86 and EM, % purity 99.92 was kindly supplied as a gift sample by Emcure Pharmaceutical Pvt. Ltd. Pune. These samples were used without further purification. Tablet used for analysis was TENVIR-EM (Batch No. X81241) manufactured by Cipla Ltd. Goa, India containing TE 300 mg and EM 200 mg per tablet. Method A: Ratio derivative The method involves dividing the spectrum of mixture by the selleck chemical Crenolanib standardized spectra of each of the analyte and deriving the ratio to obtain spectrum that is dependent of concentration of analyte used as a divisor. Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different TE standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of EM (8 ��g/ml) as shown in [Figure 1a] and the first derivative of these spectra traced, are illustrated in [Figure 1b].

Wavelength 271.07 nm was selected for the quantification of TE in TE + EM mixture. The ratio and ratio derivative spectra of the solutions of EM at different concentrations were obtained by dividing each with the stored standard spectrum of the TE (12 ��g/ ml) [Figure [Figure2a2a and andb,b, respectively]. Wavelength 302.17 nm was selected for the quantification of EM in TE + EM mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of TE and EM in tablets was calculated by using following equations.

[16] Figure 1 Ratio spectra (a) and first derivative of the ratio spectra (b) of (a) 3, (b) 6, (c) 12, (d) 18, and (e) 21 ��g/ml solution of TE when 8 ��g/ ml solution of EM is used as divisor. Figure 2 Ratio spectra (a) and first order derivative of the ratio spectra (b) of (a) 2, (b) 4, (c) 8, (d) 12, and (e) 14 ��g/ml solution of EM when 12 ��g/ml solution of TE is used as divisor. At 271.07 nm: CTE = d/d�� [ATE/AEM] �C Intercept (C)/ Slope (m), (1) At 302.17 nm: CEM = d/d�� [AEM/ATE] �C Intercept (C) / Slope (m). (2) Method B: Derivative method The method involves obtaining the first derivative spectrum of the series of the solution of mixtures of TE + EM in ascending and descending concentration.

From the observations of the derivative spectrum, derivative amplitudes responsible for TE and EM were selected and wavelength for each amplitude was noted. These wavelengths were further confirmed by checking the first order derivative amplitude of the mixed standard solutions of these drugs in the given ratio. Mixed standard AV-951 solutions were prepared in the range of 3-21 TE (and 2-14 ��g/ml for EM) were used for the study. Wavelengths 306.88 and 224.38 nm were selected for the quantification of TE in TE + EM mixture and EM in TE + EM mixture, respectively [Figure 3].