{"id":7135,"date":"2026-10-30T14:49:48","date_gmt":"2026-10-30T14:49:48","guid":{"rendered":"https:\/\/www.waters.com\/blog\/?p=7135"},"modified":"2026-10-30T14:49:49","modified_gmt":"2026-10-30T14:49:49","slug":"the-next-frontier-in-nitrosamines-testing-detection-and-risk-assessment-in-packaging-and-iv-infusion-bags","status":"publish","type":"post","link":"https:\/\/www.waters.com\/blog\/the-next-frontier-in-nitrosamines-testing-detection-and-risk-assessment-in-packaging-and-iv-infusion-bags\/","title":{"rendered":"The Next Frontier in Nitrosamines Testing: Detection and Risk Assessment in Packaging and IV Infusion Bags"},"content":{"rendered":"\n<p class=\"wp-block-paragraph\">Nitrosamine analysis continues to push analytical labs to new levels of sensitivity, while the evolving regulatory landscape is expanding the conversation beyond traditional testing. As attention turns to other N-nitroso species (i.e., o-NNOs) and potential extractables and leachables (E&amp;L) from packaging materials, such as IV bags, labs face new challenges in detection, quantification, and method development.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><a href=\"https:\/\/event.on24.com\/wcc\/r\/5452504\/0AF2258FE836520D565CD21531D16E3A?partnerref=Blog\">In a recent webinar<\/a>, nitrosamines expert Jason Brown of Brown Pharma Consulting joined Dr. Marian Twohig, Consulting Scientist at Waters to explore this changing landscape. From emerging nitrosamine considerations and the intersection with E&amp;L to practical LC-MS\/MS strategies for achieving demanding limits for small dialkyl nitrosamines, they examined where the industry has been, the challenges laboratories face today, and what may come next.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">This blog continues the conversation by answering some of the questions that attendees asked during the webinar.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_901a07-a1 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_901a07-a1 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_901a07-a1\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: Can you provide some more details around the use of a delay column and its effect on retention time, pressure, and compatibility with an analytical column?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: <a href=\"https:\/\/www.waters.com\/nextgen\/global\/library\/application-notes\/2026\/the-use-of-a-delay-column-to-mitigate-persistent-interference-peaks-in-the-trace-level-quantitation-of-ndba-in-normal-saline-intravenous-infusion-bags-using-uhplc-ms-ms.html\">The retention properties of the delay column <\/a>should be equal to or greater to those of the analytical column to ensure sufficient delay of the interfering component, thus preventing co\u2011elution with the analyte. The overall retention time difference observed after the delay column is added to the flow path will be proportional to the volume of the packed bed column, the volume of the connecting tubing, and the flowrate of the pump.<sup>1<\/sup> The observed pressure difference will also depend on the added components. In the experimental details described in the webinar, the delay column had a particle size of 5 \u00b5m, with dimensions of 2.1 x 50 mm and an experimental flowrate of 300 \u00b5L\/min, bringing the method pressure to less than 6000 psi. The added pressure was well within the maximum pressure of the analytical UHPLC pump, which is 15,000 psi.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_467c17-b6 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_467c17-b6 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_467c17-b6\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: For extraction of nitrosamines from IV bags, how do you decide the volume of sample to be used for extraction?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: In this iteration of the work, we analyzed the IV bag solutions in their intended for-use conditions, enabling the detection of low ng\/mL detection of the small molecule nitrosamines.<sup>2<\/sup> If the application volumes of the solutions contained in the IV bags are high, a concentration step might be necessary to achieve the sensitivity required to meet regulatory limits. In this case, SPE or liquid-liquid extraction could be used to concentrate the target nitrosamines, thus ensuring the required sensitivity level is achieved by the detector used for the analysis.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">The small molecule nitrosamines vary in their polarity. Therefore, the SPE chemistry used should be suitable to retain and provide good recovery for all the nitrosamines in the testing method.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_454733-f6 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_454733-f6 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_454733-f6\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: Do you have suggestions to improve the retentivity of polar nitrosamines in the analytical method?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: For robust method development of polar compounds using reversed-phase liquid chromatography (RPLC), the target analyte should exhibit a minimum retention factor (k\u2032) of 1.0 to ensure adequate separation from the column void volume. Ideally, a k\u2032 value of 2.0 or greater should be achieved to provide improved retention and method robustness.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">The retention factor, also referred to as the capacity factor (k\u2032), is a measure of the extent to which an analyte is retained on the chromatographic column. It reflects the amount of time the analyte spends interacting with the stationary phase relative to the mobile phase. Higher k\u2032 values indicate stronger interactions with the stationary phase, resulting in increased retention. As k\u2032 describes the elution position of an analyte relative to the void volume, it is a useful parameter for assessing the likelihood of interference from matrix components or other compounds that elute near the void and may impact analyte detection.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Several approaches can be used to increase analyte retention. Selection of a stationary phase with enhanced polar retention characteristics is often beneficial. Examples include columns specifically designed for polar analytes, such as <a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/columns\/xselect-columns.html\">Waters XSelect HSS T3<\/a>, <a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/columns\/atlantis-premier-columns.html\">Atlantis Premier BEH C<sub>18<\/sub> AX<\/a>, and <a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/columns\/cortecs-columns.html\">CORTECS T3 Columns<\/a>. These phases can provide improved retention of polar compounds, including under highly aqueous mobile-phase conditions.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Method development should begin by reducing the proportion of organic solvent in the mobile phase. Where column chemistry permits, operation under 100% aqueous conditions can further promote retention of polar analytes. Adequate column re-equilibration is also critical, with a minimum of 10 column volumes at the initial mobile-phase conditions recommended between injections.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Achieving sufficient retention improves method robustness by ensuring the analyte is well separated from the void volume, thereby reducing the potential impact of early-eluting interferences on analyte sensitivity, accuracy, and reproducibility. If acceptable retention cannot be obtained for the target nitrosamines using RPLC, alternative retention mechanisms should be considered. For example, hydrophilic interaction liquid chromatography (HILIC) may provide improved retention and selectivity for highly polar compounds.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_7ec13b-0d .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_7ec13b-0d .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_7ec13b-0d\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: How did you arrive at the acceptable ion ratios to be used for the final method? Does the software help with monitoring these ratios in routine analysis?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: <a href=\"https:\/\/www.waters.com\/nextgen\/global\/products\/informatics-and-software\/waters_connect-for-quantitation.html\">waters_connect for Quantitation Software<\/a> can be configured to measure and monitor MRM ion ratios. The expected MRM ion ratios can be specified in the processing method or, alternatively, reference standards can be designated in the sample list and used to automatically calculate the expected ion ratios. Rule sets are configured in the software and used to flag when result parameters, including MRM ion ratios, are outside the specified acceptable criteria for a component. In the rule set, the tolerances applied to the measured versus the expected ion ratio are specified. If a sample produces peaks outside the specified tolerances, it will be flagged (on a component-by-component basis) as having an exception. The Ion Ratios Check column can be seen in the data summary table along with the pass\/fail status applied to the detected components.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_02f06e-bd .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_02f06e-bd .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_02f06e-bd\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: Have you had any discussions with regulators around the use of delay columns?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: We have not discussed the use of a delay column with the regulatory agencies. The use of a delay column was included in the method development strategy to enable the removal of an interfering peak eluting at the same retention time and contributing a signal from every MRM transition of NDBA, which would have caused ambiguity in the detection of NDBA in the final assay.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_025f77-f7 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_025f77-f7 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_025f77-f7\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: Is the agency accepting the justifications for higher specification limits for NDBA in high-volume parentals based on toxicological evaluation and lifetime exposure strategy?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: At the time of the recording, no. However, a recent FDA guidance changed that stance. So, Less-Than-Lifetime limit adjustments are now accepted. This must be done on a product-by-product basis and based on treatment duration. To be clear, this guidance applies to all marketed drugs, not just parenterals.<sup>3<\/sup><\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_b63d64-f7 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_b63d64-f7 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_b63d64-f7\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: If my lab already does nitrosamine analysis, can we perform E&amp;L analysis, too? What products and processes are really in scope?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: The specific skills needed to perform non-targeted analysis differ greatly from those needed in targeted nitrosamine analysis. Additionally, while there is some equipment overlap, it\u2019s rarely complete. This even extends to MS needs, where full spectral capabilities are a necessity. The efforts to <a href=\"https:\/\/www.waters.com\/nextgen\/global\/applications\/chemical-and-materials-science\/extractables-and-leachables-analysis-solutions.html\">make a typical MS lab into an E&amp;L-specific lab<\/a> are difficult to overcome without a large budget and substantial investment into the analysts.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Scope is a great question. The new draft Container Closure System Guidance from the FDA expanded this, recently.<sup>4<\/sup> So, all drug substances and drug products are in scope. Additionally, all manufacturing process steps for large molecule and biologics are in scope. Essentially, that only leaves out excipient raw materials and small molecule production. I would look and hope for that to change, as it relies on some assumptions I would not agree with, but I would not expect to see that change soon. Keep in mind that even items &#8220;in scope&#8221; must go through risk identification and risk assessment steps. Both steps remove components from the workflow, leading to laboratory analysis.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_143a13-fb .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_143a13-fb .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_143a13-fb\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: What tests should\/can\/must we request from our packaging material suppliers for nitrosamine risk assessments for tablets, syrups, suppositories, and injectables?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: Right now, the risk of leachable nitrosamines from packaging is believed to be entirely related to nitrocellulose use in labels directly affixed to the primary packaging and, again, is believed to be contained to blisters and infusion bags. Some have suggested including questions regarding potential nitrosamines in technical requests to packaging and labelling suppliers. As part of a risk-based approach, this is easily justified.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_3bd35b-2b .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_3bd35b-2b .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_3bd35b-2b\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: What are the risks associated with nitrosamines formation inside the body? Is there any guidance on this?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: This is an interesting and complex topic. Certainly, there is a real, understood possibility of nitrosamine formation in the GI tract. There have been a few studies that have actively looked into this. To my knowledge, there have not been any exhaustive studies using modern LC-MS technologies. While the GI tract certainly has all the factors necessary for nitrosation: temperature, pH, and nitrite quantities, and there are also primary amines, which will react with the nitrite preferentially. I led a proof-of-concept study a few years ago that showed large-scale nitrosamine formation in multiple biorelevant dissolution media. However, it is impossible to fully replicate the in vivo complexities. It should also be noted that many vulnerable amines are present in many common foods.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">To the point, much more is needed to fully understand patient risk. Contributions from gastric formation, including food, is relevant.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_6d99ee-f2 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_6d99ee-f2 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_6d99ee-f2\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Q: Recently published papers have shown the impact of NOx on nitrosamine formation at small, analytical-scale samples. Does this impact the interpretation of data for large-scale bulk material?<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">A: Rok Gharek and team authored a couple of great papers on this subject, and I had the opportunity to discuss this directly with him and one of his coauthors.<sup>5<\/sup> Part of the study explored how NOx saturated various levels of a tube filled with an API or excipient to show that it was not just surface exposure. Their conclusion was that while NOx can penetrate deep into various levels of a bulk container, that the available NOx was consumed by the outermost layers of the bulk.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Honestly, a good resampling of the bulk material, using a sample thief to take from the core, is the only way forward. Some work should be considered in the lab to determine which materials are even at risk, as the papers referenced showed that some materials are at much higher risk than others.<\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_67be95-c1 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_67be95-c1 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_67be95-c1\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"text-decoration: underline;\"><strong><a href=\"https:\/\/event.on24.com\/wcc\/r\/5452504\/0AF2258FE836520D565CD21531D16E3A?partnerref=Blog\">Watch the full webinar<\/a><\/strong> <\/span>to get more details about the changing analytical landscape for nitrosamines testing.<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\"><strong><em>Special thank you to Jason Brown of Brown Pharma Consulting for his presentation in the webinar and for his assistance answering these questions.<\/em><\/strong><\/p>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_421906-a3 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_421906-a3 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_421906-a3\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">References<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li><a href=\"https:\/\/www.waters.com\/nextgen\/global\/library\/application-notes\/2026\/the-use-of-a-delay-column-to-mitigate-persistent-interference-peaks-in-the-trace-level-quantitation-of-ndba-in-normal-saline-intravenous-infusion-bags-using-uhplc-ms-ms.html\">The Use of a Delay Column to Mitigate Persistent Interference Peaks in the Trace Level Quantitation of NDBA in Normal Saline Intravenous Infusion Bags Using UHPLC-MS\/MS<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/www.waters.com\/nextgen\/global\/library\/application-notes\/2026\/identification-and-quantitation-of-n-nitrosamines-in-normal-saline-intravenous-infusion-bags-using-uhplc-ms-ms.html\">Identification and Quantitation of N-Nitrosamines in Normal Saline Intravenous Infusion bags Using UHPLC-MS\/MS<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/www.fda.gov\/media\/195007\/download?attachment\">Application of Less-Than-Lifetime Adjustment to Nitrosamine Impurities<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/www.fda.gov\/regulatory-information\/search-fda-guidance-documents\/container-closure-systems-human-drugs-and-biological-products\">Container Closure Systems for Human Drugs and Biological Products<\/a><\/li>\n\n\n\n<li>Grahek, R., et al. (2026). &#8220;Excipient nitrite levels are not static material attributes: Nitrite generation from environmental NOx and implications for drug product manufacturing.&#8221; <em>Journal of Pharmaceutical Sciences<\/em> 115(11).<\/li>\n<\/ol>\n\n\n<style>.wp-block-kadence-spacer.kt-block-spacer-7135_a10426-04 .kt-block-spacer{height:60px;}.wp-block-kadence-spacer.kt-block-spacer-7135_a10426-04 .kt-divider{border-top-width:1px;height:1px;border-top-color:#eee;width:80%;border-top-style:solid;}<\/style>\n<div class=\"wp-block-kadence-spacer aligncenter kt-block-spacer-7135_a10426-04\"><div class=\"kt-block-spacer kt-block-spacer-halign-center\"><hr class=\"kt-divider\"\/><\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><em>Views expressed here are of the primary and contributing authors alone, and do not represent the views of Waters Corporation. Please perform your own due diligence while making any decisions based on regulatory guidelines.<\/em><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Nitrosamine analysis continues to push analytical labs to new levels of sensitivity, while the evolving regulatory landscape is expanding the conversation beyond traditional testing. 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