A number of different procedures require the use of HPLC validation methods, as you’ll discover when you begin courses in pharmaceutical school. Some of these procedures include:
- Identification tests
- Testing for impurities (limit tests or quantitative tests)
- Assay procedures
- Dissolution tests
- Particle size determination
Each of these tests uses a specific set of validation characteristics to determine if the substance is suitable for the intended use. During the High Performance Liquid Chromatography (HPLC) course at AAPS, you’ll be introduced to this popular method of compound analysis that’s used in various industries such as the pharma, cannabis, biopharma, food, chemical, environmental, and healthcare industries. During the Advanced Method Validation and Technology Transfer certification module, you’ll explore various best practices towards validation methods. In this blog, we’ll be exploring three parameters: specificity, linearity, and range. Continue reading to learn about these three HPLC critical parameters.
Specificity should be considered when using HPLC validation for identification tests, determining impurities, and assays. Depending on the objective of the analysis, different procedures to determine specificity may be used, and sometimes even combined, to reach the desired outcome.
For example, for impurity tests, specificity is analyzed by comparing closely eluted peaks. Analyst will review the chromatogram and then compare impurity profiles. Various characteristics are analyzed depending on the specific method used, but can include:
- Visual comparison
- Retention times
- Peak shapes
For identification tests, on the other hand, the specificity can be determined by comparing results with the profile of known reference material to identify the similarity or difference to the known material. These standards are set by reference material from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use for the industry.
When you complete pharmaceutical school, you’ll have experience performing advanced HPLC validation methods from a state-of-the-art lab. Method development and method validation will both be vital components of your education, setting you up for success when you enter the workforce.
When considering the validation method of linearity, you’ll want to look for a directly proportional, linear relationship between the test results across a given range. In order to do this, you’ll need a minimum of 5 tested concentrations to plot the results and come to a conclusion according to current standards laid out for quality guidelines. If there is a linear relationship present, then you can move on to a statistical analysis of the results to finalize your results. You can even determine the deviation of any of your collected points from the regression line to evaluate the linearity further.
Building off of your linearity studies, you can begin to determine a suitable range for the content. This could be another useful HPLC validation method to perform after your pharmaceutical certification program. The range is defined as the upper and lower concentrations that have been demonstrated with acceptable precision, accuracy, and linearity using the documented methodology.
Generally, there are suggested or probable limits that can give you an idea of the range you may be considering, which could include a minimum span of 70-120% of the test concentration meeting the criteria stated above. It is important to figure out the baseline probable range for a concentration before completing the tests to ensure a timely, effective test and deter detector signal saturation.
Overall, while it may be hard to validate methods and determine the best approach, you’ll get the hang of each of the parameters and their uses as you progress in your certification modules with AAPS.
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