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.
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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
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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.
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Agrawal, V., Baghel, R., Singh, A.K. et al. Development and Validation of LC–MS Method for the Estimation of N-Acetyl-Tryptophan and its Impurities Under Stress Conditions. Chromatographia 82, 1467–1477 (2019). https://doi.org/10.1007/s10337-019-03776-z
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DOI: https://doi.org/10.1007/s10337-019-03776-z