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Method development and Validation Parameters of UV- A Commentary

Durga aruna R*

Department of Pharmacy, Vagdevi College of Pharmacy, Gurazala, AP, India

*Corresponding Author:
Durga aruna R,
Department of Pharmacy,
Vagdevi College of Pharmacy,
Gurazala, AP, India

Received: 04 May 2015 Accepted: 27 May 2015 Published: 02 June 2015

Visit for more related articles at Research & Reviews: Journal of Pharmaceutical Analysis


Investigative routines improvement must be approved to give solid information to administrative entries. These strategies are vital for various purposes, including testing for quality control discharge, testing of steadiness tests, testing of reference materials and to give information to bolster details.


UV spectrophotometer, Reagents, Beer’s Law, Lambert’s Law



UV-Visible spectrophotometry is one of the most frequently employed technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measure the ratio, or function of ratio, of the intensity sof two beams of light in the U.V-Visible region are called Ultraviolet-Visible spectrophotometers. 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 [1].

Beer’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases exponentially with the number of absorbing molecules. In other words, absorbance is proportional to the concentration [2,3].

Lambert’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases exponentially as it passes through a medium of homogeneous thickness. A combination of these two laws yields the Beer-Lambert law [4-9].


A Shimadzu UV–visible spectrophotometer (UV scaled down 1700, Shimadzu Corporation, Kyoto, Japan) was utilized for all absorbance estimations with coordinated quartz cells [10-13].

Materials & Methods

All chemicals and reagents were of logical evaluation.

Determination of wavelength of greatest retention

Method Development

It is the procedure of demonstrating that a logical strategy is adequate for utilization to quantify the centralization of an API in a particular exacerbated measurement structure which permits disentangled strategies to be utilized to confirm that an investigation methodology, precisely and reliably will convey a dependable estimation of a dynamic fixing in an intensified arrangement [14-25].


Validation is a procedure of building up narrative proof showing that a system, procedure, or movement did underway or testing keeps up the craved level of consistence at all stages. The scientific strategy approval is vital for diagnostic technique improvement and tried widely for specificity, linearity, exactness, accuracy, range, location limit, quantization cutoff, and vigor [26-34]. In synopsis, systematic strategy advancement and acceptance permits to affirm that a precise and dependable intensity estimation of a pharmaceutical planning can be performed.

Validation parameters

The goal of the diagnostic strategy ought to be plainly comprehended since this will administer the approval attributes which need to be assessed. Average approval attributes which ought to be considered are recorded underneath [35,36]

• Linearity

• Precision

• Repeatability

• Accuracy

• Specificity

• Detection Limit

• Quantitation Limit

• Range


Linearity of system was controlled by get ready standard arrangements at diverse focus levels. The alignment bend of medication over the focus run 15-90 μg/mL was plotted and its linearity was assessed by straight relapse investigation [37-40].


The exactness of an explanatory system portrays the closeness of individual measures of an analyte when the method is connected more than once to numerous aliquots of a solitary homogeneous volume of an organic lattice. Accuracy is further subdivided into inside of keep running, inside of day exactness… or repeatability, which evaluates exactness amid a solitary systematic run, and between-keep running, between day repeatability … additionally named transitional exactness, which measures the exactness with time (regularly days) and may include diverse experts, gear, and reagents. Reproducibility is a third level of accuracy that is evaluated by method for a between lab trial (institutionalization) and is not piece of the showcasing approval dossier.

In the writing, the accuracy of systematic strategies is frequently inadequately reported (as a result of poor estimations)

Precision ought to be measured utilizing at least five determinations (recreates) every focus. At least three focus levels (low, medium and high) in the scope of the normal focuses is suggested.

The accuracy of a systematic strategy is normally communicated as the difference, standard deviation or coefficient of variety of a progression of estimations.

Precisions are helpfully communicated by the relative standard deviation (RSD)


Inter-day precision

Repeatability is the capacity to discover a quality as close as could reasonably be expected to the same worth (10 μg/mL) with the 5 imitates over the 3 days; we have 3 appraisals of this difference (0.294, 0.866 and 0.3099) and their mean is 0.4905 i.e. the WMS [41-45].

For all intents and purposes this fluctuation is communicated as a RSD (%CV) with:

This is the most reduced conceivable variability connected with this strategy of estimation. From everyday the figured means can be marginally diverse.

Variance of repeatability (or within day variance) is composed. It is the most minimal conceivable difference for a given logical run (same day, same investigator, and so forth.). It is evaluated by the lingering of the ANOVA that is completed. It is connected with the duplicate to-reproduce variety.


The variety emerging utilizing the same estimation process among diverse instruments and administrators, and over more time periods.


Accuracy of the system is found out by standard expansion strategy at 3 levels. The exactness of the system was controlled by computing recuperation of GFX by the technique for expansion. Known measure of GFX at 25%, 50%, 100%, and 150% was added to a prequantified specimen arrangement. The recuperation studies were completed in the tablet in triplicate each in the vicinity of placebo. The mean rate recuperation of GFX at every level was at least 99% and not more than 101 [46,47]. The recuperation studies were completed by including distinctive sums (80%,100%,120%) of the unadulterated medication to the preanalyzed plan. The arrangements were arranged in triplicates and the % recuperation was computed [49-50].