Development and Validation of the Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer Method for Quantification of Methylenecyclopropylglycine in Litchi Fruits Using the Standard Addition Method

  • Somya Asthana
  • Kajal Karsauliya
  • Sumita Dixit
  • Anurag Tripathi
  • Arvind Kumar
  • Sheelendra Pratap SinghEmail author
  • Mukul DasEmail author


Recently, the presence of methylenecyclopropylglycine (MCPG) found in litchi seeds and ripe and unripe litchi fruit pulp proved to be a breakthrough finding as it causes hypoglycemic encephalopathy leading to death of under nourished/starved children. Earlier methods of isolation and detection of MCPG in litchi fruit were tedious and time-consuming. To reduce the complexity, we developed a better and straightforward methodology using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) and QuEChERS technique to quantify MCPG without derivatization in litchi samples. The UPLC system included a Thermo C8 column with a mobile phase 0.1% formic acid in acetonitrile and water (gradient flow) at a flow rate of 0.3 ml/min. MCPG levels were determined using an API 4000 mass spectrometer with electrospray ionization (ESI) source. Other parameters were also determined following the quantitation by multiple reaction monitoring mode. The method was validated for selectivity, specificity, linearity, limit of quantification (LOQ), limit of detection (LOD), intra-inter day precision and accuracy, stability, and ruggedness. Calibration curve ranged from 7.8 to1000 ng/ml linearly while the LOD value for MCPG was 0.66 ng/ml. The percent accuracy and precision ranged from 92.98 to 105% and 1.78 to 8.43%, respectively. Validated method was applied for the determination of MCPG in litchi samples which provided a better and novel method for the determination of MCPG.


Methylenecyclopropylglycine Hypoglycemic encephalopathy QuEChERS technique Derivatization Mass spectrometer 



The authors are grateful to the director of CSIR-IITR for taking keen interest in the present study. One of us (SA) is thankful to CSIR for the award of Senior Research Fellowship. Thanks are due to Mr. S. K. Purushottam, Dr. Gyan Bhushan, and Dr. Arun Shah for the collection of litchi seeds and semi-ripe and ripe litchi fruits. The CSIR-IITR communication number of this manuscript is 3526.

Compliance with Ethical Standards

Conflict of Interest

Somya Asthana declares no conflict of interest. Kajal Karsauliya declares that she has no conflict of interest. Sumita Dixit declares that she has no conflict of interest. Anurag Tripathi declares that he has no conflict of interest. Arvind Kumar declares no conflict of interest. Sheelendra Pratap Singh and Mukul Das, the corresponding authors of this manuscript, declare no conflict of interest.

Ethical Approval

This article does not include any study with human or animal subjects, performed by any of the authors.

Informed Consent

Not applicable.


  1. Asthana S, Dixit S, Srivastava A, Kumar A, Singh SP, Tripathi A, Das M (2019) Methylenecyclopropyl glycine, not pesticide exposure as the primary etiological factor underlying hypoglycemic encephalopathy in Muzaffarpur, India. Toxicol Lett 301:34–41CrossRefGoogle Scholar
  2. Board NH (2015) Statewise area, production, and productivity of Litchi: Indian Horticulture Database 2014. Ministry of Agriculture Government of IndiaGoogle Scholar
  3. Commission E (2014) Analytical quality control and validation procedures for pesticide residues analysis in food and feed. In: SANCO (ed.)Google Scholar
  4. Das M, John TJ (2017) Lychee-associated acute hypoglycaemic encephalopathy outbreaks in Muzaffarpur, India. Lancet Glob Health 5:e859–e860CrossRefGoogle Scholar
  5. Das M, Asthana S, Singh S et al (2015) Litchi fruit contains methylene cyclopropyl-glycine. Curr Sci 109:2195–2197CrossRefGoogle Scholar
  6. Dinesh D, Pandey K, Das V et al (2013) Possible factors causing acute encephalitis syndrome outbreak in Bihar, India. Int J Curr Microbiol App Sci 2:531–538Google Scholar
  7. Gray D, Fowden L (1962) α-(Methylenecyclopropyl) glycine from Litchi seeds. Biochem J 82:385–389CrossRefGoogle Scholar
  8. Isenberg SL, Carter MD, Hayes SR, Graham LA, Johnson D, Mathews TP, Harden LA, Takeoka GR, Thomas JD, Pirkle JL, Johnson RC (2016) Quantification of toxins in soapberry (Sapindaceae) arils: hypoglycin A and methylenecyclopropylglycine. J Agric Food Chem 64:5607–5613CrossRefGoogle Scholar
  9. John TJ (2003) Outbreak of killer brain disease in children: mystery or missed diagnosis? Indian Pediatr 40:863–869Google Scholar
  10. John TJ, Das M (2014) Acute encephalitis syndrome in children in Muzaffarpur: hypothesis of toxic origin. Curr Sci 106:1184–1185Google Scholar
  11. Lehotay S (2007) AOAC official method 2007.01 pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate. J AOAC Int 90:485–520Google Scholar
  12. Li H, Wang C, Zhu Q, du H, Guan S, Wang F, Zhang W, Fan W, Chen Z, Yang G, Aboul-Enein HY (2016) Reduction of matrix effects through a simplified QuEChERS method and using small injection volumes in a LC-MS/MS system for the determination of 28 pesticides in fruits and vegetables. Anal Methods 8:5061–5069CrossRefGoogle Scholar
  13. Liu Y, Shi X-W, Liu E-H, Sheng LS, Qi LW, Li P (2012) More accurate matrix-matched quantification using standard superposition method for herbal medicines. J Chromatogr A 1254:43–50CrossRefGoogle Scholar
  14. Melde K, Buettner H, Boschert W, Wolf HPO, Ghisla S (1989) Mechanism of hypoglycaemic action of methylenecyclopropylglycine. Biochem J 259:921–924CrossRefGoogle Scholar
  15. Melde K, Jackson S, Bartlett K, Sherratt HSA, Ghisla S (1991) Metabolic consequences of methylenecyclopropylglycine poisoning in rats. Biochem J 274:395–400CrossRefGoogle Scholar
  16. Qingqing Du TW, Wang Z, Jiang X, Wang L (2015) Rapid determination of glutamine in human plasma by high-performance liquid chromatographic–tandem mass spectrometry and its application in pharmacokinetic studies. J Chromatogr Sci 53:79–84CrossRefGoogle Scholar
  17. Sahni GS (2012) Recurring epidemics of acute encephalopathy in children in Muzaffarpur, Bihar. Indian Pediatr 49:502–503CrossRefGoogle Scholar
  18. Sahni GS (2013) The recurring epidemic of heat stroke in children in Muzaffarpur, Bihar, India. Ann Trop Med Public Health 6:89–95CrossRefGoogle Scholar
  19. Samuel PP, Muniaraj M, Thenmozhi V, Tyagi B (2013) Entomo-virological study of a suspected Japanese encephalitis outbreak in Muzaffarpur district, Bihar, India. Indian J Med Res 137:991–992Google Scholar
  20. Shah A, John TJ (2014) Recurrent outbreaks of hypoglycaemic encephalopathy in Muzaffarpur, Bihar. Curr Sci 107:570–571Google Scholar
  21. Shrivastava A, Kumar A, Thomas JD, Laserson KF, Bhushan G, Carter MD, Chhabra M, Mittal V, Khare S, Sejvar JJ, Dwivedi M, Isenberg SL, Johnson R, Pirkle JL, Sharer JD, Hall PL, Yadav R, Velayudhan A, Papanna M, Singh P, Somashekar D, Pradhan A, Goel K, Pandey R, Kumar M, Kumar S, Chakrabarti A, Sivaperumal P, Kumar AR, Schier JG, Chang A, Graham LA, Mathews TP, Johnson D, Valentin L, Caldwell KL, Jarrett JM, Harden LA, Takeoka GR, Tong S, Queen K, Paden C, Whitney A, Haberling DL, Singh R, Singh RS, Earhart KC, Dhariwal AC, Chauhan LS, Venkatesh S, Srikantiah P (2017) Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study. Lancet Glob Health 5:e458–e466CrossRefGoogle Scholar
  22. Sonawane LV, Poul BN, Usnale SV et al (2014) Bioanalytical method validation and its pharmaceutical application—a review. Pharm Anal Acta 5:2CrossRefGoogle Scholar
  23. Standard B (2008) Foods of plant origin—determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE–QuEChERS-method. InGoogle Scholar
  24. USFDA (2013) Guidance for industry: bioanalytical method validation. Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Rockville, MD, USA. InGoogle Scholar
  25. Van de Merbel NC (2008) Quantitative determination of endogenous compounds in biological samples using chromatographic techniques. Trends Anal Chem 27:924–933CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Somya Asthana
    • 1
    • 2
  • Kajal Karsauliya
    • 3
  • Sumita Dixit
    • 1
  • Anurag Tripathi
    • 1
  • Arvind Kumar
    • 2
  • Sheelendra Pratap Singh
    • 3
    Email author
  • Mukul Das
    • 1
    Email author
  1. 1.Food Toxicology Division, Food, Drugs and Chemical Toxicology GroupCSIR-Indian Institute of Toxicology Research (CSIR-IITR)LucknowIndia
  2. 2.School of Biotechnology, Institute of ScienceBanaras Hindu University (BHU)VaranasiIndia
  3. 3.Analytical Chemistry Laboratory, Regulatory Toxicology GroupCSIR-Indian Institute of Toxicology Research (CSIR-IITR)LucknowIndia

Personalised recommendations