Trace-Level Quantification and Identification of Mutagenic and Cohort-of-Concern N-nitroso-mirabegron Impurity (NDSRIs) using UFLC-ESI-MS/MS in Mirabegron

Sandip Vadariya¹, Jigar Patel²*, Haresh Patel¹, Hitin Hirpara³

¹Analytical Research Development, Cohance Lifesciences Limited API RandD Centre,

Ankleshwar, 393002, India.

²Deputy Director (Technical), Sophisticated Instrumentation Centre for Applied Research and Testing – SICART, Vallabh Vidyanagar, Anand, 388120, India.

³Research and Development Department, Cohance Lifesciences Limited API RandD Centre,

Ankleshwar, 393002, India.

*Corresponding Author E-mail: pramukhprit@gmail.com

ABSTRACT:

Currently, all the pharmaceutical industries and related industries have been dealing with issue from nitrosamine drug substance related impurities (NDSRIs) due to their potential health risks. Regulatory agencies provide guidelines for essential detail for generation pathways of their formation, severity, and permissible limits for human consumption. This research presents a trace-level quantification and identification, rapid, and high-sensitive LC-MS/MS technique for N-nitroso-mirabegron (NDSRIs) impurity in mirabegron with a LOQ of 0.8 µg/mL. The QSAR results are positive for N-nitroso-mirabegron, and the cohort of concern (CoC) suggests possible genotoxicity. Inert Sustain PFP (4.6mm x 150mm), 5µ HPLC column and mobile phase A contain 0.63 g/L of ammonium format, methanol, and acetonitrile in a 4:3:3 (v/v/v) ratio while mobile phase B contain 0.2% formic acid in methanol. Positive ionization in Electron spray ionization (ESI) and multiple reaction monitoring (MRM) detection uses for identification and quantification. Linearity range of 0.8µg/mL to 12µg/mL with regression coefficient >0.990, precision (%RSD = 1.8, %RSD of Method precision = 3.01 and Intermediate Precision = 5.47). Accuracy within the range 84.55 – 91.64% within limit. While screening mirabegron samples for NDSRIs, the presence of N-nitroso-mirabegron was detected.

KEYWORDS: Analytical Methods and Validation, LC-MS/MS, Mirabegron, Nitrosamine, NDSRIs.

1. INTRODUCTION:

Mirabegron is prescribed in the management of overactive bladder (OAB) and class of this drug is sympathomimetic and β3-adrenoceptor agonist. In mid-2012, the US Food and Drug Administration (FDA) approved the mirabegron for a treating overactive bladder, characterized by symptoms such as urge urinary incontinence, urgency, and increased urinary frequency¹.

It is practically insoluble in water and in heptane, slightly soluble in anhydrous ethanol. Any substance containing an N-NO functional group is referred to as an N-nitrosamine, which has been classified as a cohort of concern (CoC). In humans, when exposed to significant amounts over an extended period, that can lead to cancer due to potential nature. The regulatory bodies USFDA, WHO, EMA, TGA, ANVISA and Health CANADA release the guidelines to control, identification and control strategy. As per ICH M7, nitrosamine impurities are a potential genotoxic risk and are categorized as a CoC^2-3. Since 2018 to till date, Nitrosamine impurities observed in sartans, ranitidine, metformin, nizatidine, metformin, orphenadrine, varenicline, quinapril, Acyclovir, propranolol, Dabigatran and Amitriptyline higher than limit^4-8. The formation of N-nitrosamines is possible, when active pharmaceutical ingredients (APIs) or API fragments undergo the Nitrosation process in the presence of secondary (di) or tertiary (tri) amines and nitrosating agents (i.e. Sodium nitrite), such as nitrites in residual present in additives used during drug preparation^9-10. In the manufacturing of active pharmaceutical ingredients (API) and drug products (formulation), it is possible to generate potential genotoxic impurities (PGIs) and nitrosamine impurities from various components such as byproducts, reagents, and intermediates¹¹. The USFDA has published that certain drug substances and products have been observed to contain impurities linked to nitrosamines, specifically API-structure-derived complex nitrosamines, also known as NDSRIs¹², these impurities generated during the manufacturing process, stability/storage of the active pharmaceutical ingredient (API) and formulation (drug product). It is a type of nitrosamine that shares structural similarities with the API. Research also indicates that NDSRIs may develop because of residual nitrite impurities present in additives at residual or parts per million levels (ppm). It has been found that the nitrite impurities are detect in certain common excipients, such as water, significantly increases the possible presence of NDSRIs in a particular drug product^13-15. The tough challenges of identifying and quantifying of nitrosamine impurities due to the poor ionization, lower mass number of impurities (m/z) and sample matrix interference, when in NDSRIs similar structure with API, unviability of impurity standard. Routine testing and control of impurities by analytical method in creating an accurate analytical method for routine testing can be difficult due to the strict limits set for detecting these impurities^16-17. NDSRIs impurities lack carcinogenicity and mutagenicity study data and limit of these impurities is not easily available, the limit of NDSRIs is derived from AI from the CPCA score from which an AI limit can be determined^12,18-20. The primary objective behind limiting the safe daily dose consumption of NDSRIs impurities and ensuring their absence in the drug substance is to safeguard human health and prevent potential risks of exposure to carcinogenic substances. Therefore, rigorous efforts are made to develop sensitive and reliable analytical techniques to accurately measure and control nitrosamine impurities in pharmaceutical products. Following stringent regulations and employing advanced analytical approaches, the industries related to pharmaceutical industry aims to prioritize the safety and well-being of consumers. Recently, many pharmaceutical products recalled due to contamination from Nitrosamine impurity and NDSRIs impurities. It is the extremely serious and alarming reasons for recall the pharmaceutical product due to the detection of higher the specification levels of nitrosamine impurities^21-25.

1.2 Quantitative structure-activity relationship (QSAR) and N-Nitroso-Mirabegron AI Calculation.

As per ICH M7(R2), requirement classification QSAR predictions of Mirabegron, N-nitroso-mirabegron (Rand S Isomers) performed using Nexus 2.5.1 and the results are presented in Table 1. N-nitroso-mirabegron impurity, Derek is plausible, while Sarah is Cohort of Concern and positive. According to ICH M7 guidelines, the Threshold of Toxicological Concern (TTC) limit cannot be justified to the "CoC" group due to its higher potent then compared to other known carcinogens. Recently published EDQM Acceptable intakes (AIs) established for N-nitrosamines guidelines (15/02/2024, EMA/72902/2024 /Rev. 3, Non-clinical Working Party (NcWP))¹⁰, The potency category and score and AI of N-nitroso-mirabegron is 3 and 400ng/day respectively³⁰. The daily dose of Mirabegron (BEG) is 50mg/day. From the below calculation formula, the limit of N-nitroso-mirabegron in Mirabegron (BEG) is 8.0ppm.

Limit (ppm) = AI/MDD (AI= Acceptable intake, MDD = maximum daily dose of an API (in mg)

Figure 1. Structures of (a) Mirabegron API, (b) N-nitroso-mirabegron

2. EXPERIMENTAL:

2.1. Grade of Reagents and Chemicals:

LC/MS-MS grade reagents and solvents procured and used which purity >99.8%. Ammonium formate was purchased from MERCK; acetonitrile and methanol was used from J. T. Baker (LLC, USA) make; formic acid was procured from Fischer Chemicals (Czech Republic); and water was used from Milli-Q. N-nitroso-mirabegron (Potency: 99.84%) was procured from SimSon Pharma Limited, Mumbai, India, and Mirabegron was used in-house (Cohance Lifesciences Limited, API RandD Unit-II, Ankleshwar, India).

2.2. Buffer and Mobile Phase and Diluent Preparation:

Preparation of Buffer solution:

Weigh 0.63g of ammonium formate accurately into 1000mL of water, sonicate to dissolve, and mix well.

Preparation of Mobile phase A:

A mixture containing buffer solution, methanol, and acetonitrile in a ratio of 4:3:3 (v/v/v) and mix well. Filter and degas.

Preparation of Mobile phase B:

Transfer accurately 2.0mL formic acid into 1000mL of methanol and mix well. Filter and degas.

Preparation of Diluent and blank:

For diluent and blank solution used mobile phase A.

2.3. Preparation of standard solutions:

0.02µg/mL of N-nitroso-mirabegron impurity standard prepared as follows:

Transfer a suitable amount of N-nitroso-mirabegron impurity standard to a suitable volumetric flask and dissolve in diluent. Dilute with diluent to volume and final concentration should be 0.02µg/mL.

2.4. Sample preparation (2500µg/mL):

Weight and transfer 125mg of test sample into a 50mL volumetric flask, then add 20mL of diluent sonicate to dissolve the solid (If necessary, add more diluent), and make up with diluent and mix well.

2.5. LC-MS/MS Operating Conditions:

The method development and chromatographic separation was performed using a UFLC (Shimadzu, Japan), coupled with an LCMS-8040 (Shimadzu, Japan), LCMS-MS triple quadrupole with an ESI (electrospray ionization) interface. N-nitroso-mirabegron impurity ionized in positive mode with molecular ions as (M+H)⁺ at m/z 425.85 and represent in figure 2. Table 2 represents the MRM and ESI source parameters. The final condition for the identification and detection of N-nitroso-mirabegron using by UFLC-MS/MS parameters is listed in Table 3.

Table 2. Optimized MRM

Precursor m/z	Product m/z	Dwell time	Q1 Pre Bias	CE	Q3 Pre Bias
425.85	260.15	100	-16.0	-17.0	-29.0
425.85	247.10	100	-22.0	-19.0	-27.0
425.85	146.25	100	-16.0	-26.0	-29.0

Table 3 Optimized final UFLC-MS/MS condition

UFLC System chromatographic parameter
Mobile Phase A:	0.63% g/L ammonium formate in Water, Methanol, and Acetonitrile, 4:3:3 (v/v/v)
Mobile phase B: Wavelength:	0.2% Formic acid in MeOH 247nm
Flow Rate:	0.6ml/min
Injection Volume:	25µl
Auto Sampler temp:	15°C
Needle Wash Solution:	Water: Acetonitrile [40:60 (v/v)]
Column oven temp.:	30°C
Column:	Inert Sustain PFP (4.6 mm x 150 mm), 5µ
Gradient elution Program	Time (Min)	Mobile phase-A (%)	Mobile phase-B (%)
	0.01	100	0
	8	100	0
	11	15	85
	30	15	85
	35	100	0
	45	100	0
MS/MS System
Type and Mode:	MRM (Positive)
Detector Start time:	2.0 min
Detector End time:	12.0 min
Interface:	ESI
Nebulizing gas flow:	3.00 L/min
Desolvation Line (DL) temperature:	250 °C
Heat block temperature:	400 °C
Drying gas (N₂) flow:	15 L/min
Interface voltage:	Relative to Tuning file (4.5 KV)
Event time:	0.309 sec
FCV valve MS program:	Sr. No.	Time (min)	Command
	1	0.00	FCV2
	2	3.00	FCV2
	3	11.00	FCV2

Figure 4: Spiked Sample Solution (at specification level)

Figure 5: Sample Solution (PDA and MRM)

3.2. Method Validation:

The optimized and finalized analytical method was validated as per USP general chapter <1225> validation of compendial procedures, ICH Q2, and in a 21CFR part 11 compliance environment. The validation of the analytical method was confirmed by considering specificity (SPC), linearity (LIN) and Range, LOD, LOQ, method (MP) and intermediate precision (IP), accuracy (ACC), and solution stability (SS).

3.2.1 Specificity and System Precision:

Specificity of this method established by the ability of the LC-MS chromatographic system to differentiate retention time between the diluent peaks and individual impurity and Mirabegron peak. A blank solution, Standard solution (six replicates), Sample solution (Unspiked), and Sample solution (Spiked) were prepared and injected. Sample solution (Unspiked) and Sample solution (Spiked) were determined and the results exposed that there is no interference of blank and other peaks with N-nitroso-mirabegron peak retention time and % RSD of six replicate standard solution 1.80%. Therefore, the method can be termed as a specific and precise. The MRM chromatograms of blank, standard solution, and spiked sample solution can be found in Figure 3-5.

3.2.2 Determination of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) and LOQ Precision:

Visual detection and signal-to-noise ratio method (S/N) approach are employed for the establishment of the LOD (S/N: 3) and LOQ (S/N: 10) for the N-nitroso-mirabegron impurity. The standard solution was further diluted with known concentration for the LOD and LOQ determination and injected into LC-MS/MS. S/N values were derived using the RMS algorithm. The LOQ solution precision was proven by the six consecutive injections from the same vial for the N-nitroso-mirabegron. Table 4 presents the summary data.

3.2.3. Linearity and Range:

The linearity and range established in the concentration range between 0.002µg/mL to 0.03µg/mL (0.8µg/mL – 12 µg/mL) for N-nitroso-mirabegron impurity. Prepare a single solution preparation and single injection for each level. Obtain a linearity curve by plotting the concentration on X-axis and area responses on Y-axis. Determine the correlation coefficient (r) and summaries in Table 4. Analyte peak areas Vs concentrations of impurity correlation is represented in Figure 6.

Figure 6. Linearity plot of N-nitroso-mirabegron from LOQ to 150 % levels

3.2.4. Accuracy and Recovery Study:

The accuracy is performed by standard addition method in triplicate preparation at three different levels 0.002, 0.02, and 0.03µg/mL (LOQ, 100%, and 150% level respectively). Samples were prepared in triplicate at each level, and each level sample solution was injected in single. The acceptance criteria for recovery were 70 % - 130% as per the guideline. The percentage recoveries for N-nitroso-mirabegron impurity are presented in Table 4.

3.2.5. Precision (Repeatability and Ruggedness):

Method precision was established by analyzing six spiked sample preparations (0.02µg/mL and 8µg/mL wrt test concentration) under the same conditions as per standard test procedure for N-nitroso-mirabegron using the same lot of the API sample. Furthermore, for the intermediate precision study, the same procedure was followed as followed in Method Precision, but it was repeated on a different day, by a different analyst, and using the same amount of sample. N-nitroso-mirabegron impurity value for preparation, mean and their % RSD were calculated. Acceptance criteria for both study % RSD of the recovered individual impurity of six sample preparations (Spiked at specification level) and Cumulative %RSD of MP and IP should not be more than 20.0. All achieved values for the precision were found as per acceptance criteria and tabulated in Table 4.

3.2.6. Solutions Stability:

The solution stability study was carried out up to 35hrs. The spiked and un-spiked sample solutions (at specification level) were placed in the autosampler at 15 ˚C in LC vial for 35h and calculated the area ratio against 0hrs (Initial un-spiked and Spiked sample). There were no significant changes observed after 35 h for N-nitroso-mirabegron impurity. Therefore, we confirmed the impurity in the sample and spiked the sample solution to stable for at least 35 hours.

3.2.7. Batch Analysis:

Batch analysis was performed with three samples using a validated UFLC-MS/MS analytical method to accurately determine the N-nitroso-mirabegron. The amount of N-nitroso-mirabegron in three samples was found in the range of 0.42 - 0.46ppm which was lower than LOQ.

Table 4. Summary

Method validation parameter and Acceptance criteria

Results

Specificity.

Interference from blank and impurities

There is no interference of blank and other impurities peaks with the N-nitroso-mirabegron peak.

Determination of LOD, S/N value (≥3)

Determination of LOQ, S/N value (≥10)

405

Precision at LOQ

% content (n = 6, % RSD < 20.0)

2.06

Linearity and Range.

correlation coefficient (r) (>0.990)

Range: 0.8 ppm to 12 ppm

Slope: 118946.0056

Intercept: 9335.5281

correlation coefficient (r):0.9996

Accuracy.

Between 70.0 % - 130.0 %.

the level at LOQ mean / RSD: 88.62 / 3.20

the level at 100 % mean / RSD: 86.97 / 2.87

the level at 150 % mean / RSD: 90.92 / 0.97

Method Precision (MP).

% content (n = 6, % RSD < 20.0)

% Mean/ RSD: 86.53 / 3.01

Intermediate Precision (IP).

% content (n = 6, % RSD < 20.0)

% content (n = 12, % RSD < 20.0)

% Mean/ RSD: 91.29 / 5.47

% Mean/ RSD: 88.91 / 5.10

Solution Stability

The area ratio value at all-time intervals should be between 0.80 to 1.20.

Sample and Spiked solution stable up to 35hrs at 15°C.

5. CONCLUSION:

As guidelines published from EDQM (15/02/2024, EMA/72902/2024/Rev. 3, Non-clinical Working Party (NcWP)), the potency score and AI of N-nitroso-mirabegron are 3 and 400ng/day, respectively¹⁰. N-nitroso-mirabegron QSAR studies found the Class 3 category with a CoC, indicating potential genotoxicity (PGI). As published, literature and guidelines indicate NDSRIs impurities are generated during the manufacturing and storage of API. Therefore, it is necessary to quantification and identification the nitrosamine impurities at low concentrations and LOQ should be below or equal to 10%. For the residual level quantification and identification of N-nitroso-mirabegron, a rapid and precise UFLC-MS/MS technique was developed and validated. This technique provided accurate and extremely sensitive measurement of N-nitroso-mirabegron. This analytical method was validated as per guidelines and shows lower level LOD (0.28ppm) and LOQ (0.8ppm), which indicates the method is sensitive to detection of impurity. This validated method is widely used in routine monitoring of N-nitroso-mirabegron in mirabegron with the limit of 8.0ppm. In-house manufacture API screening with validated methods and results were found in the range of 0.42-0.46ppm, which was lower than LOQ (10% of specification levels).

6. CONFLICTS OF INTEREST:

The author declares no conflicts of interest.

7. ACKNOWLEDGEMENTS:

The authors wish to extend their gratitude to the management of The CVM University, Gujarat, INDIA, and Cohance Lifesciences Limited, Ankleshwar, INDIA for supporting and providing facilities for analysis of this work. They also thank their colleagues in the analytical laboratory for their cooperation in carrying out this work.

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Received on 03.02.2025 Revised on 15.07.2025

Accepted on 18.10.2025 Published on 03.04.2026

Available online from April 06, 2026

Research J. Pharmacy and Technology. 2026;19(4):1492-1498.

DOI: 10.52711/0974-360X.2026.00214

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