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

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 ana...

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Bibliographic Details
Published in:Chromatographia 2019-10, Vol.82 (10), p.1467-1477
Main Authors: Agrawal, Vaishali, Baghel, Ruchi, Singh, Ajay K., Pathak, Dharam Pal, Sandal, Nidhi
Format: Article
Language:English
Subjects:
Acidic oxides
Analytical Chemistry
Chemistry
Chemistry and Materials Science
Chromatography
Degradation
Elution
Formulations
Impurities
Ions
Laboratory Medicine
Linearity
Original
Oxidation
Pharmacy
Proteomics
Spectrometry
Thermal stress
Tryptophan
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Summary: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 ( R 2  = 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
ISSN:0009-5893
1612-1112
DOI:10.1007/s10337-019-03776-z