N-nitrosamines are organic compounds bearing a nitroso functional group, and the majority have been identified as mutagenic and carcinogenic in animal studies, making them potentially carcinogenic to humans. Interestingly, roughly 18 percent are considered non-carcinogenic, but the class as a whole demands rigorous control. These compounds can form during manufacturing when amines interact with nitrosating agents such as nitrite, which can originate from contaminated solvents, reagents, raw materials — and, notably, from excipients themselves. Cellulose derivatives, lactose, povidone, and magnesium stearate have all been identified as potential nitrite sources. When nitrite from excipients nitrosates the active pharmaceutical ingredient or its related substances, a distinct subclass forms: N-nitrosamine drug substance-related impurities, or NDSRIs, which now account for approximately 90 percent of reported cases.

The most infamous example remains N-nitroso-varenicline, the first NDSRI ever identified, detected in the smoking cessation drug Chantix (Champix in Europe) at levels exceeding the established safety threshold. The product was recalled and withdrawn from the market in 2021. Crucially, N-nitroso-varenicline is absent from the varenicline raw material — it forms in the finished product through nitrosation at the secondary amine functional group in the presence of nitrite from excipients. This case demonstrated that even a perfectly pure API can become contaminated during storage, fundamentally changing how the industry thinks about drug quality.

In response, the European Medicines Agency launched its “Call for review,” mandating that marketing authorization holders systematically investigate their products through a three-step procedure. Step 1 requires a comprehensive documentary risk evaluation for each API or finished product. Step 2 demands confirmatory testing if any risk is identified. Step 3 calls for risk mitigation measures and a variation to the marketing authorization if N-nitrosamines are confirmed. The Liege team’s case study centers on an oral solid dosage form — uncoated tablets — where the documentary risk analysis flagged excipients as potential nitrite sources, triggering the need for confirmatory testing. Because the API lacks secondary amine functional groups, the investigation focused exclusively on small-molecule N-nitrosamines rather than NDSRIs.

The analytical challenge is formidable. Regulatory sensitivity requirements are determined by the acceptable limit for each N-nitrosamine in a specific product, calculated by dividing the acceptable intake (in nanograms per day) by the maximum daily dose (in milligrams per day). For this product, the maximum daily dose is 16 milligrams per day, meaning some N-nitrosamines must be detectable at parts-per-billion levels. Both the EMA and the FDA establish acceptable intake values using the carcinogenic potency categorization approach, which classifies N-nitrosamines into five categories based on chemical structure. Novartis has even developed an automated tool that assigns the category and corresponding acceptable intake value for NDSRIs based solely on SMILES-encoded structural information.

Building on a previously developed Quantitative Structure Retention Relationship approach, the team proposed and validated three complementary LC-MS/MS methods under the ICH Q2 (R2) guidelines. The methods employ a three-level limit test approach — calibrating at 10, 30, and 100 percent of each N-nitrosamine’s acceptable limit — and together enable detection of up to 17 small-molecule N-nitrosamines within a single-sequence analysis. The 17 target compounds span a wide structural range, from N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to N-nitrosomorpholine (NMOR), N-nitrosodiphenylamine (NDPhA), and N-nitroso-diethanolamine (NDELA). The methods differ only in mobile phase composition or APCI probe temperature: Method 1 operates at pH 2.7 with a 250 °C probe for thermally robust compounds, Method 2 at pH 2.7 with a 150 °C probe for thermolabile analytes, and Method 3 at pH 5.0 with a 150 °C probe to resolve co-elution issues with the API observed at lower pH.

Sample preparation involved crushing uncoated tablets into fine powder, adding stable isotope-labeled internal standards, and extracting with methanol under ultrasonication followed by centrifugation. A second extraction step with 1 percent formic acid solution removed residual water-soluble excipients. All preparations were conducted under light-protected conditions due to the photosensitivity of N-nitroso compounds. Validation across four independent series with two operators demonstrated high selectivity, adequate sensitivity with detection limits below 10 percent of the acceptable limit for all compounds, and acceptable recovery within the 85–115 percent range. One persistent analytical challenge emerged: a consistent carry-over related to NDBA (N-nitrosodi-n-butylamine) was traced to the equipment itself rather than solvents or contamination events, and was managed through a dedicated system suitability test ensuring the carry-over peak never exceeded 10 percent of the NDBA signal at the lowest calibration level.

Confirmatory testing was then applied to 12 batches manufactured between 2021 and 2024 — three newly produced batches and nine retained samples stored under controlled stability conditions of 25 °C and 60 percent relative humidity. The results were striking: most N-nitrosamines were not detected at all throughout the product’s shelf life, supporting the omission of their specifications and eliminating the need for further testing. One impurity, N-nitrosamine 12, was detected in 6 of 12 batches but consistently remained below 10 percent of its acceptable limit, so it too requires no further testing. However, one impurity — N-nitrosamine 6 — told a different story. At release, its content remained below 10 percent of the acceptable limit. By 12 months, it exceeded 10 percent but stayed below 30 percent. By 24 months, it consistently surpassed 30 percent, though it remained strictly below the acceptable limit even at 36 months.

This evolving profile suggests N-nitrosamine 6 originates from API degradation during storage. The product remains compliant with its specification at the end of shelf life, so risk mitigation measures are not required — but quantitative routine analysis remains necessary. The team outlined potential strategies to enable skip testing: using low-nitrite excipients (though this may increase manufacturing costs), lowering the storage temperature from 25 °C to 5 °C to slow degradation kinetics, or redesigning packaging — for example, replacing primary packaging with a nitrocellulose-free lidding foil for blister packs. The case study demonstrates that controlling N-nitrosamines is not a one-time exercise but an ongoing surveillance commitment, particularly for impurities that emerge from degradation over time. As the industry continues to grapple with these genotoxic contaminants, platform methods like those developed at Liege could help centralize analytical efforts and facilitate testing across diverse products — a critical step toward safeguarding public health.

Subject of Research: Pharmaceutical analysis

Article Title: A case study on N-nitrosamine investigation in a marketed drug: from analytical procedure validation to risk mitigation decision

Article References: Zhang, Y., Houari, S., Van Laethem, T., Kumari, P., Ziemons, E., Hubert, P., & Hubert, C. (2026). A case study on N-nitrosamine investigation in a marketed drug: from analytical procedure validation to risk mitigation decision. Frontiers in Analytical Science, 6, Article 1772719. https://doi.org/10.3389/frans.2026.1772719

Image Credits: AI Generated

DOI: 10.3389/frans.2026.1772719

Keywords: N-nitrosamines, LC-MS/MS, pharmaceutical impurities, ICH Q2 (R2), drug safety, confirmatory testing, risk mitigation, Frontiers in Analytical Science, case, N-nitrosamine, investigation, marketed

Researchers at the Intensive Care Unit of Hospital Metropolitano Doutor Célio de Castro retrospectively analyzed 178 adult patients who completed brain death protocols between December 2017 and December 2023. Of these, 141 patients underwent transcranial Doppler ultrasonography, or TCD, as the complementary exam, while 37 were evaluated with electroencephalography or cerebral angiography. The findings, published in the journal Neurocritical Care, show that TCD use was independently associated with a 53.9 percent reduction in the median time needed to complete the brain death protocol, a difference so large that it reshapes the logistics of end-of-life care and organ procurement.

The numbers are striking. In the TCD group, the median protocol duration was nine hours, with an interquartile range of five to twenty-three hours. In the non-TCD group, the median stretched to thirty-five hours, with an interquartile range of twenty to sixty-six hours. After adjusting for age, sex, comorbidity burden measured by the Charlson Comorbidity Index, illness severity measured by the Simplified Acute Physiology Score III, calendar year, and whether the protocol was opened during business hours or after hours, TCD use carried an incidence rate ratio of 0.46, meaning the protocol took less than half as long. The only other independent predictor of protocol duration was whether the process began during business hours, underscoring how staffing availability shapes this delicate timeline.

Transcranial Doppler ultrasonography is a bedside technique that uses low-frequency ultrasound pulses through the temporal bone and other acoustic windows to measure blood flow velocity in the major arteries of the circle of Willis, including the middle cerebral, anterior cerebral, and basilar arteries. The technique was first described in 1982, when researchers demonstrated that flow velocity in the basal cerebral arteries could be recorded noninvasively. In the context of brain death, rising intracranial pressure progressively exceeds the perfusion pressure, producing a characteristic sequence of Doppler waveforms: first a blunting of diastolic flow, then oscillating or reverberating flow in which blood moves back and forth with each heartbeat without net forward perfusion, then tiny systolic contraction spikes, and finally the disappearance of detectable flow altogether. Demonstration of these arrest patterns in both anterior and posterior circulations, on two examinations separated by an interval defined by national guidelines, provides evidence of cerebral circulatory arrest.

The diagnostic credentials of the technique are well established. A meta-analysis published in Intensive Care Medicine in 2006 reported high specificity for confirming brain death, and a subsequent systematic review and meta-analysis in the American Journal of Neuroradiology reached similar conclusions, with reported sensitivity around 88 percent and specificity approaching 98 percent in pooled analyses. Brazilian guidelines issued in 2012 formally endorsed transcranial ultrasound as a confirmatory test for brain death, and the country’s Federal Council of Medicine incorporated it into the regulatory framework through Resolution 2,173 of 2017. Unlike angiography, TCD requires no transport to a radiology suite, no iodinated contrast, and no exposure of potentially donor organs to nephrotoxic agents. Unlike electroencephalography, it does not require prolonged recording sessions or interpretation delays, and it sidesteps the technical confounders of electrical artifacts in the intensive care environment.

The efficiency gains extended beyond the protocol itself. Length of stay in the intensive care unit after protocol initiation was significantly shorter in the TCD group, with a median of one day compared with two days in the non-TCD group, and an adjusted incidence rate ratio of 0.51. In a health system where intensive care beds are chronically scarce and expensive, each day of occupancy by a patient who has already died by neurologic criteria carries an opportunity cost measured in the patients waiting for that bed. The researchers also examined whether the choice of confirmatory exam influenced organ donation, and found that it did not: donation rates were statistically indistinguishable between the groups, at 35 percent in the TCD group and 38 percent in the non-TCD group. Faster protocols, in other words, did not come at the expense of donation outcomes, though they also did not visibly increase them in this cohort.

The study’s statistical approach was deliberately conservative. Because protocol duration and length of stay are skewed, count-like variables, the team used negative binomial regression rather than ordinary linear models, adjusting for the full panel of demographic, clinical, and logistical covariates. The groups were comparable at baseline in age, comorbidity, severity scores, and sex, with all baseline comparisons showing no significant differences. Supplementary correlation analyses reinforced the main findings: TCD use and business-hours protocol opening showed the strongest negative correlations with protocol duration, while illness severity, age, and comorbidity played smaller or negligible roles. Notably, the underlying diagnoses differed between groups, with subarachnoid hemorrhage more frequent among patients who did not receive TCD, at 49 percent versus 26 percent, and ischemic stroke more frequent in the TCD group, at 28 percent versus 11 percent. This pattern likely reflects a practical reality of neurosonology: adequate acoustic windows are harder to obtain in some patients, and certain clinical scenarios push teams toward angiography or electroencephalography instead.

The authors are careful about the limits of their conclusions. This was a single-center, retrospective study at a Brazilian tertiary hospital where confirmatory testing is mandatory, and they caution that generalizability to settings without such requirements is limited. In countries where confirmatory tests are optional, the operational calculus differs, since the protocol can sometimes be completed with repeated clinical examinations alone. The findings also hinge on the availability of trained practitioners; TCD interpretation in the setting of suspected brain death demands specific expertise in recognizing the arrest waveforms and in distinguishing them from technical limitations such as poor insonation windows. The authors argue that their results support broader implementation of TCD in countries with mandatory confirmatory testing, contingent on that trained practitioner availability being secured.

The implications ripple outward to two of the most pressing issues in critical care. The first is organ donation, where every hour of delay increases the risk of hemodynamic instability, cardiac arrest, and loss of viable organs, and where families awaiting a definitive answer endure prolonged uncertainty at the bedside. The second is intensive care capacity, where protocols that conclude in nine hours rather than thirty-five return beds to the queue of critically ill patients faster. A bedside ultrasound exam that costs a fraction of a digital subtraction angiography study and requires no patient transport may thus be one of the highest-leverage tools available in the delicate interval between devastating brain injury and the formal pronouncement of death. As brain death determination frameworks worldwide undergo revision, including the recent consensus guideline from American neurologic and critical care societies, this Brazilian dataset offers a reminder that the operational details of how death is confirmed can matter almost as much as the criteria themselves.

Subject of Research: Use of transcranial Doppler ultrasonography as a confirmatory exam in brain death determination protocols

Article Title: Transcranial Doppler Ultrasonography as a Complementary Exam in Brain Death Determination: Impact on Protocol Duration and ICU Length of Stay—A Single-Center Study in Brazil

Article References: de Freitas, M. R., de Lima, A. F., & Coutinho, C. F. A. (2026). Transcranial Doppler Ultrasonography as a Complementary Exam in Brain Death Determination: Impact on Protocol Duration and ICU Length of Stay—A Single-Center Study in Brazil. Neurocritical Care. https://doi.org/10.1007/s12028-026-02641-w

Image Credits: AI Generated

DOI: 10.1007/s12028-026-02641-w

Keywords: transcranial Doppler, brain death, brain death protocol, organ donation, intensive care unit, cerebral circulatory arrest, neurocritical care, TCD ultrasonography, ICU length of stay, Brazil, confirmatory testing, negative binomial regression