**4.1 Method development**

Prior the development of any method for the analysis of certain compound or formulation, there are many factors must be considered before developing the method and applying it to the intended use. The first step include collecting information about the analyte itself (the analyte structure and its physicochemical properties). The mode of detection should be selected (e.g. UV detection). Sample preparation which may include centrifugation, sonication and filtration. The type of the diluent also plays an integral role in the analysis as it should be transparent and does not interfere in the analysis. The stability of the prepared solution, the mobile phase; stationary phase and mode of elution in case of chromatographic elution. All these factors and much more should be considered, optimized and the developed method is then validated and applied for the analysis.

### **4.2 Spectrophotometric methods**

#### *4.2.1 UV/VIS spectrophotometry*

Absorption spectrophotometry is the measurement of an interaction between electromagnetic radiation and the molecules, or atoms, of a chemical substance [15]. Techniques frequently employed in pharmaceutical analysis include UV, visible, IR and atomic absorption. Spectrophotometric measurement in the visible region was referred to as colorimetry.

The procedure of UV-unmistakable spectrophotometry includes the estimation of the measure of bright (190–380 nm) or noticeable (380–800 nm) radiation consumed by a substance in arrangement. Retention of light in both the UV and unmistakable area of the electromagnetic range happens when the vitality of the light matches the vitality required to instigate an electronic change and it is related with vibration and rotational progress in the atom. There are two systems of utilizing spectroscopic estimations in medication examination, the total and the similar strategies for measure, and the one utilized relies upon which side of the Atlantic Ocean you complete the investigation. In the UK and Europe the Beer-Lambert condition will in general be utilized in what is known as the outright technique for examine. In this strategy the absorbance is estimated tentatively and the Beer-Lambert condition is comprehended for c, the medication fixation. Hence, the British Pharmacopeia and European Pharmacopeia quote A1% 1 cm qualities in medication monographs. In the US Pharmacopeia, the near strategy for test is liked. In this sort of examine a standard arrangement of the medication to be investigated is readied, the absorbance of the example and the standard are estimated under indistinguishable conditions, and the centralization of the example is determined from the relationship:

$$\begin{array}{l}\text{estimate unune unusungusnance conuunons,} \\ \text{e is determined from the relationship:} \\ \begin{array}{l}\text{Atest} \\ \hline \text{[test]} = \frac{\text{Astd}}{\text{[std]}} \end{array} \end{array} \tag{1}$$

**31**

band.

*Drug Analysis*

*DOI: http://dx.doi.org/10.5772/intechopen.88739*

*4.2.2 Derivative spectrophotometry*

The first derivative (1

curvature of the 0

is referred to as the fundamental, zero order or 0

A = *f*(λ) dA/dλ = *f*(λ) d2

spectrum against wavelength. The second derivative (2

amplitude than those with broad bandwidth [15].

Zero order first order second derivative

D spectrum against wavelength.

bipolar function is characteristic of all odd-order derivatives.

with wavelength against wavelength, i.e. a plot of the slope of the fundamental

The first order derivative spectrum of an absorption band is characterized by a maximum, a minimum and a crossover point at λmax of the absorption band. This

The second derivative spectrum is characterized by two satellite maxima and an inverted band of which the minimum corresponds to the λmax of the fundamental

A derivative spectrum is therefore gives better resolution of overlapping bands than the corresponding fundamental spectrum and may permit the accurate determination of the λmax of the individual bands. Secondly, it discriminates in favor of substances of narrow spectral band width against those with broad bandwidth. And consequently, substances with narrow spectral bandwidth display larger derivative

These advantages of enhanced resolution and band width discrimination found

in derivative spectrophotometry permit the selective determination of certain

tive [18].

extraction procedure may not be 100%. On the off chance that an inner standard is utilized, misfortunes in test will be reflected by comparative misfortunes in the standard and the proportion of test to standard ought to stay consistent. Inner measures are especially utilized in chromatographic examination (particularly gas chromatography and elite fluid chromatography), where fluctuations in instrumental parameters (for example flow rate of versatile stage) influence precision. In certain spectroscopic examinations a comparable way to deal with the utilization of inner benchmarks is utilized. This is the strategy of standard augmentations and includes expansion of expanding volumes of a standard arrangement of the analyte to a fixed volume of the example and development of an alignment diagram. The diagram in a standard expansion examine is of positive incline however converges they-pivot at a positive estimation of absorbance. The measure of medication in the example is found by extrapolation of the alignment chart back to the point where the line crosses the x-pivot (for example at the point when y 0 in the condition of the line). The strategy for standard increments is generally utilized in nuclear spectroscopy (for example assurance of Ca2+ particles in serum by nuclear emanation spectrophotometry) and, since a few aliquots of test are examined to create the alignment chart, should expand the exactness and accuracy of the measure. The chief advantage of colorimetric and spectrophotometric methods is that they provide a simple means for determining minute quantities of substances [16, 17]. Although spectral interference (degradation products, excipients, etc.) can often occur, the selectivity and sensitivity of these methods can be improved by employing an instrumental technique such as derivative spectrophotometry.

In derivative spectrophotometry the absorbance (A) of a sample is differentiated with respect to wavelength (λ) to generate the first, second or higher order deriva-

In the context of derivative spectrophotometry, the normal absorption spectrum

D spectrum.

A/λ2

D) spectrum is a plot of the rate of change of absorbance

= *f* ∆(λ), etc.

D) spectrum is a plot of the

Where [test] is the centralization of the example and [std] is the convergence of the readied standard. The relative strategy for test has the bit of leeway that it very well may be utilized regardless of whether the medication experiences a substance response during the measure (for example development of a shaded subsidiary to permit estimation in the obvious district of the range), yet experiences the hindrance that a credible example of the medication being referred to must be accessible for examination. When doing medication examines by spectroscopy it is frequently important to set up a scope of groupings of a standard example of the analyte and measure the absorbance of every arrangement. At the point when these information are plotted, a straight line of positive incline ought to be acquired that goes through the inception. Developing diagrams of this sort not just confirms that the Beer-Lambert law applies to the test at the wavelength of estimation yet additionally enables the chart to be utilized for alignment purposes. An answer of obscure fixation is set up in the very same manner as the benchmarks and its absorbance is estimated at a similar wavelength as the principles. This absorbance is then perused off the alignment chart and the fixation is determined. Standard arrangements arranged independently from the example along these lines are known as outer models. An increasingly thorough system includes the utilization of inside models. An inside standard is an exacerbate that is comparative in compound structure and physical properties to the example being investigated. The inner standard ought to be added to the example being referred to before extraction or measure initiates and is then present in the example framework all through the consequent test. In the measure of complex examples, some example pre-treatment is normally required and the recuperation of the example from the

#### *Drug Analysis DOI: http://dx.doi.org/10.5772/intechopen.88739*

*Pharmaceutical Formulation Design - Recent Practices*

Absorption spectrophotometry is the measurement of an interaction between electromagnetic radiation and the molecules, or atoms, of a chemical substance [15]. Techniques frequently employed in pharmaceutical analysis include UV, visible, IR and atomic absorption. Spectrophotometric measurement in the visible

The procedure of UV-unmistakable spectrophotometry includes the estimation of the measure of bright (190–380 nm) or noticeable (380–800 nm) radiation consumed by a substance in arrangement. Retention of light in both the UV and unmistakable area of the electromagnetic range happens when the vitality of the light matches the vitality required to instigate an electronic change and it is related with vibration and rotational progress in the atom. There are two systems of utilizing spectroscopic estimations in medication examination, the total and the similar strategies for measure, and the one utilized relies upon which side of the Atlantic Ocean you complete the investigation. In the UK and Europe the Beer-Lambert condition will in general be utilized in what is known as the outright technique for examine. In this strategy the absorbance is estimated tentatively and the Beer-Lambert condition is comprehended for c, the medication fixation. Hence, the British Pharmacopeia and European Pharmacopeia quote A1% 1 cm qualities in medication monographs. In the US Pharmacopeia, the near strategy for test is liked. In this sort of examine a standard arrangement of the medication to be investigated is readied, the absorbance of the example and the standard are estimated under indistinguishable conditions,

and the centralization of the example is determined from the relationship: \_ Atest [test]

= \_ Astd [std]

Where [test] is the centralization of the example and [std] is the convergence of the readied standard. The relative strategy for test has the bit of leeway that it very well may be utilized regardless of whether the medication experiences a substance response during the measure (for example development of a shaded subsidiary to permit estimation in the obvious district of the range), yet experiences the hindrance that a credible example of the medication being referred to must be accessible for examination. When doing medication examines by spectroscopy it is frequently important to set up a scope of groupings of a standard example of the analyte and measure the absorbance of every arrangement. At the point when these information are plotted, a straight line of positive incline ought to be acquired that goes through the inception. Developing diagrams of this sort not just confirms that the Beer-Lambert law applies to the test at the wavelength of estimation yet additionally enables the chart to be utilized for alignment purposes. An answer of obscure fixation is set up in the very same manner as the benchmarks and its absorbance is estimated at a similar wavelength as the principles. This absorbance is then perused off the alignment chart and the fixation is determined. Standard arrangements arranged independently from the example along these lines are known as outer models. An increasingly thorough system includes the utilization of inside models. An inside standard is an exacerbate that is comparative in compound structure and physical properties to the example being investigated. The inner standard ought to be added to the example being referred to before extraction or measure initiates and is then present in the example framework all through the consequent test. In the measure of complex examples, some example pre-treatment is normally required and the recuperation of the example from the

(1)

**4.2 Spectrophotometric methods**

region was referred to as colorimetry.

*4.2.1 UV/VIS spectrophotometry*

**30**

extraction procedure may not be 100%. On the off chance that an inner standard is utilized, misfortunes in test will be reflected by comparative misfortunes in the standard and the proportion of test to standard ought to stay consistent. Inner measures are especially utilized in chromatographic examination (particularly gas chromatography and elite fluid chromatography), where fluctuations in instrumental parameters (for example flow rate of versatile stage) influence precision. In certain spectroscopic examinations a comparable way to deal with the utilization of inner benchmarks is utilized. This is the strategy of standard augmentations and includes expansion of expanding volumes of a standard arrangement of the analyte to a fixed volume of the example and development of an alignment diagram. The diagram in a standard expansion examine is of positive incline however converges they-pivot at a positive estimation of absorbance. The measure of medication in the example is found by extrapolation of the alignment chart back to the point where the line crosses the x-pivot (for example at the point when y 0 in the condition of the line). The strategy for standard increments is generally utilized in nuclear spectroscopy (for example assurance of Ca2+ particles in serum by nuclear emanation spectrophotometry) and, since a few aliquots of test are examined to create the alignment chart, should expand the exactness and accuracy of the measure. The chief advantage of colorimetric and spectrophotometric methods is that they provide a simple means for determining minute quantities of substances [16, 17]. Although spectral interference (degradation products, excipients, etc.) can often occur, the selectivity and sensitivity of these methods can be improved by employing an instrumental technique such as derivative spectrophotometry.