ABSTRACT
For many years, it is known as Nitrosamines are responsible for carcinogenicity in human. These nitroso‐impurities were found detected in plastics, food items, cigarette, insect killer, water and alcoholic beverages. In 2018 EMA and USFDA claimed the presence of N‐nitrosodimethylamine in valsartan used for the treatment of hypertension. Consequently, to assess the adverse effects of nitrosamine generation during storage, in the course of production, or through contaminated supply chains, regulators have issued many guidelines. Hence, there is a crucial need for accurate evaluation technique to measure nitroso‐impurities in pharmaceutical products.
According to the Carcinogenic Potency Categorization Approach, the allowable intake is 400 ng/day, which translates to an allowable limit of 16 ppm for a 25 mg daily dose. A study was then performed at 1.5 ppm relative to sample concentration, which is less than 10% of this limit. The main aim of this development to optimize and prove suitability of a new liquid chromatography–tandem mass spectrometry method for quantitatively analyzing 3‐amino‐N‐nitrosopiperidine (NTPA) in Alogliptin Benzoate (AGP), achieving detectable response of 0.03 ppm and quantifiable response value of 0.1 ppm relative to sample concentration. Current method was found to be linear with the coefficient of regression of 0.9979. Also, spiking study was performed by the calculating percentage spiked NTPA in the drug, which ranged from 96.67% to 100.14%. The results confirmed that the methodology was reliable, precise, robust and reproducible to quantify NTPA at 1.5 ppm in samples of 2.75 mg/mL concentration.
Keywords: 3‐amino‐N‐nitrosopiperidine (NTPA), Alogliptin benzoate (AGP), liquid chromatography tandem mass spectrometry, nitrosamine, validation
1. Introduction
AGP is an orally administered medication used to treat type‐2 diabetes by improving glycemic control. It belongs to the class of Dipeptidyl Peptidase‐4 inhibitor and has trade name Nesina in the US and Vipidia in Europe (Alkather et al. 2021). It has comparatively low glucose‐lowering activity, lower chances of increase in body weight and hypoglycemia observed is also rare, just like other gliptin class members. In case metformin fails to achieve adequate control of diabetes in patients, it is often combined with AGP or other gliptins to enhance therapeutic efficacy (Feng et al. 2007). With over a million prescriptions, AGP was ranked to the 295th most commonly prescribed medication in 2020, indicating that despite the FDA's warning, many patients and their doctors believed the drug's advantages outweighed its drawbacks (Scott 2010).
The current literature and regulatory documents might not fully account for every impurity ‐including organic, inorganic, potential‐genotoxic, and mutagenic ones that could be present in the drug substance of AGP. Inadequate impurity control can endanger human health by compromising the drug's quality, safety, and efficacy. As a result, it is essential to rigorously examine each synthetic step and every reagent to find possible impurities, and to set clear quality standards for all raw materials and solvents.
NTPA can potentially be generated as an impurity through a reaction between 3‐amino piperidine and N‐substituted amines used during AGP synthesis. Related structure of AGP and its possible nitrosamine impurity NTPA generation from starting material are shown in Figure 1.
FIGURE 1.
Structure of Alogliptin and Generation of 3‐amino‐N‐nitrosopiperidine.
Therefore, occurrence of nitrosamine impurity like NTPA in AGP may adversely affect human health. In addition to being corrosive and irritating to the skin and eyes, it also act as carcinogen (Chaudhary et al. 2024).
About, 150 years ago, in 1870 Nitrosamines were first studied by Otto Witt (Witt 1878). However, in 1956 Magee and Barnes had described probable carcinogenicity of N‐nitrosodimethylamine (Magee and Barnes 1956). This sparked a growing interest in development of analytical method to check carcinogenic impurities in various products. Various human body parts such as nasal mucosa, tongue, oesophagus, lung, liver, bladder, stomach, and pancreas are known to develop tumors due to exposure to Nitrosamines (Daripelli et al. 2025). In the pharmaceutical industry, nitrosamine impurities were first detected in valsartan drug products in 2018 (Manchuri et al. 2024). Thereafter, in July 2018 the US‐FDA and the European Medicines Agency, announced that a new class of carcinogenic impurities NDMA and NDEA are present in generic Active Pharmaceutical Ingredients and products of Angiotensin Receptor Blockers (Tuesuwan and Vongsutilers 2021). Regulatory bodies around the world announced a number of recalls of these drugs to mitigate the risk associated with the nitrosamine impurities. Subsequently, many other drug products such as metformin hydrochloride and ranitidine were also reported to contain trace levels of nitrosamine impurities (U.S. Food and Drug Administration 2021; U.S. Food and Drug Administration 2023a; EMA 2019). In order to limit the probable carcinogenic risk, and control nitrosamine impurities in Active Pharmaceutical Ingredients, regulatory agencies have published acceptable intakes for the nitrosamine impurities (U.S. Food and Drug Administration 2023b; Therapeutic Goods Administration 2025). The acceptable intake threshold for nitrosamines was established by linear extrapolation method based on the tumorigenic dosage that results in TD₅₀ above baseline levels obtained while performing trials in animals. These values correspond to an estimated lifetime cancer risk of 1 in 100,000. The resulting acceptable intake for N‐nitrosodimethylamine is 96 ng/day, 26.5 ng/day for N‐nitrosodimethylamine and 127 ng/day N‐nitrosomorpholine (EMA 2025; EMA 2023). Similarly, regulatory bodies such as EMA, TGA and Health Controlling department of Canada had proposed allowable intake for Nitroso impurity NTPA as 400 ng/day for AGP as per CPCA potency category 3 as shown in Table 2. Based on this demand the study was designed to develop and validate LCMS/MS method to determine nitrosamine impurity in AGP (Health Canada 2025; ICH 2006; ICH 2023).
TABLE 2.
Potency Score Breakdown.
| Alpha Hydrogen Potency Score | |
|---|---|
| Count of α‐hydrogen atoms present on each carbon | α‐Hydrogen Score |
| 2,2 | 1 |
| Alpha‐hydrogen Score (α‐HS) | 1 |
| Deactivating Feature Score | |
| Deactivating Feature | Individual Deactivating Feature Score |
| 6‐membered ring having N‐Nitroso group | 2 |
| Total deactivating (DFS) | 2.00 |
| Total activating (AFS) | 0.00 |
| Final Potency Score | |
|
Potency Score (PS = α‐HS + DFS + AFS) |
Potency Category |
| 1 + 2 + 0 | 3 |
| Total CPCA Score (PS) | 3 |
There have been few reports on validation and analysis of potential genotoxic impurities of AGP (Zhang et al. 2015; Gharat et al. 2025; Patel et al. 2025; Gharat et al. 2026). Additionally, there are some literature reports highlighting the use of well‐known hyphenated approaches to identify genotoxic impurities in pharmaceutical products (Birla et al. 2024). AGP's enantiomeric purity has been validated and determined using a chiral HPLC (Rao et al. 2014). In 2016, LC‐QTOF mass spectrometric techniques were also established for the isolation and characterization of related compounds in AGP (Lu et al. 2016). However, some literature shows synthesis of 3‐Aminopiperidine and its derivatives (Nienburg 1937). According to certain research, 3‐Amino‐piperidine is isolated as degradation product in gliptins (Gumieniczek and Berecka‐Rycerz 2023; Zhang et al. 2016). These analytical techniques deficient to determine NTPA content. According to our literature survey, no other study has been conducted to analyze NTPA genotoxic impurity in AGP. In order to confirm product efficacy and patient's care, the analysis such impurities in drugs or APIs is essential. Therefore, to comply with the regulatory requirements, a sensitive and reliable analytical method is required for the identification and measurement of NTPA in AGP. Accordingly, the present study aims to check the presence of NTPA in AGP samples using Liquid chromatography with Mass Spectrophotometer. The method developed and validated was found to be specific, robust, precise and linear. Hence, it is intended for use.
2. Reagents and Methodology
2.1. Standards, Reagents and Drug Samples
Materials used included AGP bulk drug sample and NTPA impurity, both provided by Indoco Remedies Limited, Navi Mumbai, Maharashtra, India. Methanol from JT Baker and Ammonium acetate from Merck Chemicals were also utilized.
2.2. Instrumentation
A Sartorius analytical balance was used for all standard and sample weighing. LCMS/MS studies were conducted on a Waters Xevo TQMS with a Waters Acquity UPLC, USA. The instrument, which featured a triple quadrupole mass analyzer and an electrospray ionization (ESI) source with positive mode, was operated using MassLynx software. Specific instrument settings included a gas temperature of 150 °C, a cone gas flow of 150 L/Hr, a nebulizer pressure of 7 bar, a cone voltage of 20 V, and a capillary voltage of 1 kV. Instrument setting includes (A) Chromatographic conditions, (B) Gradient time‐composition programme, (C) Method events and (D) Method conditions in Table 1.
TABLE 1.
Instrument setting.
| (A) Chromatographic conditions | |
|---|---|
| Parameter | Condition |
| Phase Solution A | Ammonium containing acetate buffer with pH having 9.5 |
| Phase Solution B | Methanol |
| Ratio of the mobile phase | Binary gradient |
| Blank | Diluent |
| Diluent | Water: Methanol (80:20) |
| Column Temperature | 40 °C |
| Column | YMC, S‐5um,12 mm (4.6 mm X 150 mm) |
| Analysis Injection requirement | 10.0 μL |
| Auto sampler Compartment cooling | 15 °C |
| Flow level | 1.0 mL per minute |
| Run Time | 20 min |
| (B) Gradient time‐composition programme | |
|---|---|
| Event Series | Event Condition |
| Event 1 | 0.00 min Flow state LC |
| Event 2 | 11.5 min Flow state Waste |
| (C) Method events | ||
|---|---|---|
| Time (minutes) | Phase Solution A (Percentage) | Phase Solution B (Percentage) |
| 0 | 100 | 00 |
| 3 | 100 | 00 |
| 5 | 90 | 10 |
| 7 | 70 | 30 |
| 9 | 50 | 50 |
| 11 | 20 | 80 |
| 13 | 10 | 90 |
| 17 | 100 | 0 |
| 20 | 100 | 0 |
| (D) Method conditions | |||
|---|---|---|---|
| Tool | Unit applied | Tool | Unit applied |
| Ion source | ESI positive | Desolving Gas level | 1000 Litre per hour |
| Source heating | 150° | Desolvation temperature | About 600° |
| Source offset | 5 V | Polarity | Positive |
| Capillary setting | 1 KiloVolt | Collision flow | 0.130 mL |
| Cone setting | 22 Volt | Nebuliser flow | 7 Bar |
| Cone gas level | 300 Litre per hour | Collision power | 6 Electron Volt |
2.3. Analytical Solutions
A Stock solution for the NTPA was prepared at 275 μg/mL and diluted appropriately to get working standard solution of (0.0055 μg/mL) in diluent (Water/Methanol:80/20). Further, 1 mL of Stock solution was added to get Stock solution A at 2.75 μg/mL. Aliquots of Standard Stock Solution A (0.07, 0.50, 1.00, and 1.50 mL) were added to obtain final spiking concentrations of approximately 0.10, 0.73, 1.46, and 2.18 ppm, respectively, with respect to the sample concentration. AGP Sample solutions were prepared by dissolving approximately 27.5 mg of sample in 10 mL of diluent.
3. Results and Discussion
3.1. Setting up Cone Voltage and Collision Energy
Best values for cone voltage is 22 Volts and collision energy is 6 eV obtained in initial assessment on LCMS. In this stage potential precursor and product mass are obtained.
3.2. Carcinogenic Potency Categorization Approach (CPCA)
Potency Score Breakdown is tabulated in below Table 2.
4. Method Development
4.1. Preliminary Trials
In preliminary trials, to get developed technique of LCMS/MS for NTPA were difficult. Various stationary phases such as Inertsil ODS‐3 (100 mm x 3 mm x 3 μm), Inertsil ODS‐3 (100 mm x 3.9 mm x 3 μm), and Inertsil ODS‐3 (100 mm x 3.9 mm x 5 μm) were tried but no response was achieved. Different liquid phases were employed in both condition isocratic as well as gradient at acidic and basic pH but failed to get good response for NTPA.
4.2. Developed LCMS/MS Method
Developed technique for NTPA was obtained on Tandem mass spectroscopic instrument with the help of liquid phases. In this successful development stationary phase of YMC S‐5um, 12 mm (4.6 mm X 150 mm) and binary liquid phases of Ammonium acetate buffer with pH = 9.5 with Methanol to enrich ionization were used. In this method, Capillary set value was finalized at 1 kilovoltage by performing experiments on 0.5, 1 and 1.5 kilovoltage and set value for cone finalized at 22 voltage by testing experiments on 10, 20 and 30 voltage to get best elution for NTPA. This method suitability further confirm by performing analytical validation.
5. Analytical Technique Validation
An analytical method was rigorously validated in accordance with the ICH Q2R2 guideline (Chan et al. 2004). The validation encompassed a thorough evaluation of several key performance characteristics including: Specificity, LOD & LOQ determination, Linearity from LOQ, Precision, LOD observation, Accuracy, Robustness, Stability study and Batch analysis (ICH 2023; EMA 2018).
5.1. Specificity
In this parameter, results for NTPA impurity at working solution, AGP sample solution and Spiked NTPA in AGP sample solution were studied. After achieving best elution with highest resolution of all three peaks containing Benzoic acid, AGP sample and NTPA as stated in Figure 2. Also, NTPA eluted without any matrix interference from AGP sample or diluent used as stated in Figure 3. Results stated in Table 3 confirms Specificity of the method.
FIGURE 2.
A) Diluent as Blank B) NTPA Standard as Working Standard C) Test Sample as AGP D) NTPA Spiked in Test sample of AGP.
FIGURE 3.
LC‐UV Chromatogram of AGP and NTPA at 278 nm.
TABLE 3.
Retention time of NTPA.
| In Graph | Detection at minutes |
|---|---|
| Working Standard | 7.591 |
| Diluent | Absent |
| Limit level doping in AGP | 7.595 |
| AGP Sample | Absent |
5.2. Determination of Detection and Quantitation Value From Linearity
The detection and quantitation has been obtained from linearity study and are 0.03 ppm and 0.1 ppm relative to sample concentration respectively. The linear regression (R (Birla et al. 2024)) plotted and described in Figure 4A.
FIGURE 4.
(A) Detection and Quantitation values of NTPA (B) Linear regression study of NTPA.
From spiking experiments at multiple concentration range about 5.0 to 200.0% of the limit level method found to be linear with % Y intercept +4.57. Linearity curve plotted peak response against multiple NTPA spiking solution are stated in (Figure 4 (B)).
5.3. Precision
Preciseness of sample solutions prepared was evaluated by conducting repeatability studies within and between days. Multiple (for 6 number) doping of NTPA in AGP samples at limit level of 1.5 ppm relative to sample concentration were tested. %RSD for both repeatability studies within and between days are found within acceptance limit. This proves method found to be precise and results are tabulated in Table 4A.
TABLE 4.
(A) Precision (B) Precision at different levels (C) LOD Observation (D) Accuracy (E) Batch analysis results of NTPA.
| (A) Preciseness | (B) Preciseness at various % | |||
|---|---|---|---|---|
| Criteria: ≤15% Relative Standard of deviation | Criteria: ≤15% Relative Standard of deviation | |||
| Replicates | NTPA Content relative to sample concentration in ppm (Precision set) | NTPA Content relative to sample concentration in ppm (Intermediate set) | Spiked % of the limit level | %Relative Standard Deviation for NTPA Content |
| 01 | 1.429 | 1.455 | 7% | 7.09 |
| 02 | 1.421 | 1.470 | 100% | 2.65 |
| 03 | 1.416 | 1.476 | 150% | 1.59 |
| 04 | 1.410 | 1.470 | 200% | 0.79 |
| 05 | 1.401 | 1.470 | (C) LOD Observation | |
| 06 | 1.398 | 1.469 | Criteria: NTPA peak should be present | |
| Average | 1.413 | 1.468 | ||
| Std.Dev | 0.01 | 0.01 | Analysis‐1 | 1661 |
| %RSD | 0.84 | 0.48 | Analysis‐2 | 1640 |
| %Cumulative Deviation | 1.99 | Analysis‐3 | 1765 | |
| (D) Spiking study | ||||
| Criteria: Percentage recovery results ranging between 80 and 120 | ||||
| Spiked % > |
(LOQ level) Spiked 7% of the limit level |
Spiked 50% of the limit level | Spiked 100% of the limit level | Spiked 150% of the limit level |
| Obtained Recovery % > | 100.63 | 97.06 | 98.11 | 96.79 |
| 99.91 | 97.52 | 97.79 | 96.92 | |
| 99.88 | 97.20 | 97.19 | 96.29 | |
| Mean Recovery % | 100.14 | 97.26 | 97.70 | 96.67 |
| (E) Batch Analysis | ||||
| Criteria: NMT 1.5 ppm relative to sample concentration | ||||
| Lot | Lot Number | Content NTPA in ppm relative to sample concentration | ||
| 1 | AGP/VQ/CRD/24/001 | Below Quantitation Limit | ||
| 2 | AGP/VQ/CRD/24/002 | Below Quantitation Limit | ||
| 3 | AGP/VQ/CRD/24/003 | Below Quantitation Limit | ||
5.4. Precision at Different Levels
Precision was assessed by spiking NTPA into solutions at the LOQ, 100%, 150%, and 200% of the limit level. Table 4B presents the calculated percent relative standard deviation for the spiked NTPA content at each of these levels.
The calculated %RSD for the NTPA content complied with the Q2R2 validation guideline. This demonstrates that the method is precise across the tested concentration levels: LOQ, 100%, 150%, and 200% of the limit level.
5.5. LOD Observation
We prepared an NTPA solution at the LOD concentration of 0.03 ppm relative to sample concentration and performed three replicate injections. The results of this analysis are shown in Table 4C. Visual detection of the NTPA peak in all three replicates validated the method's LOD as approximately 2.0% of the limit level.
5.6. Spiking Study
Spiking study was performed at different percentage condition and results obtained at Quantitation 7, 50, 100 and 150 percentage of the limit level. Spiking results ranging between 96.67 and 100.14 percentage. It confirms method accuracy is in line with the validation acceptance criteria as tabulated in Table 4D.
5.7. Solution Study at Different Time Intervals
NTPA content in test sample of AGP and NTPA spiking sample of AGP were analyzed. Observed NTPA in both with and without spiking sample of AGP found in line with acceptance criteria. Results indicates sample solution prepared in methodology are stable as tabulated in Table 5.
TABLE 5.
NTPA Content data at time intervals.
| Time Interval In hours | NTPA Content in Test Sample relative to sample concentration in ppm | Time Interval In hours | NTPA Content in Spiked Test Sample relative to sample concentration in ppm |
|---|---|---|---|
| Initial | BQL | Initial | 1.473 |
| 15 | BQL | 18 | 1.640 |
| 24 | BQL | 27 | 1.801 |
| 33 | BQL | 37 | 1.811 |
| %RSD | Not Applicable | %RSD | 9.48 |
Criteria: ≤15% Relative standard of deviation.
5.8. Robust Study
Robust study has been evaluated by analyzing doped 1.5 ppm NTPA standard in AGP sample relative to sample concentration at multiple method variation. Deviation and Cumulative deviation has been determined against six replicates results obtained in method precision as shown in Figure 5. Charts clearly indicates method is found robust when variation in parameters were applied (Criteria: ≤15% Relative standard of deviation).
FIGURE 5.
Robustness chart of NTPA.
5.9. Batch Analysis
In method validation three batches has been confirmed for its quality with respect to NTPA impurity and results confirmed that samples of AGP defined in Table 4E are shows compliance for the test.
5.10. Mass Spectrometry Results
During mass ionization fragment observed at a peak 100.01 m/z (MNTPA‐NO)+ belongs to mass of NTPA as stated in Figure 6.
FIGURE 6.
Mass spectra of NTPA.
During method development analysis ionization pattern indicates precursor ion at 130.00 m/z (MNTPA + H)+ considered as identifier and the product ion at 100.01 m/z (MNTPA‐MNO)+ for NTPA can be used for quantification purpose in this study.
6. Conclusion
Based on validation results obtained on developed Liquid chromatography tandem mass spectrometry method for trace analysis of NTPA in AGP. It is concluded that methodology is linear, accurate, precise, robust and successful that meets regulatory guidelines. Method is intended for use in commercial samples of AGP to check quality of the product.
Funding
The authors have nothing to report.
Conflicts of Interest
The authors declare that there are no financial or personal relationships with other people or organizations that could inappropriately influence this work.
Acknowledgements
No external funding was received for this work. We would like to express our gratitude to Indoco Remedies Limited, Navi Mumbai, Maharashtra, India, for their support and for providing the AGP samples and NTPA standard used in this study.
Contributor Information
Pallavi T. Roy, Email: pallavichem@gmail.com.
Nitin A. Mirgane, Email: mirgane@gmail.com.
Data Availability Statement
Data underlying the results presented in this study are available from the authors upon request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data underlying the results presented in this study are available from the authors upon request.