Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 8E) docx

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Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 8E) docx

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422 METHOD DEVELOPMENT Figure 8-47. Resolution map for all critical pairs. Color scale on the left indicates the minimum resolution that is predicted for a particular color in the resolution map. The x axis is the gradient time and the y axis is the temperature. The crosshair can be moved to obtained the predicted conditions for optimal resolution of all critical pairs. See color plate. Figure 8-48. (T op) Drylab-predicted chromatogram versus (bottom) verification run. 8.5.6.12 Running Forced Degradation Samples. Prior to initiating method development as was mentioned in Section 8.4, the structure must be analyzed to predict most probable degradation products. For this compound in this case study, it has an amide functionality, so most probable impurity is the carboxylic acid impurity. It was indeed determined by a forced degradation study (acidic conditions) that it was the major acid hydrolysis degradation product. Using the method that was optimized by Drylab, a forced degrada- tion sample was run that was stressed at 50°C for 1 week in pH 1 diluent. It was noted that a major impurity was formed (Figure 8-49) and was determined to be the carboxylic acid impurity by MS analysis. This compound contains an amide and is prone to hydrolysis. Scheme 2 shows the potential degradation pathway. Therefore to enhance the retention of this potential degradation product the initial organic composition of the gradient was reduced from 35% to 25 v/v% acetonitrile (Figure 8-49). The carboxylic acid impurity has an enhanced retention at lower organic composition and had adequate resolu- tion between active and impurity eluting after main component was still obtained. METHOD DEVELOPMENT APPROACHES 423 Scheme 2. Using this optimized method shown in Figure 8-49 that starts at 25 v/v% acetonitrile, LC-MS studies were performed to determine the [M + H] + ion of the impurity that has been resolved from the main peak. The mass spectrum of Product M was taken and was shown to be spectrally homogeneous. The mass spectrum of the impurity (RRT 1.04) that has now been resolved from the main peak was also taken. The UV and the total ion chromatograms are shown in Figure 8-50. This impurity, RRT 1.04, has the same [M + H] + ion that was co-eluting with the main component in the initial separation on the C8 424 METHOD DEVELOPMENT Figure 8-49. Effect of organic composition on the retention of carboxylic acid degra- dation product. Column: Waters Sunfire C18, 3.5 µm, 150 × 4.6 mm. (A) 10 mM NH 4 OAc, pH 5.8. (B) Acetonitrile: Flow, 1.5 mL/min; temperature, 35°C. Figure 8-50. Chromatographic conditions: Column: Sunfire C18, 3.5 µm, 150 × 4.6 mm. Mobile phase: 10 mM NH 4 OAc, pH 5.8; acetonitrile, 25% acetonitrile to 75% acetoni- trile over 10 min and 5-min hold at 75% acetonitrile. Wavelength: 247 nm, column tem- perature, 35°C, flow, 1.3 mL/min; injection volume, 10 µL; MS conditions flow split 10 : 1. ESI: + ion mode, single quadrupole, Z-Q. Capillary, + 3.5 kV; cone, 25V; source tem- perature, 150°C; desolvation temperature, 400°C; cone gas flow, 113 L/hr; desolvation gas flow, 419 L/hr. column (Figure 8-41). This confirms that the optimized method was able to resolve the impurity from the main component. 8.5.6.13 Final Optimization. The method could then be further optimized for speed by increasing the flow rate and decreasing gradient time (t g ) pro- portionally or by decreasing gradient range (∆%) and decreasing t g pro- portionally. These are further discussed in Chapter 17, Section 17.3. The temperature could be increased as well, but the chromatographer should be aware that although increases in speed may be realized, the selectivity may change with modifications in the temperature. Other potential improvements in the method could include using a smaller (e.g., 3mm) i.d. column while using the same length column and particle size of the packing material. A 3.0-mm-i.d. column can be used to reduce solvent waste, since columns with smaller diameters have reduced column volume and require use of lower flow rates, and therefore they can decrease solvent waste by at least 60%. A simple calculation to achieve equivalent retention on a smaller-i.d. column at the same linear velocity is shown in equation (8-2). For example, if the original method used a 15-cm × 4.6-mm column with a 1.5 mL/min flow rate, what would be the equivalent flow rate for a 15-cm × 3- mm column? Using the following equation, this can be calculated. (8-2) If you divide the 1.5-mL/min flow rate by 0.425, then a flow rate of 0.64 mL/min is obtained. Moreover, use of lower flow rates can lead to enhanced ionization efficiency using ESI (no flow splitting). Increasing the flow rate increases droplet size, which decreases the yield of gas-phase ions from the charged droplets. 8.5.6.14 Case Study 3: Concluding Remarks. There is no cookbook for method development. The strategies presented an approach that could be taken for effective method development and optimization. In summary, a steep gradient was used initially to predict the suitable isocratic conditions for deter- mining the most suitable pH for the method. The retention behavior of active as a function of pH (isocratic) was determined. The best mobile-phase pH for further gradient experiments was determined. Also, ACD was shown to be able to estimate the pK a of the molecule; and by applying the rules based on pH shift of the mobile phase and pK a shift of the analyte upon addition of organic component, the optimal pH for analysis was predicted. In order to elu- cidate if there was co-elution of impurities with the target analyte spectral homogeneity was assessed using both PDA and LC-MS. If possible, use AMDS/Dry Lab for method optimization and then use MS to confirm the sep- aration of active species from possible co-eluting species. MS/MS analysis can Π Π rL rL r r 2 30 2 46 30 46 0 425 . . . . . -i.d.column -i.d.column -i.d.column 2 -i.d.column 2 == METHOD DEVELOPMENT APPROACHES 425 be performed for further structural elucidation of the impurities. Deuterated experiments can be performed to support structural assignments. 8.5.7 Case Study 4: Structural Elucidation Employing a Deuterated Eluent The fine structural details of analytes could be further defined by a deuterium- exchange experiments that measures the number of exchangeable protons in each molecule. The number of exchangeable protons in a molecule can be determined based on the mass shift. This technique allows an understanding of which protons are susceptible to exchange, but also can be used to differ- entiate compounds of the same molecular weight that have a different number of exchangeable protons. Deuterium exchange provides strong evidence to support degradation product and synthetic by-product elucidation. Take, for example, the two compounds 5-aminoindazole and 1-aminoindan, both of which have an [M + H] + of 133.9. An HPLC method was developed to sepa- rate these two compounds (Figure 8-51). The mass spectra for each compound are shown in Figures 8-52A and 8-52B. Both of these compounds show [M + H] + ions of 133.9. If an analytical chemist were to discern between these two compounds, MS/MS analysis could be performed, but if only a single quadrupole instru- ment was available, what additional experiments could the analytical chemist perform on the single quadrupole instrument? The use of deuterated mobile phases could be used. Since 5-aminoindazole has three exchangeable protons and 1-aminoindan has only two exchangeable protons, by using a deuterated mobile phase the [M + D] + species would be different. Both these compounds were run by the same HPLC method as in Figure 8-51, but with a deuterated mobile phase (70% D 2 O : 30% MeCN). The mass spectra of the two components were taken from this chromatographic run. In Figure, 8-53, 426 METHOD DEVELOPMENT Figure 8-51. HPLC separation of 5-aminoindazole (R T at 1.23 min) and 1-aminoindan (RT at 2.28 min), 70% H 2 O:30% MeCN. Flow, 0.5 mL/min; Waters symmetry shield, 50 × 4.6 mm, 5 µm. the mass of the first component on the chromatogram increases by 5, indicat- ing the presence of three exchangeable protons (one for each hydrogen atom replaced with D and two from D + ), which suggests that this analyte is 5- aminoindazole. In Figure 8-54 the mass of the second compound increases by 4; therefore only two exchangeable protons (one for each hydrogen atom replaced with D and two from D + ) are present, suggesting that the analyte is 1-aminoindan. The assignments of each species were confirmed by running individual standards of each compound. Knowledge of the number of labile H atoms in a molecule is useful for assisting in the elucidation of proposed impurity structures. METHOD DEVELOPMENT APPROACHES 427 Figure 8-52. ESI positive ion mode MS spectra. (A) Mass spectra of 1-aminoindan (ESI positive ion mode). (B) Mass spectra of 5-aminoindazole (ESI positive ion mode). 428 METHOD DEVELOPMENT Figure 8-53. Mass spectra of 5-aminoindazole in deuterated mobile phase. Figure 8-54. Mass spectra of 1-aminoindan in deuterated mobile phase . . by Drylab, a forced degrada- tion sample was run that was stressed at 50°C for 1 week in pH 1 diluent. It was noted that a major impurity was formed (Figure. DEVELOPMENT APPROACHES 425 be performed for further structural elucidation of the impurities. Deuterated experiments can be performed to support structural

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