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 The product was recalled and withdrawn from the market in 2021.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

The triple combination is becoming increasingly common in the management of type 2 diabetes, and for good reason. Each drug attacks high blood sugar through a different biological route: metformin suppresses glucose production in the liver and improves insulin sensitivity, gliclazide prods the pancreas to release more insulin, and dapagliflozin forces the kidneys to flush excess glucose out through the urine. Combining them in a single pill improves patient compliance and delivers better glycaemic control than any one agent alone. But that therapeutic synergy creates an analytical headache, because a quality-control laboratory must be able to verify the exact amount of each ingredient in every batch before the tablets reach patients.

The difficulty lies in chemistry. Metformin is a small, strongly polar, highly hydrophilic molecule with a log P value of roughly minus 2.64, which means it barely interacts with the oily, hydrophobic surface of a reverse-phase column and tends to zip through unretained. Dapagliflozin and gliclazide, by contrast, are far more lipophilic, with log P values around 2 or higher, so they cling to the stationary phase much longer. On top of that, the dose disparity is enormous: commercial tablets contain 500 to 1000 milligrams of metformin, 40 to 80 milligrams of gliclazide, and a mere 5 to 10 milligrams of dapagliflozin, so any single method must accurately quantify compounds whose concentrations in solution differ by orders of magnitude.

Led by Rajendra Patel, with Nishith Teraiya and Anjali Prajapati, all of the K.B. Institute of Pharmaceutical Education and Research in Gandhinagar, the team systematically tuned every variable that governs chromatographic behavior. They tested water-methanol and water-acetonitrile mixtures in various ratios, but unbuffered systems produced broad, asymmetrical peaks with poor resolution. The breakthrough came from adding a volatile 20 millimolar ammonium acetate buffer adjusted to pH 3.0 with ortho-phosphoric acid. At that acidity, metformin remains fully protonated and water-loving, so it elutes first, while the more lipophilic dapagliflozin and gliclazide linger progressively longer on the hydrophobic C18 stationary phase, producing clean, well-resolved peaks.

The final recipe is elegantly simple. Separation occurs on a Kromasil C18 column, 250 millimeters long with a 4.6 millimeter internal diameter and 5 micrometer particles, using a mobile phase of the acetate buffer and acetonitrile mixed 35:65 by volume, pumped at a constant 0.8 milliliters per minute in isocratic mode. A UV detector set at 224 nanometers, a wavelength where the absorption spectra of all three drugs overlap acceptably, monitors the eluate. Under these conditions metformin appears at 2.47 minutes, dapagliflozin at 3.90 minutes, and gliclazide at 6.46 minutes. Because the solvent composition never changes during the run, the method avoids the complexity, variability, and extra solvent consumption of gradient elution, keeping both analysis time and operating costs low.

Validation followed the International Council for Harmonisation’s Q2 (R2) guidelines, the global benchmark for analytical procedures. Calibration curves were linear across 125 to 375 micrograms per milliliter for metformin, 2.5 to 7.5 for dapagliflozin, and 15 to 45 for gliclazide, with correlation coefficients all above 0.997. Recovery experiments, in which known amounts of standard drug were spiked into tablet samples at 80, 100, and 120 percent of target, yielded recoveries between 98 and 102 percent for every analyte, demonstrating negligible interference from tablet excipients. Precision testing, repeated within a single day and across three separate days, produced relative standard deviations consistently below 2 percent, the accepted threshold for repeatability in pharmaceutical analysis.

The robustness trials are particularly reassuring for routine laboratories. The researchers deliberately nudged the method’s settings, slightly altering mobile phase composition, flow rate, detection wavelength, buffer pH, and column temperature, and found that the separation held up every time, with peaks remaining symmetrical, well resolved, and statistically unchanged. Sample solutions stored for 48 hours at room temperature and at refrigerator temperature showed no degradation and no loss of assay value, meaning laboratories can prepare samples in advance without compromising results. Limits of detection and quantification, calculated from the regression statistics of the calibration lines, confirmed the method is sensitive enough for the low-dose dapagliflozin component.

The most dramatic evidence for the method’s stability-indicating power came from forced degradation studies, which regulators require to prove that an analytical method can spot decomposition before it spots nothing at all. The team exposed the drugs to 0.1 normal hydrochloric acid for one hour, 0.1 normal sodium hydroxide for two hours, 3 percent hydrogen peroxide for one hour, 40 degrees Celsius heat for one hour, and direct sunlight for two hours. Each stressor told a chemical story: gliclazide degraded most under acid, consistent with hydrolysis of its sulfonylurea linkage; dapagliflozin proved most vulnerable to light, its aromatic glucoside structure susceptible to photo-oxidation; and metformin, shielded by its highly polar biguanide scaffold, stayed comparatively stable even under heat. Crucially, in every case the degradation products eluted as separate peaks that never overlapped the three drug peaks.

Applied to a real commercial tablet containing 10 milligrams of dapagliflozin, 60 milligrams of gliclazide, and 500 milligrams of metformin, the method returned assay values of 101.52, 98.19, and 99.64 percent respectively, comfortably within pharmacopoeial expectations and confirming that the technique works outside the pristine world of pure standards. That real-world performance matters because regulators and manufacturers increasingly rely on such methods for batch release, shelf-life determination, and post-market surveillance. A method that is simultaneously accurate, robust, and stability-indicating gives quality-control chemists a single, streamlined workflow where they previously might have needed multiple runs or separate methods for each drug pair.

The broader significance extends past one formulation. As fixed-dose combinations multiply across modern pharmacotherapy, the analytical community faces growing pressure to verify ever more complex mixtures with fewer resources. This study demonstrates that careful exploitation of fundamental properties, lipophilicity, ionization state, and spectral overlap, can tame even a three-drug combination spanning a hundredfold dose range in a simple isocratic run. For the millions of patients taking triple antidiabetic therapy, the work represents an invisible but vital layer of assurance: every tablet’s contents can now be confirmed, and its chemical deterioration detected, in under seven minutes of chromatography.

Subject of Research: Development and validation of a stability-indicating RP-HPLC method for simultaneous quantification of dapagliflozin, gliclazide, and metformin in antidiabetic combination tablets

Article Title: Development of a robust stability-indicating RP-HPLC method for simultaneous estimation of triple antidiabetic combination containing dapagliflozin, gliclazide and metformin

Article References: Patel, R., Teraiya, N., & Prajapati, A. (2026). Development of a robust stability-indicating RP-HPLC method for simultaneous estimation of triple antidiabetic combination containing dapagliflozin, gliclazide and metformin. Discover Chemistry, 3(1), Article 518. https://doi.org/10.1007/s44371-026-00978-x

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00978-x

Keywords: RP-HPLC, dapagliflozin, gliclazide, metformin, type 2 diabetes, stability-indicating method, forced degradation, pharmaceutical analysis, ICH Q2 (R2), chromatography, quality control, fixed-dose combination