, Volume 82, Issue 10, pp 1467–1477 | Cite as

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


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


LC–MS N-acetyl tryptophan Stress Impurities Degradation products 



Multiple Reaction Monitoring




Human serum albumin


International Conference on Harmonization










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


Reversed phase




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.

Informed Consent

Not applicable.

Supplementary material

10337_2019_3776_MOESM1_ESM.png (157 kb)
Supplementary material 1 (PNG 156 kb)
10337_2019_3776_MOESM2_ESM.png (67 kb)
Supplementary material 2 (PNG 67 kb)
10337_2019_3776_MOESM3_ESM.png (213 kb)
Supplementary material 3 (PNG 212 kb)
10337_2019_3776_MOESM4_ESM.png (187 kb)
Supplementary material 4 (PNG 186 kb)
10337_2019_3776_MOESM5_ESM.png (209 kb)
Supplementary material 5 (PNG 209 kb)
10337_2019_3776_MOESM6_ESM.png (174 kb)
Supplementary material 6 (PNG 174 kb)
10337_2019_3776_MOESM7_ESM.png (206 kb)
Supplementary material 7 (PNG 205 kb)
10337_2019_3776_MOESM8_ESM.png (187 kb)
Supplementary material 8 (PNG 186 kb)
10337_2019_3776_MOESM9_ESM.png (188 kb)
Supplementary material 9 (PNG 187 kb)
10337_2019_3776_MOESM10_ESM.png (186 kb)
Supplementary material 10 (PNG 185 kb)
10337_2019_3776_MOESM11_ESM.png (187 kb)
Supplementary material 11 (PNG 186 kb)
10337_2019_3776_MOESM12_ESM.png (189 kb)
Supplementary material 12 (PNG 189 kb)
10337_2019_3776_MOESM13_ESM.png (187 kb)
Supplementary material 13 (PNG 187 kb)
10337_2019_3776_MOESM14_ESM.png (173 kb)
Supplementary material 14 (PNG 172 kb)
10337_2019_3776_MOESM15_ESM.png (200 kb)
Supplementary material 15 (PNG 200 kb)
10337_2019_3776_MOESM16_ESM.png (167 kb)
Supplementary material 16 (PNG 167 kb)
10337_2019_3776_MOESM17_ESM.png (195 kb)
Supplementary material 17 (PNG 194 kb)
10337_2019_3776_MOESM18_ESM.png (211 kb)
Supplementary material 18 (PNG 210 kb)
10337_2019_3776_MOESM19_ESM.png (185 kb)
Supplementary material 19 (PNG 184 kb)
10337_2019_3776_MOESM20_ESM.png (60 kb)
Supplementary material 20 (PNG 60 kb)


  1. 1.
    Yu MW, Finlayson JS (1984) Quantitative determination of the stabilizers octanoic acid and N-acetyl-DL-tryptophan in human albumin products. J Pharm Sci 73:82–86. CrossRefGoogle Scholar
  2. 2.
    Zhu T, Chen Z, Zhan C-Y, Wang H-J, Mao L, Lian H-Z (2015) Determination of acetyl-tryptophan in human albumin by reversed-phase high performance liquid chromatography. Asian J Chem. Google Scholar
  3. 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. CrossRefGoogle Scholar
  4. 4.
    Hogan KL, Leiske D, Salisbury CM (2017) Characterization of N-acetyl-tryptophan degradation in protein therapeutic formulations. J Pharm Sci 106:3499–3506. CrossRefGoogle Scholar
  5. 5.
    Nelis HJ, Lefevere MF, Baert E, D’Hoore W, De Leenheer AP (1985) Chromatographic determination of N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. J Chromatogr 333:381–387CrossRefGoogle Scholar
  6. 6.
    Shrivastava A, Gupta VB (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 2:21CrossRefGoogle Scholar
  7. 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. CrossRefGoogle Scholar
  8. 8.
    Thompson M, Ellison SL, Wood R (2002) Harmonized guidelines for single laboratory validation of methods of analysis. Pure Appl Chem 74:835–855CrossRefGoogle Scholar
  9. 9.
    Kruve A, Rebane R, Kipper K, Oldekop ML, Evard H, Herodes K, Ravio P, Leito I (2015) Tutorial review on validation of liquid chromatography-mass spectrometry methods: part I. Anal Chim Acta 870:29–44. CrossRefGoogle Scholar
  10. 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.
  11. 11.
    Nebsen M, Elzanfaly ES (2016) Stability-indicating method and LC–MS–MS characterization of forced degradation products of sofosbuvir. J Chromatogr Sci 54:1–10. CrossRefGoogle Scholar
  12. 12.
    Rao RN, Ramachandra B, Vali RM, Raju SS (2010) LC–MS/MS studies of ritonavir and its forced degradation products. J Pharm Biomed Anal 53:833–842. CrossRefGoogle Scholar
  13. 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.
  14. 14.
    Simat TJ, Steinhart H (1998) Oxidation of free tryptophan and tryptophan residues in peptides and proteins. J Agric Food Chem 46:490–498. CrossRefGoogle Scholar

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

Personalised recommendations