shortest analysis time was Box–Behnken design (BBD) with three replicates at the centre point (middle level). The independent variables were investigated and their low, Medium, and high levels described in Table below the QbD trials. The evaluated Responses (dependent variables) were the no. Of theoretical plates (Y1), assay (Y2), and tailing factor (Y3). Prediction of the optimum Composition was carried out using overlay plotting, brute Force method, and numeric approach of desirability function. Overlay Plot (i.e., combined contour plot) option in the software was Also embarked upon to locate the optimum composition. Within This optimal area, an optimum chromatographic condition was located by trading off different responses. The prognosis of the Optimum analytical condition was also conducted using numerical Optimization technique/with help of Design Expert software. The Box-Behnken Design Validation Fifteen runs were done, selected from grid search data, prepared asper the chosen composition(s), and evaluated for the critical Quality attributes (CQA), viz. Number of theoretical Plates (TP), Assay, and tailing factor (TF). The predicted and observed Responses were compared, and linear correlation plots were constructed Percent bias (error) was calculated with respect to the observed responses and the residual plots were also constructed For TP, assay, and TF.¹⁶

4.3 Method Validation:

4.3.1 Linearity:

Figure No. 3: Linearity of Aripiprazole

4.3.2. Accuracy (recovery):

Table no. 2: Result of recovery data for Aripiprazole

Method

Level

(%)

Amt.

taken

(ug/ml

Amt. Added

(ug/ml

Absorbance

Mean*

±S.D.

Amt.

recovered

Mean *±

S.D.

% Recovery

Mean *±

S.D.

80%

18.09 ±

0.0013

8.09 ±

0.0013

101.11 ±

0.17

HPLC

100%

19.76 ±

0.022

9.76 ±

0.022

97.60 ±

0.22

120%

21.88 ±

0.015

11.88 ±

0.015

99.00 ±

0.13

Accuracy of RP-HPLC method is ascertained by recovery studies performed at different levels of concentrations (80%, 100% and 120%). The % recovery was found to be within 98-101%

4.3.3 System suitability parameters: (Repeatability)

Table No. 3: Repeatability studies on RP-HPLC forAripiprazole

METHOD	Concentration of Aripiprazole(mg/ml)	Peak area	Amount found (mg)	% Amount found
HPLC METHOD	40	2251.056	40.24	100.60
		2256.075
	Mean	2253.57
	SD	3.55
	%RSD	0.16

Repeatability studies on RP-HPLC for Aripiprazole found to be the %RSD was less than 2%, which shows high percentage amount found in between 100% to 102% indicates the analytical method that concluded

4.3.4 Precision:

Table no.4: Result of Intraday and Inter day Precision studies on RP-HPLC and UV method Aripiprazole

DRUG	Method	Conc. (µg/ml)	Interday Precision		Intraday Precision
DRUG	Method	Conc. (µg/ml)	Mean± SD	%Amt Found	Mean± SD	%Amt Found
Aripiprazole	HPLC	20	1144.81 ±0.02	97.40	1146.68 ±1.39	97.57
		30	1688.74 ±1.67	98.88	1689.23 ±1.86	98.91
		40	2253.45 ±1.34	100.59	2257.07 ±3.63	100.76

*Mean of each 3 reading for RP-HPLC method

Intraday and Inter day Precision studies on RP-HPLC and UV method for Aripiprazole which shows the high precision % amount in between 97% to 101% indicates to analytical method that concluded.

4.3.5 Robustness:

The mobile phase composition was changed in (±1 ml/min-1) proportion and the flow rate was varied by (±1ml/min-1), and wavelength change (±1ml/min-1) of optimized chromatographic condition. The changes were did flow rate (±1ml/min-1), PH of mobile phase composition (±1ml/min-1), and Wavelength (±1ml/ min-1). %RSD for peak area was calculated which should be less than 2%. Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

4.3.6 LOD and LOQ:

The LOD and LOQ of Aripiprazole was found to be 0.049 (ug/mL) and 0.149(ug/mL), analytical method that concluded.

4.3.7 Analysis of Marketed formulation:

Table no.5: Analysis of marketed formulation.

Drug	Amt.Found	%Label Claim	SD	%RSD
HPLC	29.6609	98.87	0.059	0.199
HPLC	29.74461	99.15	0.197	0.199

Analysis of marketed formulation were also %Label Claim was found to be 100-101% Satisfactory are concluded.

4.3.8 Degradation Study:

Table no.6: Result of Degradation Study of Aripiprazole.

parameters	Drug name	Area	% assay	% degradation	% RSD
Acid	APZ	860.111	50.99	49.01	0.12
Base	APZ	21.68	1.29	98.71	0.15
oxidative	APZ	0	0.00	100.00	0.29
Neutral	APZ	1683.42	99.78	0.22	0.001

5. CONCLUSION:

Simple, rapid, accurate and precise RP-HPLC have been developed and validated for the routine analysis of Aripiprazole in API and tablet dosage forms. Both methods are suitable for the simultaneous determination of Aripiprazole in Single-component formulations without interference of each other. The developed methods are recommended for routine and quality control analysis of the investigated drugs in two component pharmaceutical preparations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms.

6. REFERENCES:

1. Marchand D. H., Snyder L. R., and Dolan J. W. S.L. : Journal of Chromatography A, 2008, vol. 1191.

2. Bk., Sharma. Instrumental Methods of Chemical Analysis. S.l.: meerut: Goel Publishing House, 2000. 7.

3. Gurdeep R. Chatwal, Sham K. Anand. Instrumental Methods of Chemical Analysis. New Dehli: New Delhi: Himalaya Publishing House, 2008. 5.

4. Gandhi Noopurk, Shahdarshil B, Maheshwari Dilip G 2016 “Aripiprazole and clozapine: A review of spectroscopic and chromatographic method” Asian Journal of Pharmaceutical Technology and Innovation 5 (22) 27-39

5. Mallikarjuna Rao, Nagasarapu and Gowrisankar Dannana, 2017, New stability indicating high performance liquid chromatography method for the estimation of Aripiprazole in bulk and their formulation. Indian Journal of Drugs 5 (3), 116-123 ISSN: 2348-1684.

6. Gennaro A.R., Remington. The Sciences and Practice of Pharmacy. Baltimore, Maryland, USA : Luppincott, Williams and Wilkins, 2000.

7. Douglas AS., Holler FJ., Crouch SR. Principle of Instrumental Analysis, 6^thed, Thomson Publication, 2007: p.1.

8. Ahmed Naved, Shaikhobaid, Barrawazaateka, Khan Sarfarazzaheer. Zahid 2017 development and validation of rapid HPLC method for determination of aripirazole in bulk drug and pharmaceutical formulation. Journal of Innovations in Pharmaceutical and Biological Science ISSN : 2349-2759 vol 4 (3) 15-19

9. Sharma BK., Instrumental Methods of Chemical Analysis, 23^rded, Goel Publishing House, Meerut, 2002: p.7-8.

10. https://en.wikipedia.org/wiki/Analytical_chemistry

11. Skoog DA, West DM, Holler FJ. Analytical chemistry – An Introduction, 6^thed. Saunder College Publishing. p.3.

12. Qbd Approach to Analytical RPHPLC. Devesh A. Bhatt, Smita I. Rane., Dhule : International Journal of Pharmacy and Pharmaceutical Sciences, 2011, Vol. 3. ISSN 0975-1491

13. Willard HH, Merit LL, Dean JA, Settle FA. Instrumental Methods of Analysis.7^thed. New Delhi: CBS Publishers and Distributors; 1996.p.513.

14. Gurdeep R. Chatwal, Sham K. Anand. Instrumental Methods of Chemical Analysis. New Dehli : Himalaya Publishing House, 2008. 5.

15. Beckett AH, Stenlake JB.Practical Pharmaceutical Chemistry. New Delhi : CBS Publishers and Distributors, 2002.

16. Kasture AV, Wadodkar SG, Mahadik KR, More HM. A Textbook of Pharmaceutical Analysis. 10^th ed. Vol. II. Pune: Nirali Prakashan; 2004.p.4.

Received on 17.08.2021 Modified on 26.11.2021

Asian J. Pharm. Ana. 2022; 12(1):29-34.

DOI: 10.52711/2231-5675.2022.00006

Generic name	Aripiprex
IUPAC Name	7-[4-[4-(2, 3-dichlorophenyl) piperazin-1-yl] butoxy]-3, 4-dihydro- 1H-quinolin-2-one
chemical class	Diazinanes
Therapeutic class	Antidepressant, D2 receptor antagonist¹³
Molecular weight	448.4 g/mol
Chemical formula	C₂₃H₂₇Cl₂N₃O₂
Melting point	137-140° C¹³
Biological half life	72 hrs
Volume of distribution	4.9 L/Kg
Clearance	0.8 ml/min/kg
Bioavailability	87%
Protein binding	>99%
Mechanism of action	The exact mechanism of antipsychotic action of aripiprazole has not been fully elucidated but, like that of other atypical antipsychotic agents (e.g.,olanzipine, risperidone, ziprasidon), may involve the drug's activity at dopamine D2 and serotonin type (5-HT1A) and type 2 (5-HT2A) receptors.^14,15.