Chemical impurity analysis is critical in medical device and pharmaceutical development because impurities can impact the safety and efficacy of final therapeutic products. Examples of chemical impurities include: starting raw materials, by-products, intermediates, degradants, reagents, ligands, catalysts, heavy metals, residual metals, or any other materials or process aids in the production process.
Regulations for pharmaceutical impurities include USP general chapter <1086> “Impurities in Drug Substances and Drug Products” and USP <476> “Organic Impurities in Drug Substances and Drug Products”. You can learn more about these chapters here. USP <232> and <233> provide guidelines for elemental impurities.
A Hydrocortisone Case Study:
EKG was approached by a client to assess their reference standard for Hydrocortisone Impurity G (the hydrocortisone aldehyde) which, after some time, was displaying a number of unidentified peaks on their assay method. The client wanted to know what these peaks were and wanted a method developed to observe them in hydrocortisone cream. The method would then need to be validated for ongoing stability analysis, should they end up being compounds of interest to the client.
HPLC DAD chromatogram of client-provided Hydrocortisone Impurity G, showing three primary peaks of interest.
Our first goal was to elucidate the structure of the three most significant peaks observed in the chromatograms of the reference standard. We began by mimicking the client’s method on our UHPLC, coupled with a Time-of-Flight mass spectrometer (to learn about how neat this instrument is and the many applications it has for our clients, read more here). Using the high-resolution mass spectra generated, we determined the structure of the three peaks. Molecular masses were assigned based on identification of the M+H+ and 2M+H+ ions in positive mode as shown below.
We found the largest peak to be the hydrate of impurity G. The other two peaks corresponded to molecular weights associated with the replacement of an -OH group with methanol, forming a methoxy compound. As this exchange forms a stereo-center (denoted with a * below), there are two UPLC observable (diastereomeric) peaks associated with this structure.
The compounds of interest, with their molecular weights.
Our second goal was to develop a sample preparation technique to test hydrocortisone creams. After extensive exploration of extraction methods, we landed on a specific methanol:water:acetonitrile solvent ratio. The extraction involved vortexing, water baths for temperature maintenance, and filtration techniques.
By spiking cream samples and analyzing a number of replicates, we ensured the instrumental method was sensitive enough to detect even minute amounts of these impurities and specific enough to distinguish these peaks from the sample matrix. Using extracted ion chromatograms to look specifically for the established m/z values, now that we knew which exact masses to target, we could confirm which impurities we were observing.
Finally, now that we had a method for the detection of compounds related to Hydrocortisone Impurity G, we moved into validation of the method for ongoing stability analysis, as needed.
EICs (extracted ion chromatograms) confirm observation of the impurities in the cream samples. The double peak in the first chromatogram is due to the presence of two diastereomer peaks.
Impurities Testing at EKG Labs
EKG Labs provides impurity testing services to the pharmaceutical and medical device industries. We can perform impurity identification in accordance with FDA and ICH guidelines for impurities. Using our state-of-the-art instrumentation, EKG Labs has the experience to isolate, analyze, and identify impurities. We have experience demonstrating precise recovery even in most challenging matrices and can extract your unique sample as necessary. EKG Labs can also develop and validate a risk-based, product-specific method in order to monitor one or more targeted compounds. At EKG Labs we work with you to provide a customized solution to your impurity identification and analysis needs.
About EKG Labs
EKG Labs is an analytical service provider for the pharmaceutical and medical device industries. Our wide variety of analytical services supports product development, regulatory filings, and analytical chemistry investigations. Specialty analytical services include: extractables, leachables, chemical characterization, impurity identification, method development, method validation, and other investigational activities.