The development of analytical methods for assay and impurity profiling of new chemical entities (NCEs) in pharmaceutical analysis is a complex task that no single method development approach can satisfy. New drug substance high performance liquid chromatography (HPLC) methods must resolve a wide range of potential degradants and manufacturing impurities while being appropriately sensitive, selective, precise, accurate, and robust. An unbiased and well-designed approach is key to rapidly developing optimal chromatographic conditions based on data-rich analysis.

This article offers an overview of what to consider when developing chromatographic methods and how a step-by-step, unbiased approach can pay off in reduced timelines and costs with robust results.

Method development matters
The goal of method development is to design an HPLC method that operates reliably in a good manufacturing practice (GMP) environment. In the early stage of method development, it is important to understand analyte behavior in both the mobile and stationary phases. Be aware of the functional groups on the analyte, and whether the compound is acidic, basic, or neutral. Reactive functional groups and other properties may affect solubility, stability, and column retention. Comprehensive chemical knowledge can help elucidate critical method attributes and aid in troubleshooting if poor performance occurs.
Though understanding the chemical properties of the compound is crucial for method development, performing unbiased and systematic screening activities is essential to developing a robust method predicated on a wealth of data.  

Common method development pitfalls
While no single answer exists for all development processes, there are some common pitfalls to avoid:

1. Letting “educated guesswork” drive method development. Do not expect an approach that showed success for a similar compound to be appropriate for a new analyte—each compound presents a unique impurity profile, degradants, and residual process intermediates that require special attention. Although chemical properties (such as acidic or basic groups) have a role in designing analytical methods, a practical and objective procedure should be pursued. Attempting to develop an appropriate method while minimizing your method development tools (see below) by relying on “educated guesswork” can lead to an inefficient iterative process of method tuning, while producing little improvement. 
2. Not establishing goals and reasonable expectations. Before beginning a method development project, define the goals for resolution, specificity, sensitivity, and other chromatographic properties. These can be set according to the phase of development for the drug compound. Consider potential degradation pathways, process impurities, and stereochemistry of the compound prior to initiating method development to help identify related substances that may be difficult to chromatograph and detect. It may be more efficient—for timelines and costs—to utilize several simple and robust methods, as opposed to developing a single overly complicated method.
3. Delaying robustness studies (or, settling for a method that just works). Robustness studies ensure that the method performs reliably under minor variations. Observing chromatographic changes during a systematic method development process can inherently establish method robustness. Understanding which parameters are critical to quality during the development process will prevent validation of methods that operate on the fringes of acceptability. Building robustness into the early method development process can ultimately save time and money over the lifecycle of the drug, from the early stages through commercialization.   

Method development tools
Detectors
Before comprehensively evaluating the chemical interactions of the analyte with the HPLC phases, the detection mechanism can be a significant factor to consider. Different detectors can have vastly different sensitivity attributes and should be chosen based on chemical properties of the analytes. For compounds with a UV-chromophore, a photo diode array detector (PDA) assists with selecting the ideal detection wavelength for the active and its related substances. A multi-detector setup using both a PDA and a universal detector ensures that all species are captured. Common universal detectors include charged aerosol detectors (CAD), evaporative light scattering detectors (ELSD), refractive index detectors (RID), and mass spectrometers. Fluorescence detectors may also be used if chemical properties allow for this unique detection.

Columns (Stationary Phase)
A variety of commercial column chemistries are available that can provide different elution profiles (or selectivity) for a mixture of compounds. While C18 phases are common in the industry, advancements in the last few decades have produced more unique phases such as pentafluorophenyl (PFP) or phenyl-hexyl phases that may provide enhanced resolution. Among the major phase classifications, there are other manufacturer-related features to consider such as silica porosity, end capping, core-shell technology, and charged surface hybrid structures.

For example, pore size can have a large impact on the resolution of an analyte mixture, depending on the size and structure of the compounds. Carbon load may also be considered if the compound or matrix retains too long (or not long enough).

Ideally, an HPLC equipped with multiple column compartments is utilized to produce a rich source of data which allows for comparison of resolution between analytes and impurities across an array of screened column phases. After a lead set of columns are identified, a forced degradation analysis can further aid in selection of a superior column chemistry, and later the appropriate dimensions. 

Solvent Selection (Mobile Phase)
Concomitantly with column screening, an HPLC setup with multiple solvent lines can afford advantages to rapid method development. Evaluating various organic solvents during column screening provides copious information about the selectivity of the analytes with each stationary and mobile phase combination. The solvent selectivity triangle may be consulted when evaluating the advantages of various strong eluents and their influence on selectivity. Methanol, tetrahydrofuran, and acetonitrile are commonly recommended strong solvents with complementary acidic, basic, and polar natures. When selecting mobile phases to screen, first verify that combinations of weak and strong solvents are miscible, and any buffering salts are fully soluble. Additionally, the UV-cutoff of the mobile phase solvents should be suitable for the detection wavelength to avoid baseline instability and poor sensitivity.    

Furthermore, systematic changes to temperature, gradient, and mobile phase pH help elucidate the interactions of analytes on the column—and add to method robustness characterization! These factors may be considered simultaneously while screening mobile phases. Together, the mobile phase and column screening approach provides a wealth of objective data regarding resolution, retention, peak shape, and sensitivity.

Mobile Phase Modifiers
While phosphoric acid, formic acid, or trifluoroacetic acid (TFA) are commonly added modifiers, others may be necessary to establish resolution of analytes. Make decisions on mobile phase additives based on analyte structure properties. For example, acidic modifiers (or low-pH buffers) can protonate surface silanols and alter cationic interactions, reducing peak tailing in certain circumstances.

Also, consider the detection mechanism. If a universal detector is used, avoid non-volatile additives (such as sodium or phosphate salts). In low-UV detection methods, TFA and formic acid may not be appropriate for adequate sensitivity.

Besides acidic modifiers, other common modifiers include:

• Bases: Even with a good track record in LC methods, bases can be a challenge because they may modify the overall charge state of the analyte. Operating a method above the pKa of a basic species is useful for stable compounds when there is a need to increase the on-column retention. Organic bases are not widely used in the pharmaceutical industry for developing assay-related substance methods because of a tendency for molecules to hydrolyze, or be reactive, at high pH. The pH limit of the column must also be considered if using a basic modifier.
• Ion-pairing agents: In cases where separation remains elusive, an ion-pairing agent may be necessary. Ion-pairing agents, such as TFA, have a strong affinity for oppositely charged compounds, minimizing undesirable secondary interactions (resulting in tailing) or improving retention. Other ion-pairing agents such as alkyl sulfonic acids have an ionic end and a non-polar tail that is strongly retained by the hydrophobic column stationary phase. This leaves a charged functional group available to modify interactions of the analyte with the stationary phase and offers greater retention of oppositely charged species. Certain ion-pairing additives are not a first choice in development because of increased equilibration times, higher UV-cutoffs, and permanent alteration to columns.
• Buffers: As mobile phases, buffers can control the ionization state of compounds. When optimized for pH and concentration, they can alter selectivity of analytes and improve resolution. The stability and pKA of compounds need to be considered when selecting a suitable buffer system to avoid rapid degradation during analysis or peak splitting due to the compound being in multiple charge states.

A systematic approach
When starting method development, generic gradient and detection screening (detection wavelength selection and injection volume) is often a first step. This ensures adequate sensitivity and retention are achieved before initiating an exhaustive column and mobile phase screen. Intermediates and impurity markers are blended into a “resolution solution” to provide the most data during screening. Forced degradation early in the process can also be performed, and is particularly helpful when related substances are not yet characterized or available.

Mobile phase screening, including THF, methanol and acetonitrile can then be carried out based on the solvent selectivity triangle. Various buffered mobile phases and mobile phase additives are also tested. Several promising columns generally emerge from this method development workflow. Those columns are then taken through gradient ramping, temperature screening, and flow rate adjustments. The final method is then optimized and ready for pre-validation activities, which includes the final forced degradation evaluation.

Robust methods deliver
Using a critical approach to column and mobile phase selection screening, with systematic, stepwise optimization to work robustness into methods from early stages will pay off across the life of a program. Rapid and data rich method development delivers sensitive, selective, precise, accurate, and robust methods that today’s manufacturers can rely on. 

Method Development Without Impurity Markers When there are no impurity markers available, like in the case of new drug candidates, several options can be considered: Dirty lots: The earliest batches—where the manufacturing process still needs developing—are taken and called dirty lots. They are used in the column screening approach, in addition to the forced degradation studies. At Cambrex, mass spectrometry detection is also used to provide more information about the impurity profiles and enable peak tracking. Forced degradation: During a column screening approach, forced degradation studies are incorporated to verify resolution of key related species. This includes acid, base, photo, oxidative, and thermal stress. Mass spectrometry: Pharmaceutical development is complex. If available, additional detection via mass spectrometry is often an advantage (though it cannot be used in some methods, with non-volatile mobile phase components). By incorporating column screening and mass spectral detection, various key peaks can be tracked, enhancing the overall screening approach.

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Dr. Daniel Kirschner is executive director of analytical services at the Cambrex Durham, NC site specializing in analytical services for drug substances and drug products at all stages of the clinical and commercial development lifecycle.