INTRODUCTION:

High Performance Liquid Chromatography (HPLC) is one of the most commonly used analytical techniques in industry. It is used to separate and analyze compounds over the mass-transfer of analysis between stationary phase and mobile phase. The technique of HPLC uses a liquid mobile phase to separate the components of a mixture. The components themselves are first dissolved in a solvent and then required to flow (via the mobile phase) complete a column (stationary phase) in high pressure. The mixture is determined into its components within the column and the amount of resolution is dependent upon the interaction between the solute components and the column stationary phase and liquid phase. The interaction of the solute with the mobile and stationary phases can be worked through different choices of both solvent and stationary phases.^[2]

Figure.1. Structure of Paliperidone

Paliperidone, also known as 9-hydroxyrisperidone, is a dopamine antagonist of the atypical antipsychotic class of medications. It is the primary active metabolite of the antipsychotic resperidone.

Paliperidone is used to treat mania and at lower dosages as maintenance for bipolar disorder. It is also used for schizophrenia and schizoaffective disorder. A few mutual paliperidone side effects include drowsiness, headaches, and a rapid heart rate. Less common side effects of this medicine (occurring in less than 5 percent of people) can contain fatigue, shakiness, and dry mouth. Certain side effects of paliperidone are more serious and should be reported to your healthcare provider. These more serious side effects can include large or rapid weight gain, anxiety, and very severe constipation.^[3]

Active substance:

Paliperidone is a white to yellow non-hygroscopic powder. The molecular formula is C23H27FN4O3 and the relative molecular mass is 426.49.

Chemical name of paliperidone is (±)-3-[2-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]ethyl]-6,7,8, 9-tetrahydro-9-hydroxy-2-methyl-4Hpyrido[1, 2-a] pyrimidin-4-one.

Its dissociation constants are pKa1 = 8.2 (piperidine moiety) and pKa2 = 2.6 (pyrimidine moiety). Solubility in water is 0.003g/100ml, rising to to 2.3g/100ml in 0.1 N HCl, however in ethanol it is 0.076g/100 ml. The logP is a neutral molecule in a 1-octanol and aqueous buffered solution is 2.39, whereas the logP of its formulation in phosphate solution of pH 7.0 is 1.02.

Paliperidone has one chiral center and is synthesized as a racemic mixture. There are Two polymorphs observed, polymorph I and II and in addition to a hydrate and a solvate.^[12]

Schizophrenia is a complex, chronic psychiatric disorder characterized by active psychosis (e.g., delusions, hallucinations, and disorganized thoughts), as well as deficits in cognitive and psychosocial functioning.1,2 This debilitating disorder is associated with reduced quality of life and shortened life span. 2 While psychotherapeutic interven¬tions can improve insight and functional abilities in individuals with schizophrenia, pharmacotherapy is a fundamental component of any treatment plan.3 Unfortunately, complete remission of symptoms is unlikely. Consequently, the ultimate goals of treatment are to improve functional capabilities, minimize residual symptoms during periods of remission, and decrease relapse frequency and duration.^[11]

Analysis:

Analysis is important in any product or service, but in drug it is very important as it involves life. In assessment to general consumer products, in drugs there is and there can be only quality/standard product and no other product. This derived from series of tests from quality control, starting from raw materials in process during manufacturing of the drug, finished product is the right requirement to the patients so quality of drugs should be taken in maintenance. These tests may differ from single entity or combination of several potent drugs in formulation these tests of quality control can be appropriate to the following types:

1 Chemical methods

2 Physicochemical methods

3 Microbiological methods

4 Biological methods

Analysis can be mainly Classified:

1. Quantitative analysis

The Volumetric analysis is merely a titration centered in a neutralization reaction as well as precipitation or a complex forming reaction along with a titration based in a redox reaction. The Complex forming titration is a reaction that incomes place between metal ions and a standard solution that is in the utmost cases EDTA (ethylene diamine tetra acetic acid).

2. Qualitative analysis:

Qualitative Analysis can takings place without Quantitative Analysis, but Quantitative Analysis needs the identification (qualification) of consider for which numerical evaluations are given.^[1]

Analytical chemistry:

It is the branch of Science that usages development technologies in defining the composition by analytical technique. We can accomplish both qualitative along with quantitative results. Analytical instruments play a major role in the process to achieve high quality and reliable analytical data. Hence everyone in the analytical laboratory should be worried about the quality assurance of equipment.

Analysis is vigorous in any product or service also vital in drug because it includes life.^[1] It is the analysis of separation, quantification and chemical additives identification of herbal plus synthetic materials constituted through one or more compounds or factors. Analytical chemistry is separated into two predominant classes, a qualitative evaluation that is to say the identification with regard to the chemical additives exists in the sample, however quantitative evaluation evaluations the amount of positive aspect or compound surrounded by the substance, i.e. the sample.^[7]

Analytical chemistry deals with method of determining the chemical composition of sample. It is primarily concerned about determining the qualitative and quantitative composition of material. It is a scientific discipline used to study the chemical composition, structure and behavior of matter. The term chemical analysis may be defined as the application of a process or series of process to identify or quantify a substance, the component of a solution or mixture or the structure of chemical compounds. It is involved in all the stages from drug discovery, development, action, safety, formulation, use, quality control, packaging, storage, marketing etc. Any drug or dosage form for human use must have excellent quality and purity, free from impurities. This dosage form directly affects the human life and behavior so their analysis is important which is carried out using analytical methods. Analytical method Development is the heart of analytical chemistry. It involves development and validation of new analytical method for the purpose of testing samples. Sample testing is done by using UV, IR, HPLC, HPTLC, GC-MS, and LC-MS etc. Analytical chemistry has since long, occupied an important in the development of science and technology. It is very broad and embraces a wide range of natural, chemical and instrumental technique and procedure.^[4]

Pharmaceutical products formulated by using more than one drug, usually mentioned to as combination products, are planned to come across before unmet patients need Analytical Method Development and Validation 56by conjoining the therapeutic effects of two or more drugs in one product. These combination products can current intimidating challenges to the analytical chemist accountable for the development and validation of analytical methods. The permitted test methods that result as of these methods are used through quality control laboratories to make sure the identity, purity, potency, and performance of drug products.^[6]

The frequent novels drugs are organism presented and are constantly growing day by day. Therefore it is completely authoritative to develop novel methods and introduced them for controlling their quality. Modern pharmaceutical analysis needs the ensuing requirements.

1 It should take a minimal time and should be economical.

2 The accuracy must accept the guidelines of Pharmacopoeia.

3 The selected method should be precise as well as selective.

Purpose of analytical method development:

The reasons for the novel methods development of drug analysis are as bellow:

a) When there is no authorized drug or drug combination available in the pharmacopoeias.

b) When there is no well-mannered analytical process for the present drug in the literature due to patent regulations.

c) When there are no analytical methods for the preparation of the drug due to the interferingproduced by the formulation excipients.

d) Analytical methods for the quantitation of the analyte in biological fluids are create to be unapproachable.

e) The current analytical procedures may requirement costly reagents and solvents. It may also involve troublesome extraction and separation procedures.[^9]

Basic principles for new method development for drug analysis:

1 The drug or drug combination may not be official in any pharmacopoeias

2 A proper analytical procedure for the drug may not be available in the literature due to patent regulations

3 Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients

4 Analytical methods for the quantitation of the drug in biological fluids may not be available

5 Analytical methods for a drug in combination with other drugs may not be available

6 The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.^[10]

Various International regulative Agencies have set the standard and fixed the protocol to match the reference for granting approval, authentication and registration. Some of the famous organizations governing the quality standards are:

1) United States Food and Drug Administration (US FDA).

2) Current Good Manufacturing Practice (cGMP) regulations.

3) Good Laboratory Practice (GLP) regulations.

4) The Pharmaceutical Inspection Cooperation Scheme’s (PIC/S).

5) Pharmaceutical Inspection Cooperation Scheme (PIC/S).

6) The International Conference for Harmonization (ICH).

7) Quality Manual ISO/IEC 17025 issued by International Organization for Standardization.

8) World Health Organization (WHO)^[4]

Fig.2 Flow chart of method development [5]

Fig.3 Flow chart showing different steps in analytical method development ^[5]

Steps in Method Development:

1. Physicochemical Properties of drug molecule

2. Selection of Chromatographic conditions

3. Developing the approach of analysis

4. Preparation of sample

5. Method Optimization

6. Method validation^[2]

A) Physicochemical Properties of Drug Molecule:

a) Chemical properties:

Chemical structure of the known and expected product chemical structures are good inputs for initiating the method development and it will give scientific approach for the method development. Draw the comparative difference between impurities, starting materials, by-products intermediate and degradation products with final products.

b) pH and pKa value of compound:

The nature and polarity of the compound is assumed based on pH and pKa values. The compound is half ionized, when pH is equivalent to pKa. Almost all the pH related change occurs within the ± 1.5 units of the pKa values. Outsides the range the compound is either ionized or non-ionized, and its retention does not change much with pH

B) Selection of Chromatographic Conditions:

During initial method development, a set of initial conditions (column, mobile phase and detectors) is selected. In most cases, these are based on reversed phase separations on a C18 column with UV detection. A choice on developing an isocratic or else a gradient method should be prepared at this point. ^[2]

a) Selection of column:

The principle part of a HPLC system is the column. Changing a column will have the great effect on the resolution of analytes during method development. An appropriately selected column can produce a good chromatographic separation and it provides accurate and reliable analysis.^[2]

Table .1. Various types of columns and their applications^[5]

Column	Phase	Solvents	Application
C18	Octadecyl	ACN, MeOH, H2O	General, nonpolar
C8	Octyl	ACN, MeOH, H2O	General, nonpolar
Phenyl	Styrl	ACN, MeOH, H2O	Fatty acids, double bond
Column	Phase	Solvents	Application
Cyano	Cyanopropyl	ACN, MeOH, H2O, THF	Ketones, aldehydes
Amino	Aminopropyl	ACN, MeOH, H2O, THF, CHCl3, CH2Cl2	Sugars, anions
Column	Phase	Solvents	Application
Diol	Dihydroxy hexyl	ACN, MeOH, H2O, THF	Proteins
SAX	Aromatic quaternary amine	SALT Buffers, ACN, MeOH, H2O	Anions
SCX	Aromatic sulfonic acid	SALT Buffers, ACN, MeOH	Cations
DEAE	Alkyl ether, ethyl 2°amine	SALT Buffers, ACN, MeOH, H2O	Protein cations

b) Chromatographic conditions:

Selection of appropriate chromatographic conditions is the next step in the method development. This includes selection of temperature, selection of composition of mobile phase, pH of mobile phase and flow rate etc. A decision of developing either an isocratic or a gradient method should be made at this point. In most cases, these are based on reversed-phase separations on a C18 column with UV detection.^[12]

c) Optimization of Mobile phase:

When samples contain ionizable compounds, the mobile phase pH can be one of the most important variables in the controls in the control of retention in a reversed-phase HPLC (RP-HPLC) separation [2]In RP chromatography, water and buffer are weak solvent and CAN, methanol, THF is strong solvents and this are most widely use solvents. Other considerations when selecting solvents:

Methanol – High viscosity may limit use of smaller particle size or longer columns at elevated flow rates.

Acetonitrile – Relatively high cost. THF – UV absorbance at low wavelengths; high viscosity[13]

d) Column temperature:

The HPL Ccolumn temperature is a challenge because it can have changeable effects on selectivity. It is difficult to decide if the use of elevated temperatures will help or hinder a specific separation. For complex separations, improvements in one portion of the chromatogram are almost always accompanied by decreases in another part of the same chromatogram.^[14]

e) Selection of wavelength and detector:

All listed molecules UV/Visible and FT-1R spectrums are required to select the UV detector nm for all molecules. FTIR spectral data is the main source for understanding the functional groups activity. ^[15]

C) Sample Preparation:

The sample should preferably be dissolved in the initial mobile phase. If this is not possible due to stability or solubility problems, formic acid, acetic acid or salt can be added to the sample to increase solubility. The excipients do not generally effect the separation hence the volume of the sample filled is small compared to the column volume. The simply effect once large sample volumes are suitable may be an extra peak or two eluting in the void volume afterward sample injection. Sample preparation is avital part of HPLC analysis, intended to offer a reproducible and homogenous solution that is suitable for injection onto the column.^[17] The purpose of sample preparation is a sample aliquot that, is comparatively free of interferences, the damage of column, and Iswell-suited with the intended HPLC method that is, the sample solvent will dissolve in the mobile phase without disturbing sample retention or resolution. Sample preparation originates at the point of collection, spreads to sample injection onto the HPLC column. All of these operations form an important part of sample preparation and have a critical effect on the accuracy, precision, and convenience of the final method^[16]

D) Method Optimization:

The experimental conditions should be elevated to developpreferred separations and sensitivity after execution appropriate separations. The stability with assay experimental conditions will be accomplishedover planned or systemic examination of the parameters by pH (if ionic),gradient, flow rate, mobile phase components and ratio, temperature, sample amounts, Injection volume and diluents solvent type.^[18]

The mobile and stationary phase compositions need to be taken into account. Optimization of mobile phase parameters is all the timedeliberated first as this is considerable easier and convenient than stationary phase optimization. To decrease the number of trial chromatograms elaborate, only the parameters that are to be expected to have a significant effect on selectivity in the optimization must be studied. ^[15]

E) Method Validation:

Validation should in this way be measured in the associatedconditions:

· Completely new procedure.

· Latest equipment.

· The Procedure as well as equipment which have been used tosuit different needs and,

· Procedure where the finished result test is a poor and undependable marker of product quality.^[21]

Important stages in validation:

The action identifying with validation studies can be categorized mainly into three stages:

Stage 1

This includes pre-validation qualification stage which covers all exercises identifying with product studies and improvement, formulation pilot batch testing, scale-up research, exchange of innovation to business scale groups, arrangement stability conditions, and dealing of in-process, finished pharmaceutical formulations, qualification of equipment, master documents, and process limit.^[4]

Stage 2

This involves process validation phase. It is intended to check that every installed limit of the vital process parameter is substantial and that satisfactory products can be created even below the worst situation.

Stage 3

It is also called as the validation maintenance stage, it involves constant review of all procedure related archives, including validation of the review reports, to guarantee that there have been no modifications, departure, failures, and alteration to the production procedure and that all standard operating procedures (SOPs), involving change control procedures, had been observed. At this phase, the approval team involving people representing all essential departments also guarantees that there have been no modifications/deviations that ought to have brought about requalification and revalidation.^[19]

Types of validation:

Validation is classified into following types:

Fig, 4 Validation types^19]

A. Equipment validation:

The main concept of validation is to provide a high degree of reported approval that the equipment and the procedure obey to the written guidelines. The degree (or intensity) is dictated by the complexity of the device or system. The validation should give the essential data and test methods required to give that the device and technique meet determined prerequisites. Equipment Validation includes the following:

a. Installation qualification (IQ):

IQ guarantees all crucial processing, packaging system, and ancillary items are in compliance with the installation. It checks that the equipment has been established or installed as per the manufacturer’s suggestion in a systematic way and positioned in surrounding appropriate for its meant purpose ^[22]

Installation qualification points include

· Equipment layout character which is the material of creationcleanability and several others.

· Installation conditions like wiring, functionality, utility and so onwards.

· Calibration, preventative protection, cleansing plans.

· Safety characteristics.

· Supplier documentation, prints, illustrations, and hand operated.

· Software documentation.

· Enlist the spare components.

· Environment-related conditions like clean room necessities,

Humidity and temperature^[23]

b. Operational qualification (OQ): OQ performed to give a high level of degree of affirmation that the equipment works as proposed.^[22]Process control limits like temperature, time, stress, line velocity, set up conditions, and so on.

Software parameters:

Crude material details.

· Process operating methods.

· Material managing necessities.

· Process change control.

· Training.

· Short-term balance and capability of the technique.

· The use of statistically valid procedures inclusive of screening examinations to optimize the technique can be utilized throughout this stage.^[23]

c. Performance qualification (PQ):

PQ checks that the device is repeatable and it is uniformly producing a quality item.^[22]

PQ concern consists of:

· True product, procedure parameters, and process established in OQ.

· Adequacy of the product.

· Guarantee of methodcapability as made up in OQ.

· Process repeatability, prolonged process stability^[23]

B. Process validation:

The process validation is a component of the coherent prerequisites of a quality management system. [11] Process Validation is the most essential and perceived parameters of current good manufacturing practices. The objective of a quality system is to produce items that are matched with their proposed use uniformly. Process approval is a key component in guaranteeing that these standards and objective are met.^[23]

a. Prospective validation:

It is described as the well-known reported program that a device does what it indicated to do base on pre-planned protocols. This validation is normally performed previously for distribution both of a newer item or item made under a revised production process.^[24]

The equipment or facilities list is to be utilized (including calculation, observing or recording equipment) togetherby its calibration status.

· Finished dosage forms for discharge.

· List of analytical techniques, as suitable.

· Proposed in-process controls with specification criteria.

· Additional testing to be finalized, with specification limits and analytical agreement, as appropriate.

· Sampling design.

· Techniques for recording and assessing outcomes.

· Functions and obligations.

· Proposed timetable.^[25]

b. Concurrent validation:

It is same as prospective validation with the exception of the working firm, will offer the product at the time of qualification runs, to the society at its market cost, and furthermore like retrospective validation. This type of validation includes in-process observing of vital processing steps and product checking out. This helps to produce and reported proof to demonstrate that the manufacturing technique is in a condition of control.^[24]

c. Retrospective validation:

It is characterized by the established reported confirmation that a system does what it implies to do on the audit and investigation of historical data. This is accomplished by the survey of the ancient manufacturing testing information to demonstrate that the procedure has always remained in controldistribution. Retrospective validation is adequate for well-established procedures and will be wrong where there have been current modifications within the composition of the product, working methods or device.^[24]

C. Cleaning validation:

Cleaning validation is a reported proof with a high level of confirmation that can uniformly clean a system or equipment to already determined and specification criteria. Cleaning approval is aninformed procedure that establishes the efficacy and uniformity in cleaning pharmaceutical production equipment. The aim of cleaning approval is to check the viability of the cleaning system for the expulsion of product deposits, additives, degradants,excipients, or cleaning agents and in a the control of potential microbial contamination.

Necessity for cleaning validation:

To check the viability of cleaning techniques and to make sure that no risks are related to cross-contamination of API or detergents.^[26]

Revalidation:

Revalidation gives the proof that modifications in the procedure, as well as the procedure condition that are presented don't unfavorably influence process attributes and product quality. Organizations, facilities, equipment and methods which consist of cleaning, should to be periodically considered to affirm that they stay valid. Where no remarkable modifications have been made to the approved status, a review with proof that facilities, organizations, equipment and procedures address the recommended necessities satisfies the need for revalidation

Revalidation becomes vital in specific circumstances. A small number of of the modifications that need validation are stated below:

· Modifications in crude materials.

· Modifications in the equipment.

· Modifications in the source of active crude material producer.

· Alteration of packing material.

· Modification of the procedure.

· Modifications inside the plant/facility.

· A selection is no longer to carry out revalidation studies have to be completely justified and reported.^[25]

Importance of validation:

· Assured high quality.

· Time boundation.

· Optimization of the method

· Least batch product failure, improved efficiency, manufacturing, and productivity.

· Quality cost decreased.

· Rejection decreased.

· Yield increases.

· Fewer complaints about process related issues.

· Fast and realistic start-up of new equipment’s.

· Increased worker consciousness of the process.^[27]

Parameters of analytical method validation:

1) Accuracy

2) Precision

a) Repeatability

b) Intermediate Precision

c) Reproducibility

3) Specificity

4) Detection Limit

5) Quantitation limit

6) Linearity

7) Range

8) Stability

9) Robustness

10) Ruggedness

11) System Suitability^[2]

1) Accuracy:

The accuracy of a method may differ through the range of probable assay values and therefore must be determined at few different protection levels. The accuracy should refuge at least 3 concentrations (80, 100 and 120%) in the estimated range.

Accuracy may also be determined by relating test results with those achieved using additional validated test method. Dosage form assays normally deliver accuracy within 3‐5% of the true value. The ICH documents report that accuracy should be estimated using a minimum of nine determinations beyond a minimum of three concentration levels, covers the definite range (i.e. three concentrations and three replicated determination for every single concentration).^[20]

2)Precision:

The correctness of an analytical procedure states the nearness of agreement (degree of scatter) between a group of measurements achieved from altered sampling of a uniform sample underneath the recommended conditions Precision may be taken into consideration at 3 levels:

a) Repeatability:

It expresses the exactness below a similar operating condition over a brief interval of time and also referred as intra-assay precision. A minimum of six replicates test preparation of a similar or consistent sample ready at the 100% check

b) Intermediate precision:

It expresses the exactness under inside research laboratories, in distinct days, through distinct analyst, on distinct instruments/equipment. Two different analysts every making six sample solutions, as per stated method

c) Reproducibility:

It refers to the precision between different analytical labs. Every research facility set up an aggregate of six sample solutions, according to the analytical technique.^[19]

3) Specificity:

For every stage of development, the analytical technique should demonstrate specificity. The method was should have the power to clearly consider the analyte of interest where within the presence of all probable parts, which can include degradants, excipients or sample matrix, and sample blank peaks.^[28]

An ICH guideline describes specificity as ability to measure clearly the analyte in the presence of other compounds that may be possible to be present. Typically these might be impurities, degradants, matrix, etc. The definition has the following implications:

· Identification test: It should be capable to distinguish compounds of closely related structure which are estimated to be present i.e., to satisfy identity of an analyte.

· Purity test: To confirm that the analytical technique accomplished permits an accurate statement of content of the impurity of an analyte i.e. residual solvents content, related substances, heavy metals, etc.

· Assay: To arrive at an accurate result, this permits a correct report on the potency or content of analyte in a sample.^[9]

4) Limit of detection (LOD):

Lowest quantity of an analyte which may be detected by the chromatographically separation however it is not necessary that this quantity will quantify as a precise value. A blank resolution is injected and peak to peak quantitative noise relation we have to calculate from blank chromatograms. Then, calculate the concentration at the signal to quantitative noise relation is concerning 3:1.

LOD can be expressed as

LOD = 3.3SD/S

Where, SD = Standard deviation of response,

S = Slope of calibration curve.

The LOD value of betahistine hydrochloride (BET) and prochlorperazine maleate (PRO) is 0.29μg/ml and 0.34μg/ml respectively. LOD of the paracetamol was studied by the signal to noise ratio, and the result was found to be 120μg/ml.^[19]

5) Limit of Quantitation (LOQ):

It is characterized by the least quantity of an analyte that can be quantified with exactness and precision.

LOQ can be communicated as

LOQ = 10SD/S

Where SD = Standard deviation of response,

S = Slope of calibration curve.

The LOQ value of betahistine hydrochloride (BET) and prochlorperazine maleate (PRO) is 0.957μg/ml and 1.12μg/ml respectively. LOQ of the paracetamol was studied by the signal to noise ratio, andthe result was found to be 360μg/ml.^[19]

6) Linearity:

Linearity may be characterized as the capacity of an analytical technique to produce outcomes which are directly related to the concentration of an analyte in the solution.^[29]

For simultaneous estimation of nitazoxanide and ofloxacin linearity perform as five different concentrations of standard mixtures prepared, 50%, 75%, 100%, 125%, 150% were injected, and chromatogram was recorded. The correlation coefficient was calculated and was observed to be greater than 0.99 for both the drugs which are within the limit.^[30]

7) Range:

It can be characterized as the interval amongst upper and lower quantities of analyte in the sample. Minimum of the specified range to be 80% to 120% of the test sample for the assay test.^[31]

The range is generally expressed in the similar units as the test results (for example percentage, parts per million) achieved by the analytical method.

· For Assay - 80 to 120% of test concentration

· Content uniformity of test concentration - 70 to 130%

· Dissolution - Q-20% to 120%

· Impurities - reporting level – 120% of impurity specification limit

· Assay & Impurities - Reporting level to 120% of assay specific.^[2]

8) Ruggedness:

Ruggedness is the degree or measure of reproducibility under different situations such as in different laboratories, different analyst, different machines, environmental conditions, operators etc.^[19]

9) Robustness:

In the liquid chromatography there are some examples of characteristic variations are:

· Effect of variations of pH in a mobile phase

· Effect of variations in mobile phase composition

· Different columns (different lots and/or suppliers)

· Temperature

· Flow rate^[2]

10) Stability:

Solution stability is stability of standard and extracted sample solution (ready to inject) as of the sample or matrix and evaluated as per definite method, and it must be stored correctly in room temperature and refrigerated condition depending upon the stability of the sample and standard solution. The stability of standard and sample solution should be established in room temperature and refrigerated, if refrigerated before analyzing it should be thawing to room temperature. A minimum two preparation of standard and sample solution should be prepared and analyzed as per specified method.^[2]

Fig. 5: Flow Chart of Stability^[10]

11) System Suitability:

As per the USP, system suitability tests are an essential part of chromatographic methods. These tests are used to confirm that resolution as well as reproducibility of the system issuitable for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, and samples constitute an integral system that can be estimated as an entire.^[2]

Table 2: Acceptance criteria of system suitability parameters^[19]

Sr. No.

Parameter name

Acceptance criteria

Reference

Tailing Factor

Number of theoretical plate

Resolution

RSD

>2000

>1.5

HPLC Method Development:

High Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques. More than 85% of general pharmaceuticals are analyzed by HPLC. The technique of chromatography was originally developed by Russian Botanist M.S. Tswett in 1903 but after that, plenty of revolutions and amendments were done and it is still going on. HPLC is the separation module which contain mainly stationary phase and mobile phase having opposite polarity equipped with high pressure pumps and the separation is achieved by the interaction of stationary phase and the mobile phase. A proper choice of stationary phase and mobile phase is essential to reach desired separation. The pH of mobile phase, different types of buffer, column temperature, sample diluents, detection wavelength and many more of the variables which play a major role in method development. [5]

Separation goals:

Table.3. Separation goals in brief^[5]

Goal	Comment
Resolution	Precise and rugged quantitative analysis requires that RS be greater than 1.5
Separation time	< 5-10 min is desirable for routine procedures
Quantization	≤ 2% for assays ≤ 5% for less-demanding analyses ≤ 15% for trace analyses
Pressure	< 150 bar is desirable < 200 bar is usually essential (for a new column)
Peak height	Narrow peaks are desirable for large signal/noise ratios
Solvent consumption	Minimum mobile phase use per run is desirable

Instrumental requirements of HPLC:

· Pumps- solvent delivery system

· Mixing unit, gradient controller and solvent degassing

· Injector- manual or auto injectors

· Guard column

· Analytical columns

· Detectors

REFERENCES:

1. Hema and Swetha Reddy, A Review on new Analytical method Development and Validation by RP-HPLC, International Research Journal of Pharmaceutical and Biosciences, 2017, 4 (3): 41-50

2. Prajakta G. Thete & Ravindra B. Saudagar, Review on analytical Method development and validation by RP-HPLC, Indo American Journal of Pharmaceutical Sciences, 2018, 05(05): 4897- 4907

3. Hardeet al., A validated Inherent Stability–Indicating HPLC Method for estimation of Paliperidone in the Bulk drug and Pharmaceutical dosage form, World Journal of Pharmacy & Pharmaceutical Sciences, 2015, 4(8): 1937-1948

4. Doltade et al, Analytical Method Development and Validation: A Review, Journal of Drug Delivery & Therapeutics. 2019, 9(3):563-570

5. Ravisankar et.al, A review on analytical method development, Indian Journal of Research in Pharmacy and Biotechnology, 2014, 2(3): 1183-1195

6. Chapter 2, Analytical Method Development and Validation, 55- 94

7. Sharma et al., A Review on Analytical Method Development and Validation, International Journal of Applied Pharmaceutics, 2018, 10(6): 8-15

8. Chauhan et al., Analytical Method Development and Validation: A Concise Review, Journal of Analytical &Bioanalytical Techniques, 2015, 6(1): 01-05

9. Panchumarthy Ravisankar, Ch. Naga Navya, D. Pravallika, D. Navya Sri, A Review on Step-by-Step Analytical Method Validation, IOSR Journal of Pharmacy, 2015, 5(10): 07-19

10. RajendraPatil, Tushar Deshmukh, Vijay Patil, and Kishanchand Khandelwal, Review on Analytical Method Development and Validation, Research and Reviews: Journal of Pharmaceutical Analysis, 2014, 3(3): 1-10

11. Alicia Lopez, Jose Rey, Role of paliperidonepalmitate 3-monthly in the management of schizophrenia: insights from clinical practice, Neuropsychiatric Disease and Treatment 2019, 15; 449–456

12. European public assessment report, Scientific Discussion, EMEA, 2007, 1- 58

13. John Dolan, A Guide to HPLC and LC-MS Buffer Selection; ACE HPLC column, 1-20

14. Philip J. Koerner, General Principles of HPLC Method Development; Part 1, General Chromatographic Theory slide, 2013; 1-53

15. Sowjanya P., Subashini D., Rakesh S., A Review on Method Development and validation by HPLC, RRJPPS, Jan-Mar 2015; 4(1)

16. Willard H. H., Merritt L. L., Dean J. A., Settle F. A., Instrumental Methods of Analysis, Seventh Edition, CBS Publishers and Distributors Pvt. Ltd., 118-148, 598-607

17. P. D. Sethi; High Performance Liquid Chromatography, Quantitative Analysis of Pharmaceutical Formulations; 1^st edition, New Delhi: CBS Publishers And Distributors, 2001; 1, 3-11, 116-120

18. Ashwini B. Sambherao, Bhushan A. Bhairav and Dr. R. B. Saudagar, Analytical method development and validation by RP-HPLC and UV spectrophotometric methods, European Journal of Biomedical and Pharmaceutical sciences, Vol. 4(10); 1-6

19. M. A. Potdar; Pharmaceutical Quality Assurance, Nirali Prakashan, 2007, 8.28-8.31.

20. Vichandran V, Shalini S, Sundram K. M., Harish Rajak, Validation of analytical methods – strategies & importance, International Journal of Pharmacy and Pharmaceutical Sciences, 2(3); 18-22

21. ICH Q2 (R1), Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization, IFPMA, Geneva, 2005

22. Jatto E, Okhamafe AO, an overview of pharmaceutical validation and process controls in drug development, Trop J Pharm Res, 2002, 1; 115-22

23. Verma P, Madhav NS, KR Gupta V, A review article on pharmaceutical validation and process controls, Pharma Innovation, 2012, 1; 51-60

24. Md Alamshoaib, Pharmaceutical process validation: an overview, J Adv Pharm Edu Res, 2012, 2; 185-200

25. Ahir KB, Singh KD, Yadav SP, Patel HS, Poyahari CB, Overview of validation and basic concepts of process validation, Sch Acad J Pharm, 2014, 3; 178-90

26. Nandhakumar L, Dharmamoorthy G, Rameshkumar S, Chandrasekaran S, an overview of pharmaceutical validation: quality assurance viewpoint, Int J Res Pharm Chem, 2011, 1; 1003-14

27. Goyal D, Maurya S, Verma C, Cleaning validation in the pharmaceutical industry-an overview, Pharma Tutor, 2016, 4; 14-20

28. Lavanya G, Sunil M, Eswarudu MM, Eswaraiah MC, Harisudha K, Spandana BN et al. Analytical method validation: an updated review, Int J Pharm Sci Res, 2013, 40; 1280

29. Tiwari G, Tiwari R, Bioanalytical method validation: an updated review, Pharm Methods, 2010, 1: 25-8

30. Ravichandran V, Shalini S, Sundram KM, Harish R,. Validation of analytical methods–strategies and importance, Int J Pharm Pharm Sci, 2010, 18-2

31. Nirupa G, Tripathi UM, RP-HPLC analytical method development and validation for simultaneous estimation of two drugs nitazoxanide, ofloxacin and its pharmaceutical dosage forms, Int J ChemTech Res, 2012, 5; 775-83

32. Shweta K, Anita S, A review on analytical method validation, Int J Pharm Res Rev 2016, 5; 30-6

33. Khemchand Dadareet al, Application of RP-RRLC methods for estimation of paliperidone in tablet dosage forms, Journal of Chemical and Pharmaceutical Research, 2012, 4(6); 3154-3157

34. Ashok Kumar K. et al, Analytical Method Development and Validation of Paliperidone in Bulk and Tablet Dosage Form by Reverse Phase HPLC Method, Asian Journal of Research in Chemistry and Pharmaceutical Sciences,2013, 1(2); 71 - 78.

35. F´abio Barbosa, Luciano Mantovani, C´assia V. Garcia, and Andreas S. L. Mendez, Studies on Paliperidone in OROS Tablets: Extraction Procedure and Chromatographic Analysis, International Scholarly Research Network Chromatography, 2012; 1-9

36. Sharma et al, A Review on Analytical Method Development and Validation, International Journal of Applied Pharmaceutics, 2018, 10(6); 8-15

Received on 31.12.2019 Modified on 24.01.2020

Research J. Science and Tech. 2020; 12(1):23-35.

DOI: 10.5958/2349-2988.2020.00003.0