**5. Method validation (validation of analytical results)**

After optimizing the experimental variables for maximum complex formation and extraction, some analytical performance characteristics such as linearity, limits of detection (LOD) and quantification (LOQ), accuracy and precision, robustness and ruggedness, and selectivity were investigated using standard solutions of drugs under study.

#### **5.1 A calibration curve (linearity)**

In quantitative analyzes using spectroscopic methods, the standard curve is always needed. Where the active substance of the pure drug is subjected to the same optimal conditions for the samples under study and the absorbance was measured at the maximum length. This is followed by plotting the absorbance measurements against the concentrations of the samples. A straight line passing through the origin is obtained if Beer's law is obeyed. This curve may then be used in the subsequent determination of the constituent under the same conditions.

#### **5.2 Sensitivity of the method (LOD and LOQ)**

Knowledge of the sensitivity of the color is important and the following terms are commonly employed for expressing the sensitivity. For more sensitive spectrophotometric methods, <sup>ɛ</sup> is ˃ <sup>1</sup> � 104 L. mol�<sup>1</sup> . cm�<sup>1</sup> and values of ɛ ˂ <sup>1</sup> � <sup>10</sup><sup>3</sup> l. mol�<sup>1</sup> . cm�<sup>1</sup> correspond to less sensitive methods. Sandell's sensitivity [62] refers to the number of μg of the constituent determined, converted to the colored product, which is a column solution of cross section 1 cm<sup>2</sup> , shows an absorbance of 0.001 (expressed as μg/cm<sup>2</sup> ). Limits of detection LOD and LOQ are the smallest amount of an analyte that can be determined and quantified by a particular method. The LOD and LOQ values were calculated using the formulae:

$$\text{LOD} = \frac{\text{3.3S}}{m} \text{ and } \text{LOQ} = \frac{10 \text{S}}{m} \tag{9}$$

where *S* is the standard deviation of replicate (*n* = 7) absorbance of blanks and *m* is the slope of the calibration curve.

#### **5.3 Precision and accuracy**

The purpose of carrying out a determination is to obtain a valid estimate of a true value. When one considers the criteria according to which an analytical procedure is selected, precision and accuracy are usually the first to come to mind. Precision and accuracy together determine the error of an individual determination. They are among the most important criteria for judging the results generated by the analytical procedure.

*Spectrophotometric/Titrimetric Drug Analysis DOI: http://dx.doi.org/10.5772/intechopen.109364*

To evaluate the precision and accuracy of the methods, standard drug solution at three concentration levels was subjected to analysis on the same day (intra-day) in seven replicates and on five consecutive day (inter-day) by preparing all solutions afresh each day. Mean (�) and standard deviations (SD) were obtained by backcalculated drug concentration at each level. Accuracy and precision were evaluated in terms of relative error (RE) and relative standard deviation (RSD), respectively.

#### **5.4 Robustness and ruggedness**

Robustness is the measure of its capacity to remain unaffected by small, but deliberate, variations in parameters of the method and indicates its reliability during normal usage, while ruggedness represents the degree of reproducibility of examined results, found by analyzing the same samples under condition variables. The assay procedure was repeated after making a small incremental variation in the optimized condition such as the pH of buffer and reagent volume, and the effect of these variations was investigated to assess the robustness of the method. To evaluate ruggedness, the determination was performed by a single analyst using three instruments in the same laboratory and also by three analysts using a single instrument. Each study was performed on three levels of analyte.

#### *5.4.1 Selectivity*

It can be defined as the degree to which a method can quantity the analyte accurately in the presence of interferes. The selectivity of the developed methods was examined using placebo blank and synthetic mixture analyses. To a certain amount (mg) of the placebo blank (talc, starch, sucrose, lactose, and other compounds) prepared, accurately known amount (mg) of pure drug was added, mixed thoroughly and the mixture extract was prepared as usual; and then steps described under the procedure for dosage forms were followed. The % recovery of pure drug in the mixture was computed, which is taken as a measure of selectivity.

#### **5.5 Accuracy by recovery experiments (standard-addition method)**

Accuracy by recovery experiments: To ascertain the accuracy of the proposed methods, recovery experiments were performed *via* the standard addition technique. If the % of recovery calculated using the formula given below is satisfactory, confidence in the accuracy of the procedure is enhanced.

$$\% \text{(recovery)} = \frac{\sum XY - \sum X \sum Y}{\sum X^2 - \left(\sum X\right)^2} \tag{10}$$

where X = amount of the constituent added in μg (spectrophotometry) or mg (titrimetry), Y = amount of the constituent found, μg or mg.

#### **5.6 Evaluation of accuracy and precision by comparison of two methods**

To evaluate the accuracy and precision of the method, one often compares the method being developed or the "test method" with an existing method called the reference, standard or official method [63]. Student's t-test (comparison of two

means); suppose that a sample is analyzed by two different methods, each repeated several times and that the mean values obtained are different, student's t-test will tell, with a given probability, whether it is worthwhile to seek an assignable cause for the difference between the two means. The test gives a yes or no answer to the correctness of the null hypothesis with a certain confidence, such as 95% or 99%. The procedure is as follows: suppose that sample has been analyzed by two different methods (test and reference methods) yielding means *X1* and *X2* and standard deviations *S1* and *S2*, *n1* and *n2* is the number of individual results obtained by two methods, *t* is calculated using the following formula:

$$t = \frac{X\_1 - X\_2}{S} \sqrt{\frac{n\_1 n\_2}{n\_1 + n\_2}} \tag{11}$$

Here, it is presupposed that *S1* and *S2* are the same. If *S1* and *S2* are different, S is calculated using the following formula:

$$S = \sqrt{\frac{\Sigma (X\_1 - X\_1)^2 + \Sigma (X\_2 - X\_2)^2}{n\_1 + n\_2 - 2}} \tag{12}$$

F-test (comparison of two standard deviations); using the formula: F=S<sup>2</sup> T/S2 R.

where S<sup>2</sup> <sup>T</sup> is the variance of the test method, S2 <sup>R</sup> is the variance of the reference method.

F-test uses for the calculation of F-ratio (larger variance/smaller variance). If the calculated F-value is in the table [64, 65], one can conclude that the methods are not significantly different in precision at a given confidence level.

### **6. Conclusion**

Realizing the importance and usefulness of these two techniques; titrimetry and spectrophotometry and valuing their unique features, the author has attempted to explain of applications these simple and inexpensive techniques for the determination of different pharmaceutical formulations. The advantages and superior performances of these two techniques; titrimetry and spectrophotometry compared with the existing techniques are rapidly, simplicity, sensitivity, and use of inexpensive reagents and chemicals.

Modern methods of analysis (LCMS, GCMS, NMR, and Mass) involve sophisticated and costly equipment and pose problems of maintenance. Hence, they may not be within the reach of most of the laboratories and small-scale industries, which produce bulk drugs and pharmaceutical formulations. Among various techniques, titrimetry and spectrophotometry, still enjoy a significant role in the assay of several classes of drugs at macro, semi-micro (titrimetry), micro, or nanogram (spectrophotometry) levels. They are simple, economically viable, and easy to carry out. Visible spectrophotometry is the simplest of the spectrophotometric techniques and it is in wide use in the quantitative analysis of active substances. The spectrophotometric procedure is also recommended in Pharmacopoeial monographs such as Indian Pharmacopeia, British Pharmacopeia, USP, EP, etc. Hence, spectrophotometry is generally preferred in small-scale industries and most laboratories for routine quality assurance

*Spectrophotometric/Titrimetric Drug Analysis DOI: http://dx.doi.org/10.5772/intechopen.109364*

because of its overwhelming advantages, such as speed, simplicity, cost-effectiveness, specificity/selectivity, and sensitivity. Titration is also a simple technique giving accurate and precise results. The non-aqueous titration with visual or potentiometric end point detection has maintained its importance in pharmaceutical analysis and has been accepted by a majority of modern pharmacopeias as an official analytical method.
