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Development and Validation of LC–MS Method for the Estimation of N-Acetyl-Tryptophan and its Impurities Under Stress Conditions

  • Vaishali Agrawal
  • Ruchi Baghel
  • Ajay K. Singh
  • Dharam Pal Pathak
  • Nidhi SandalEmail author
Original
  • 4 Downloads

Abstract

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.

Graphic Abstract

Keywords

LC–MS N-acetyl tryptophan Stress Impurities Degradation products 

Abbreviations

MRM

Multiple Reaction Monitoring

DiOia

Dioxyindolylalanine

HSA

Human serum albumin

ICH

International Conference on Harmonization

Kyn

Kynurenine

NAT

N-acetyl-tryptophan

NFK

N-formyl-kynurenine

Oia

Oxyindolylalanine

PIC

H,1,2,3,3a,8,8a-hexahydro-3a-hydroxypyrrolo[2,3-b]indole2-carboxylic acid

RP

Reversed phase

Trp

Tryptophan

AUC

Area under curve

Notes

Acknowledgements

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.

Informed Consent

Not applicable.

Supplementary material

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of CBRN DefenceInstitute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organization (DRDO)New DelhiIndia
  2. 2.Delhi Institute of Pharmaceutical Sciences and ResearchNew DelhiIndia

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