Qianyang Huang (Presenter)
Cleveland State University
Authorship: Qianyang Huang (1), Xiang Zhou (1), Danting Liu (1), Baozhong Xin (2), Karen Cechner (2), Heng Wang (2), and Aimin Zhou (1)
(1) Department of Chemistry, Cleveland State University, Cleveland, OH (2) DDC Clinic, Center for Special Needs Children, Middlefield, OH
| Short Abstract In this study, a novel reverse phase UPLC/MS/MS method for determination of three monosialoganglioside species, GM1, GM2, and GM3, in human plasma has been developed and validated. This assay employed DMTMM & PAEA chemical derivatization for signal enhancement and D3-labeled monosialogangliosides as internal standards (IS). The analytes and ISs were extracted from plasma using protein precipitation procedure, cleaned up with liquid-liquid extraction, and derivatized with DMTMM & PAEA. Thereafter, the samples were injected into a Shimadzu Nexera UHPLC system interfaced to an AB Scix Qtrap 5500 mass spectrometer that operating in ESI positive and Multiple Reaction Monitoring (MRM) mode to achieve highly sensitive and specific detection. |
Long Abstract
Introduction
Gangliosides are a large subfamily of glycosphingolipids that present abundantly on the plasma membrane of neuronal and glial cells of vertebrates. These molecules are structurally characterized by a distinctive oligosaccharide moiety attached to a ceramide portion with variable length on the fatty acid chains. Physiologically, they are believed to play critical roles in the regulation of various receptor-mediated cell signaling pathways and cellular events. Disruption in their metabolic pathways pathologically leads to the pathogenesis of numerous neurodegenerative disorders, such as Parkinson disease [1-2] and Alzheimer disease [3]. Genetic defects in the ganglioside biosynthesis pathway may be devastating. GM3 synthase deficiency (GSD) is a newly identified neurological disorder that has been prevalently found in the Amish population around United States [4-6]. Although the pathological mechanism remains to be understood, the condition is severe, characterized with infantile onset of severe irritability, failure to thrive, developmental stagnation, cortical blindness, profound intellectual disability and intractable seizures. In order to better understand the etiology of such diseases, analytical assays with sufficient specificity and sensitivity for determination of gangliosides in relevant biological matrices are highly demanded.
Methods
In this study, a novel reverse phase UPLC/MS/MS method for determination of three monosialoganglioside species, GM1, GM2, and GM3, in human plasma has been developed and validated. This assay employed DMTMM & PAEA chemical derivatization for signal enhancement and 2D3-labeled monosialogangliosides as internal standards (IS) for signal normalization in LC-ESI-MS. The analytes and ISs were extracted from plasma using protein precipitation procedure, cleaned up with a mixture of water/methanol/chloroform, dried under nitrogen purging, and derivatized with DMTMM & PAEA. Thereafter, the samples were injected into a Shimadzu Nexera UHPLC system interfaced to an AB Scix Qtrap 5500 mass spectrometer that operating in ESI positive and Multiple Reaction Monitoring (MRM) mode to achieve highly sensitive and specific detection.
Results
Considering the m/z from the majority of singly charged molecular ions of monosialogangliosides were beyond the detection range of most of commercially available triple-quadrupole instruments, including our Qtrap 5500 mass spectrometer, and they showed low preference to undergo dual protonation/deprotonation to generate doubly charged molecular ions in both positive and negative ESI, we introduced a novel chemical derivatization method with DMTMM & PAEA to increase the abundance of their doubly charged molecular ions in positive ESI. The sensitivity of monosialoganglioside species in positive ESI was observed to be enhanced for 15-20 times after derivatization. In addition, more than 15 different components were chromatographically resolved from each other by using a C8 UPLC column as stationary phase and a mixture of water/methanol/acetonitrile/formic acid as mobile phases within an 11min run. Moreover, calibration curves ranging from 10~2000, 10~2000, and 80~16000 ng/ml with correlation coefficients of 0.9981, 0.9989, and 0.9977 were established for measurements of monosialogangliosides GM1, GM2, and GM3, respectively. Thereafter, we validated this quantitative assay based on the FDA guideline for precision, accuracy, stability, and extraction recovery. The relative percent error and coefficient of variation from measurements were determined to be below 11 and 11% for each monosialoganglioside species. The extraction recovery was found to be above 80% for each monosialoganglioside species using our sample preparation strategy. The loss of derivatized analytes during storage was revealed to be insignificant (<10%) under the studied conditions. The validated method has been successfully applied to measure monosialoganglioside levels in the plasma from different human subject, including normal adults, GSD carriers, and GSD patients.
Conclusions
In summary, we developed and validated a novel quantitative assay for determination of monosialoganglioside species in human plasma using LC/MS and chemical derivatization. This method employed the UPLC system in gradient elution for chromatographic separation and Multiple Reaction Monitoring (MRM) for mass spectrometric detection, which has been successfully applied to the measurement of ganglioside levels in plasma samples from GSD patients, carriers, and normal human subjects. Regarding its high specificity, sensitivity, and throughput, this method could be applicable for therapeutic evaluation and clinical diagnosis of other ganglioside-related neurodegenerative disorders in the near future.
References & Acknowledgements:
Reference
[1]E.A. Fazzini, The effect of gangliosides on a neurotoxin (MPTP) induced model of Parkinson's Disease in mice., 1989, pp. 175 pp.
[2]P.E. Di, J. Fantini, H. Chahinian, M. Maresca, N. Taieb, and N. Yahi, Altered Ion Channel Formation by the Parkinson's-Disease-Linked E46K Mutant of α-Synuclein Is Corrected by GM3 but Not by GM1 Gangliosides. Journal of Molecular Biology 397 (2010) 202-218.
[3]T. Ariga, M.P. McDonald, and R.K. Yu, Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease-a review. Journal of Lipid Research 49 (2008) 1157-1175.
[4]M.A. Simpson, H. Cross, C. Proukakis, D.A. Priestman, D.C.A. Neville, G. Reinkensmeier, H. Wang, M. Wiznitzer, K. Gurtz, A. Verganelaki, A. Pryde, M.A. Patton, R.A. Dwek, T.D. Butters, F.M. Platt, and A.H. Crosby, Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nature Genetics 36 (2004) 1225-1229.
[5]F. Farukhi, C. Dakkouri, H. Wang, M. Wiztnitzer, and E.I. Traboulsi, Etiology of vision loss in ganglioside GM3 synthase deficiency. Ophthalmic Genetics 27 (2006) 89-91.
[6]H. Wang, A. Bright , B. Xin et al, Cutaneous dyspigmentation in patients with ganglioside GM3 synthase deficiency. Am J Med Genet 161A (2013) 875-879.
Acknowledgements
We thank the families for their patience and support. The study was supported in part by Zverina Family Fund and Fonterra to DDC Clinic and Dissertation Research Award from Cleveland State University. The UPLC/MS/MS system used for this study was purchased by a grant from the Major Research Instrumentation Program (MRI) of National Science Foundation (NSF) under grant number CHE-0923308.
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