The Onyx Approach to HPLC Method Development
The Onyx Approach to HPLC Method Development
High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique widely employed in the pharmaceutical industry. A validated HPLC Assay and Impurities method is one of the most compelling results on a Certificate of Analysis for an API. Therefore, a good method development strategy, a process that involves optimising various parameters to achieve accurate and reliable results, is fundamental to producing a validated HPLC method. Developing an HPLC method for an API which is also used to analyse raw materials and intermediates in the reaction scheme can be challenging due to the complexity of the compounds and the wide range of functional groups present. This blog describes the HPLC method development process utilised at Onyx and explores key considerations and strategies to produce a ‘fit-for-validation’ HPLC method.
Before embarking on method development, it is crucial to fully understand the fundamentals of HPLC. HPLC separates and quantifies components in a sample matrix based on their interactions with a stationary, and mobile phase. The stationary phase is typically a packed column, whereas the mobile phase is a liquid that carries the sample through the column. Parameters like column type, mobile phase composition, flow rate, and detector settings play a pivotal role in method development.
Mobile Phase pH
To ensure that the HPLC method is applicable across all phases of manufacture, samples of the API, intermediates, and identified impurities are required. These samples are prepared at a nominal concentration and screened on various mobile phases. The mobile phases span a wide range of pH’s from acidic solutions like trifluoroacetic acid (pH approx. 1) and formic acid (pH 2–3) through to more neutral buffers with a pH of up to 9, all of which will be part of the screening process. Typically, a computationally derived measurement of the pKa of the API is used to determine the optimal pH. Throughout this screen, generic HPLC parameters are used. These parameters can vary from project to project, but they must be kept consistent throughout the mobile phase screening. The different analytes are assessed for their retention, selectivity, and peak efficiency.
Stationary Phase Selection
The choice of stationary phase is fundamental to HPLC analytical method development. During the mobile phase screening, all available analytes are evaluated on both a C18 and Phenyl stationary phase across the full range of mobile phase pH values. This examination provides a more comprehensive understanding of each analyte’s different properties and interactions due to the varying selectivity and retention observed across different stationary phases. Using the initial findings, a selection of Onyx’s dedicated Method Development HPLC columns are chosen for stationary phase screening. Factors such as particle size, pore size, and column length are taken into consideration, and a column switching valve is utilised to allow rapid screening. The column screening is followed by another evaluation of retention, selectivity, and peak efficiency of the different analytes.
Optimising Mobile Phase Composition
The mobile phase composition significantly influences separation efficiency. Different solvent ratios and alternative organic phases are employed to achieve optimal peak resolution and sensitivity. An understanding of the chemistry of the different analytes aids in the selection of appropriate solvents. pH and buffer concentration will impact the ionisation and retention time of the analytes, so at this stage in the method development, buffer concentrations and pH are finely tuned to enhance the separation of the analytes. Sometimes, compromises need to be made due to the complexities of the processes and multiple functional group transformations that can occur in a synthetic route. Usually, the best conditions for the API are pursued as this is the compound which the method will be validated for. The compatibility of the buffer with the detector is a key consideration at this stage. Mass Spectrometry (MS) is a powerful tool in process development therefore only MS compatible mobile phase additives are investigated.
Temperature and Flow Rate
The pressure of the HPLC system plays a significant role in the resolution of analytes. Temperature and flow rate both affect system pressure and by extension, resolution Therefore optimising these elements enhances resolution and reproducibility. MS is used for troubleshooting problems in process development, therefore Onyx methods aim to be below 350 bar, to account for the additional backpressure of an MS detector.
Detector and Detector Settings
Most methods developed for Assay and Impurities determination at Onyx are UV methods. It is vital that the UV Spectra of all the analytes are thoroughly assessed, and a suitable detection strategy is established. For example, some analytes may have differing UV properties, requiring the calculation of response factors to account for these variations. The analysis of the API should be performed at a peak maximum on the UV spectrum of the API to ensure good reproducibility and accuracy of the method.
Preliminary Validation and Forced Degradation
Once a method is developed, it is essential to validate its performance. All methods developed at Onyx will undergo preliminary validation to ensure an appropriate working concentration is assigned for the analysis. The UV response at the working concentration must be linear, and the response should be sufficient to give adequate sensitivity to low-level impurities. These properties are confirmed in the preliminary validation before the method undergoes a formal validation.
It is also important that the developed method is stability indicating. Therefore, a forced degradation study is also performed to highlight any issues that may arise when analysing the API routinely. This study provides confidence that the method will detect any degradation that may occur during a stability study on the API. Degradation conditions that should be covered in such a study include hydrolysis (acid and base), thermal decomposition, oxidation and photolysis.
Conclusion
HPLC method development for pharmaceuticals is both an art and a science, requiring a deep understanding of chromatographic principles and hands-on experience. By carefully optimising parameters such as stationary phase selection, mobile phase composition, flow rate, and detector settings, an analyst can develop robust and reliable HPLC methods for a wide range of applications. Continuous learning, systematic experimentation, and attention to detail are key to mastering this essential skill in analytical chemistry.