11


be adhered to during the development process. This reduces downstream problems in method transfer, either in-house or to outside contractors.


Matching Parameters to Purpose


Answering these questions would be an empty exercise if the answers didn’t inform method development. To turn the identifi ed purpose into concrete method parameters, defi ne method specifi cations and choose screening conditions.


Method specifi cations state clearly what the project needs to achieve before any work begins. The questions shared here will help to identify a broad category for the project: preparative scale-up, quantitative method development, or investigative study.


Each category has different requirements. For example, preparative scale-ups are concerned with separation purely of the compound of interest. The goal is to get high recovery and purity in a reasonable time, and other factors - like resolving individual peaks - can be sacrifi ced. But what constitutes ‘reasonable’ time or recovery? These should be defi ned in the specifi cations, depending on the information already gathered.


For quantitative method development, benchmarks must be set for quality of quantitation. If percent weight-weight is desired, pure standards must be available. There may be governmental or third-party guidelines on quantitative accuracy, or those may need to be decided, again based on the project aims.


Finally, for investigative studies (e.g., forced degradation studies), detailed information about the samples will help to design the method-development plan. It is best to work from a single combined sample, rather than multiple samples. With fewer samples, it is simpler to track compounds across multiple chromatographic conditions. Consider what impurities are probably being formed and how to determine whether the method detects all impurities. Never assume that a single UV/Vis detector, whether diode-array or single- wavelength, will capture all compounds. Systems with multiple detectors (e.g., light scattering, corona-charged aerosol, fl uorescence, and MS) are more thorough.


After the specifi cations are set, the experimental work begins. For effi cient screening and optimisation, use the information gathered in pre-development to select parameter ranges for screening.


Figure 2 shows all the parameters that affect analytical-method performance. Informed decisions should be made about all of these; selecting parameters to investigate based on guesses or past habits will only prolong method-development time.


Figure 4: An example optimisation workfl ow.


Detector choice is yet another factor that is infl uenced by both analyte physicochemical properties and other requirements. Obviously, a detector should be chosen based on expected compound properties, e.g., whether a compound is UV-absorbing. (Eluent properties should also be considered, as they may infl uence compound properties. For example, absorbance spectra shift with solvent pH.) But project purpose also enters the equation: if measuring degradants is important, there should be some understanding of how the compound might degrade and how that might affect measured signals in different detectors. It is often a good idea to start a development project with multiple detectors. This ensures that all compounds are detected and thus mass balance can be achieved.


A factor-by-factor explanation of all the parameters in Figure 2 is beyond the scope of this paper. Yet as these examples illustrate, informed consideration is necessary for effi cient method development.


Optimising Parameters


Picking parameter ranges for screening is only the start of the experimental work. A rational approach to method development explores the selected parameter space to fi nd optimal and robust areas.


Figure 4 shows a standard screening and optimisation plan. Rather than optimising all the variables at once, the chromatographer handles them in waves - a more effi cient and manageable approach.


In the fi rst wave, the column, buffer, and solvent are screened. This step should narrow down choices, though it needn’t leave only one option. If two or more columns perform to the determined specifi cations, it may be worthwhile to take multiple columns forward.


Once column, buffer, and solvent are selected, optimise gradient and temperature. Simulation tools can help here. Given a few chromatograms collected at different gradients and temperatures, software can simulate chromatograms across the parameter space and fi nd areas of optimal resolution.


This stepwise process will make method development more effi cient, but the experienced chromatographer will keep some perspective on the entire project. Certain steps taken early


on will make methods more likely to pass later steps of validation and risk assessment. For example, start working with limit-of-detection samples early. This will fl ag any issues with sensitivity before work is sunk into a method that will never meet specifi cations.


By the end of optimisation, the method-development team should have settled on a complete set of method parameters.


Validating Performance


Approaching pre-development and development methodically should put chromatographers in a good place for validation - with a method that is likely to pass validation and robustness testing.


Figure 2: Many parameters affect the success of the fi nal chromatographic method.


For example, the solvents and pHs to be screened should be selected based on analyte physicochemical properties. Often, experimental data is not readily available, so software tools can be used to help. Commercial software tools provide useful predictions such as solubility, logP, logD, and pKa.


Figure 3 shows one such tool, ACD/Method Selection Suite. The predicted logD of a compound is graphed against pH (left). Based on this, the percentage of analyte in the dominant ionic form is also graphed (right). The dominant species changes with pH, but the important point is to choose an eluent pH on the plateaus of this curve. Otherwise minor shifts in pH will vastly affect the method.


Before validation, assess the forced degradation results (if applicable) and check mass balance. Write up a risk assessment: a living document that addresses the most likely points of failure. This document should be regularly reviewed and updated, especially with the results of robustness and method-transfer exercises.


Then move to validating the method, checking: 1. Sample concentration and injection volume 2. Analytical-wavelength selection 3. Relative-response-factor determination 4. Analytical solution stability 5. Specifi city, resolution, linearity, reporting limit, and limit of detection 6. Method of quantifi cation


During the validation process, draw upon earlier data where reasonable. A large quantity of data is generated during development. If development experiments are well designed, and the resulting data is properly processed, annotated, and stored, it can be used to answer validation questions. For example, development data can be used to investigate peak-shape changes or retention-time drifts over time, reducing the number of validation experiments required.


If performance can be proven, then the method-development project has been a success. But so long as a method is in use, development never really ends. Whenever the use case changes - if raw materials change, the synthesis pathway is altered, or the samples are different - reassess the method.


Figure 3: Predictive software can be used to visualise logD (left) and dominant ionic form (right) changes with pH.


To take another example, column selection depends upon both compound properties and project considerations. Compound properties obviously affect stationary phase selection: non-polar analytes should be separated on C8, C18, or similar stationary phases; low-to- medium polarity analytes on diol, amino, cyano, phenyl, or C4; and medium-to-higher polarity analytes on silica, amino, or cyano. But the project purpose should also be considered. What will happen to the method after development? If it will be transferred to a lab with only HPLC, then it would be pointless - and even counterproductive - to develop the method on a UHPLC column. If the method is going to be used long-term, then ideally the original column type should be available for the entire duration. Even with the same column type, batch-to-batch variation in column packing can affect separation. Before fi nalising method conditions, it is a good idea to run the separation on multiple batches to monitor resolution and specifi city changes.


Conclusion


Method development is about more than separating compounds. To develop a method that will achieve the ultimate goals of a team or organisation, a chromatographer should start with a thorough understanding of the method purpose. The information gathered at this step feeds into downstream steps of planning and parameter selection. Starting the development process intelligently makes optimisation more effi cient and validation more likely to be passed. When a chromatographer understands and implements all steps of this process, they are on the way to a complete method.


References


1. A.M. Sabir, M. Moloy, B. Parminder S. Int. Res. J. Pharm. 4 (2013) 39. 2. S. Sule, S. Ambadekar, D. Nikam, A. Sule, S. Bhure. World J. Pharm. Res. 3 (2014) 258.


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