3A.2.10 Robustness
A method is robust if it is unaffected by small changes in operating conditions. To evaluate the robustness of developed analytical method few parameters were deliberately changed. The parameters included analysis at different temperatures viz. 250C and 300C, at 0.8 and 1.0 mL/min flow rate, using C18 columns from different manufacturers one from Qualisil BDS, USA (4.6 x 250 mm x5µ) and the other from LC-GC, India (4.6 x 250mm x5µ). Each of the three selected factors were changed one at a time to estimate the effect. Replicate injections (n= 6) of standard solution (10µg/mL) were injected. (Table no.3A.3)
3A.2.11 Stability
The stability of TMX in the mobile phase was investigated by analyzing the standard of TMX (10µg/mL) …show more content…
Thus different analytical methods were developed including HPLC technique, using various mobile phases. In the development of analytical method it is extremely important to understand the solubility of an analyte. Tamoxifen citrate is very slightly soluble in water (0.3mg/mL at 20°C; pH 3.0-3.5) with high log P value (6) and is soluble in methanol (50 mg/ml). It also shows pH dependent solubility. (Vamsi,2013)
The wavelength selected for analysis was 265nm to reduce baseline noise at absorption maximum (257nm) of Tamoxifen citrate. Mobile phase composition or solvent strength plays an important role in RP-HPLC. An HPLC column packed with stationary phase of octadecyl carbon chain (C18)-bonded silica was used. A polar mobile phase such as mixture of organic solvent methanol and buffer was used. The hydrophobic molecules in the polar mobile phase show more affinity to the hydrophobic stationary phase and these molecules can be eluted from the column by decreasing the polarity of the mobile phase for reducing hydrophobic interactions. The more hydrophobic molecule will be strongly bound to the stationary phase and the concentration of organic solvent required to elute the molecule will be higher. Hence buffer concentrations of 10 and 20 % were selected …show more content…
Risk assessment identifies the critical method variables, the parameters that impact the Analytical Target Profile. Once the technique is identified, Analytical Quality by design focuses on method development and includes detailed assessment of the risks associated with variability.
In chromatographic method, the variability due to HPLC instrument configuration, column selection and injection volumes are kept controlled in the experimental strategy, while rest of the variables such as pH, buffer and % ratio of mobile phase are assigned to test the robust behavior and for the establishment of method operable design region. FDA has suggested conducting method operable design region together with method validation as most recommended. Once this is defined, appropriate method controls are put in place and method validation is carried out. The design of experiments provides a base for scientific understanding of relation between quantities of input variables, Critical method parameters and output response which will show considerable effect on method performance and Analytical Target
A method is robust if it is unaffected by small changes in operating conditions. To evaluate the robustness of developed analytical method few parameters were deliberately changed. The parameters included analysis at different temperatures viz. 250C and 300C, at 0.8 and 1.0 mL/min flow rate, using C18 columns from different manufacturers one from Qualisil BDS, USA (4.6 x 250 mm x5µ) and the other from LC-GC, India (4.6 x 250mm x5µ). Each of the three selected factors were changed one at a time to estimate the effect. Replicate injections (n= 6) of standard solution (10µg/mL) were injected. (Table no.3A.3)
3A.2.11 Stability
The stability of TMX in the mobile phase was investigated by analyzing the standard of TMX (10µg/mL) …show more content…
Thus different analytical methods were developed including HPLC technique, using various mobile phases. In the development of analytical method it is extremely important to understand the solubility of an analyte. Tamoxifen citrate is very slightly soluble in water (0.3mg/mL at 20°C; pH 3.0-3.5) with high log P value (6) and is soluble in methanol (50 mg/ml). It also shows pH dependent solubility. (Vamsi,2013)
The wavelength selected for analysis was 265nm to reduce baseline noise at absorption maximum (257nm) of Tamoxifen citrate. Mobile phase composition or solvent strength plays an important role in RP-HPLC. An HPLC column packed with stationary phase of octadecyl carbon chain (C18)-bonded silica was used. A polar mobile phase such as mixture of organic solvent methanol and buffer was used. The hydrophobic molecules in the polar mobile phase show more affinity to the hydrophobic stationary phase and these molecules can be eluted from the column by decreasing the polarity of the mobile phase for reducing hydrophobic interactions. The more hydrophobic molecule will be strongly bound to the stationary phase and the concentration of organic solvent required to elute the molecule will be higher. Hence buffer concentrations of 10 and 20 % were selected …show more content…
Risk assessment identifies the critical method variables, the parameters that impact the Analytical Target Profile. Once the technique is identified, Analytical Quality by design focuses on method development and includes detailed assessment of the risks associated with variability.
In chromatographic method, the variability due to HPLC instrument configuration, column selection and injection volumes are kept controlled in the experimental strategy, while rest of the variables such as pH, buffer and % ratio of mobile phase are assigned to test the robust behavior and for the establishment of method operable design region. FDA has suggested conducting method operable design region together with method validation as most recommended. Once this is defined, appropriate method controls are put in place and method validation is carried out. The design of experiments provides a base for scientific understanding of relation between quantities of input variables, Critical method parameters and output response which will show considerable effect on method performance and Analytical Target