Development and Validation of LC–MS Method for the Estimation of N-Acetyl-Tryptophan and its Impurities Under Stress Conditions
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A highly sensitive and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS–MS) assay was developed and validated for the quantification of N-acetyl tryptophan (NAT) in formulations and to identify impurities under different stress conditions. N-acetyl tryptophan was analyzed using a reversed-phase gradient elution after treatment under acidic, basic, oxidative, hydrolytic and thermal stress conditions. Linearity in the calibration curve was obtained at a concentration range of 10–100 µgmL−1 (R2 = 0.9916). The lower limits of detection and quantification were 3.53 and 10.69 µgmL−1. The degradation of NAT was observed maximum under oxidation stress (52.84%) and minimum under thermal stress (10.22%). Three major degradation products were formed under acidic and basic stress conditions, of which tryptophan was the major one. Thermal stress yielded a single major impurity at m/z 230. Water hydrolysis could form dihydroxy-N acetyl tryptophan at m/z 279. Oxidation stress led to the formation of seven major degradation products. The most effective stress condition was found to be oxidative which leads to 52.84% degradation of the drug followed by acidic stress (34.64%) and basic stress (15.66%). The present study showed an accurate, precise and sensitive LC–MS–MS method for the systematic investigation of NAT and its impurities in formulations.
KeywordsLC–MS N-acetyl tryptophan Stress Impurities Degradation products
Multiple Reaction Monitoring
Human serum albumin
International Conference on Harmonization
Area under curve
The authors would like to thank Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, New Delhi for providing all the necessary facilities and requirement to complete this review.
Compliance with Ethical Standards
Conflict of Interest
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. There are no conflicts of interest.
Research Involving Human Participants and/or Animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- 3.Fang L, Parti R, Hu P (2011) Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography-mass spectrometry method for their quantitation. J Chromatogr A 1218:7316–7324. https://doi.org/10.1016/j.chroma.2011.08.044 CrossRefGoogle Scholar
- 7.Lalitha Devi M, Chandrasekhar KB (2009) A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant. J Pharm Biomed Anal 50:710–717. https://doi.org/10.1016/j.jpba.2009.05.038 CrossRefGoogle Scholar
- 10.US Food and Drug Administration (2018) Bioanalytical method validation, guidance for industry, US Department of Health and Human Services Food and Drug Administration, Rockville, MD, pp 1–41. https://www.fda.gov/media/70858/download
- 13.ICH (2003) ICH harmonised tripartite guideline: stability testing of new drug substances and products, Q1A (R2) current step 4 version. Geneva, pp 1–24. https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A_R2/Step4/Q1A_R2_Guideline.pdf