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Chapter 1
UHPLC Method Development
Shujun Chen and Alireza Kord
1.1 INTRODUCTION
UHPLC has been gradually adopted in industrial labs, especially the pharmaceutical industry due to its high resolution, high speed, and solvent saving, since its introduction in early 2004 (1). A UHPLC method using a sub-2 μm column could reduce the analysis time by up to 80% and save the mobile phase consumption by at least 80% compared with an HPLC method using a conventional 3.5 μm column without sacrificing separation performance (2). In addition, the much shorter run time significantly reduces UHPLC method development scouting time (1).
HPLC method development principles can be applied to UHPLC method development, although detailed procedures may differ. In addition, many existing HPLC methods used in the pharmaceutical industry can be converted to UHPLC methods. In practice, a UHPLC method may need to be converted to HPLC when a UHPLC system is not available.
This chapter provides an overview of the UHPLC method development process and the conversion process of an HPLC method to UHPLC or vice versa. It mainly focuses on analytical reversed phase UHPLC method development of small molecules. A general process and detailed steps are discussed as well as practical examples given.
1.2 METHOD DEVELOPMENT
There are many publications on HPLC method development strategies (3–10). These strategies can be applied to UHPLC method development, although instrumentation and columns are different. The UHPLC method development process includes gathering sample information and defining method goals, scouting columns and mobile phases, analyzing scouting results and selecting separation conditions, optimizing the method, and validating the method. Steps in common UHPLC method development processes are summarized in Figure 1.1. Each step is described and discussed in the following sections.
1.2.1 Gather Sample Information and Define Method Goals
1.2.1.1 Gather Sample Information
Sample information is very useful for method development. The sample information includes the process used to generate the sample, chemical structures and physical and chemical properties of sample components, and their toxicity.
Understanding the process is helpful to method development and is achieved by talking to a chemist or formulator who provides the sample. In the pharmaceutical industry, there is rarely a totally unknown sample. In process chemistry, active pharmaceutical ingredient (API), intermediate (IM), and starting material (SM) are made from synthetic schemes. Most impurities in the sample are known, although some impurities may be unpredictable. In the pharmaceutical process, drug products are manufactured by mixing API with excipients. The major components of the sample are known, although some degradation products may not be predictable.
In addition, knowledge of chemical structures and physicochemical properties of sample components is useful for method development. The chemical structures of possible sample components provide data on molecular weights and functional groups. Special attention should be paid to acidic, basic, aromatic, and other functional groups from which pKa, solubility, chromophore, and stability can be inferred if data of physical and chemical prosperities is not available. Based on pKa, a suitable column and mobile phase buffer pH can be selected for a robust method. The solubility and stability of major components can be used to select a suitable diluent and mobile phase. The UV chromophore can be used to select a suitable detection wavelength.
It is very important to read material safety data sheet (MSDS) before working on samples. Some samples may be toxic and should be handled in a safe manner recommended by MSDS.
1.2.1.2 Define Method Goals
There are four major types of tests in the pharmaceutical industry. These are identification tests, quantitative tests for impurities' content, limit for control of impurities, and quantitative tests of active moiety (11). A quantitative method, for example, an assay method, can be used as an identification method. In addition, many methods can be used for quantitative tests for both assay and impurities' contents.
Different types of methods have different goals. The method goals are usually defined as specificity, accuracy, precision, sensitivity, and robustness, or more specifically as resolution, linearity, recovery, repeatability, and quantitation limit for an assay and impurity method. The method goal for an identification test is often defined as “specificity.” This chapter mainly focuses on the assay and impurity method.
The same type of methods may have different goals for a different sample. For an API sample, its impurities must be separated from API and each other, and the limit of quantification (LOQ) for the impurities must be lower than the reporting threshold recommended in ICH Q3A(R2) guideline (12). For SM or IM, its impurities must be separated from SM or IM and from each other, and LOQs are determined by the criticality of the impurities. For a drug product, its impurities must be separated from its API, excipients, and each other, and LOQ must be lower than the reporting threshold recommended in ICH Q3B(R2) guideline (13).
In addition, method goals also change with the phase of each project. For an early-phase project, robustness and ruggedness are not required for an API method. However, for a late-phase project, robustness and ruggedness are required for an API method. The comparison of method goals between early- and late-phase projects is made in Table 1.1.
Table 1.1 Comparison of API Method Goals Between Early- and Late-Phase Projects
| Specificity/resolution | Resolution ≥ 1.2
Discrimination factor ≥ 0.5 | Resolution ≥ 1.5
Discrimination factor ≥ 0.8 |
| Accuracy/linearity | Quantitation limit (QL) to at least 120% of the analyte (main and specified impurities) | Quantitation limit (QL) to at least 120% of the analyte (main ... |
