Why does Esomeprazole contain nitrosamine impurities? Secondary amine trap in benzimidazole structure

Key points of this article

① S-enantiomer esomeprazole is the S-enantiomer of omeprazole. It was approved by the FDA on February 20, 2001 (NDA 21-153, trade name Nexium) [1].

② The secondary amine (N1-H) on the benzimidazole ring can generate N-nitrosoesomeprazole (C₁₇H₁₈N₄O₄S) [2] under nitrosation conditions. This is a structural vulnerability shared by similar benzimidazole proton pump inhibitors.

③ ICH M7(R2) (adopted on April 3, 2023) lists nitrosamines as a "cohort of concern", and its acceptable intake is measured in nanograms/day, which is more than 1 order of magnitude stricter than the conventional impurity threshold [3,4].

④ CATO provides 147 kinds of reference substances for this system in stock, covering free base, magnesium salt, sodium salt, deuterated internal standard and N-nitrosamine impurities, supporting the whole process control from process development to registration application.

01

BACKGROUND

From the “purple pill” to single enantiomers

In 1989, the FDA approved the first proton pump inhibitor omeprazole (trade name Prilosec), which is a racemate of half and half R- and S-enantiomers. On February 20, 2001, the FDA approved AstraZeneca'sesomeprazole magnesium(Trade name Nexium, NDA 21-153) [1].

C4X-111299 esomeprazole (free)

The core that determines its pharmacological activity is the chiral sulfoxide center (S configuration), and the N1 position on the benzimidazole ring is the source of impurity risks to be discussed in this article: free base CAS number 119141-88-7, divided into Subformula C₁₇H₁₉N₃O₃S, chemical name (S)-5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole.

02

MECHANISM

Structural origin: how benzimidazole secondary amine is nitrosated

The benzimidazole ring of esomeprazole carries a hydrogen atom at the N1 position, which belongs to the secondary amine structure in the heterocyclic system. In acidic environments and the presence of nitrite or other nitrosation precursors (commonly found in contaminated raw materials, process water or packaging materials), the N-H site can react with nitrosation reagents to formN-nitrosoesomeprazole——The IUPAC name is (S)-6-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1-nitroso-1H-benzo[d]imidazole, molecular formula C₁₇H₁₈N₄O₄S, molecular weight 374.41[2]. Compared with the parent substance, this impurity happens to be the product in which "N1-H" is replaced by "N1-NO", and the structure is clearly assigned.

This structural vulnerability is not unique to esomeprazole - omeprazole, lansoprazole, pantoprazole, and rabeprazole are all benzimidazole proton pump inhibitors, all with "benzimidazole-secondary amine" as the core skeleton, and therefore share similar nitrosation risk sites. This is different from the nitrosation sites and causes of Sartan antihypertensive drugs and ranitidine [5,6] - for APIs of different structural categories, the nitrosamine risk assessment must be based on their specific chemical sites or process links, and the same set of conclusions cannot be simply applied.

⚠ ICH M7 Tip: FDA clearly states in its nitrosamine control guidance that APIs containing tertiary amine groups generally have a lower risk of nitrosamine formation than APIs containing secondary amine groups [7]. The secondary amine structure on the benzimidazole ring of esomeprazole is exactly what needs to be focused on during the process route design and risk assessment stages.

03

CLASSIFICATION

Impurity Profile and Regulatory Classification: From “Milligram Level” to “Nanogram Level”

Most of the impurity profiles of esomeprazole follow ICH For conventional process impurities and degradation products managed by the Q3A(R2) percentage threshold system (the European Pharmacopoeia and the United States Pharmacopoeia have included named impurities such as impurities A and D), the reporting/identification/quality control thresholds are 0.05% and 0.10 respectively. % (or 1.0 mg/day, whichever is lower), 0.15% (or 1.0 mg/day, whichever is lower) [8]. Calculated based on the upper limit of the commonly used dose of 40 mg/day, the identification threshold of 0.10% is approximately equivalent to 40,000 nanograms/day. In addition, the synthetic route may also introduce potentially genotoxic impurities with structural warnings - a study in "Chromatographia" in 2025 reported that there are six such impurities (RC16, RC17, RC18, RC20, RC21, RC23, all containing benzene ring or pyridine ring structures) in the starting materials of esomeprazole magnesium API [9]. They are both under the jurisdiction of ICH M7 but the limit system is different from nitrosamines.

Nitrosamine impurities (including "drug related nitrosamine impurities" such as N-nitrosoesomeprazole, or NDSRI) belong to the "cohort of concern" compounds defined by ICH M7(R2). The general toxicological concern threshold (TTC, 1.5 micrograms/day) is not applicable, but exclusive limits at the nanogram/day level are used. In the absence of compound-specific toxicological data, regulatory agencies generally use the CPCA (Carcinogenic Potency Classification Method) to classify nitrosamines into five levels, and the corresponding acceptable intakes (AI) are as follows [10]:

Compared with the 40,000 nanograms/day (common impurity identification threshold) mentioned earlier, the AI ​​limit of nitrosamine impurities ranges from 27 times to 2200 times lower - this is the fundamental reason why benzimidazole PPIs such as esomeprazole need to "create a new system" for impurity control.

04

ANALYSIS

Analysis method: How to detect nanogram-level impurities

Methodological research in "Chromatographia" in 2025 established a HPLC-MS/MS quantitative method for six potential genotoxic impurities in esomeprazole magnesium raw materials: Inertsil ODS-SP C18 column (4.6×150mm, 5μm, column temperature 40℃), using 0.5% formic acid aqueous solution and methanol as the mobile phase, flow rate 0.5mL/min, electrospray positive ion multiple reaction monitoring (ESI+) MRM) testing, all verification parameters meet the standards [9]. The analysis of trace amounts of nitrosamines generally uses more sensitive mass spectrometry detectors, such as triple quadrupole or high-resolution orbitrap mass spectrometry [11]; in terms of enantiomeric purity control, there are also reports in the literature that chiral HPLC was used to directly separate omeprazole and its chiral impurities, and was used to determine the enantiomeric purity of esomeprazole magnesium trihydrate [12]. Deuterated internal standard (such as esomeprazole sodium-d3) is a commonly used matrix effect correction method in LC-MS/MS quantitative analysis.

05

REGULATORY

Regulatory Filing Considerations: A Timeline for Nitrosamine Regulation

For registration applications such as ANDA, NDA, and CEP, the current logic is "risk assessment first, confirmatory testing follow-up": risk assessment is completed based on the process route and structural characteristics. If risk points are identified, confirmatory testing is required, and the control strategy is included in the quality standards of APIs [17,18]; this logic also applies to registration and re-registration applications under China's NMPA system.

06

CATALOG

Representative reference substance of CATO esomeprazole system

The following isCATORepresentative controls for esomeprazole system (part):

The above are only representative reference substances for the esomeprazole system.CATOThe current stock covers 147 impurities, salt forms, isotopically labeled compounds and related intermediates of this system. Please contact CATO for the complete catalog.

07

FAQ

FAQ

Q1: What is NDSRI? What is the difference between it and ordinary small molecule nitrosamines?

The structure of small molecule nitrosamines (such as NDMA, NDEA) has nothing to do with the API itself, and usually has a fixed AI limit, for example, NDMA is 96 ng/day, and NDEA is 26.5 ng/day [19]; ​​while NDSRI (drug body-related nitrosamine impurity) is highly similar to the structure of the API, such as N-nitrosoesomeprazole, which usually lacks exclusive toxicology data and requires the use of structure prediction methods such as CPCA to determine the limit [10]. CATO’s spot N-nitrosamine esomeprazole impurity 142 falls into the latter category.

Q2: Will nitrosamine impurities be detected in esomeprazole?

uncertain. The presence of sites that can be nitrosated does not mean that the finished product will inevitably be detected - it also depends on factors such as whether there are sources of nitrosating agents in the process (such as contaminated raw materials, reagents, or packaging materials). This is why both FDA and EMA require risk assessments to be conducted first, and then confirmatory testing to be carried out as appropriate [17,18].

Q3: Can CATO provide the reference substances and internal standards required for quantitative analysis of nitrosamine impurities in esomeprazole?

Can.CATONitrosamine reference standards such as N-nitrosamine esomeprazole impurity 142 and deuterated internal standards such as esomeprazole sodium-d3 are available from stock, which can be directly used for HPLC-MS/MS method development and verification, covering the entire process requirements from methodology establishment to routine release testing.

Q4: What are the essential differences between ICH M7(R2) requirements for nitrosamine impurities and ordinary process impurities?

Common process impurities follow the percentage threshold system of ICH Q3A/B (reporting/identification/quality control thresholds are usually in the range of 0.05% to 0.15%, and are converted in combination with the maximum daily dose); nitrosamines belong to the "cohort of concern" compounds defined by ICH M7(R2), and the general toxicological concern threshold of 1.5 μg/day is not applicable. Instead, exclusive or category limits of 18 to 1500 ng/day are used [3, 4, 10].

08

REFERENCES

References

[1] FDA. Clinical Pharmacology Review, Esomeprazole Magnesium, NDA 21-153. Approved 02/20/2001. fda.gov/media/71541

[2] N-Nitroso Esomeprazole. Veeprho Pharmaceutical Reference Standards. veeeprho.com/impurities/n-nitroso-esomeprazole

[3] ICH. M7(R2): Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. Step 4, adopted 3 April 2023.

[4] ICH. Addendum to M7(R2): Application of the Principles of the ICH M7 Guideline to Calculation of Compound-Specific Acceptable Intakes. Step 4, adopted 3 April 2023.

[5] Liu J, et al. Development of a sensitive and stable GC-MS/MS method for simultaneous determination of four N-nitrosamine genotoxic impurities in sartan substances. J Anal Sci Technol. 2021;12:3.

[6] FDA. Information about Nitrosamine Impurities in Medications. fda.gov/drugs/drug-safety-and-availability

[7] FDA. Control of Nitrosamine Impurities in Human Drugs, Guidance for Industry. fda.gov/media/141720

[8] ICH Q3A(R2): Impurities in New Drug Substances.

[9] Li WS, Zhang ZM, Wang XY, Sun CX, Liu Z, Ma X, Qiao Z, Zhu HB, Han LF. Determination of Potential Genotoxic Impurities in Esomeprazole Magnesium by HPLC-MS/MS. Chromatographia. 2025;88(11):891-903. doi:10.1007/s10337-025-04444-1

[10] FDA. Carcinogenic Potency Categorization Approach (CPCA). fda.gov/media/183710

[11] FDA. NDSRI Method Performance Criteria presentation. fda.gov/media/187987

[12] Zanitti L, Ferretti R, Gallinella B, La Torre F, Sanna ML, Mosca A, Cirilli R. Direct HPLC enantioseparation of omeprazole and its chiral impurities: application to the determination of enantiomeric purity of esomeprazole magnesium trihydrate. J Pharm Biomed Anal. 2010;52:665-671.

[13] Understanding EMA and FDA Regulations on Nitrosamine Control. ProPharma Group. (Related background on NDMA impurity detected in valsartan in 2018)

[14] EMA. Nitrosamine impurities (referral procedure advice to MAHs). 26 September 2019.

[15] FDA Updates Investigations into Impurities in APIs. DCAT Value Chain Insights. Reports on ranitidine NDMA contamination incident in September 2019.

[16] Center for Drug Evaluation of the State Drug Administration. "Technical Guiding Principles for Research on Nitrosamine Impurities in Chemical Drugs (Trial)". May 8, 2020.

[17] FDA. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs), Guidance for Industry. August 2023.

[18] FDA. Control of Nitrosamine Impurities in Human Drugs, Guidance for Industry, Revision 2. September 2024.

[19] FDA. Control of Nitrosamine Impurities in Human Drugs, Guidance for Industry, Table 1 (AI limits for NDMA, NDEA, NMBA, NMPA, NIPEA, NDIPA).

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