Trends and Challenges in Nitrosamine Testing: Part Three – Building a Method

If starting from scratch, how would you go about building a method for nitrosamine analysis?
 

Alan Thompson: When starting from scratch, we would tend to exercise a methodical process as outlined below. This instruction mainly pertains to samples analyzed by LC-MS. For more volatile samples, a GC-MS may be deemed more appropriate, but a similar approach would be adopted:

1. Identify a suitable solvent for the API and impurity. A higher API concentration will improve the eventual sensitivity of the method. 
2. Prepare the nitrosamine impurity in the appropriate solvent and infuse directly into the mass spectrometer. Identify the most suitable compound fragment and optimize source parameters, fragmentation energies and multiple reaction monitoring (MRM) parameters to increase the sensitivity and selectivity of the impurity.
3. Prepare an API sample and spike in the nitrosamine impurity at the required quantitation limit. Create an LC-MS/MS method using generic LC parameters compatible with mass spectrometry (e.g. Mobile Phase A: 0.1% Formic Acid in Water, Mobile Phase B: 0.1% Formic Acid in Acetonitrile, C18 UHPLC column) and the MS parameters identified in step 2. Use UV detection to monitor the retention time of the API. Optimize the LC parameters and sample preparation, and ensure adequate resolution between the nitrosamine impurity and the API and sensitivity of the method. Different MS sources (APCI, ESI) can also be trailed at this stage, whilst method segments should be introduced to ensure that only the nitrosamine impurity enters the MS. Feasibility tests can also be run with a view to moving to method validation.
4. If the compound under investigation is in a formulated drug product, the next step is to explore and optimize sample extraction, and to monitor the impact of excipients on sample recovery. If sample recovery cannot be achieved, it may be necessary to introduce internal standards at this point. 

Jingyue Yang: I’d start by firstly determining the method’s analytical target profile; i.e., figuring out the limit of quantitation (LOQ) based on the recommended acceptable intake limit of NDSRIs and the maximum daily dosage of the drug product – to ensure I understood the intended use of the method. Next, I’d focus on achieving LC separation of NDSRIs from the API or other major components, with reverse-phase chromatography and common mobile phases as my initial choices for separation.

Meanwhile, I would select an appropriate ionization mode and evaluate the fragmentation pattern of the NDSRI to optimize the mass spectrometric conditions for sensitive detection, including the ionization settings and scan mode. I’d then assess the lowest concentration of the NDSRI standard that can be detected. Comparing this value to the target LOQ helps to determine whether further optimization for detectability is necessary.

Next, I’d work on refining and optimizing sample preparation, which involves determining the “apparent” API concentration, selecting an appropriate extraction solvent and developing procedures to ensure efficient extraction. Finally, I would start evaluating the method for validation characteristics.

Naiffer Romero: Building a robust analytical method for the detection and quantification of nitrosamines from the ground up typically begins by identifying the specific nitrosamines of concern and defining clear performance criteria (e.g., detection limits, accuracy, specificity). Next, comes selection of an appropriate analytical platform (often gas or liquid chromatography coupled with mass spectrometry) based on the target analytes’ physical and chemical characteristics. The development phase includes preparing or sourcing well-characterized reference materials, establishing sample preparation and extraction protocols, and systematically optimizing instrument parameters to ensure sensitivity and selectivity. Rigorous validation – covering specificity, linearity, precision, accuracy, and robustness – then confirms the method’s reliability before routine use.

In this process, the United States Pharmacopeia (USP) plays a pivotal support role. USP has allocated resources to developing new nitrosamine impurity reference materials, ensuring that laboratories worldwide have access to high-quality standards for testing and method validation. USP also hosts an analytical method repository as part of the Nitrosamines Exchange knowledge community, fostering collaboration and knowledge sharing among professionals tackling nitrosamine challenges. These tools and resources from USP help streamline the entire method development and validation process, ultimately promoting more robust and accurate nitrosamine detection across the industry.

Are current technologies adequate?
 

Alan Thompson: Current technologies have demonstrated that they can adequately quantify commonly known nitrosamines to existing proposed limits, and thus far we’ve been able to develop methods for all samples which have come our way. However, that is not to say that there are compounds or drug products which won’t present challenges. Every formulated product requires a unique approach depending on varying factors including solubility, resolution and compatibility of any existing methods with mass spectrometry.

Kevin Parker: While top-end mass spectrometers are often capable of reaching the low levels of detection required, methods also exist in which preconcentration of the analyte is possible. There have been quite a few publications showing the utility of solid phase extraction (SPE) in concentrating n-nitrosamines to enable detection of very dilute samples. This is particularly useful in cases when liquid formulations are used and the concentration of analytes is already low in the drug product before being diluted into an HPLC/MS friendly diluent. Solid-phase extraction techniques can also be useful in cases where drug product matrices cause ion suppression or interfere with analysis, whereby their removal can quickly boost detection limits.

Continuing to push the limit of detection is always going to be key in the development of new technology.

Look out for the fourth installment, where we’ll reveal insights on the current regulatory landscape for the pharma industry and advice for adhering to guidelines

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