Introduction: Co-eluting impurities are two or more chemical entities that elute, or come out, at similar or equivalent retention times when analyzed by chromatographic techniques such as HPLC and GC. Co-eluting impurities can pose several challenges to product development. For example, co-eluting impurities can cause an “exaggerated” response. This is because two or more impurities can create a blended peak and can be mistaken for a single impurity. Ultimately, this can cause confusion and increased difficulty when trying to identify the peak.
Furthermore, many regulated industries require identifying, monitoring, and reporting of impurities over a certain threshold in final products. A co-eluting peak may exceed the regulatory threshold and may trigger a mandatory, in-depth investigation and monitoring. However, modification of analytical methods may separate the impurities, demonstrating that in-depth monitoring is not required for the individual peaks. There are many ways to separate co-eluting impurities, including the following case study performed at EKG Labs.
Case Study: A client approached EKG Labs with their active pharmaceutical ingredient (API) that had co-eluting impurities. This was a concern because the co-eluting peaks were difficult to quantify and appeared to be reaching ICH reporting thresholds, thus forcing additional investigations and monitoring.
EKG Labs began by duplicating the client’s current method in-house to ensure the method was transferrable. The following chart demonstrates the client’s reproduced method and multiple co-eluting peaks.
The goal was to separate the group of peaks surrounding the API detected at approximately 20 minutes. EKG Labs then modified the column technology:
EKG Labs then optimized the mobile phase and gradient parameters:
Conclusion: In order to achieve goals set by the client (separating the small unknown impurity peak on the shoulder of the API), as well as to better separate co-eluting impurities in the same region, an exhaustive evaluation and screening of various parameters was performed. Parameters such as column technology, stationary phase pore size, mobile phase pH, and multiple mobile phase gradients were tested. Ultimately, the impurity peak which originally was co-eluting with the API was resolved with a resolution to greater than 3. This was achieved by controlling the tailing factor of the API peak with the new column technology and by developing a mixed gradient/isocratic method.
Additional Analytical Support for Impurities: In addition to separating co-eluting impurities, EKG Labs can help clients overcome other impurity-related challenges. We also provide chemical characterization, impurity identification and failure analysis for a variety of industries such as combination products, medical devices, pharmaceuticals and consumer products. If you are experiencing a challenge with impurities or need analytical services to support your product development, please contact us at 810-354-5229.
About EKG Labs
EKG Labs is an analytical service provider for the medical device and pharmaceutical industries. Our analytical services support regulatory filings, product development, and analytical chemistry investigations. Specialty analytical services include: extractables, leachables, biocompatibility, characterization, impurity identification, method development, method validation, deformulation, and other investigational activities.