EKG Labs performed a short-term stability study specifically to evaluate several additives in a nutrient solution with surgical applicability. After finishing an extensive literature search to get a sense of compound stability and establish starting points for some of the more challenging additives to analyze, EKG developed a minimal number of methods for optimal separation and evaluation of the compounds. By spiking placebo samples, EKG assessed the recoveries of the methods and determined them suitable. All methods were developed for reverse phase chromatography, but, because the sample matrix was very polar, instrumental parameters were carefully optimized and some unusual HPLC columns were utilized to retain the additives.
Among the additives of interest in this study was N-acetyl cysteine, which was utilized in solution as an antioxidant. EKG Labs observed some interesting interactions with cysteine in solution during the R&D method development phase. The presence of cysteine was evaluated using an ion exchange column and N-acetyl cysteine spikes into placebo across a wide range of concentrations. At very low concentrations, one peak was detected and distinctly identified as cysteine. However, as concentrations of spiked N-acetyl cysteine increased, the peak area at this retention time only increased to a point, after which a second peak began to be observed and to increase proportionally.
Example chromatogram: First peak (9.344) hypothesized to be cysteine, second peak (10.140) hypothesized to be the disulfide, third peak was a different additive of interest to the client. Disulfide cysteine shown.
EKG’s technical team hypothesized that two different chemical processes were taking place: de-acetylation into cysteine followed by oxidation to form disulfide bonds. Because the lab was able to adjust sample preparation in order to target concentrations in an appropriate range to use only the areas of the second peak, no further development was performed at the time.
Stability Studies at EKG Labs
While EKG Labs is not primarily set up for executing the storage portion of long-term stability studies, we do work closely with a local pharmaceutical stability storage facility and are happy to manage such programs for our clients. At each pull time, we coordinate the transfer of samples back to our lab under appropriate conditions and within a satisfactory timeframe in order for us to perform the necessary testing of the stability samples. Alternatively, many clients will routinely send us samples from their own established stability facilities for testing under unique EKG in-house developed and validated methods. Internally, we have non-humidity controlled chambers at both 37°C and 50°C which we utilize for our extractable/leachable programs and for short-term stability studies (generally three months or less).
As demonstrated in this case study, EKG Labs routinely develops methods for the identification, quantification, and continuous monitoring of a wide variety of additives, impurities, and degradation products. We also have significant experience developing methods for low concentration additives in complex matrices.
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. EKG regularly conducts stability evaluations and impurity investigations in challenging matrices, developing and validating methods to monitor chemicals of interest or potential concern.
Additional specialty analytical services include: extractables, leachables, a variety of USP testing, chemical characterization portions of biocompatibility (ISO 10993), and other investigational activities.
If you are experiencing a challenge with development and validation of a difficult method or need analytical services to support your product development, please contact us at 810-354-5229.