In this study, we synthesized and characterized N-nitrosochlordiazepoxide, a potential genotoxic impurity originating from chlordiazepoxide hydrochloride, assessing its toxicity via quantitative structure-activity relationship (QSAR) analysis. Characterization techniques including UV spectroscopy and HPLC demonstrated purity at 97.0%, complemented by Fourier transform infrared spectroscopy analysis indicating characteristic peaks at 1,500 cm-1 (nitroso group) and 945 cm-1 (N-O bond), as well as mass spectrometry (m/z 329.2 [M+H]+) and NMR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, utilizing an HPLC octyldecylsilane column, revealed ion transitions from (Q1) m/z 329.20 to (Q3) m/z 281.90, 299.25, and 240.80. Method validation confirmed linearity (0.18–3 ppm) following The International Conference of Harmonization Q2 (R1) guidelines, with LOD and LOQ determined at 0.18 ppm and 0.375 ppm, respectively. Robustness studies showed <3% RSD with minor adjustments, and method precision exhibited <2.3% RSD for the main fragment (Q3) m/z 281.90, with intermediate precision below 1.8% RSD. Method accuracy was verified by recovery rates between 92.01% and 104.55%, and solution stability was confirmed for 24 hours at room temperature. Notably, analysis of three batches of chlordiazepoxide hydrochloride samples detected no traces of N-nitrosochlordiazepoxide, underscoring the enhanced sensitivity and accuracy of this LC-MS/MS method in pharmaceutical analytical procedures, crucial for ensuring product safety and compliance.
Birudukota S, Mangalapu B, Ramakrishna RA, Halder S. Development and application of an LC-MS/MS method for the detection of N-nitrosochlordiazepoxide as a potential genotoxic impurity. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2025.199616
1. López-Muñoz F, Álamo C, García-García P. The discovery of chlordiazepoxide and the clinical introduction of benzodiazepines: half a century of anxiolytic drugs. J Anxiety Disord. 2011 May 1;25(4):554–62. http://doi.org/10.1016/j.janxdis.2011.01.002
2. Greenblatt DJ, Shader RI, MacLeod SM, Sellers EM. Clinical pharmacokinetics of chlordiazepoxide. Clin Pharmacokinet. 1978 Oct;3:381–94. http://doi.org/10.2165/00003088-197803050-00004
3. Salzman G, DiMascio A, Shader RI, Harmatz JS. Chlordiazepoxide, expectation and hostility. Psychopharmacologia. 1969 Jan;14:38–45. http://doi.org/10.1007/BF00401533
4. Shemer A, Tykocinski O, Feldon J. Long term effects of chronic chlordiazepoxide (CDP) administration. Psychopharmacology. 1984 Jun;83:277–80. http://doi.org/10.1007/BF00464794
5. McNaughton N, Morris RG. Chlordiazepoxide, an anxiolytic benzodiazepine, impairs place navigation in rats. Behav Brain Res. 1987 Apr 1;24(1):39–46. http://doi.org/10.1016/0166-4328(87)90034-9
6. Zbinden G, Bagdon RE, Keith EF, Phillips RD, Randall LO. Experimental and clinical toxicology of chlordiazepoxide (Librium®). Toxicol Appl Pharmacol. 1961 Nov 1;3(6):619–37. http://doi.org/10.1016/0041-008X(61)90028-X
7. Le Vann LJ. Chlordiazepoxide, a tranquillizer with anticonvulsant properties. Can Med Assoc J. 1962 Jan 1;86(3):123.
8. Rickels K, Downing RW, Howard K. Predictors of chlordiazepoxide response in anxiety. Clin Pharmacol Therap. 1971 Mar;12(2part1):263–73. http://doi.org/10.1002/cpt1971122part1263
9. File SE. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods. 1980 Jun 1;2(3):219–38. http://doi.org/10.1016/0165-0270(80)90012-6
10. Gardos GA, DiMascio A, Salzman C, Shader RI. Differential actions of chlordiazepoxide and oxazepam on hostility. Arch Gen Psychiatry. 1968 Jun 1;18(6):757–60. http://doi.org/10.1001/archpsyc.1968.01740060117014
11. Azima H, Arthurs D, Silver A. The effects of chlordiazepoxide (Librium) in anxiety states: a multi-blind study. Can Psychiatric Assoc J. 1962 Feb;7(1):44–50. http://doi.org/10.1177/070674376200700107
12. Lorr M, McNair DM, Weinstein GJ. Early effects of chlordiazepoxide (Librium) used with psychotherapy. J Psychiatric Res. 1963 Dec 1;1(4):257–70. http://doi.org/10.1016/0022-3956(63)90001-3
13. Choi DW, Farb DH, Fischbach GD. Chlordiazepoxide selectively augments GABA action in spinal cord cell cultures. Nature. 1977 Sep 22;269(5626):342–4. http://doi.org/10.1038/269342a0
14. Salzman C, Kochansky GE, Shader RI, Porrino LJ, Harmatz JS, Swett CP. Chlordiazepoxide-induced hostility in a small group setting. Arch Gen Psychiatry. 1974 Sep 1;31(3):401–5. http://doi.org/10.1001/archpsyc.1974.01760150103015
15. Hollister LE, Motzenbecker FP, Degan RO. Withdrawal reactions from chlordiazepoxide (“Librium”). Psychopharmacologia. 1961 Jan;2:63–8. http://doi.org/10.1007/BF00429621
16. Reddy AV, Jaafar J, Umar K, Majid ZA, Aris AB, Talib J, et al. Identification, control strategies, and analytical approaches for the determination of potential genotoxic impurities in pharmaceuticals: a comprehensive review. J Sep Sci. 2015;38:764–79. http://doi.org/10.1002/jssc.201401143
17. Zhong Z, Chen Y, Xia H, Chen Z, Cheng, Q. Development and validation of a sensitive GC-MS/MS method for the determination of five potential genotoxic impurities in abiraterone acetate. J Chromatogr Sci. 2022;60:105–10. http://doi.org/10.1093/chromsci/bmab052
18. Grigori K, Loukas YL, Malenovi´c A, Samara V, Kalaskani A, Dimovasili E, et al. Chemometrically assisted development and validation of LC-MS/MS method for the analysis of potential genotoxic impurities in meropenem active pharmaceutical ingredient. J Pharm Biomed Anal. 2017, 145, 307–14. http://doi.org/10.1016/j.jpba.2017.06.061
19. Christofi M, Markopoulou CK, Tzanavaras PD, Zacharis CK. UHPLC-fluorescence method for the determination of trace levels of hydrazine in allopurinol and its formulations: validation using total-error concept. J Pharm Biomed Anal. 2020;187:113354. http://doi.org/10.1016/j.jpba.2020.113354
20. Rao T, editor. Genotoxicology of N-nitroso compounds. Berlin, Germany: Springer Science & Business Media; 2013 Nov 11.
21. International Agency for Research on Cancer. Some N-nitroso compounds. World Health Organization (WHO) Lyon, France. IARC Monographs on the Evaluat; 1978.
22. Brambilla G, Cajelli E, Finollo R, Maura A, Pino A, Robbiano L. Formation of DNA-damaging nitroso compounds by interaction of drugs with nitrite. A preliminary screening for detecting potentially hazardous drugs. J Toxicol Environ Health. 1985 Jan 1;15(1):1–24. http://doi.org/10.1080/15287398509530632
23. Brambilla G, Martelli A. Genotoxic and carcinogenic risk to humans of drug–nitrite interaction products. Mutat Res. 2007 Jan 1;635(1):17–52. http://doi.org/10.1016/j.mrrev.2006.09.003
24. Eichholzer M, Gutzwiller F. Dietary nitrates, nitrites, and N-nitroso compounds and cancer risk: a review of the epidemiologic evidence. Nutr Rev. 1998 Apr 1;56(4):95–105. http://doi.org/10.1111/j.1753-4887.1998.tb01721.x
25. Loh YH, Jakszyn P, Luben RN, Mulligan AA, Mitrou PN, Khaw KT. N-nitroso compounds and cancer incidence: the European prospective investigation into cancer and nutrition (EPIC)–Norfolk study. Am J Clin Nutr. 2011 May 1;93(5):1053–61. http://doi.org/10.3945/ajcn.111.012377
26. Bartsch H, Ohshima H, Pignatelli B, Calmels S. Endogenously formed N-nitroso compounds and nitrosating agents in human cancer etiology. Pharmacogenetics. 1992 Dec 1;2(6):272–7. doi: http://doi.org/10.1097/00008571-199212000-00005
27. Mirvish SS. Effects of vitamins C and E on N-nitroso compound formation, carcinogenesis, and cancer. Cancer. 1986 Oct 15;58(S8):1842–50. http://doi.org/10.1002/1097-0142(19861015)58:8+<1842::AID-CNCR2820581410>3.0.CO;2-%23
28. Knekt P, Järvinen R, Dich J, Hakulinen T. Risk of colorectal and other gastro-intestinal cancers after exposure to nitrate, nitrite and N-nitroso compounds: a follow-up study. Int J Cancer. 1999 Mar 15;80(6):852–6. http://doi.org/10.1002/(SICI)1097-0215(19990315)80:6<852::AID-IJC9>3.0.CO;2-S
29. Bogovski P, Bogovski S. Special report animal species in which n-nitroso compounds induce cancer. Int J Cancer. 1981 Apr 15;27(4):471–4. http://doi.org/10.1002/ijc.2910270408
30. Lijinsky W. Chemistry and biology of N-nitroso compounds. Cambridge, UK: Cambridge University Press; 1992 Apr 23.
31. Özhan G, Alpertunga B. Genotoxic activities of drug-nitrite interaction products. Drug Chem Toxicol. 2003 Jan 1;26(4):295–308. http://doi.org/10.1081/DCT-120024844
32. Coulston F, Olajos EJ. Toxicology of N-nitroso compounds. Ecotoxicol Environ Saf. 1982 Feb 1;6(1):89–96. http://doi.org/10.1016/0147-6513(82)90083-5
33. Johnson GE, Dobo K, Gollapudi B, Harvey J, Kenny J, Kenyon M, et al. Permitted daily exposure limits for noteworthy N-nitrosamines. Environ Mol Mutag. 2021 Jun;62(5):293–305. doi: http://doi.org/10.1002/em.22446.
34. Brambilla G, Robbiano L, Martelli A, Cajelli E, Allavena A, Mazzei M. Genotoxicity of N-nitrosochlordiazepoxide in cultured mammalian cells. Toxicol Appl Pharmacol. 1989 Mar 1;97(3):480–8. http://doi.org/10.1016/0041-008x(89)90253-6
35. Robbiano L, Carlo P, Finollo R, Brambilla G. DNA damage induced in rats by oral administration of chlordiazepoxide plus sodium nitrite or of N-nitrosochlordiazepoxide. Toxicol Appl Pharmacol. 1990 Jan 1;102(1):186–90. http://doi.org/10.1016/0041-008X(90)90095-C
36. Mereto E, Campart GB, Ghia M, Petrogalli F. Oral administration of chlordiazepoxide plus sodium nitrite investigated for tumor initiating activity in the rat liver. Cancer Lett. 1990 Aug 1;53(1):61–5. http://doi.org/10.1016/0304-3835(90)90011-L
37. Takeda Y, Kanaya H. Formation of nitroso compounds and mutagens from transquilizers by drug/nitrite interaction. Cancer Lett. 1981 Mar 1;12(1-2):81–6. http://doi.org/10.1016/0304-3835(81)90041-0
38. Agency, E.M. Guideline on the limits of genotoxic impurities. In evaluation of medicines for human use. London, UK: European Medicines Agency; 2006; pp. 1–8. https://www.ema.europa.eu.>scientific-guideline
39. Guidance, F.D. Genotoxic and carcinogenic impurities in drug substances and products: recommended approaches; US Food and Drug Administration. Washington, DC: Department of Health and Human Services; 2008. https://www.federalregister.gov>documents>2008/12/16
40. Guideline, I.H. Assessment and control of dna reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk M7. In Proceedings of the Geneva, Switzerland: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH); , 31 March 2017. https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential-carcinogenic-risk-scientific-guideline
41. Fda US. Guidance for industry-recommended acceptable intake limit for nitrosamine drug substances-related impurities (NDSRIs). US Department of Health and Human Services, Food and Drug Administration, Silver Spring, Maryland, USA. Centre for Drug Evaluation and Research (CDER) August 2023, Pharmacology/Toxicology. https://www.fda.gov>search-fda-guidance-documents
42. Mazzei M, Balbi A, Roma G, Di Braccio M, Robbiano L. HPLC analysis of the nitrosation products of chlordiazepoxide. Farmaco. 1989 Sep;44(9):883–91.
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