Review on Analytical Method Development & Validation by Using UV-Visible Spectroscopy

Mr. Shrinivas Kapale*, Mr. Gopal Lohiya, Dr.Kranti Satpute

Dayanand Education Society’s, Dayanand College Of Pharmacy,Latur -413512, Maharashtra

*Correspondence: kapaleshri007@gamil.com   Tel.: (+91 8261880859)

INTRODUCTION

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. The objective of any analytical measurement is to obtain consistent, reliable and accurate data. Validated analytical methods play a major role in achieving this goal. The results from method validation can be used to judge the quality, reliability and consistency of analytical results, which is an integral part of any good analytical practice. Validation of analytical methods is also required by most regulations and quality standards that impact laboratories. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. There is a scope, therefore to develop newer analytical methods for such drugs. Analytical methods development and validation play important roles in the discovery, development, and manufacture of pharmaceuticals.

Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients, need analytical method development and validation by combining the therapeutic effects of two or more drugs in one product. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. Identification and quantification of impurities is a crucial task in pharmaceutical process development for quality and safety. Related components are the impurities in pharmaceuticals which are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during stability testing, or develop during formulation or upon aging of both API and-formulated  APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products. Various analytical methodologies are employed for the determination of related components in pharmaceuticals. There is a great need for development of new analytical methods for quality evaluation of new emerging drugs.

UV-Visible spectroscopy:

In UV-visible spectroscopy, the amount of light absorbed at each wavelength of UV and visible region of electromagnetic spectrum is measured. This absorption spectroscopy uses electromagnetic radiations between 200 nm to 800 nm and is divided into the ultraviolet (UV,200-400 nm) and visible (VIS, 400-800 nm) regions (Kumar S, 2006). The principle of UV-Visible spectroscopy is based on the absorption of ultraviolet light or visible light by sample or chemical substance which results in the production of different spectra. When a molecule absorbs UV radiation, the electron present in that molecule undergo excitation, this causes transition of electron within a molecule from a lower level to a higher electronic energy level and the ultraviolet emission spectra arise from the reverse type of transition. Most commonly used solvents in UV spectroscopy are water, methanol, ethanol, ether, chloroform,carbon tetrachloride, cyclohexane and dichloroethane. Applications of UV spectroscopy are detection of functional groups, detection of conjugation, detection of geometrical isomers and detection of impurities [27]

Instrumentation of UV-Visible spectroscopy:

A. Radiation sources: Most commonly used radiation sources are tungstan lamp, hydrogen discharge lamp, deuterium lamp, xenon discharge lamp.

Fig. 1: UV-Visible spectroscopy

B. Wavelength selector: The monochromator is used to disperse the radiation according to the wavelength. The basic elements of a monochromator are an entrance slit, a dispersing element and an exit slit.

C. Sample cell: In UV-Visible spectroscopy sample containers are used to hold liquid sample are called as cells or cuvettes. Cuvettes are made from quartz.

D. Photo detector: Most commonly used detectors in UV spectrophotometer are barrier layer cell, photocell and photomultiplier tube.

E. Readout device: The output from the detector is suitably amplified and then displayed on a readout device.[27]

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 nonpharmacopoeial 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. 2: Life cycle of the analytical method

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 themedication), 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:

• Is this technique possesses the needful sensitivity?

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

• Is the accuracy and precision doable with this technique?

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

• Is the time requires to perform this technique applicable [3]?

Steps for developing a method:

Various steps 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, optical isomerism, etc., are collected.

• The standard analyte is equal to 100% purity is acquired.  Necessary arrangement is to be created for the proper storage (refrigerator, desiccators, 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, atomic absorption spectroscopy, etc.), high-performance liquid chromategraphy and gas chromatography so on and, are however about once coordinated with the stability of samples [2].

Requirement of the technique:

Requirement of analytical methodology is essential to build up the analytical fig. of advantage like linearity, selectivity, specificity, range, accuracy, precision, LOD, LOQ etc. shall be outlined [2].

 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 relevant books, journals, united states pharmacopeia/national formulary(USP/NF), association of official agricultural chemists (AOAC) and american society for testing and materials (ASTM)publications and it is extremely convenient to look Chemical Abstracts Service automatic computerized literature [2].

Selecting the method:

• Utilizing the data obtained from the literature, the methodology is evolving since the method is being modified wherever needed. Sometimes, it is important to acquire 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 [2].

Proper instrumentation and initial studies:

 Installation qualification (IQ), operation qualification (OQ), and performance qualification (PQ) of instrument pertinent to research standard methodology is examined by an appropriate set up of instruments [2].

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 documente with relation to dead ends [2].

Proper documentation of analytical fig. of merits:

The true determined analytical fig. of benefit consisting of LOD, LOQ, cost, linearity and evaluation time and planning of samples, etc. are also recorded [2].

 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 [2].

Estimation of percent recovery of real samples and

demonstration of quantitative sample analysis:

Percentage recovery of spiked, actual standard medication into a sample grid which includes no analyte is evaluated. Optimization to reproducibility of recuperation from test to test 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 [2]

Validation:

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 impliesfor 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:

• Completely new procedure.

• Latest equipment.

• Procedure and equipment which have been adjusted to suit altered needs and,

• Procedure where the finished result test is a poor and undependable marker of product quality [4].

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, 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 10 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. 3: Validation types [9]

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:

 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 judgments, the analysts have the correct instruction, capabilities, 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 [15].

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 [15].

 Preparation of sample:

 Preparation of the sample is a key component to effective method validation. It has been mentioned that sample planning represents 60 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 [15].

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, converter, 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 properties of the analytical species, the concentration of the analytes in the sample, sample matrix, speed, cost, and so forth [15].

 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 [15].

Importance of validation

• Assured high quality.

• Time boundation.

• Optimization of the method.

• Minimum batch product failure, enhanced 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.

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.

 The validation parameters as per ICH guidelines are described below:

Accuracy:

 Accuracy is expressed as the nearness of agreement between the values found and values that are already available. It can also be defined as the closeness between the true value and the observed value. It is sometimes called as trueness.

• Degree to which the determine value of analyte corresponds to the true value.
• Accuracy can vary over the expected concentration range.
• It should be determined using a working or reference standards in the 80% – 120% level of expected range.
• Accuracy is determined by :

1. Analyzing a sample of known concentration and comparing with the true value.
2. Spiking a blank (Sample having all components except the analyte) and comparing with the expected result.
3. Standard addition method in which the sample concentration is determined. A known amount of analyte is added and the concentration is once again determined. The difference of the two concentration values is compared with the actual value of added analyte.

• Accuracy is also defined by the comparison of test results with those obtained using another validated test procedure.

Precision:

Precision is a key parameter in analytical method validation that measures the closeness of agreement between a series of measurements obtained from multiple samplings of a homogeneous sample under prescribed conditions. Precision ensures the consistency and reproducibility of an analytical method.

It is critical for assessing the reliability of results in quality control and research applications.

Precision expresses closeness of a series of measurements of the same sample under identical conditions

High degree of precision does not necessarily means a high degree of accuracy.

Precision is expressed as variance, standard deviation or as coefficient of variation of a series of measurements.

Minimum of five replicate sample determinations should be carried out. [23].

• 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 [24].

Types of repeatability:

Measurement repeatability

The closeness of results obtained when the same measurement procedure, operators, system, operating conditions, and location are used on the same sample over a short period of time. 

Gage Repeatability and Reproducibility (GR&R)

A method for evaluating the precision of a measurement system by checking its consistency across different operators and tests. There are three types of GR&R studies: crossed, nested, and expanded. 

Intermediate precision

The precision obtained within a single laboratory over a longer period of time. It's also known as within-lab reproducibility.

Replicability

The ability to make an observation using a different measuring system, dataset, team, and location on multiple trials. 

• 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 .[24]

Specificity:

 For every 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, excipients/sample matrix, and sample blank peaks. Specificity was performed to determine the retention time of each drug in a mixture and in the sample.[25]

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 3:1.

LOD can be expressed as

LOD = 3.3SD/S

Where,

 SD = Standard deviation of response,

 S    = Slope of calibration curve [27].

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 [28].

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

• Visual inspection,

• Signal to noise ratio,

• Standard deviation of the blank, and

• Regression line at low concentrations [29].

Linearity:

Linearity refers to the ability of analytical procedures to produce results in direct proportion to the concentration range of analyte in samples within the required concentration levels.

Linearity should be determined using a minimum of 6 standards whose concentration spans from 80% to 120% of expected concentration level.

Linearity report should include slope of line, linear range and correlation coefficient data. Correlation coefficient r should be greater than or equal to 0.99 in the working 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 [31].

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. [32].

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 high-performance liquid chromatography are pH, drift rate, the temperature of the column and mobile phase composition [33].

 System suitability parameters:

 System suitability test is used to check the sensitivity, resolution, and reproducibility of the chromatographic system are well for the analysis to be done. The factors mainly used in system suitability are tailing factor, a number of the theoretical plate, retention time, resolution, etc. [34].

CONCLUSION:

The analytical method developed using the UV-Visible spectrophotometer proved to be a simple, cost-effective, and reliable approach for the quantitative analysis of [specific analyte] in [specific matrix]. By optimizing critical parameters such as wavelength selection, solvent choice, and calibration range, the method demonstrated excellent linearity, accuracy, precision, and sensitivity, meeting the standards outlined by ICH guidelines. The validated method showed robustness and reproducibility, making it highly suitable for routine quality control and analytical applications. The study underscores the utility of UV-Visible spectrophotometry as a versatile and efficient tool for analytical method development across various fields, including pharmaceuticals, environmental monitoring, and biological research.

REFERENCES:

1.         Hema, Swati Reddy G. A review on new analytical method development and validation by RP-HPLC. Int Res J Pharm Biosci 2017;4:41-50.

2.         Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on stepby-step analytical method validation. IOSR J Pharm 2015;5:7-19.

3.         Patel A, Dwivedi N, Kaurav N, Bashani S, Patel S, Sharma HS, et al. Chemical analysis of pharmaceuticals: a review. J Med Pharm Innov 2016;3:4-7.

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

5.         Pathuri R, Muthukumaran M, Krishnamoorthy B, Nishat A. A review on analytical method development and validation of the pharmaceutical technology. Curr Pharm Res 2013;3:855-70.

6.         Patil R, Deshmukh T, Patil V, Khandelwal K. Revie\w on analytical method development and validation. Res Rev J Pharm Anal 2014;3:1-10.

7.         Chauhan A, Mittu B, Chauhan P. Analytical method development and validation: a concise review. J Anal Bioanal Tech 2015;6:1.

8.         Mahar P, Verma A. Pharmaceutical process validation: an overview. Int J Pharm Res Biosci 2014;3:243-62.

9.         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;4:1280.

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

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

12.     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.

13.     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.

14.     Bhardwaj SK, Dwivedi K, Agarwal DD. A review: HPLC method development and validation. Int J Anal Bioanal Chem 2015;5:76-1.

15.     Araujo P. Key aspects of analytical method validation and linearity evaluation. J Chromatogr B 2009;877:2224-34.

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

17.     Lodhi B, Padamwar P, Patel A. Cleaning validation for the pharmaceuticals,biopharmaceuticals, cosmetic and

1.         neutraceuticals industries. J Innov Pharm Biol Sci 2014;1:27-38.

18.     Murthy DN, Chitra K. A review article on cleaning validation. Int J Pharm Sci Res 2013;4:3317.

19.     Srivastava RK, Kumar SS. An updated review: analytical method validation. Eur J Pharm Med Res 2017;4:774-84.

20.     Patel Paresh U. Development and validation of simultaneous equations method for estimation of betahistine dihydrochloride and prochlorperazine maleate in tablet dosage form. Inventi Rapid: Pharm Analysis and Quality Assurance; 2013.

21.     Nirupa G, Tripathi UM. RP-HPLC analytical method development and validation for simultaneous estimation of two drugs nitazoxanide, ofloxacin and its pharmaceutical dosage

2.         forms. Int J ChemTech Res 2012;5:775-83.

22.     Devi TP, Setti A, Srikanth S, Nallapeta S, Pawar SC, Rao JV, et al. Method development and validation of paracetamol drug by RP-HPLC. J Med Allied

23.     Nayudu ST, Suresh PV. Bio-analytical method validation–a review. Int J Pharm Chem.

24.     Daksh S, Goyal A, Pandiya CK. Validation of analytical methods– strategy and significance. Int J Res Dev Pharm Life Sci 2015;4:1489-97.

25.     Tijare LK, Rangari NT, Mahajan UN. A review on bioanalytical method development and validation. Asian J Pharm Clin Res 2016;9:6-10.

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