Objective: To develop an analytical method and validate it for the given unknown drug sample using UV-Visible Spectrophotometer.

                  

Principle Involved in UV Spectrophotometer

        Ultraviolet absorption spectroscopy is generally used for the quantitative determination of compounds that absorb in UV region. This determination is based on Beer’s Law which is as follows:-

                          A = - log T = log Io/It = € c b

        Where,

                 € is the extinction coefficient

                 c is the concentration and

                 b is the length of the cell used in UV spectrophotometer

        A straight line should be obtained on plotting concentration VS absorbance if the Beer’s law holds good. This plot may be used as calibration curve for determining the unknown concentrations of solutes.

Preparation of Primary Stock: 

        10 mg of drug was weighed accurately and dissolved in 10ml of  solvent system to get a concentration of 1mg/ml.

Preparation of Secondary Stock: 

        1ml of the primary stock was taken and diluted to 10ml with the solvent system to get a concentration of 100μg/ml.

Procedure:

1. From the secondary stock 10 μg/ml was prepared and analyzed under JASCO UV-Visible Spectrophotometer for detection of maximum wavelength and absorbance was recorded.
2. Based on the reading obtained, serial dilutions were made and analyzed to determine the calibration range.

Preparation of Sample solutions :

( The dilutions were prepared in triplicate and absorbance was measured )

Observations :

(1) Calibration curve / Linearity - Range

(2) Accuracy Study

Accuracy was  performed by taking triplicates of LQC, MQC,  HQC as 8, 22, 37 μg/ml.

(a) Accuracy

(b) Standard Addition

Concentration of prepared drug + excipient (D+E) solution = 20 μg/ml

Concentration of prepared pure drug (D) solution                     = 20 μg/ml

cx=(b*cs)/(m*vx)

where,

        cx= concentration of drug + excipient (D+E) solution

        cs= concentration of pure drug  (D) solution

        b = intercept = 0.191

        m= slope = 0.056

        vx = volume of D+E solution taken

cx = (0.191*20)/(0.056*4) = 17.05 μg/ml

% Bias = {(20-17.05)/20}*100 = 14.75%

% Recovery = 100 - 14.75 = 85.25%

     

(c) Placebo Spiking

Concentration of prepared pure drug (D) solution = 20 μg/ml

(3) Precision

a) Intraday :

b) Interday:

(4) Robustness :

The robustness was studied by changing the proportion of solvent system i.e. methanol:water  =80:20 and samples of LQC, MQC, HQC were  taken and analyzed.

(5) Stock Solution Stability

It was performed at 0, 6, 12 & 24 hrs taking LQC, MQC & HQC as  8, 22 & 37 μg/ml.

(6) LOD and LOQ

LOD = 3.3*slope/ std dev of intercept

          = 3.3* 0.026/0.014224

          = 1.805404  μg/ml

LOQ = 10*slope/ std dev of intercept

          = 10* 0.026/0.014224

          = 5.47092  μg/ml

(7) Selectivity

        The study was performed using lactose, starch, talc , magnesium stearate and HPMC that are included in the tablet formulation.

Result:

1. The λmax of the given drug sample was determined as 296 nm using methanol:water = 7:3 as the solvent system. A linear relationship was obtained when a graph was plotted between Absorbance and Concentration.
2. The Correlation coefficient(R2) between Absorbance and Concentration plot was found to be 0.997.
3. The linearity-range was found to be 5 - 40 µg/ml at a wavelength of 296 nm.
4. Accuracy was determined as % recovery which was found to be greater than 90%. Accuracy was found to be greater than 85% & 86% when standard addition & placebo spiking methods were used respectively.
5. Precision was determined as % RSD which was found to be less than 8%.
6. Robustness was studied and % recovery found to be greater than 80%.
7. The stock solution was to be stable from the stock solution stability results.
8. LOD and LOQ were calculated by standard deviation method which was found to be as 1.805404 μg/ml and 5.47092 μg/ml respectively.  
9. Selectivity  study was carried out using  tablet excipients and there was no interference at λmax  of the given drug.