Development of MRM based ultra-performance liquid chromatography in combination with a mass spectrometry method for the absolute quantification of targeted phospholipid in the zebrafish model
1
Issued Date
2022
Copyright Date
2022
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xiv, 83 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Medical Biochemistry and Molecular Biology))--Mahidol University, 2022)
Suggested Citation
Thapa, Kajol, 1996- Development of MRM based ultra-performance liquid chromatography in combination with a mass spectrometry method for the absolute quantification of targeted phospholipid in the zebrafish model. Thesis (M.Sc. (Medical Biochemistry and Molecular Biology))--Mahidol University, 2022). Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/113833
Title
Development of MRM based ultra-performance liquid chromatography in combination with a mass spectrometry method for the absolute quantification of targeted phospholipid in the zebrafish model
Author(s)
Advisor(s)
Abstract
Phospholipids (PLs), or synonymously called polar lipids, are the low-abundance lipids that are the main constituents of biological membranes. The structural complexity (different head groups, fatty acyl chains, linkages, and unsaturation) of PLs can give rise to thousands of isomers/isobars. Considering the complexity of PLs, a number of different approaches have been proposed in lipidomics earlier, ranging from high-throughput technologies such as shotgun to highly sensitive tandem mass spectrometry (LC-MS/MS). However, none of the currently available methods can quantify all PLs with higher sensitivity. Thus, this study aims to develop a highly sensitive multiple reaction monitoring (MRM) based ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-TQ-MS) method for the absolute quantification of targeted PLs. In brief, the mixture of twelve lipid standards and an internal standard (IS) was extracted using MTBE/MeOH (5:1 v/v). Then, the polar and neutral lipids were separated using liquid-liquid extraction (LLE) using MeOH/NaCl (80:20, v/v) and hexane/MeOH (98:2, v/v). Finally, the optimized protocol was applied to quantify target PLs from the liver of healthy zebrafish (10 mg, n=3). The % recovery of the MTBE extraction protocol for the IS was between 80-100% (%RSD = 6.98, n=10). The % recovery of lipid standards from the LLE protocol for PC(16:0/18:0), PE(16:0/18:1), PG(16:0/18:1), PC(P-18:0/18:1), PS(18:0/18:2), SM(d18:1/17:0), and PI(18:1/18:1) ranged from 80-98% (%RSD = 2.51-9.70, n=4). Absolute quantification of targeted polar lipids revealed that the concentration of our targeted polar lipids in normal-feed zebrafish ranges from 1.8-177.3 nmol/mg dry weight of the liver sample. Among these, PC(16:0/18:1) was the most abundant glycerophospholipid, followed by PG(16:0/18:1). Interestingly, some of these lipids were not detected in rat liver in an earlier study, possibly because of the low sensitivity of the previously proposed method. Overall, our results showed good recovery of targeted PL standards, reflecting the high efficacy of the extraction method. Furthermore, the lower values of the limit of detection (0.00047-0.00406 μM) and limit of quantification (0.0016-0.01354 μM) validate the increased sensitivity of the proposed method. The high sensitivity of our method suggests that it is highly reliable for the extraction, separation, and quantification of polar lipids from zebrafish liver samples. Besides, this protocol can be applied to quantify lipids in higher animal models such as mice, primates, and humans to answer different biological questions.
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Medicine Siriraj Hospital
Degree Discipline
Medical Biochemistry and Molecular Biology
Degree Grantor(s)
Mahidol University