INTRODUCTION:

In modern method High performance liquid chromatography was firstly referred to as high pressure liquid chromatography. An analytical chemistry technique which is used to separate, identify and quantity each component in a mixture is called as HPLC.

HPLC unit consist of solvent reservoirs, solvent degasser , mixing vessel for delivery of the mobile phase, high pressure pump, gradient value, switch value in inject position ,switching value in load position, pre column ( guard column), sample injection 100p, analytical column, detector ( i.e IR , UV) , data acquisition , waste or fraction collector.

HPLC depends on its pumps to pass a pressurized liquid and a sample mixture through a column which is filled with adsorbent thats leads to the separation of sample compound. The adsorbent is an active compound of the column; that is typically a granular material made up of a solid particles; which is usually 2-50 um in size.

The sample mixture are separated from each compound due to their different degrees of interaction with the adsorbent particles. The pressurized liquid which is referred as a mobile phase is typically a mixture of solvent ( eg. Water, methanol, acetonitrile, buffer, etc). The compound and temperature of mobile phase play’s a major role in the process of separation .

HPLC has its application in chemical, bio-science, pharmaceutical, consumer product, environmental clinical, etc.

Rivaroxaban:

Rivaroxaban is an anticoagulant used in the treatment and prohibition of blood clots. Specifically it is used to treat deep vein thrombosis and pulmonary emboli and prevent blood clots in atrial fibrillation and following hip or knee surgery. Rivaroxaban is an alternative to Warfarin. As compared to Warfarin it has lower interactions with other medications. Route of administration is oral.

The activity of the drug is carried out by blocking the activity of the clotting protein factor Xa.

Common side effect is bleeding. It is prohibited in pregnancy and breastfeeding. Other serious side effects may include spinal hematoma and anaphylaxis. Rivaroxaban was patented in 2007 and approved for medical use in the United States in 2011. In the United States, it will not be available as a generic medication until 2024.

It’s marketed preparation is Xarelto.

Rivaroxaban is the first available orally active drug which is direct factor Xa inhibitor. Rivaroxaban is an oxazolidinone derivative which helps in enhancing the inhibiting of both the free Factor Xa and Factor Xa bound in the prothrombinase complex. Inhibition of Factor Xa disturbs the intrinsic and extrinsic pathway of the blood coagulation cascade, which inhibit both the thrombin formation and development of thrombi.

The tablet of Rivaroxaban is available in dose of 10 mg of Rivaroxaban and Excipients q.s Colours (ferric oxides USPNF –red &titanium dioxide-white IP). To date, there is no official pharmacopoeia monograph of research paper which indicates determination of Rivaroxaban by High performance thin layer chromatography.

Review of literature revealed that various spectrophotometric methods [4-7], bioanalytical methods involving UPLC- MS/MS[8],UHPLC- MS MS[9],colorimetric method[10],RP-HPLC[11- 14] have been reported for the estimation of Rivaroxaban from its tablet dosage form.

Analytical method development:

New methodologies are being progressed for evaluation of the novel product when there are no definitive techniques are present. This new novel techniques are developed to reduce the value besides time for higher precision and strength To investigate the presence of either pharmacopoeial or non-pharmacopoeial product. These methodologies are optimized and valid through initial runs. Substitute ways are planned and place into practice to exchange the existing procedure within the comparative laboratory information with all accessible advantages and disadvantages.

Necessity of method development:

Drug evaluation exhibits the establish characterization of the drugs in combination like drug and organic fluids. At some point of producing technique and development of drug the principal purpose of analytical strategies is to generate data regarding efficiency, impurity, stability, and effect of manufacturing parameters to verify that the production of drug product increase.

Analyst before the development of new technologies, consider the below indicate criteria:

· Is this technique possesses the need full sensitivity?

· Is this method sufficiently selective for direct use without interference by means of the opposite element within the sample?

· Are the reagents and equipment required on this method available or obtained at a sensible price?

· Is the time requires to perform this technique appropriate^[2].

Steps for developing a method:

Various point are involved in the development of an analytical method are as follows:

Characterization of analyte and standard

· All the known necessary data concerning the analyte and its structure that is to mention the physical and chemical properties such as solubility, boiling point, viscosity, colour, size etc., are collected.

· The standard analyte equal to 100% purity is acquired. Necessary arrangement is to be created for the proper storage (refrigerator and freezer).

· In the sample matrix, when multiple parts are to be measured, the amount of elements is observed duly presenting the information and the accessibility of standard are calculated.

· Techniques like spectroscopy (UV-Visible, FTIR, etc.), HPLC and GC so on and, are however about once coordinated with the stability of samples^[1].

Requirement of the technique:

Requirement of analytical methodology is essential to build up the analytical fig. of advantage like accuracy, linearity, precision selectivity, specificity, range, LOD, LOQ etc. shall be outlined^[1].

Literature survey and prior methods:

All the data of literature related to the drug are reviewed for its physical and chemical properties, manufacturing, solubility and applicable analytical ways with reference to related books, journals,(USP/NF), (AOAC) and (ASTM) publications and in which it is extremely convenient to look Chemical Abstracts Service automatic computerized literature^[1].

Selecting the method:

· Utilizing the data obtained from the literature, the methodology is developing since the method is being modified wherever needed. Sometimes, it is important to gain additional instrumentation to create, alter or replicate and validate existing procedures for analytes and tests.

· If there are not any past appropriate ways available to investigate the analyte to be examined^[1].

Proper instrumentation and initial studies:

(IQ) (OQ) and (PQ) of instrument pertinent to research standard methodology is examined by an appropriate set up of instruments^[1].

Optimization:

While performing optimization, once a parameter is modified at a time, and a group of conditions are differentiated, before utilizing trial and error approach. This work is needed for accomplished basing on a scientific organized method plan duly all necessary points and documented with relation to dead ends^[1].

Proper documentation of analytical fig. of merits:

The true determined analytical fig. of benefit consisting of LOD, LOQ, cost, linearity and evaluation time, etc. are also recorded^[1].

Evaluation of produced technique with actual specimen:

The specimen solution needs to prompt specific, complete recognition of the peak interest of the medication other than all different matrix parts^[1].

Estimation of % recovery of real samples and demonstration of quantitative sample analysis:

% recovery of spiked, actual standard medication into a sample grid which includes no analyte is evaluated. from test to test optimization to reproducibility of recuperation must have appeared. It is not always essential to get 100% restoration so far as the outcomes are reproducible to perceive with a high level of assurance ^[1].

Validation:

Validation is an idea that had been developed in the U. S. in 1978. The idea of validation has extended during that time to grasp an extensive variety of activities from analytical approaches utilized for the quality control of medication to computerized systems for clinical trials, marking. Validation is not endorsed by regulatory specifications. It is best seen as a critical and necessary part of cGMP.

Validation essentially implies for analysis of validity or activity of demonstrating viability. it's a work force effort wherever it entails humans from numerous departments of the corporate. it's required for any new or amended technique to verify that it's capable of giving constant results, once it's once used by totally different| completely different} operators victimization similar instrument inside identical or utterly different laboratories ^[5]. Validation is an essential component of QA. It includes the efficient investigation of systems and procedures aimed toward deciding if they execute their planned capacities sufficiently determined.

Validation considered in accompanying circumstances such as below:

· Complete new procedure.

· Latest instrument.

· Procedure and instrument which have been adjusted to suit altered need sand.

· Procedure where the finished result test is a poor and undependable marker of product quality^[3].

Important stages in validation:

Validation studies can be categorized mainly into three stages:

Stage 1

It includes pre-validation qualification stage which covers all exercises identifying with product studies and improvement, scale-up research, setup of stability conditions, exchange of innovation to business scale groups, pilot batch testing, and managing of in-process, finished pharmaceutical product, qualification of equipment, master documents,^[3].

Stage 2

This phase includes process validation phase. It is intended to check that every installed limit of the process parameter is significant and that satisfactory products can be created even below the worst situations^[3].

Stage 3

This stage is called as the validation maintenance stage, which requires constant review of all procedure related archives, including validation of the review paper, to guarantee that there have been no modifications, departure, and alteration to the production procedure and that all SOPs, involving change control procedures, had been observed. The approval team involving people representing all essential departments also guarantees that there have been no modifications that ought to have brought about requalification and revalidation at this phase ^[3].

Types of validation:

Validation is classified into following types:

Equipment validation:

The key concept of validation is to give a high degree of reported confirmation that the instrument and the procedure conform to the written guidelines. The degree 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 point.

Installation qualification (IQ):

IQ guarantees all crucial processing, and ancillary items are in compliance with the installation. It checks that the equipment has been established or installed as per the manufacturer’s requirement in a systematic way and positioned in surrounding appropriate for its meant purpose ^[5].

Installation qualification points include:

· Equipment layout character that is the material of construction clean ability and many other parameter.

· Installation situations like wiring, utility and so forth.

· Calibration, preventative protection, clean sing plans.

· Safety characteristics.

· Supplier documentation, illustrations, and hand operated.

· Software document.

· List of spare components.

· Environment-related conditions like clean room necessities, humidity, and temperature^[6].

Operational qualification (OQ):

Operational qualification performed to give a high level of degree of affirmation that the equipment works as proposed ^{[ 5].}

OQ concerns consist of:

· Process control limits like temperature, time, stress, set up conditions, and soon.

· Software parameters.

· Crude material information.

· Process operating methods.

· 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^[6].

Performance qualification (PQ):

Performance qualification checks that the device is repeatable and it is uniformly producing a quality item ^[5].

PQ concern consists of:

· procedure parameters, and process set up in Operational qualification.

· Adequacy of the product.

· Guarantee of technique ability as built up in Operational qualification.

· Process repeatability, prolonged process stability^[6].

Process validation:

The process validation is a component of the coherent prerequisites of a quality management system ^[6]. Process Validation is the most essential and perceived parameters of cGMP. 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.

· Process validation is reported evidence which gives a high level of affirmation that a particular procedure will produce a product meeting its determined prerequisites. It mainly involves the following^[3]

Prospective validation:

It is described as the well-known reported program that a device does what it indicated to do based 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 ^[8].

In this validation, the protocol is accomplished before the procedure is placed into company use ^[7].

Prospective validation need to incorporate, however, not be limited to the subsequent:

· Short depiction of the procedure.

· Summary of the important processing steps to be evaluated.

· Equipment list is to be utilized collectively with 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 completed, with specification limits and analytical approval, as suitable.

· Sampling design and modification

· Techniques for recording outcomes.

· Functions and obligations.

· Proposed timetable^[9]

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 ^[8].

This approval includes in-process observing of essential processing steps and product testing. This creates and recorded proof to demonstrate that the production procedure is in a condition of the control.

· In remarkable conditions, it might be acceptable not to finish the validation program before routine manufacturing begins.

· The choice to complete simultaneous approval must be supported, archived and accepted by authorized personnel.

· Documentation prerequisites for simultaneous validation are similar as designated for prospective validation^[9].

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 control. This kind of approval of a procedure for an item already in distribution. 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^[8]

Few basic components of retrospective validation are:

· Batches are produced for a definite duration. The number of lots discharged per year.

· Batch size/strength/year/time.

· Master manufacturing/packaging data.

· Current particulars for active ingredients/finish materials. List of process deviations, corrective actions, and modification to production archives.

· Data for stability study for a few batches^[9].

Revalidation:

Revalidation gives the proof that modifications in the procedure, as well as the procedure condition that are presented don't suitable influence process attributes and product quality. Organizations, facilities, equipment and methods which include cleaning, ought to be periodically assessed to affirm that they stay valid. Where no extraordinary 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^[9].

Revalidation becomes vital in specific circumstances. Few of the modifications that require validation are mentioned below:

· Modifications in raw materials.

· Modifications in the instrument.

· Modifications in the source of active raw material producer.

· Alteration of packing material.

· Modification of the process.

· Modifications inside the plant.

· A selection is no longer to carry out revalidation studies have to be completely justified and reported ^[9].

Analytical method validation:

Validation of an analytical approach is established through laboratory research, that the execution attributes of the procedure meet the wants for the proposed scientific application.

Validation is required for any new or altered procedure to verify that it is fit for giving predictable and dependable outcomes, once used by various administrators by usage of comparable instrumentation inside the similar or absolutely distinct laboratories^[10]. Method validation is a reported program that offers with that the processing system will give a high level of affirmation to meet its predicated acceptance basis ^[4].

It consists of mainly five different steps which are as follows:

· Qualification of the system: System qualifications permit to check that the instrument is appropriate for the planned investigation, the materials are appropriate to be used in analytical measurement, the analysts have the correct instruction, and foregoing documentation such as analytical inclusive of analytical approaches, proper authorized protocol with pre-set up standards have been reviewed. On the off chance that the general qualifications of a device are overlooked, and trouble arises, the source of the issue will be hard to recognize ^[11].

· Sampling: Sampling assists in the choice of a representative part of the fabric which is along these lines subjected to evaluation. The selection of a suitable sampling technique is of significant importance since it gives assurances that the sample chose is really illustrative of the material as a whole for the purpose of important statistical inferences. Inside the statistical literature, there is a considerable collection of work on sampling techniques, anyway the relative expenses and time engaged with every technique ought to be assessed ahead of time ^[11].

· Preparation of sample: Preparation of the sample is a key component to effective method validation. It has been mentioned that sample planning represents 70% to 80% of the work action and working expenses in an investigative lab. The literature on the preparation of the sample is enough and properly documented. In any case, the investigator ought to recall that the choice of a particular preparation technique relies upon concentrations of analytes, sample matrix, size of the sample and the instrumental method ^[11].

· Analysis of sample: The evaluation is associated with the instrument utilized to extract qualitative or quantitative data from the samples with an adequate vulnerability level. The investigation could be predictable, in a great sense, as the device has 3 interconnected fundamental components, namely input, and output. The input and output are assigned by the letters x and y, and they represent the concentration and response individually. The selection of a specific analysis depends on many considerations, for example, the chemical and physical properties of the analytical species, the concentration of the analytes in the sample, matrix, speed, cost, and so forth ^[11].

· Assessment of data: The essential reason behind information assessment is to outline and pick up knowledge into a specific informational index by utilizing numerical and statistical techniques. Data assessment permits extracting valuable data and reaching inferences about the inputs and outputs, and in particular about the validation procedure in general ^[11].

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 a reported procedure that demonstrates the efficacy and consistency in cleaning pharmaceutical production instrument. The goal of cleaning approval is to check the viability of the cleaning system for the expulsion of product deposits, additives or cleaning agents and in a the control of potential microbial contamination.

It is vital to validate cleaning techniques for the subsequent motives:

· Pharmaceutical products and API can be contaminated by other products and microbes.

· It is an administrative prerequisite in pharmaceutical product manufacture the worry is the same-guarantee that the equipment is properly clean and safety and quality is kept up.

· It is likewise guaranteed from an inside control and consistency perspective the quality of manufacture.

· To protect product integrity.

· To reuse the equipment ^[12,13].

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 ^{[12, 13]}.

Cleaning validation protocol:

· The goal of the validation procedure.

· Obligations regarding performing and endorsing the validation study.

· Equipment details.

· The time interval between the end of production and the start of the cleaning techniques.

· Cleaning methods to be utilized for every product, each manufacturing device or each piece of instrument.

· The quantity of the cleaning cycle to be perform continuously.

· Routine checking instrument.

· Sampling techniques, including the basis for why a specific sampling technique is utilized.

· Defined sampling areas.

· Information on recovery studies, where suitable.

· Analytical techniques including LOD and LOQ. The acceptance criteria, along with including the method of reasoning for setting specified limits ^[14].

Importance of validation:

Increased worker consciousness of the process^[4].

· Assured high quality.

· Optimize the method.

· Minimum batch product failure, enhanced efficiency and productivity.

· Quality cost reduced.

· Rejection reduce.

· Yield increasing.

· Fast and realistic start-up of new instrument.

Validation parameters:

The main aim of method validation is to produce proof that the method will what it is supposed to do, accurately, reliable^[4]. The validation parameters as per ICH guidelines are described below:

Accuracy:

It can also be defined as the closeness between the true value and the observed value. It is sometimes called as trueness, and it could be determined by usingat least 9 determinations over a minimum of 3 concentration over the specified range^[15].

Accuracy of PRO and BET was studied by the standard addition method at three different levels (80%, 100%, and 120%). To the pre-analyzed sample a known amount of drug was added and percentage recovery calculated and this method was used for accuracy, the recovery was found to be 99.38% for BET and 99.11% for PRO^[16].

Accuracy of paracetamol was studied by preparing standard solution of different concentrations (10, 20, 30μg/ml) and injected to check the % recovery. The percent recovery of the drug was found in the range of 98.8 to 102.0% respectively for all concentrations^[18].

Precision:

From different sampling of a uniform sample underneath the prescribed conditions the exactness of an analytical procedure expresses the nearness of agreement between a group of measurements^[19]. Precision may be taken into consideration at three levels:

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^[20].

Intermediate precision:

It denotes the exactness under inside research laboratories, in distinct days, through distinct analyst, on distinct instruments. Two different analysts each preparing 6 sample solutions, as per specified method^[21].

Reproducibility:

It expresses the precision between different analytical labs. Every research facility set up an aggregate of 6 sample solutions, according to the analytical technique^[20].

The precision of PRO and BET method was determined by inter-day and intra-day variation (% RSD). Intra-day precision was performed by analyzing standard drug solutions within the calibration range, 3 times on the same day. Inter-day precision was performed by analysing drug solutions within the calibration range on 3 different days over a period of 7 days. The low % RSD values of inter-day and intra-day variation for BET and PRO, revealed that the method is precise^[17].

The precision of paracetamol was checked by injecting a solution of 100µg/ml for 6 times in same days, different days, and in a different time interval on the same day. The % RSD was found to be less than 3%, which showed good precision^[18].

Specificity:

For a single stage of development, the analytical technique should demonstrate specificity. The technique was should have the power to unequivocally assess the analyte of interest whereas within the presence of all expected parts, which can encompass degradants, sample matrix, and sample blank peaks^[22].

Specificity was performed to determine the retention time of each drug in a mixture and in the sample. The retention time of standard drugs individually was determined, and it was found to be 3.750 min and 1.533 min for nitazoxanide and ofloxacin and retention time of both drugs in the standard mix was found to be 3.760 min for nitazoxanide and 1.542 min for ofloxacin respectively^[17].

Limit of detection (LOD):

Lowest quantity of an analyte which may be detected by the chromatographical 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

LOD can be expressed as LOD = 3.3SD/S

Where,

SD = Standard deviation of response,

S = Slope of calibration curve ^[21].

LOD of the paracetamol was studied by the signal to noise ratio, and the result was found to be 100µg/ml ^[16].

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 ^[23].

The LOQ value of BET and PRO is 0.957µg/ml and 1.12µg/ml respectively ^[16].

LOQ of the paracetamol was studied by the signal to noise ratio, and the result was found to be 360µg/ml ^[18]. Some usual techniques, methods for the assessment of LOD and LOQ are as follows:

· Visual inspection,

· Standard deviation of the blank and

· Regression line at low concentrations^[24].

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^[25].

Standard solution of BET and PRO was taken in a 10 ml volumetric flask and diluted with 0.1 N HCL to get the final concentration in the range of 5 to 25µg/ml for BET and 3 to 15µg/ml for PRO. Prepared 6 times in this calibration range and absorbance determined at the respective wavelength for each drug alone. The results show good linearity between absorbance and concentration in the prescribed concentration range for both the drugs^[16].

For simultaneous estimation of nitazoxanide and ofloxacin linearity perform as five different concentrations of standard mixtures prepared, 25%, 50%, 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^[17].

The linearity of paracetamol was performed by preparing different concentrations (5, 10, 20, 40,80 and 120μg/ml) from a stock solution of 10mg/ml. The solution of 20µl was injected into column three times. Linearity of paracetamol was found in the concentration range of 6.25-100μg/ml with a correlation coefficient of 0.999^[26].

Range:

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^[26].

Ruggedness:

The degree or measure of reproducibility under different situations such as in different laboratories, different analyst, different machines, environmental conditions, operators etc^{. [26].}

In the simultaneous estimation of nitazoxanide and ofloxacin, ruggedness was performed by different analyst and in different laboratories in different days to checks for any variation in the chromatography tech. The % RSD for area and retention time was calculated for determination^[15].

Robustness:

It is characterized by the level of ability of an analytical technique, to stay similar by minute purposely change in the technique parameter. The different technique parameters which can be modified in HPLC are pH, the temperature of the column and mobile phase composition^[27].

Methods:

1. In one of the study scientists worked on RP-HPLC method development and validation for estimation of rivaroxaban in pharmaceutical dosage forms, at 40ﹾC Phenomenex Luna 5 µm C18 100 Å LC Column (250 x 4.6 mm) was used. Isocratic elution was performed using Acetonitrile: Water (55:45 v/v) mixture. The flow rate was 1.2 mL min-1. UV detection was at 249 nm. Rivaroxaban and Internal standard (Caffeine) were eluted within 2.21 and 3.37 minutes, respectively. The method was accurate, robust, precise and rapid^[28].

2. In paper of Development of Validated RP-HPLC method for estimation of Rivaraxoban in pharmaceutical formulation, a HIBAR 5µ C18 column (250 X 4.6 mm) was used as a stationary phase. The mobile phase used was Potassium Dihydrogen Orthophosphate: Acetonitrile in the ratio 60:40. Validation parameters such as Linearity, Accuracy, Precision, Robustness, LOD, LOQ were studied^[29].

3. In the paper of Development and Validation of Stability Indicating RP-HPLC Method for Rivaroxaban and Its Impurities, the chromatoghrapic separation was done using ZorbaxSB C18 (250 mm X 4.6 mm, 3.5µ) HPLC column using Acetonitrile and water in the ratio 820:180 as a mobile phase. Validation parameters such as Specificity, Linearity, Accuracy, Precision, Robustness, LOD, LOQ were studied^[30].

4. In the paper of Development of New Analytical Method and Validation for Quantitative Estimation of Rivaroxaban in Formulation and Bulk Drug, isocratic elution was employed on a Aglient Eclipse C8 LC Column (150 x 4.6 mm) with 5µm pore size was used at 40ﹾC. UV detection wavelength was at 251nm. Mobile phase used was Triethyl amine: Acetonitrile 85:15 v/v (pH 11.6). At an interval of 5 minutes 20µl sample was injected. Validation parameters such as Linearity, Accuracy, Precision, Range, Specificity, Selectivity, LOD and LOQ were studied^[31].

5. In one of the paper study was done on Photolytic-Thermal Degradation Study And Method Development Of Rivaroxaban By RP-HPLC. Chromatographic separation was achieved using a phenomenex C18 (250×4.6 mm, 5μm), 100°A particle size columns. A flow rate was at 1 ml/min and using a PDA detector to monitor the elute at 250 nm. The mobile phase consisted of Methanol: Acetronitrile (50:50 v/v). The method was validated for specificity, linearity, precision, accuracy and robustness^[32].

6. One of the scientists worked on RP-HPLC method development and validation for estimation of rivaroxaban in pharmaceutical dosage forms. For isocratic elution Phenomenex Luna 5 µm C18 100 Å LC Column (250 x 4.6 mm) was used at 40ﹾC. Mobile phase used was ACN: Water (55:45 v/v) mixture. UV detection was at 249 nm. The flow rate was 1.2 mL min-1. Validation parameters observed were specificity, linearity, precision, accuracy, robustness, recovery, selectivity and reproducibility^[33].

7. The isocratic separation was achieved on stainless steel Purospher® STAR Hibar® C18 analytical column (250 mm × 4.6 mm i.d., 5.0 μm). Mobile phase used was acetonitrile and water in the ratio 80:20 v/v. A flow rate was of 1 mL/min. Detection was carried out using a UV detector at 247 nm. The method was validated for accuracy, precision, linearity, LOD, LOQ and robustness. By using the above parameters one of methods was developed^[34].

8. One of the scientists had studied on Assay comparison of rivaroxaban by new HPLC method with an existing method in tablet dosage form. The chromatoghrapic separation was done by C18 column (Phenomenex 250 x 4.6 mm, 5 μm maintained at 35°C) at 251 nm. Mobile phase used was ACN: Water in the ratio 55:45 v/v at a flow rate of 1.2 ml/min. Validation parameters observed were specificity, linearity, precision, accuracy, robustness, recovery, selectivity and reproducibility^[35].

CONCLUSION:

This article given an idea for Analytical Method Development and Validation of Rivaroxaban and what is validation, its type, why it is necessary, how to develop a method and how to carry out the validation procedure to demonstrate that the technique is able for its proposed reason. All validation parameter such as ruggedness, robustness, specificity, Range, LOD, LOQ, linearity are defined well with example of definite drug. Validation is a necessary technique in the pharma sector and it is used to state that the quality is worked into the procedures supporting the development of drug and production.

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Received on 02.01.2020 Modified on 31.01.2020

Research J. Science and Tech. 2020; 12(1):36-46.

DOI: 10.5958/2349-2988.2020.00004.2