UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis

UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis RAMA KRISHNA MYNENI, Govardhan rao Thalluri, Ramakrishna K This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8397946/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract The detection and stringent control of N-nitrosamine impurities in pharmaceutical products is a critical imperative for regulatory agencies and the global pharmaceutical industry, driven by the potent genotoxic and carcinogenic potential inherent to these compounds. This research work describes the development and rigorous validation of a highly sensitive Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQ MS) method specifically designed for the quantification of N-Nitroso-N-Desmethyl Methadone in Methadone Hydrochloride (HCL) Tablets (10 mg). Chromatographic separation was successfully achieved utilizing an ACQUITY UPLC BEH Shield RP 18 stationary phase under an isocratic elution program composed of ammonium acetate buffer and acetonitrile. Mass spectrometric detection was executed via electrospray ionization in positive mode (ESI+) employing Multiple Reaction Monitoring (MRM) to guarantee maximal selectivity for the two isomeric peaks of the impurity. The analytical method was validated in strict conformity with USP and ICH Q2(R1) guidelines. Linearity was demonstrated over a concentration range of 0.33 ng/mL to 6.67 ng/mL, corresponding to 10% to 200% of the established specification limit. The limit of quantification (LOQ) was confirmed at 0.33 ng/mL (0.33 ppb), verifying the method's capacity to quantify trace levels significantly below the acceptable intake. Precision assessments yielded a cumulative relative standard deviation (RSD) of 3.7%, while accuracy studies demonstrated mean recoveries ranging from 87.5% to 93.5%. The proposed method represents a robust, specific, and reliable analytical tool for the routine quality control of Methadone HCL, ensuring patient safety through the effective monitoring of mutagenic impurities. N-Nitroso-N-Desmethyl Methadone Methadone HCl UPLC-MS/MS Genotoxicity Method Validation Trace Analysis Figures Figure 1 1. Introduction 1.1 Regulatory Landscape and N-Nitrosamine Crisis in Pharmaceuticals In recent years, the pharmaceutical industry has been profoundly impacted by the identification of N-nitrosamine impurities in a spectrum of widely used drug substances [1–3]. N-nitrosamines constitute a class of chemical compounds recognized as probable human carcinogens, a classification that has precipitated widespread product recalls and necessitated the enforcement of rigorous regulatory control strategies [4]. The International Council for Harmonisation (ICH) M7(R1) provides a guideline for the assessment and control of DNA reactive (mutagenic) impurities to mitigate potential carcinogenic risks to patients [5]. Concurrently, the United States Pharmacopeia (USP) has promulgated General Chapter Nitrosamine Impurities , which delineates the analytical prerequisites for monitoring these contaminants [6]. 1.2 Methadone Hydrochloride: Pharmacological Context and N-Nitrosamine Formation Methadone Hydrochloride, a synthetic opioid agonist, is extensively utilized in the management of severe pain and opioid dependence. Structurally, methadone contains amine which, in the presence of nitrosating agents such as nitrites ubiquitously present in excipients, water, or manufacturing equipment can undergo N-nitrosation to yield N-Nitroso-N-Desmethyl Methadone [9–10]. Given the structural homologies to other known potent mutagenic nitrosamines, stringent regulatory limits are imperative. An acceptable daily intake (ADI) limit of 400 ng/day has been established for this impurity [12]. Considering the maximum daily dosage of Methadone HCL is 120 mg/day, this ADI translates to a specification limit of 3.33 ppm in the drug product. 1.3 Analytical Challenges and Technological Solutions Conventional analytical techniques generally fail to provide the requisite sensitivity and selectivity necessary to quantify these impurities at the parts-per-billion (ppb) level within complex sample matrices [13]. Consequently, mass spectrometry (MS) hyphenated with liquid chromatography, specifically Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQ MS), has been established as the gold standard for such trace analyses [14–15]. This work presents the development and validation of a UPLC-TQ MS method for the determination of N-Nitroso-N-Desmethyl Methadone in Methadone HCL Tablets (10 mg). The validation encompasses the evaluation of critical parameters including specificity, linearity, accuracy, precision, and robustness in accordance with ICH Q2(R1) guidelines [17]. 2. Materials and Methods 2.1 Materials and Reagents The reference standard for N-Nitroso-N-Desmethyl Methadone was secured with a certified potency of 96.84%. Validation studies utilized Methadone HCL Tablets (10 mg) as the drug product. To minimize background interference during mass spectrometric detection, all reagents employed were of high analytical purity. Acetonitrile (HPLC grade), Ammonium Acetate (ACS grade), and Formic Acid (ACS grade) were sourced from established commercial vendors (Sigma Aldrich). High-purity water was generated via a Milli-Q water purification system. Sample filtration was conducted using nylon membrane filters with a pore size of 0.45 µm. IUPAC Name N-Nitroso-N-Desmethyl Methadone (N-methyl-N-(5-oxo-4,4-diphenylheptan-2yl) nitrous amide Impurity) Figure 1 : Chemical structure of N-Nitroso-N-Desmethyl Methadone 2.2 Instrumentation and Chromatographic Configurations Analytical measurements were performed using a Waters Acquity I Class UPLC system interfaced with a Xevo TQS Micro Triple Quadrupole Mass Spectrometer. Chromatographic separation was executed on an ACQUITY UPLC BEH Shield RP 18 column (1.7 µm particle size, 2.1 x 100 mm dimensions) maintained at a constant temperature of 35°C. The mobile phase system comprised: Mobile Phase A : 5 mM ammonium acetate and 0.1% formic acid in water (0.5 g ammonium acetate in 1000 mL water + 1.0 mL formic acid). Mobile Phase B : Acetonitrile. The method employed a flow rate of 0.2 mL/min with a mobile phase composition of 35% Mobile Phase B and 65% Mobile Phase A. The injection volume was 5 µL, and the autosampler temperature was regulated at 10°C to ensure sample stability (Chromatographic conditions Table S 1). 2.3 Mass Spectrometric Detection Parameters The mass spectrometer operated in Electrospray Ionization (ESI) positive mode. The main operating parameters included a source temperature of 120°C and a desolvation temperature of 600°C. Desolvation gas flow and cone gas flow were optimized at 600 L/hr and 150 L/hr, respectively. Quantitation was executed in Multiple Reaction Monitoring (MRM) mode monitoring two transitions (Mass spectrometer Method Conditions Table S 2). 2.4 Preparation of Standard and Sample Solutions A diluent composed of Milli-Q water and Methanol in a 65:35 volume ratio was used for all preparations. Standard Preparation : A stock solution of N-Nitroso-N-Desmethyl Methadone was prepared in methanol. This was serially diluted to generate a working standard solution at a concentration of 3.34 ppb (0.00334 ppm), aligning with the specification level. Sample Preparation : NLT 10 tablets were weighed to determine average tablet weight. Powder equivalent to 10 mg Methadone HCL was transferred to a 10 mL flask, extracted with diluent via sonication for 20 minutes, and made up to volume. The solution was filtered through a 0.45 µm nylon filter, discarding the first 2 mL. 2.5 Method Validation Strategy The analytical method validation protocol followed the comprehensive framework established in ICH Q2(R1) guidelines, encompassing systematic evaluation of six primary validation attributes: Specificity Assessment Chromatographic analysis of blank matrices (methadone tablets free of N-Nitroso-N-Desmethyl Methadone, obtained through appropriate sample preparation procedures), pure reference standards of N-Nitroso-N-Desmethyl Methadone, and spiked matrix samples confirmed the method's ability to specifically identify and differentiate the target analyte from all potential interferents, excipients, and degradation products present in the pharmaceutical matrix. Linearity Determination A series of N-Nitroso-N-Desmethyl Methadone reference standards spanning a concentration range from 0.33 ng/mL to 6.67 ng/mL (representing 10% to 200% of the established specification limit) were analyzed in replicate to establish the linear dynamic range of the method. Linear regression analysis of the relationship between injected concentration and measured response (peak area) determined the correlation coefficient, slope, and intercept values that characterize method linearity. Limit of Quantification (LOQ) and Limit of Detection (LOD) Determination The lowest concentration of N-Nitroso-N-Desmethyl Methadone that could be reliably quantified with acceptable precision and accuracy was established as the LOQ (0.33 ng/mL, or 0.33 ppb), while the LOD representing the lowest detectable concentration was confirmed through analysis of appropriately diluted reference standards. Accuracy and Recovery Studies Methadone tablet samples were deliberately spiked with known quantities of N-Nitroso-N-Desmethyl Methadone at multiple concentration levels (typically 50%, 100%, and 150% of the specification limit) and analyzed in replicate. The percentage recovery, calculated as (measured concentration / spiked concentration) × 100%, was determined at each level to assess the method's quantitative accuracy. Precision Assessment Replicate analyses (typically n = 6) of N-Nitroso-N-Desmethyl Methadone reference standards and spiked matrix samples at established concentration levels were performed under identical conditions to determine intra-assay precision. The relative standard deviation (RSD) was calculated to quantify the magnitude of analytical variability across replicate measurements. Robustness Evaluation The method's resilience to minor but deliberate variations in critical analytical parameters (such as mobile phase pH, temperature, flow rate, and column lot variations) was systematically assessed to confirm that method performance remained within acceptance criteria despite these anticipated real-world variations. 3. Results and Analytical Performance 3.1 System Suitability and Specificity System suitability was evaluated by analyzing six replicate injections of the working standard solution. The relative standard deviation (RSD) for the peak areas of the two isomers (Peak-1 and Peak-2) was calculated to ensure the system's performance was acceptable. Additionally, bracketing standards were analyzed to verify system stability throughout the run The %RSD values were well within the acceptance limit of 15.0%, and the bracketing standards demonstrated consistent recovery, confirming the stability of the system during analysis (Table S3). 3.2 Specificity Assessment Specificity was assessed to verify the method's ability to determine the analyte in the presence of the matrix. Chromatograms of the blank diluent and placebo formulations for 10 mg tablets were acquired. No interfering peaks were observed at the retention times of the analyte isomers (Peak-1: ~4.55 min; Peak-2: ~4.94 min). This confirms that the excipients and diluent do not interfere with the quantification of the impurity (Figure S 1). 3.3 Linearity and Range Linearity was established by analyzing a series of standard solutions ranging from the Limit of Quantification (LOQ) to 200% of the specification limit (0.33 ng/mL to 6.67 ng/mL). The calibration curve was constructed by plotting the peak area against concentration. Regression analysis yielded a correlation coefficient (r) > 0.99 for both peaks, indicating excellent linearity. Regression analysis yielded a correlation coefficient (r) > 0.99 for both peaks, indicating excellent linearity (Table S 4 and Figure S 2). 3.4 Sensitivity (Limit of Detection and Quantification) Sensitivity was determined based on the Signal-to-Noise (S/N) ratio. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were established at concentrations where the S/N ratios were greater than 3 and 10, respectively. The method demonstrated high sensitivity with an LOQ of 0.33 ng/mL (0.33 ppb), which is 10% of the specification limit, ensuring reliable quantification of trace amounts (Table S 5 and Figure S 3). 3.4 Precision Method precision (repeatability) was evaluated by preparing six independent samples of the Methadone HCL Tablets (10 mg) spiked at the specification level (3.33 ppm). The %RSD of 4.7% is well within the acceptance limit of NMT 15.0%, demonstrating good repeatability (Table S 6). 3.6 Intermediate Precision Intermediate precision was assessed by repeating the analysis on a different day, by a different analyst, and using a different column. The cumulative %RSD of 3.7% (Method Precision + Intermediate Precision) confirms the ruggedness of the method (Table S 7). 3.7 Accuracy Accuracy was determined through recovery studies at three concentration levels: LOQ (10%), 100%, and 200% of the specification limit. The study was conducted in triplicate for the drug product. All mean recovery values fell within the specified acceptance ranges, confirming the method's accuracy (Table S 8 and Figure S 4). 3.8 Robustness Robustness was evaluated by deliberately varying critical parameters, including flow rate (± 0.02 mL/min) and column temperature (± 5°C). The %RSD remained below 15.0% under all conditions, indicating that the method is robust and reliable despite small variations in operating parameters (Table S 9) 3.9 Solution Stability and Filtration Solution stability was assessed by storing standard and sample solutions at room temperature for 24 hours. Additionally, a filtration study comparing centrifuged samples vs. filtered samples (0.45 µm nylon) showed a relative difference of 5%, which is within the acceptance limit of 15%, confirming no significant analyte adsorption (Table S 10). 4. Quality Control Applications and Patient Safety Implications The practical implementation of this validated analytical method in routine quality control operations provides pharmaceutical manufacturers with a powerful tool for comprehensive monitoring of N-nitroso-related mutagenic impurities in methadone hydrochloride tablets. The method's exceptional sensitivity, enabling detection and quantification at 0.33 ppb, ensures that impurity levels remain substantially below established acceptable intake thresholds, providing a substantial safety margin between detected impurity concentrations and regulatory limits. The high selectivity achieved through MRM detection prevents false positives arising from co-eluting matrix components, ensuring regulatory agencies and healthcare systems receive accurate, reliable information regarding product safety and purity. The 3.7% precision ensures that analytical decisions based on this method—including acceptance or rejection of batches—reflect genuine product characteristics rather than analytical artifacts or random method variability. The accuracy characteristics guarantee that reported impurity concentrations accurately reflect true analyte levels, critical information for establishing batch safety profiles and supporting regulatory submissions. The application of this analytical technology to methadone hydrochloride specifically addresses pharmaceutical products utilized in opioid substitution therapy and pain management contexts, where patient populations may include individuals with heightened health vulnerabilities. Ensuring the purity and safety of these medications through rigorous analytical quality control represents a significant component of comprehensive patient protection strategies. The method's capability to reliably detect and quantify trace impurities at ppb levels provides regulatory confidence that manufacturing processes maintain stringent control over chemical purity throughout production and storage intervals. 5. Authenticity and Novelty of the Analytical Method Development This research work presents the comprehensive development and rigorous validation of a UPLC-TQ MS method specifically engineered for the precise determination of N-Nitroso-N-Desmethyl Methadone in Methadone Hydrochloride Tablets (10 mg formulation strength). The analytical method validation encompasses systematic evaluation of critical performance parameters including specificity (selectivity), linearity over appropriate concentration ranges, accuracy through recovery studies, precision through replicate analysis assessments, and robustness under deliberately varied analytical conditions, all conducted in strict accordance with internationally accepted ICH Q2(R1) guidelines and complementary USP requirements. The development strategy prioritizes achievement of sensitivity levels substantially below the established specification limit, ensuring sufficient analytical margin for regulatory decision-making and quality assurance operations in routine pharmaceutical manufacturing environments. 6. Discussions 6.1 Method Development Rationale and Analytical Strategy The selection of UPLC-TQ MS as the analytical platform for N-Nitroso-N-Desmethyl Methadone quantification in methadone hydrochloride tablets reflects careful consideration of the unique analytical challenges posed by ultra-trace impurity monitoring in complex pharmaceutical matrices. The exceptional sensitivity requirement (achieving 0.33 ppb LOQ), coupled with the stringent selectivity demand necessitated by potential matrix interferences in tablet formulations containing numerous excipients, effectively eliminates conventional chromatographic methods from consideration. The UPLC-TQ MS platform uniquely combines the separation efficiency of modern ultra-high-performance liquid chromatography with the unparalleled selectivity afforded by multiple reaction monitoring mass spectrometric detection, enabling precise quantification of N-Nitroso-N-Desmethyl Methadone far below the established specification limit. The isocratic mobile phase approach, rather than gradient elution commonly employed in traditional HPLC methods, provides significant advantages for routine pharmaceutical quality control operations. Isocratic separation ensures rapid system equilibration between sequential analyses, reduces overall analytical run time, and enhances method reproducibility by eliminating the potential variability introduced by gradient programming and column conditioning cycles inherent to gradient methods. The ammonium acetate/acetonitrile mobile phase composition was specifically selected to optimize both the chromatographic behavior of the target analyte and the electrospray ionization efficiency in the mass spectrometric detection stage, reflecting an integrated analytical design that optimizes performance across the entire separation-detection continuum. The deliberate selection of the ACQUITY UPLC BEH Shield RP 18 stationary phase, a specialized reversed-phase material engineered with embedded polar functional groups that reduce problematic interactions between analytes and residual silanol sites on the column surface, provides superior peak shape and reduced tailing characteristics particularly valuable when analyzing compounds prone to non-ideal chromatographic behavior. The 1.7 µm particle size characteristic of modern UPLC columns enables higher operational flow rates and enhanced separation efficiency compared to conventional 5 µm HPLC columns, contributing substantially to the overall method sensitivity and chromatographic resolution achievable in this application. 6.2 Validation Results in Context of Regulatory Requirements The comprehensive validation results demonstrate full compliance with ICH Q2(R1) and USP guidelines, establishing the method as a scientifically robust analytical tool suitable for regulatory submissions and routine pharmaceutical quality control applications. The LOQ of 0.33 ng/mL (0.33 ppb) represents approximately 10% of the established 3.33 ppm specification limit, providing substantial analytical margin that ensures reliable confirmation of compliance with regulatory requirements and enables detection of trace impurity levels with considerable safety margin. This sensitivity level substantially exceeds the capability of conventional analytical methods and reflects the extraordinary instrumental capabilities of modern UPLC-TQ MS technology. The linearity correlation coefficient exceeding 0.99 across the twentyfold concentration range from LOQ to 200% of specification confirms that the relationship between injected concentration and analytical response remains strictly proportional throughout the validated range, enabling accurate interpolation for sample quantification across all anticipated quality control scenarios. The 3.7% RSD precision value, substantially below the typical 15% acceptance criterion for trace-level methods, demonstrates exceptional reproducibility that ensures analytical decisions regarding batch acceptance or rejection reflect genuine product characteristics rather than analytical artifacts. The 87.5–93.5% recovery range confirms accurate quantification with minimal systematic error, while the demonstrated robustness across deliberately varied analytical parameters establishes that the method will perform consistently across different laboratory environments and instrumental platforms. 6.3 Practical Quality Control Applications The validated UPLC-TQ MS method provides pharmaceutical manufacturers with a powerful analytical tool for comprehensive routine quality control monitoring of N-nitroso-related mutagenic impurities in methadone hydrochloride tablets throughout manufacturing, stability testing, and commercial distribution. The method's exceptional sensitivity enables detection and precise quantification at 0.33 ppb, ensuring that impurity levels remain substantially below established acceptable intake thresholds while providing manufacturers with early warning of potential manufacturing process deviations that might lead to impurity formation. The high selectivity achieved through MRM detection prevents false-positive results arising from coeluting matrix components, ensuring regulatory agencies and healthcare systems receive accurate, reliable information regarding product safety and purity. The demonstrated precision ensures that analytical results reliably reflect true product characteristics, critical for batch release decisions and regulatory compliance determinations. The method's robust performance across varied analytical conditions establishes confidence that quality control laboratories operating the method will achieve consistent, reliable results regardless of minor variations in laboratory environment, instrument settings, or operational procedures. This robustness is particularly important for maintaining analytical consistency across multiple manufacturing sites operating the same method and for ensuring that analytical results remain valid when the method is transferred to different laboratory facilities. The validated method serves as a scientific foundation for establishing appropriate in-process controls and specification limits that ensure consistent product quality throughout manufacturing operations. 6.4 Regulatory Implications and Public Health Significance The successful development and validation of this highly sensitive UPLC-TQ MS method addresses a critical public health imperative reflected in the rigorous regulatory requirements imposed by ICH M7(R1) and USP guidelines. The method's capability to reliably detect and quantify N-Nitroso-N-Desmethyl Methadone at parts-per-billion levels provides assurance that methadone hydrochloride tablets distributed to patients contain impurity levels far below those predicted to pose carcinogenic risk. This analytical capability represents a significant component of comprehensive regulatory strategies designed to protect public health through rigorous quality assurance of pharmaceutical products. The method's validation in accordance with internationally harmonized guidelines establishes mutual recognition across different regulatory jurisdictions, facilitating consistent quality standards for methadone products distributed globally and reducing the regulatory burden of separate method validations for different markets. The method's demonstrated performance characteristics support regulatory decision-making regarding product safety, manufacturing process validation, and stability testing protocols essential for ensuring the continued safety and efficacy of methadone formulations throughout their commercial shelf-life. Regulatory agencies can rely on analytically validated data generated through this method when making determinations regarding product approvals, manufacturing facility inspections, and compliance with established impurity specifications. 7. Conclusion A sensitive, specific, and robust UPLC-TQ MS method was successfully developed and validated for the quantification of N-Nitroso-N-Desmethyl Methadone in Methadone HCL Tablets (10 mg). The method demonstrates excellent linearity, precision, and accuracy across the validation range. The LOQ of 0.33 ppb facilitates the quantification of this genotoxic impurity well below the daily intake limit of 400 ng/day (3.33 ppm). Validation data confirms compliance with USP and ICH guidelines, establishing the method as suitable for routine quality control applications. Declarations Ethics approval and consent to participate: Not applicable as this study did not involve human subjects or animals. Consent for publication: Not applicable. Availability of data and material: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. The chromatographic data, calibration curves, and validation results are included in this published article and its supplementary materials. Competing interests: The authors declare that they have no competing interests. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Studies involving plants Not applicable as this study did not involve any plant materials Authors' contributions govardhan rao Thalluri & Ramakrishna K: Conceptualization, methodology, formal analysis, investigation, writing - original draft govardhan rao Thalluri & Ramakrishna K: Validation, data curation, writing - review & editing govardhan rao Thalluri & Ramakrishna K: Software, resources, visualization RAMA KRISHNA MYNENI: Supervision, project administration All authors have read and approved the final manuscript. Acknowledgments The authors extend their sincere gratitude to Biogenico Private Limited . for their invaluable support throughout the execution of this research. Their contributions—ranging from providing essential materials and technical expertise to facilitating experimental setups and analytical resources—played a pivotal role in the successful completion of this study. The collaboration with Hetero Labs Ltd. not only ensured access to state-of-the-art infrastructure but also fostered an environment of scientific rigor and innovation. The team deeply appreciates their commitment to advancing pharmaceutical research and their continued partnership in furthering the development of high-quality therapeutic solutions. References Schmidtsdorff S, Schmidt AH. Simultaneous detection of nitrosamines in sartans by HPLC-MS/MS. J Pharm Biomed Anal . 2019;167:138-144. Parr MK, Joseph JF. NDMA in metformin: Analysis of the recall and analytical challenges. J Pharm Biomed Anal . 2019;164:536-549. Abe Y, Yamamoto H, Hirai T, et al. Control of N-nitrosodimethylamine in ranitidine drug substance and drug products. Chem Pharm Bull (Tokyo) . 2020;68(10):927-931. Teasdale A. Nitrosamine Control: From Risk Assessment to Analytical Testing with Emphasis on Sample Preparation and Phase-Appropriate Method Validation. Org Process Res Dev . 2023;27(11):1939–1969. International Council for Harmonisation (ICH). Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk M7(R1). Geneva: ICH; 2017. United States Pharmacopeia (USP). General Chapter Nitrosamine Impurities. In: USP-NF. Rockville, MD: United States Pharmacopeial Convention; 2021. US Food and Drug Administration (FDA). Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry. Silver Spring, MD: FDA; 2021. European Medicines Agency (EMA). Assessment report: Nitrosamine impurities in human medicinal products. EMA/CHMP/428592/2019. Amsterdam: EMA; 2020. Ashworth I, Dirat O, Teasdale A, Whiting M. Potential for the formation of N-nitrosamines during the manufacture of active pharmaceutical ingredients: An assessment of the risk posed by trace nitrite in water. Org Process Res Dev . 2020;24(9):1629-1646. Smith PA, Loeppky RN. Nitrosamine formation: The role of nitrogen oxides and related species. J Am Chem Soc . 1967;89(5):1147-1157. Adhikari P, Lee J, Kim J, Kim J, Lee J. Screening of Nitrosamine Impurities in Sartan Pharmaceuticals by GC-MS/MS. Mass Spectrom Lett . 2021;12(2):31-40. Thresher A, Foster R, Ponting DJ, et al. N-nitrosamine impurity limits: A systematic approach to establishing acceptable intakes (AIs). Regul Toxicol Pharmacol . 2020;116:104749. Solanki R, Wadhwana P, Patel R, et al. UHPLC-APCI-TQ-MS analytical method capable of quantifying eight nitrosamine impurities from five different commercially available Metformin formulations. J Pharm Sci . 2023;112(7):1933-1940. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem . 2003;75(13):3019-3030. Nagdella NK, Shaik H, Subramanyam SB, et al. Development, validation, and estimation of measurement uncertainty for the quantitative determination of nitrosamines in Sartan drugs using liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. J Chromatogr Open . 2022;2:100053. Miralles P, Chisvert A, Salvador A. Determination of N-nitrosamines in cosmetic products by vortex-assisted reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with mass spectrometry. J Sep Sci . 2018;41(15):3143-3151. International Council for Harmonisation (ICH). Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva: ICH; 2005. Bylda C, Thiele R, Kobold U, Volmer DA. Recent advances in sample preparation techniques to overcome matrix effects in bioanalysis. Anal Chem . 2014;86(19):9761-9771. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":48875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structure of N-Nitroso-N-Desmethyl Methadone\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8397946/v1/7c2a247ada5309971dc369b5.png"},{"id":100414613,"identity":"fa107fe2-430f-4331-896c-25369fe75eac","added_by":"auto","created_at":"2026-01-16 13:19:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1069615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8397946/v1/4e663cf7-9c81-4812-8f71-d0fb892b0d50.pdf"},{"id":100406811,"identity":"e1ee6e26-e161-40ff-bc10-86f114fd20a9","added_by":"auto","created_at":"2026-01-16 13:03:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":660475,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryManual.docx","url":"https://assets-eu.researchsquare.com/files/rs-8397946/v1/2d68af3ae625807e49db7f69.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Regulatory Landscape and N-Nitrosamine Crisis in Pharmaceuticals\u003c/h2\u003e \u003cp\u003eIn recent years, the pharmaceutical industry has been profoundly impacted by the identification of N-nitrosamine impurities in a spectrum of widely used drug substances [1\u0026ndash;3]. N-nitrosamines constitute a class of chemical compounds recognized as probable human carcinogens, a classification that has precipitated widespread product recalls and necessitated the enforcement of rigorous regulatory control strategies [4]. The International Council for Harmonisation (ICH) M7(R1) provides a guideline for the assessment and control of DNA reactive (mutagenic) impurities to mitigate potential carcinogenic risks to patients [5]. Concurrently, the United States Pharmacopeia (USP) has promulgated General Chapter\u0026thinsp;\u0026lt;\u0026thinsp;1469\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eNitrosamine Impurities\u003c/em\u003e, which delineates the analytical prerequisites for monitoring these contaminants [6].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Methadone Hydrochloride: Pharmacological Context and N-Nitrosamine Formation\u003c/h2\u003e \u003cp\u003eMethadone Hydrochloride, a synthetic opioid agonist, is extensively utilized in the management of severe pain and opioid dependence. Structurally, methadone contains amine which, in the presence of nitrosating agents such as nitrites ubiquitously present in excipients, water, or manufacturing equipment can undergo N-nitrosation to yield N-Nitroso-N-Desmethyl Methadone [9\u0026ndash;10]. Given the structural homologies to other known potent mutagenic nitrosamines, stringent regulatory limits are imperative. An acceptable daily intake (ADI) limit of 400 ng/day has been established for this impurity [12]. Considering the maximum daily dosage of Methadone HCL is 120 mg/day, this ADI translates to a specification limit of 3.33 ppm in the drug product.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Analytical Challenges and Technological Solutions\u003c/h2\u003e \u003cp\u003eConventional analytical techniques generally fail to provide the requisite sensitivity and selectivity necessary to quantify these impurities at the parts-per-billion (ppb) level within complex sample matrices [13]. Consequently, mass spectrometry (MS) hyphenated with liquid chromatography, specifically Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQ MS), has been established as the gold standard for such trace analyses [14\u0026ndash;15].\u003c/p\u003e \u003cp\u003eThis work presents the development and validation of a UPLC-TQ MS method for the determination of N-Nitroso-N-Desmethyl Methadone in Methadone HCL Tablets (10 mg). The validation encompasses the evaluation of critical parameters including specificity, linearity, accuracy, precision, and robustness in accordance with ICH Q2(R1) guidelines [17].\u003c/p\u003e \u003c/div\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e \u003cp\u003eThe reference standard for N-Nitroso-N-Desmethyl Methadone was secured with a certified potency of 96.84%. Validation studies utilized Methadone HCL Tablets (10 mg) as the drug product. To minimize background interference during mass spectrometric detection, all reagents employed were of high analytical purity. Acetonitrile (HPLC grade), Ammonium Acetate (ACS grade), and Formic Acid (ACS grade) were sourced from established commercial vendors (Sigma Aldrich). High-purity water was generated via a Milli-Q water purification system. Sample filtration was conducted using nylon membrane filters with a pore size of 0.45 \u0026micro;m.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIUPAC Name\u003c/strong\u003e \u003cp\u003eN-Nitroso-N-Desmethyl Methadone (N-methyl-N-(5-oxo-4,4-diphenylheptan-2yl) nitrous amide Impurity)\u003c/p\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: \u003cb\u003eChemical structure of N-Nitroso-N-Desmethyl Methadone\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instrumentation and Chromatographic Configurations\u003c/h2\u003e \u003cp\u003eAnalytical measurements were performed using a Waters Acquity I Class UPLC system interfaced with a Xevo TQS Micro Triple Quadrupole Mass Spectrometer. Chromatographic separation was executed on an ACQUITY UPLC BEH Shield RP 18 column (1.7 \u0026micro;m particle size, 2.1 x 100 mm dimensions) maintained at a constant temperature of 35\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe mobile phase system comprised:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMobile Phase A\u003c/b\u003e: 5 mM ammonium acetate and 0.1% formic acid in water (0.5 g ammonium acetate in 1000 mL water\u0026thinsp;+\u0026thinsp;1.0 mL formic acid).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMobile Phase B\u003c/b\u003e: Acetonitrile.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe method employed a flow rate of 0.2 mL/min with a mobile phase composition of 35% Mobile Phase B and 65% Mobile Phase A. The injection volume was 5 \u0026micro;L, and the autosampler temperature was regulated at 10\u0026deg;C to ensure sample stability (Chromatographic conditions Table S 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Mass Spectrometric Detection Parameters\u003c/h2\u003e \u003cp\u003eThe mass spectrometer operated in Electrospray Ionization (ESI) positive mode. The main operating parameters included a source temperature of 120\u0026deg;C and a desolvation temperature of 600\u0026deg;C. Desolvation gas flow and cone gas flow were optimized at 600 L/hr and 150 L/hr, respectively. Quantitation was executed in Multiple Reaction Monitoring (MRM) mode monitoring two transitions (Mass spectrometer Method Conditions Table S 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of Standard and Sample Solutions\u003c/h2\u003e \u003cp\u003eA diluent composed of Milli-Q water and Methanol in a 65:35 volume ratio was used for all preparations.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eStandard Preparation\u003c/b\u003e: A stock solution of N-Nitroso-N-Desmethyl Methadone was prepared in methanol. This was serially diluted to generate a working standard solution at a concentration of 3.34 ppb (0.00334 ppm), aligning with the specification level.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSample Preparation\u003c/b\u003e: NLT 10 tablets were weighed to determine average tablet weight. Powder equivalent to 10 mg Methadone HCL was transferred to a 10 mL flask, extracted with diluent via sonication for 20 minutes, and made up to volume. The solution was filtered through a 0.45 \u0026micro;m nylon filter, discarding the first 2 mL.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Method Validation Strategy\u003c/h2\u003e \u003cp\u003eThe analytical method validation protocol followed the comprehensive framework established in ICH Q2(R1) guidelines, encompassing systematic evaluation of six primary validation attributes:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSpecificity Assessment\u003c/strong\u003e \u003cp\u003eChromatographic analysis of blank matrices (methadone tablets free of N-Nitroso-N-Desmethyl Methadone, obtained through appropriate sample preparation procedures), pure reference standards of N-Nitroso-N-Desmethyl Methadone, and spiked matrix samples confirmed the method's ability to specifically identify and differentiate the target analyte from all potential interferents, excipients, and degradation products present in the pharmaceutical matrix.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLinearity Determination\u003c/strong\u003e \u003cp\u003eA series of N-Nitroso-N-Desmethyl Methadone reference standards spanning a concentration range from 0.33 ng/mL to 6.67 ng/mL (representing 10% to 200% of the established specification limit) were analyzed in replicate to establish the linear dynamic range of the method. Linear regression analysis of the relationship between injected concentration and measured response (peak area) determined the correlation coefficient, slope, and intercept values that characterize method linearity.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLimit of Quantification (LOQ) and Limit of Detection (LOD) Determination\u003c/strong\u003e \u003cp\u003eThe lowest concentration of N-Nitroso-N-Desmethyl Methadone that could be reliably quantified with acceptable precision and accuracy was established as the LOQ (0.33 ng/mL, or 0.33 ppb), while the LOD representing the lowest detectable concentration was confirmed through analysis of appropriately diluted reference standards.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAccuracy and Recovery Studies\u003c/strong\u003e \u003cp\u003eMethadone tablet samples were deliberately spiked with known quantities of N-Nitroso-N-Desmethyl Methadone at multiple concentration levels (typically 50%, 100%, and 150% of the specification limit) and analyzed in replicate. The percentage recovery, calculated as (measured concentration / spiked concentration) \u0026times; 100%, was determined at each level to assess the method's quantitative accuracy.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePrecision Assessment\u003c/strong\u003e \u003cp\u003eReplicate analyses (typically n\u0026thinsp;=\u0026thinsp;6) of N-Nitroso-N-Desmethyl Methadone reference standards and spiked matrix samples at established concentration levels were performed under identical conditions to determine intra-assay precision. The relative standard deviation (RSD) was calculated to quantify the magnitude of analytical variability across replicate measurements.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRobustness Evaluation\u003c/strong\u003e \u003cp\u003eThe method's resilience to minor but deliberate variations in critical analytical parameters (such as mobile phase pH, temperature, flow rate, and column lot variations) was systematically assessed to confirm that method performance remained within acceptance criteria despite these anticipated real-world variations.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Analytical Performance","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 System Suitability and Specificity\u003c/h2\u003e \u003cp\u003eSystem suitability was evaluated by analyzing six replicate injections of the working standard solution. The relative standard deviation (RSD) for the peak areas of the two isomers (Peak-1 and Peak-2) was calculated to ensure the system's performance was acceptable. Additionally, bracketing standards were analyzed to verify system stability throughout the run\u003c/p\u003e \u003cp\u003eThe %RSD values were well within the acceptance limit of 15.0%, and the bracketing standards demonstrated consistent recovery, confirming the stability of the system during analysis (Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Specificity Assessment\u003c/h2\u003e \u003cp\u003eSpecificity was assessed to verify the method's ability to determine the analyte in the presence of the matrix. Chromatograms of the blank diluent and placebo formulations for 10 mg tablets were acquired. No interfering peaks were observed at the retention times of the analyte isomers (Peak-1: ~4.55 min; Peak-2: ~4.94 min). This confirms that the excipients and diluent do not interfere with the quantification of the impurity (Figure S 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Linearity and Range\u003c/h2\u003e \u003cp\u003eLinearity was established by analyzing a series of standard solutions ranging from the Limit of Quantification (LOQ) to 200% of the specification limit (0.33 ng/mL to 6.67 ng/mL). The calibration curve was constructed by plotting the peak area against concentration. Regression analysis yielded a correlation coefficient (r)\u0026thinsp;\u0026gt;\u0026thinsp;0.99 for both peaks, indicating excellent linearity. Regression analysis yielded a correlation coefficient (r)\u0026thinsp;\u0026gt;\u0026thinsp;0.99 for both peaks, indicating excellent linearity (Table S 4 and Figure S 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Sensitivity (Limit of Detection and Quantification)\u003c/h2\u003e \u003cp\u003eSensitivity was determined based on the Signal-to-Noise (S/N) ratio. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were established at concentrations where the S/N ratios were greater than 3 and 10, respectively.\u003c/p\u003e \u003cp\u003eThe method demonstrated high sensitivity with an LOQ of 0.33 ng/mL (0.33 ppb), which is 10% of the specification limit, ensuring reliable quantification of trace amounts (Table S 5 and Figure S 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Precision\u003c/h2\u003e \u003cp\u003eMethod precision (repeatability) was evaluated by preparing six independent samples of the Methadone HCL Tablets (10 mg) spiked at the specification level (3.33 ppm).\u003c/p\u003e \u003cp\u003eThe %RSD of 4.7% is well within the acceptance limit of NMT 15.0%, demonstrating good repeatability (Table S 6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Intermediate Precision\u003c/h2\u003e \u003cp\u003eIntermediate precision was assessed by repeating the analysis on a different day, by a different analyst, and using a different column.\u003c/p\u003e \u003cp\u003eThe cumulative %RSD of 3.7% (Method Precision\u0026thinsp;+\u0026thinsp;Intermediate Precision) confirms the ruggedness of the method (Table S 7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Accuracy\u003c/h2\u003e \u003cp\u003eAccuracy was determined through recovery studies at three concentration levels: LOQ (10%), 100%, and 200% of the specification limit. The study was conducted in triplicate for the drug product.\u003c/p\u003e \u003cp\u003eAll mean recovery values fell within the specified acceptance ranges, confirming the method's accuracy (Table S 8 and Figure S 4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Robustness\u003c/h2\u003e \u003cp\u003eRobustness was evaluated by deliberately varying critical parameters, including flow rate (\u0026plusmn;\u0026thinsp;0.02 mL/min) and column temperature (\u0026plusmn;\u0026thinsp;5\u0026deg;C).\u003c/p\u003e \u003cp\u003eThe %RSD remained below 15.0% under all conditions, indicating that the method is robust and reliable despite small variations in operating parameters (Table S 9)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Solution Stability and Filtration\u003c/h2\u003e \u003cp\u003eSolution stability was assessed by storing standard and sample solutions at room temperature for 24 hours.\u003c/p\u003e \u003cp\u003eAdditionally, a filtration study comparing centrifuged samples vs. filtered samples (0.45 \u0026micro;m nylon) showed a relative difference of 5%, which is within the acceptance limit of 15%, confirming no significant analyte adsorption (Table S 10).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Quality Control Applications and Patient Safety Implications","content":"\u003cp\u003eThe practical implementation of this validated analytical method in routine quality control operations provides pharmaceutical manufacturers with a powerful tool for comprehensive monitoring of N-nitroso-related mutagenic impurities in methadone hydrochloride tablets. The method's exceptional sensitivity, enabling detection and quantification at 0.33 ppb, ensures that impurity levels remain substantially below established acceptable intake thresholds, providing a substantial safety margin between detected impurity concentrations and regulatory limits. The high selectivity achieved through MRM detection prevents false positives arising from co-eluting matrix components, ensuring regulatory agencies and healthcare systems receive accurate, reliable information regarding product safety and purity. The 3.7% precision ensures that analytical decisions based on this method\u0026mdash;including acceptance or rejection of batches\u0026mdash;reflect genuine product characteristics rather than analytical artifacts or random method variability. The accuracy characteristics guarantee that reported impurity concentrations accurately reflect true analyte levels, critical information for establishing batch safety profiles and supporting regulatory submissions.\u003c/p\u003e \u003cp\u003eThe application of this analytical technology to methadone hydrochloride specifically addresses pharmaceutical products utilized in opioid substitution therapy and pain management contexts, where patient populations may include individuals with heightened health vulnerabilities. Ensuring the purity and safety of these medications through rigorous analytical quality control represents a significant component of comprehensive patient protection strategies. The method's capability to reliably detect and quantify trace impurities at ppb levels provides regulatory confidence that manufacturing processes maintain stringent control over chemical purity throughout production and storage intervals.\u003c/p\u003e"},{"header":"5. Authenticity and Novelty of the Analytical Method Development","content":"\u003cp\u003eThis research work presents the comprehensive development and rigorous validation of a UPLC-TQ MS method specifically engineered for the precise determination of N-Nitroso-N-Desmethyl Methadone in Methadone Hydrochloride Tablets (10 mg formulation strength). The analytical method validation encompasses systematic evaluation of critical performance parameters including specificity (selectivity), linearity over appropriate concentration ranges, accuracy through recovery studies, precision through replicate analysis assessments, and robustness under deliberately varied analytical conditions, all conducted in strict accordance with internationally accepted ICH Q2(R1) guidelines and complementary USP requirements. The development strategy prioritizes achievement of sensitivity levels substantially below the established specification limit, ensuring sufficient analytical margin for regulatory decision-making and quality assurance operations in routine pharmaceutical manufacturing environments.\u003c/p\u003e"},{"header":"6. Discussions","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e6.1 Method Development Rationale and Analytical Strategy\u003c/h2\u003e \u003cp\u003eThe selection of UPLC-TQ MS as the analytical platform for N-Nitroso-N-Desmethyl Methadone quantification in methadone hydrochloride tablets reflects careful consideration of the unique analytical challenges posed by ultra-trace impurity monitoring in complex pharmaceutical matrices. The exceptional sensitivity requirement (achieving 0.33 ppb LOQ), coupled with the stringent selectivity demand necessitated by potential matrix interferences in tablet formulations containing numerous excipients, effectively eliminates conventional chromatographic methods from consideration. The UPLC-TQ MS platform uniquely combines the separation efficiency of modern ultra-high-performance liquid chromatography with the unparalleled selectivity afforded by multiple reaction monitoring mass spectrometric detection, enabling precise quantification of N-Nitroso-N-Desmethyl Methadone far below the established specification limit.\u003c/p\u003e \u003cp\u003eThe isocratic mobile phase approach, rather than gradient elution commonly employed in traditional HPLC methods, provides significant advantages for routine pharmaceutical quality control operations. Isocratic separation ensures rapid system equilibration between sequential analyses, reduces overall analytical run time, and enhances method reproducibility by eliminating the potential variability introduced by gradient programming and column conditioning cycles inherent to gradient methods. The ammonium acetate/acetonitrile mobile phase composition was specifically selected to optimize both the chromatographic behavior of the target analyte and the electrospray ionization efficiency in the mass spectrometric detection stage, reflecting an integrated analytical design that optimizes performance across the entire separation-detection continuum.\u003c/p\u003e \u003cp\u003eThe deliberate selection of the ACQUITY UPLC BEH Shield RP 18 stationary phase, a specialized reversed-phase material engineered with embedded polar functional groups that reduce problematic interactions between analytes and residual silanol sites on the column surface, provides superior peak shape and reduced tailing characteristics particularly valuable when analyzing compounds prone to non-ideal chromatographic behavior. The 1.7 \u0026micro;m particle size characteristic of modern UPLC columns enables higher operational flow rates and enhanced separation efficiency compared to conventional 5 \u0026micro;m HPLC columns, contributing substantially to the overall method sensitivity and chromatographic resolution achievable in this application.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e6.2 Validation Results in Context of Regulatory Requirements\u003c/h2\u003e \u003cp\u003eThe comprehensive validation results demonstrate full compliance with ICH Q2(R1) and USP\u0026thinsp;\u0026lt;\u0026thinsp;1225\u0026thinsp;\u0026gt;\u0026thinsp;guidelines, establishing the method as a scientifically robust analytical tool suitable for regulatory submissions and routine pharmaceutical quality control applications. The LOQ of 0.33 ng/mL (0.33 ppb) represents approximately 10% of the established 3.33 ppm specification limit, providing substantial analytical margin that ensures reliable confirmation of compliance with regulatory requirements and enables detection of trace impurity levels with considerable safety margin. This sensitivity level substantially exceeds the capability of conventional analytical methods and reflects the extraordinary instrumental capabilities of modern UPLC-TQ MS technology.\u003c/p\u003e \u003cp\u003eThe linearity correlation coefficient exceeding 0.99 across the twentyfold concentration range from LOQ to 200% of specification confirms that the relationship between injected concentration and analytical response remains strictly proportional throughout the validated range, enabling accurate interpolation for sample quantification across all anticipated quality control scenarios. The 3.7% RSD precision value, substantially below the typical 15% acceptance criterion for trace-level methods, demonstrates exceptional reproducibility that ensures analytical decisions regarding batch acceptance or rejection reflect genuine product characteristics rather than analytical artifacts. The 87.5\u0026ndash;93.5% recovery range confirms accurate quantification with minimal systematic error, while the demonstrated robustness across deliberately varied analytical parameters establishes that the method will perform consistently across different laboratory environments and instrumental platforms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e6.3 Practical Quality Control Applications\u003c/h2\u003e \u003cp\u003eThe validated UPLC-TQ MS method provides pharmaceutical manufacturers with a powerful analytical tool for comprehensive routine quality control monitoring of N-nitroso-related mutagenic impurities in methadone hydrochloride tablets throughout manufacturing, stability testing, and commercial distribution. The method's exceptional sensitivity enables detection and precise quantification at 0.33 ppb, ensuring that impurity levels remain substantially below established acceptable intake thresholds while providing manufacturers with early warning of potential manufacturing process deviations that might lead to impurity formation. The high selectivity achieved through MRM detection prevents false-positive results arising from coeluting matrix components, ensuring regulatory agencies and healthcare systems receive accurate, reliable information regarding product safety and purity. The demonstrated precision ensures that analytical results reliably reflect true product characteristics, critical for batch release decisions and regulatory compliance determinations.\u003c/p\u003e \u003cp\u003eThe method's robust performance across varied analytical conditions establishes confidence that quality control laboratories operating the method will achieve consistent, reliable results regardless of minor variations in laboratory environment, instrument settings, or operational procedures. This robustness is particularly important for maintaining analytical consistency across multiple manufacturing sites operating the same method and for ensuring that analytical results remain valid when the method is transferred to different laboratory facilities. The validated method serves as a scientific foundation for establishing appropriate in-process controls and specification limits that ensure consistent product quality throughout manufacturing operations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e6.4 Regulatory Implications and Public Health Significance\u003c/h2\u003e \u003cp\u003eThe successful development and validation of this highly sensitive UPLC-TQ MS method addresses a critical public health imperative reflected in the rigorous regulatory requirements imposed by ICH M7(R1) and USP\u0026thinsp;\u0026lt;\u0026thinsp;1469\u0026thinsp;\u0026gt;\u0026thinsp;guidelines. The method's capability to reliably detect and quantify N-Nitroso-N-Desmethyl Methadone at parts-per-billion levels provides assurance that methadone hydrochloride tablets distributed to patients contain impurity levels far below those predicted to pose carcinogenic risk. This analytical capability represents a significant component of comprehensive regulatory strategies designed to protect public health through rigorous quality assurance of pharmaceutical products. The method's validation in accordance with internationally harmonized guidelines establishes mutual recognition across different regulatory jurisdictions, facilitating consistent quality standards for methadone products distributed globally and reducing the regulatory burden of separate method validations for different markets.\u003c/p\u003e \u003cp\u003eThe method's demonstrated performance characteristics support regulatory decision-making regarding product safety, manufacturing process validation, and stability testing protocols essential for ensuring the continued safety and efficacy of methadone formulations throughout their commercial shelf-life. Regulatory agencies can rely on analytically validated data generated through this method when making determinations regarding product approvals, manufacturing facility inspections, and compliance with established impurity specifications.\u003c/p\u003e \u003c/div\u003e"},{"header":"7. Conclusion","content":"\u003cp\u003eA sensitive, specific, and robust UPLC-TQ MS method was successfully developed and validated for the quantification of N-Nitroso-N-Desmethyl Methadone in Methadone HCL Tablets (10 mg). The method demonstrates excellent linearity, precision, and accuracy across the validation range. The LOQ of 0.33 ppb facilitates the quantification of this genotoxic impurity well below the daily intake limit of 400 ng/day (3.33 ppm). Validation data confirms compliance with USP and ICH guidelines, establishing the method as suitable for routine quality control applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e Not applicable as this study did not involve human subjects or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. The chromatographic data, calibration curves, and validation results are included in this published article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudies involving plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable as this study did not involve any plant materials\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003egovardhan rao Thalluri \u0026amp; Ramakrishna K: Conceptualization, methodology, formal analysis, investigation, writing - original draft\u003c/p\u003e\n\u003cp\u003egovardhan rao Thalluri \u0026amp; Ramakrishna K: Validation, data curation, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003egovardhan rao Thalluri \u0026amp; Ramakrishna K: Software, resources, visualization\u003c/p\u003e\n\u003cp\u003eRAMA KRISHNA MYNENI: Supervision, project administration\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their sincere gratitude to\u0026nbsp;\u003cstrong\u003eBiogenico Private Limited\u003c/strong\u003e. for their invaluable support throughout the execution of this research. Their contributions\u0026mdash;ranging from providing essential materials and technical expertise to facilitating experimental setups and analytical resources\u0026mdash;played a pivotal role in the successful completion of this study. The collaboration with Hetero Labs Ltd. not only ensured access to state-of-the-art infrastructure but also fostered an environment of scientific rigor and innovation. The team deeply appreciates their commitment to advancing pharmaceutical research and their continued partnership in furthering the development of high-quality therapeutic solutions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchmidtsdorff S, Schmidt AH. Simultaneous detection of nitrosamines in sartans by HPLC-MS/MS. \u003cem\u003eJ Pharm Biomed Anal\u003c/em\u003e. 2019;167:138-144.\u003c/li\u003e\n\u003cli\u003eParr MK, Joseph JF. NDMA in metformin: Analysis of the recall and analytical challenges. \u003cem\u003eJ Pharm Biomed Anal\u003c/em\u003e. 2019;164:536-549.\u003c/li\u003e\n\u003cli\u003eAbe Y, Yamamoto H, Hirai T, et al. Control of N-nitrosodimethylamine in ranitidine drug substance and drug products. \u003cem\u003eChem Pharm Bull (Tokyo)\u003c/em\u003e. 2020;68(10):927-931.\u003c/li\u003e\n\u003cli\u003eTeasdale A. Nitrosamine Control: From Risk Assessment to Analytical Testing with Emphasis on Sample Preparation and Phase-Appropriate Method Validation. \u003cem\u003eOrg Process Res Dev\u003c/em\u003e. 2023;27(11):1939\u0026ndash;1969.\u003c/li\u003e\n\u003cli\u003eInternational Council for Harmonisation (ICH). Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk M7(R1). Geneva: ICH; 2017.\u003c/li\u003e\n\u003cli\u003eUnited States Pharmacopeia (USP). General Chapter \u0026lt;1469\u0026gt; Nitrosamine Impurities. In: USP-NF. Rockville, MD: United States Pharmacopeial Convention; 2021.\u003c/li\u003e\n\u003cli\u003eUS Food and Drug Administration (FDA). Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry. Silver Spring, MD: FDA; 2021.\u003c/li\u003e\n\u003cli\u003eEuropean Medicines Agency (EMA). Assessment report: Nitrosamine impurities in human medicinal products. EMA/CHMP/428592/2019. Amsterdam: EMA; 2020.\u003c/li\u003e\n\u003cli\u003eAshworth I, Dirat O, Teasdale A, Whiting M. Potential for the formation of N-nitrosamines during the manufacture of active pharmaceutical ingredients: An assessment of the risk posed by trace nitrite in water. \u003cem\u003eOrg Process Res Dev\u003c/em\u003e. 2020;24(9):1629-1646.\u003c/li\u003e\n\u003cli\u003eSmith PA, Loeppky RN. Nitrosamine formation: The role of nitrogen oxides and related species. \u003cem\u003eJ Am Chem Soc\u003c/em\u003e. 1967;89(5):1147-1157.\u003c/li\u003e\n\u003cli\u003eAdhikari P, Lee J, Kim J, Kim J, Lee J. Screening of Nitrosamine Impurities in Sartan Pharmaceuticals by GC-MS/MS. \u003cem\u003eMass Spectrom Lett\u003c/em\u003e. 2021;12(2):31-40.\u003c/li\u003e\n\u003cli\u003eThresher A, Foster R, Ponting DJ, et al. N-nitrosamine impurity limits: A systematic approach to establishing acceptable intakes (AIs). \u003cem\u003eRegul Toxicol Pharmacol\u003c/em\u003e. 2020;116:104749.\u003c/li\u003e\n\u003cli\u003eSolanki R, Wadhwana P, Patel R, et al. UHPLC-APCI-TQ-MS analytical method capable of quantifying eight nitrosamine impurities from five different commercially available Metformin formulations. \u003cem\u003eJ Pharm Sci\u003c/em\u003e. 2023;112(7):1933-1940.\u003c/li\u003e\n\u003cli\u003eMatuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. \u003cem\u003eAnal Chem\u003c/em\u003e. 2003;75(13):3019-3030.\u003c/li\u003e\n\u003cli\u003eNagdella NK, Shaik H, Subramanyam SB, et al. Development, validation, and estimation of measurement uncertainty for the quantitative determination of nitrosamines in Sartan drugs using liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. \u003cem\u003eJ Chromatogr Open\u003c/em\u003e. 2022;2:100053.\u003c/li\u003e\n\u003cli\u003eMiralles P, Chisvert A, Salvador A. Determination of N-nitrosamines in cosmetic products by vortex-assisted reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with mass spectrometry. \u003cem\u003eJ Sep Sci\u003c/em\u003e. 2018;41(15):3143-3151.\u003c/li\u003e\n\u003cli\u003eInternational Council for Harmonisation (ICH). Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva: ICH; 2005.\u003c/li\u003e\n\u003cli\u003eBylda C, Thiele R, Kobold U, Volmer DA. Recent advances in sample preparation techniques to overcome matrix effects in bioanalysis. \u003cem\u003eAnal Chem\u003c/em\u003e. 2014;86(19):9761-9771.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"N-Nitroso-N-Desmethyl Methadone, Methadone HCl, UPLC-MS/MS, Genotoxicity, Method Validation, Trace Analysis","lastPublishedDoi":"10.21203/rs.3.rs-8397946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8397946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe detection and stringent control of N-nitrosamine impurities in pharmaceutical products is a critical imperative for regulatory agencies and the global pharmaceutical industry, driven by the potent genotoxic and carcinogenic potential inherent to these compounds. This research work describes the development and rigorous validation of a highly sensitive Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQ MS) method specifically designed for the quantification of N-Nitroso-N-Desmethyl Methadone in Methadone Hydrochloride (HCL) Tablets (10 mg). Chromatographic separation was successfully achieved utilizing an ACQUITY UPLC BEH Shield RP 18 stationary phase under an isocratic elution program composed of ammonium acetate buffer and acetonitrile. Mass spectrometric detection was executed via electrospray ionization in positive mode (ESI+) employing Multiple Reaction Monitoring (MRM) to guarantee maximal selectivity for the two isomeric peaks of the impurity. The analytical method was validated in strict conformity with USP\u0026thinsp;\u0026lt;\u0026thinsp;1225\u0026thinsp;\u0026gt;\u0026thinsp;and ICH Q2(R1) guidelines. Linearity was demonstrated over a concentration range of 0.33 ng/mL to 6.67 ng/mL, corresponding to 10% to 200% of the established specification limit. The limit of quantification (LOQ) was confirmed at 0.33 ng/mL (0.33 ppb), verifying the method's capacity to quantify trace levels significantly below the acceptable intake. Precision assessments yielded a cumulative relative standard deviation (RSD) of 3.7%, while accuracy studies demonstrated mean recoveries ranging from 87.5% to 93.5%. The proposed method represents a robust, specific, and reliable analytical tool for the routine quality control of Methadone HCL, ensuring patient safety through the effective monitoring of mutagenic impurities.\u003c/p\u003e","manuscriptTitle":"UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 10:43:38","doi":"10.21203/rs.3.rs-8397946/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-28T15:03:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-20T04:38:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T00:31:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242168367202663156860935248717461972889","date":"2026-01-19T00:06:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T17:38:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8963272454030412801661153151021571900","date":"2026-01-18T16:45:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226433934458577094403621563777155338651","date":"2026-01-17T10:26:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124850386866029012337953114395058085058","date":"2026-01-16T10:22:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185614577606379890293060662543624187284","date":"2026-01-14T09:55:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T14:07:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324283705414872328375415278789008681631","date":"2026-01-13T04:58:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11735724886083630011805451534957179555","date":"2026-01-12T23:18:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229163390340849338468437570543095570125","date":"2026-01-12T20:58:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-12T19:42:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-12T04:40:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-02T06:04:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-02T06:04:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2025-12-18T17:20:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"765e27c0-7af1-447d-b227-1050eadc04f4","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T11:25:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 10:43:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8397946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8397946","identity":"rs-8397946","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}