Int J App Pharm, Vol 10, Issue 6, 2018, 8-15Review Article



1Department of Pharmaceutical Quality Assurance, 2Deparment of Pharmacology, 3Department of Pharmaceutics, Mandsaur University, Mandsaur (M. P.), India
Email: [email protected]

Received: 03 Jul 2018, Revised and Accepted: 04 Sep 2018


The top objective of any pharmaceutical industry is to produce products of necessary characteristic and quality reliably, in a cost-effective manner. Development of a method is essential for discovery, development, and evaluation of medicines in the pharmaceutical formulation. The main aim of this review article was to check the development and validation of the procedure employed for the medication from the starting of the formulation to the complete commercial batch of product. At the point when an analytical technique is applied to produce outcomes for the quality of medicine associated samples, it is necessary that the outcomes are reliable. In the pharma industry, validation policy is documented for how to perform validation, types of validation and validation policy are complied with the necessities of good manufacturing practice (GMP)regulations. Validation is very important for the effective running of the pharmaceutical firms. At every stage from raw material to the finished, stability, everywhere validation was performed. The method was developed properly,and validation parameters are explained in terms of accuracy, specificity, precision, limit of detection (LOD), limit of quantitation (LOQ), ruggedness, robustness,and system suitability testing with the example of certain drugs. All validation parameters are used in the routine and stability analysis.

Keywords: Validation,Method development, Limit of quantitation, Limit of detection, Linearity, Robustness, Ruggedness


Analysis is vital in any product or service, and it is also important in drug because it involves life[1]. Analytical chemistry is the analysis of separation, quantification and chemical additives identification of herbal and synthetic materials constituted with 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, whereas quantitative evaluation estimates the amount of positive detail or compound within the substance,i.e.the sample [2].The quantity of medication brought into the marketplace is growing each year. These medicines could also be either new entities or partial structural alteration of the present one. Medicines ought to be accessible in such a type that quality as well as bioavailability, adequate plasma concentration, desired period, the onset of action, correct dose, safety, effectiveness and stability on storage of product, will be assured throughout the storage of the products[3].The advancement of a medication is a long procedure including drug invention, a research lab trial, preclinical testing, clinical testing, and regulatory registration. To additionally improve the adequacy and protection of the medication after acceptance, numerous administrative organizations, for example, the united states food and drug administration (USFDA) likewise require that the drug product is evaluated for its identification, potency, characteristics, quality, stability, and purity before it can be discharged for utilizing. Thus, pharmaceutical validation and process controls are vital in disregarding the issues that might be encountered [4]. Frequently, there is a time lag from the date of introduction of a medication into the market to the date of its consideration in pharmacopeias. This occurs because of the possible vulnerabilities within the continuous and more extensive use of those drugs, reports of ongoing toxicity (bringing about their withdrawal from the market), development of affected person resistance and improvement of higher medicine with the aim of competition. Under these situations, requirements and analytical techniques for those medicines that may not be approachable within the pharmacopoeias. It becomes necessary, so as to develop newer analytical strategies for such medications [5]. Analytical approach improvement and validation perform important functions in the discovery, improvement, and manufacturing of medications.The main aim of an analytical measure is to get consistent, realistic, and correct information. Validated analytical strategies play a significant role in achieving this goal. Outcomes from methodology validation may be used to choose the standard, reliability, and consistency of analytical results, that is associated as an integral part of any sensible analytical practice. Validation of analytical strategies is also needed by most rules and quality standards that impact laboratories [6].

Analytical method development

When there are no definitive techniques are present, new methodologies are being progressed for evaluation of the novel product. To investigate the presence of either pharmacopoeial or non-pharmacopoeial product novel techniques are developed to reduce the value besides time for higher precision and strength. These methodologies are optimized and valid through preliminary runs. Alternate ways are planned and place into practice to exchange the present procedure within the comparative laboratory information with all accessible merits and demerits.

Fig.1: Life cycle of the analytical method [7]

Necessity of method development

Drug evaluation exhibits the identity characterization and resolution of the drugs in combination like dosage forms 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 (which might be directly connected with the need of a identified dose), impurity (related to safety of the medication), bioavailability (consists of key drug traits like crystal kind, uniformity of drug and release of drug), stability(that shows the degradation product), and effect of manufacturing parameters to verify that the production of drug product is steady.

Analyst before the development of new technologies, do not forget below mention criteria:

Steps for developing a method

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


Validation is an idea that has 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 or process control, validation is established on, however not endorsed by regulatory specifications and is best seen as a critical and necessary part of current good manufacturing practice(cGMP).

The phrase validation basically implies for evaluation of validity or activity of demonstrating viability. Validation is a workforce effort where it entails humans from various departments of the plant. Validation is needed for any new or amended technique to confirm that it is capable of giving consistent and reliable results, once utilized by different operators using similar instrumentation within the same or completely different laboratories [8].Validation is an essential component of quality assurance; it includes the efficient investigation of systems, facilities, and procedures aimed toward deciding if they execute their planned capacities sufficiently and reliably as determined.

Validation should in this way be considered in the accompanying circumstances:

Important stages invalidation

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, setting up stability conditions, and managing 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 situations [4].

Stage 3

It is also called as the validation maintenance stage, it requires 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 [4].

Types of validation

Validation is classified into following types:

Fig.2: Validation types[9]

Equipment validation

Installation qualification points include

OQ concerns consist of:

PQ concern consists of:

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.

In this validation, the protocol is accomplished before the procedure is placed into industrial use [8].

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

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.

Few basic components of retrospective validation are:

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

Analytical method validation

Validation of an analytical approach is established through laboratory research, that the execution attributes of the procedure meet the requirements 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 [14].

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 [9].

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

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 [16, 17].

Cleaning validation protocol

Importance of validation

Validation parameters

The main aim of method validation is to produce proof that the method will what it is supposed to do, accurately, reliable and consistent [9]. The validation parameters as per ICH guidelines are described below:

The precision of prochlorperazine maleate (PRO) and betahistine hydrochloride (BET) method was determined by interday and intraday variation (%RSD). Intra-day precision was performed by analyzing standard drug solutions within the calibration range, three times on the same day. Inter-day precision was performed by analysing drug solutions within the calibration range on three different days over a period of seven days. The low % RSD values of interday (1.02 to 1.48% for BET at 252.9 nm and 0.67 to 0.82% for PRO at 260.15 nm) and intraday (0.77 to 1.09% for BET at 252.9 nm and 0.27 to 0.61% for PRO at 260.15 nm variation for BET and PRO, revealed that the method is precise [20].

For simultaneous estimation of nitazoxanide and ofloxacin precision performed by injecting six replicates of a sample prepared from commercial tablets and assay was calculated to determine the repeatability of retention time and a peak area of standard and samples. The percentage relative standard deviation (%RSD) values for the area of nitazoxanide and ofloxacin were 0.44 and 0.2% and RSD values for a retention time of nitazoxanide and ofloxacin were 0.44% for both the drugs[21].

The precision of paracetamol was checked by injecting a solution of 80 µg/ml for six timesin 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 [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[21].

LOD can be expressed as

LOD = 3.3SD/S

Where, SD = Standard deviation of response,

S = Slope of calibration curve [27].

The LOD value of betahistine hydrochloride (BET) and prochlorperazine maleate (PRO) is 0.29µg/ml and 0.34µg/ml respectively[20].

LOD of the paracetamol was studied by the signal to noise ratio,and the result was found to be 120µg/ml[22].

LOQ can be communicated as

LOQ = 10SD/S

Where SD = Standard deviation of response,

S = Slope of calibration curve [28].

The LOQ value of betahistine hydrochloride (BET) and prochlorperazine maleate (PRO) is 0.957µg/ml and 1.12µg/ml respectively[20].

LOQ of the paracetamol was studied by the signal to noise ratio,and the result was found to be 360µg/ml[22].

Some usual techniques, methods for the assessment of LOD and LOQ are as follows:

Standard solution of betahistine hydrochloride (BET) and prochlorperazine maleate(PRO)wastaken in a 10 ml volumetric flask and diluted with 0.1 N HCL to get the final concentration in the range of 4 to 24µg/ml for BET and 3 to 18µg/ml for PRO. Prepared six 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[20].

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[21].

The linearity of paracetamol was performed by preparing different concentrations (6.25, 12.5, 25, 50, and 100μg/ml) from a stock solution of 10 mg/ml. The solution of 20 µl 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 [22].

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. The % RSD for area and retention time was calculated for determination[21].

In the simultaneous estimation of nitazoxanide and ofloxacin, the robustness of the proposed method verified by to perform analysis under variable flow rates. The flow rate as per the developed method is 1.5 ml/min. Slight change in flow rate is 1.3 ml/min and 1.7 ml/min and chromatogram recorded. Due to a slight change in the flow rate of method shows good results and remain unaffected by that minute change. So, we can say that the method is robust [21].

For robustness of paracetamol, small modification in the flow rate, % of acetonitrile and pH of the mobile phase,the methodremains unaffected and% RSD value obtained are approximately near [22].

Table 1: Acceptance criteria of system suitability parameters

S. No. Parameter name Acceptance criteria Reference





Tailing Factor

Number of theoretical plate








T = (X+Y)/2X

Fig.3: Tailing factor [35]

Where X = Front edge of the peak and,

Y = Back edge of the peak [35].

Fig.4: Capacity factor [36]

It can be calculated as:

K= (tR–t0)/t0

Where tRis the retention time of the peak and t0 is the column dead time [27].

It is mainly utilized to examine the efficiency of the column. It can be expressed as:

N = 16(tR/W)2

Where tRis the retention time,and W is the width at the base of peak[35].

Temperature of the column,

Composition of solvents,

Nature of stationary used and,

Pressure used [37].

R = 2[(tR)A-(tR)B]/WA+WB

Where, tR1 and tR2 are retention time of second and first compounds, respectively [35].

Fig.5: Resolution [35]

In the simultaneous estimation of nitazoxanide and ofloxacin, system suitability parameterswas checked by injectingfive injections of standard mixedsolutions and two injections of the sample. The % RSD of both the drugs is less than 0.2%[21].


This article gives an idea that what is validation, its types, 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 parameters such as linearity, LOQ, LOD, Range, specificity, robustness, ruggedness and system suitability are defined well with examples of certain drugs. Validation is a necessary technique in the pharma department,and it is used to assure that the quality is worked into the procedures supporting the development of drug and production.


All the authors have contributed equally


There is no conflict of interest from all the authors


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