As regulatory standards for nitrosamines and nitrosamine drug substance-related impurities (NDSRIs) become stricter, the need for precise analytical methods grows. However, the diversity of potential contaminants, active pharmaceutical ingredients (APIs), and dosage formats often necessitates custom method development for each new assay. These methods must adeptly handle various matrices and ensure transferability to QC, all while surpassing regulatory sensitivity benchmarks.
Navigating the complexities of method development in the analysis of nitrosamine impurities is no small feat. Gleaning insights into sample preparation, chromatographic separations, and mass spectrometry can often resemble searching for a needle in a haystack. To demystify this process and offer practical advice, Separation Science turned to two distinguished experts in the field.
Duane Reiber, a Senior Principal Scientist at American Analytical Research Laboratories (AARL), brings a wealth of knowledge in mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) method development. Joining him is Marian Twohig, a Principal Scientist at Waters Corporation renowned for her expertise in LC-MS method development. Together, they shed light on the intricacies of developing robust analytical methods in a rapidly evolving regulatory landscape.
Navigating Nitrosamine Quantification
Reiber, who was employed by an international consumer healthcare company when nitrosamine impurities became an issue of concern, remembers the rapid shift in regulatory expectations and the daunting task it presented to his team.
"Our involvement started around late 2019, when the situation was escalating,” he recalls. “Over the next six to 12 months, we observed the thresholds steadily decreasing, and also discovered these limits would take into account the amount of API in the dosage forms, pushing the required sensitivity even lower. At first, meeting these levels seemed achievable, but it quickly became a challenge.”
According to both experts, it soon became apparent that a holistic approach would be needed to meet the sensitivity requirements of nitrosamine determination, particularly for smaller compounds such as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA).
“The sensitivity levels required, especially for NDMA-like compounds, are incredibly low,” says Twohig. “This presents a significant challenge in ensuring we have the right instrumentation to meet these levels, beyond just the mass spectrometer. It’s about the assay as a whole.”
These sentiments align with Reiber's experience. “When you consider consumer healthcare products, they can have a bit of everything in them,” he notes. “Yet, we’re tasked with extracting these tiny molecules at parts per trillion levels from such a complex mixture. That’s why you really need good chromatography and strong sample preparation techniques.”
Optimizing Method Development
The aim of analysts is to swiftly and effectively isolate nitrosamines from intricate matrices. However, the concept of a 'universal sample preparation' method for nitrosamine analysis remains elusive. “If you think of all the different components that nitrosamines may exist in—creams, tablets, liquids—they require very different approaches for sample preparation,” states Twohig.
"We always started with as minimal sample intervention as possible to speed up the process, given our tight six-week timeframe for developing methods and screening products,” explains Reiber. “Then, we’d look at the chromatography, especially for interferences around the compound of interest. If we had to get it cleaner, we used techniques including liquid-liquid extractions, solid phase extractions—it ran the gamut.”
Chromatography plays a pivotal role in addressing the nitrosamine challenge, with key optimization efforts focusing on this area. Twohig highlights that Waters' strategy includes providing columns with diverse chemistries, aimed at efficiently separating nitrosamines from APIs. This approach is crucial for minimizing potential ion suppression or enhancement effects, commonly referred to as matrix effects, during mass spectrometry analysis.
“The smaller nitrosamines pose a significant challenge, especially when they elute in close proximity to the drug API,” she says. “Even if we're diverting the API to waste, the sensitivity of the mass spectrometer and residual signal from the API can still impact detection sensitivity of the nitrosamines if not properly separated. The key is to start with good sample preparation but chromatography plays a major role.”
Twohig emphasizes the significance of good chromatographic peak shape, noting that poor peak shape often leads to increased manual intervention during data processing. She highlights the necessity of a final sample diluent that minimizes solvent effects, which degrade peak shape. For maintaining good peak shape, especially with polar and hard-to-retain analytes in reversed phase chromatography, she recommends using stationary phases specifically designed for retaining polar compounds.
Reiber and his team relied on a preferred collection of columns for method development, especially for the smaller nitrosamines known for their retention difficulties. He notes that flawless retention isn't a must as long as you can achieve a notable difference in other ways. “For instance, if one column presents interference, switching to another can clear the issue right up.” Introducing internal standards, he points out, can be a game changer when achieving total separation seems out of reach.
He emphasizes the role that ultra-high performance liquid chromatography (UHPLC) can play in boosting the signal of target substances, alongside techniques including solvent exchange.
“We'd usually perform a solid phase extraction followed by drying the sample down and then conducting a liquid-liquid back extraction,” says Reiber. “The smaller nitrosamines, in particular, showed fascinating solvent preferences, allowing us to extract with one solvent and back extract with another.”
Emerging Directions and Technologies
Now that most healthcare companies have established testing routines for their product portfolios, the future strategy seems to pivot towards prevention wherever possible. For instance, if a product contains an excipient known to facilitate nitrosamine production, the straightforward solution is to cease its use, explains Reiber. This approach aligns with the risk-based methodology that has characterized the industry's response to nitrosamine concerns.
Lurking in the wings, however, could be future concerns arising from mutagenic impurities in food, cosmetics, and industrial wastewater. “The general testing load will decrease, but there will always be some level of nitrosamine analysis going forward—there’s a baseline in our environment,” states Reiber.
Both experts concur that collaborative efforts in method development will remain essential. "Method development isn't about tackling issues alone; it's the integration of diverse skills that leads to solutions," Twohig emphasizes, underscoring the power of teamwork in overcoming emerging trace impurity concerns.
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This article is featured in our May 2024 publication, Pharmaceutical Purity and Precision. Find out what’s happening in the world of pharmaceutical impurity analysis and learn about the latest topics and techniques.
