Advanced LC-MS/MS method for selective quantification of nitrosamine impurities in Risperidone: Enhancing drug safety
Talanta Open, Volume 11, August 2025, 100416: Graphical abstract
The goal of the study was to develop and validate a fast, simple, and sensitive LC-MS/MS method for detecting and quantifying four nitrosamine drug substance-related impurities (NDSRIs) in Risperidone. Chromatographic separation was performed using a PFP C18 column with gradient elution and positive ESI in MRM mode for selective quantification.
The method showed excellent linearity, low detection limits, and high recovery rates, making it suitable for routine monitoring of nitrosamine impurities such as NINA, NBOP, NBP, and NB in Risperidone. This contributes to improved quality control and enhanced drug safety.
The original article
Advanced LC-MS/MS method for selective quantification of nitrosamine impurities in Risperidone: Enhancing drug safety
Hitesh Thumbar, Pankajkumar Nariya, Bhavin Dhaduk
Talanta Open, Volume 11, August 2025, 100416
https://doi.org/10.1016/j.talo.2025.100416
licensed under CC-BY 4.0
Selected sections from the article follow. Formats and hyperlinks were adapted from the original.
The presence of nitrosamine impurities in drug products has been a significant safety concerns, falling under the category of "cohort of concern" according to ICH M7 guidelines [1]. Since 2018, several drug products such as sartan, ranitidine, and metformin (Glucophage) have been recalled due to the presence of nitrosamines above acceptable intake levels. Regulatory agencies like the USFDA and EMEA have issued guidelines for controlling nitrosamine impurities in drug products that can form nitrosamines [[2], [3]]. There have been multiple recalls of different categories of drug products due to nitrosamine drug substance-related impurities (NDSRIs) exceeding acceptable intake limits [4].
Recently, nitrosamine contaminants have been found in Risperidone which is used in the treatment of schizophrenia and bipolar disorder [[5], [6], [7]]. Specifically, there is a concern about N-Nitroso Risperidone impurity-1 (NB) which can form due to the nitrosation reaction between the secondary amine and nitrous acid under acidic condition during the manufacturing process. Due to these complications, it is essential to develop highly sensitive analytical methods for the quantifying NDSRIs in Rispiridone API. The literature survey revealed that various HPLC, HPTLC and LC-MS methods have been reported for the analysis of Risperidone and its structural impurities across the different matrices such as bulk drugs, pharmaceutical formulations, and human plasma. [[8], [9], [10], [11], [12], [13], [14]]. However, most existing methods primarily focus on analyzing Risperidone and its structural impurities in various matrices without adequately addressing the regulatory requirements for controlling nitrosamine impurities in Risperidone. Currently, there is no suitable analytical method is available to detect the NDSRI impurities at trace levels in Risperidone. To address this gap, we have adopted an LC-MRM approach to enhance the sensitivity and selectivity of nitrosamine impurities in Risperidone. The developed method successfully identified four NDSRI impurities at low concentrations levels (0.12–0.46 µg/mL) within a shorter run time. This method offers a significant improvement in sensitivity compared to existing methods, making it essential for monitoring the limits of NDSRIs in Risperidone. Moreover, all crucial parameters related to the method performance, including limit of detection (LOD), limit of quantification (LOQ), specificity, recovery, reproducibility, and linearity have been established. The validated method serves as a reliable tool for routine quality control analysis and ensuring the safety and regulatory compliance of Risperidone drug substances. The structure of NDSRIs in Risperidone drug substances is presented in Table 1.
2. Materials and methods
2.3. Instrumentations
AB Sciex QTRAP mass spectrometer (Model: API-4500, Foster City, USA), Agilent 1260 infinity HPLC system consisted of quaternary pump with inbuilt degasser (G1311B), autosampler (G13129B), thermostat (G1330B ALS) and variable wavelength detector (G7114B)(Agilent Technologies, USA) were used for detection of the NDSRI impurities. Data acquisition and processing were conducted using Analyst 1.7.3 software. pH of the buffer solution was tested with a Seven Excellence (Mettler, USA) digital pH meter. Deaeration was carried out using a USB 6.5 L ultrasonic bath (PCi Analyst).
The MS/MS detection was performed using an AB Sciex QTRAP-4500 mass spectrometer equipped with an electron spray ionization (ESI) source with positive mode. The source parameters were as follows (Table 3): Ion spray voltage, 5500 V, temperature, 550 °C, nebulizer gas (GS1), 50 psi; drying gas (GS2), 50 psi. During the analysis, all the analytes were identified based on their mass-to-charge (m/z) ratios, and the precursor and product ions were identified based on their mass abundance. The experiment was carried out in multiple reactions monitoring (MRM) mode, and it was used for scanning and acquiring mass data for quantification. The MRM parameters including quantifier ion, qualifier ion, declustering potential (DP), and collision energy (CE), were depicted in Table 4.
3. Result and discussion
3.1. Method development
The objective of this study was to develop and optimize an LC-MS method for the quantification of the NINA, NBOP, NBP, and NB impurities (NDSRIs) in Risperidone drug substances. The method development was initiated by evaluating the solubility of Risperidone and NDSRI impurities in different solvents and buffer solutions including water, acetonitrile, methanol, formic acid, and ammonium acetate. For sample preparation, the Threshold of Toxicological Concern (TTC) approach was followed. According to this approach, the acceptable limit for NINA was set at a maximum of 93.75 µg/g, while the limits for NBOP, NBP, and NB were set at a maximum of 25 µg/g in the Risperidone drug substance. Due to the extremely low detection limits required for these NDSRI impurities, it was not possible to achieve the necessary limit of quantification (LOQ) at 1 % of the acceptable daily intake (ADI) using conventional HPLC methods. Therefore, we selected an LC-MS method in multi-reaction monitoring (MRM) mode to achieve the required sensitivity and precision.
In order to minimize peak broadening and achieve an optimal separation between the NDSRI impurities and Risperidone, various mobile phase compositions and columns were tested. Initially, an ACE-3 PFP C18 (150 mm x 4.6 mm, 3 µm) column was selected and mobile phase compositions such as 10 mM ammonium acetate in acetonitrile, 10 mM ammonium acetate in methanol, 0.1 % formic acid in acetonitrile, and 0.1 % formic acid in methanol were evaluated, each tested with an initial ratio of 70:30 (v/v). The flow rate was set to 0.8 mL/min with the column temperature of 40 °C using linear gradient program. The results of these trials indicated that the NBP and NB impurity peaks co-eluted at similar retention times due to their structural similarities. However, a slight improvement in the separation between the NBP and NB impurities was observed when employing a mobile phase consisting of 0.1 % formic acid in methanol. Consequently, an additional trials were conducted by adjusting the pH of the formic acid (0.2 %, pH ∼ 2.1) and modifying the initial mobile phase ratio of 85:15 (v/v). The adjustment led to improved separation of all impurity peaks however, it also extended the run time of the method. Further modification of the initial mobile phase ratio to 65:35 (v/v), successfully reduced the run time while maintaining effective separation of impurity peaks. Thus, the final optimized conditions selected were as follows: a mobile phase consisting of 0.2 % formic acid and methanol, with an ACE-3 PFP C18 column (150 mm x 4.6 mm, 3 µm) under a gradient program. The gradient program was set as time / mobile phase-B: 0/35, 10/80, 14/80, 14.5/35, and 20/35. Final optimized chromatogram was shown in Fig. 1. In addition, the results obtained from ACE-3 PFP C18 (150 mm x 4.6 mm, 3 µm) column compared with two different chromatographic columns, Kromasil C18 (150 mm x 4.6 mm, 5 µm) and Inertsil ODS 3 V (150 mm x 4.6 mm, 5 µm) to assess the robustness of the method. The results indicated satisfactory separation of all the NDSRI impurities on both columns (see in supplymetry data).
Talanta Open, Volume 11, August 2025, 100416: Fig. 1. Final optimized chromatogram.
During method development, electrospray ionization (ESI) in positive ion detection mode was selected due to its significantly higher signal intensity compared to the negative mode. MRM parameters, including decluster potential (DP) and collision energy (CE), were optimized by injecting a composite standard solution directly into the mass spectrometer to achieve maximum response for all four NDSRI impurities of Risperidone. The mass analysis of the NDSRI impurities revealed distinct fragmentation patterns. For NINA, the quantifier ion peak [M+ H]+ was observed at m/z 159.1 which further fragmented by losing an -NO molecule leading to the formation of a qualifier ion at m/z 128.2 [M + H+−30]. Similarly, NBOP exhibited a quantifier ion peak [M + H]+ at m/z 223.1. Upon losing an -NO₂ molecule, a qualifier ion formed at m/z 128.2 [M + H+−46]. In the case of NBP, the most intense peak, considered as the quantifier ion, corresponded to [M + H+−16] observed at m/z 255.2. Additionally, a qualifier ion was detected at m/z 128.2 [M + H+−46] due to the loss of an -NO₂ molecule. For NB, the quantifier ion peak [M + H]+ was identified at m/z 250.2. The ion undergoes fragmentation by losing an -NO molecule, producing in a qualifier ion at m/z 220.1 [M + H+−30]. These results highlight the unique fragmentation pathways of each impurity, providing reliable quantifier and qualifier ions for their identification and characterization. The representative MS/MS spectra of the impurities are illustrated in Fig. 2.
Talanta Open, Volume 11, August 2025, 100416: Fig. 2. Qualifier and quantifier fragmented ions in mass spectrum of NINA, NBOP, NBP, and NB.
4. Conclusion
The study successfully developed and validated a highly sensitive and selective LC-MS/MS method for the determination and quantification of four nitrosamine drug substance-related impurities (NDSRIs) in Risperidone drug substance. By optimizing both chromatographic and mass spectrometric parameters, the method achieved high sensitivity and specificity, ensuring the accurate detection of impurities at trace levels. Validation results demonstrated that the method met all regulatory requirements, exhibiting excellent linearity, precision, accuracy, and recovery for the impurities NINA, NBOP, NBP, and NB. The LC-MS/MS procedure has been successfully applied to the pharmaceutical dosage forms of Risperidone and is reliable for routine quality control analysis and serves as a robust tool for ensuring the safety and regulatory compliance of Risperidone (Risperdal) drug substances. The approach significantly contributes to the monitoring of nitrosamine impurities in pharmaceuticals, ensuring patient safety.
