(W1030-10-59) Optimization of Assay Method for a Drug with Two pKa

Purpose: The pH plays an important role in the HPLC analysis. The pH of the mobile phase has impact on the retention and separation of analytes. In some cases, the protonated and unprotonated analyte can have significant differences in responses, resulting in failure of method validation. This poster presents a case of linearity failure of an assay method for Compound A due to the differences in protonation and response of the analytes at different pH. Choosing a wavelength close to the isosbestic point can improve the linearity and meet the method validation criterion for linearity.

Methods: The Assay method of Compound A is adopted from vendor. HPLC equipped with a PDA detector was used for Assay analysis of Compound A. Below HPLC parameters were used: Column: X-Select® CSHTM C18, 150 x 4.6 mm, 3.5 μm. Mobile Phase A: 0.05% (v/v) TFA in water. Mobile Phase B: 0.05% (v/v) TFA in ACN. Flow rate: 1.0mL/min. Column temperature: 40°C±3°C. Sample temperature: 5°C±4°C. Wavelength: 250 nm.  Injection Volume: 20 µL. Diluent: 0.1% (v/v) TFA in water/ACN (70:30). Gradient method: Start with 100% mobile phase A. Increase mobile phase B to 30% over 10 minutes. Continue increasing mobile phase B up to 80% at 20 minutes. Further increase mobile phase B up to 90% at 25 minutes. Change back to 100% mobile phase A at 25.1 minutes. Maintain 100% mobile phase A until 30 minutes. To optimize the method, the linearity, and the UV spectrum difference under different mobile phase with different pH was investigated.

Results: As seen in Figure 1, linearity with the given mobile phase and given wavelength results in %y-intercept of 1.8%, which could imply bias of quantitation for the low concentration of the method. In order to improve % y-intercept, the effect of mobile phase pH to UV absorbance was investigated. Ideally, the mobile phase pH should be beyond ±2 pH units of the analyte’s pKa to have one form dominant. The vendor’s choice of a mobile phase, however, contains 0.05% TFA (pH 2.25) falling between pK_a1 (2.17) and pK_a2 (5.49) of Compound A. Based on the Henderson-Hasselbalch equation (Equation1), the mobile phase with pH 2.25 can move the equilibrium, resulting in co-existing of protonated and deprotonated forms of Compound A. To examine the changes in the forms of Compound A, UV absorbance was measured with various pH of mobile phase in Figure 2. In Figure 2, the UV absorption of compound A exhibits a pH-dependent behavior. As the pH decreases (from black to red to blue), the absorbance at 250 nm decreases while the absorbance at 360 nm increases. It shows that Compound A can exist in both forms of protonated and deprotonated and also converted to each form depending on the pH of environment. The pH dependence of absorbance plays a crucial role in UV absorbance. As the pH decreases, the absorbance maxima shift from 247.6 nm (at 0.05% TFA, pH 2.25) to 241.7 nm (at 0.35% TFA, pH 1.42). The pH dependence of the absorbance can reduce the robustness of measurements, leading to deviation of the fitting curve from original (presented by %y-intercept). The mismatch between the pH in the diluent and the mobile phase, along with change in the analyte amount of the injected linearity solutions, can create a micro-environment where pH is inconsistent inside the column. These pH variations result in changes in the ratio of protonated/deprotonated form of Compound A, which in turn affects its UV absorption. For instance, at 250 nm, Compound A exhibits a stronger response at pH 2.25 compared to pH 1.47. To address this issue, one can take advantage of isosbestic point in UV spectrum. In Figure 2, the absorbance at 280 nm remains relatively constant for both pH 1.42 and pH 2.25. As a result, the linearity plot measured at 280 nm shows a reduced %y-intercept to 0.2% in Figure 3.

Conclusion: It is important to select appropriate pH of mobile phase and the wavelength of detection by considering the pKa of compound to improve the robustness and accuracy of the analysis method.

Figure 1. The Linearity Plot of Compound A at 250 nm

Figure 2. The UV Spectrums of Compound A with Different pH of Mobile Phase


Figure 3. The Linearity Plot of Compound A at 280 nm