Please kindly go through this link,

"Development and Validation of UV-Visible Spectrophotometric Baseline Manipulation Method for Simultaneous Quantitation of Tenofovir Disoproxil Fumarate and Emtricitabine in Pharmaceutical Dosage Form"

Preparation of Standard Stock Solutions and Calibration Curve

Standard stock solution containing 200 μg/mL of TE and 300 μg/mL of EM was prepared separately in the distilled water. Individual working standard solution containing 20 μg/mL of TE was prepared from standard stock solution. Mixed standard solutions containing TE (12 μg/mL) and EM (18 μg/mL) were prepared by using standard stock solutions. The mixed standard solutions of these drugs containing 4–20 μg/mL of TE and 6–30 μg/mL of EM were prepared by serial dilutions of standard stock solutions in distilled water. Mixed standard solutions were scanned using 20 μg/mL of TE solution as blank. Instrument response at 261 nm and 289.9 nm was measured for TE and EM, respectively, and was used to prepare calibration curve. Six replicates of five mixed standard solutions were used to prepare calibration curve. As the correlation coefficient is not true indicator of linearity, Fischer variance ratio (test of linearity) was used to study the linearity of the method [5]. Test of linearity was performed by using MIP Pharmasoft 1.0, software developed and validated at MAEER’S Maharashtra Institute of Pharmacy, Pune. Absorbance of spectrum, by using the above mentioned procedure, was used to prepare calibration curves for both drugs by baseline manipulation (difference) method. Absorbance values at selected wavelengths of the curve were used to construct equations of line ((1) for TE and EM, resp.) which were used to calculate amount of analytes in unknown samples. Beer’s law obeyed in the concentration range of 4–20 μg/mL for TE and 6–30 μg/mL for EM as follows:

etc

http://www.hindawi.com/journals/jspec/2013/146580/