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Benzoylation of Nonpolar and Polar Lipid Classes in Human Plasma Characterized by RP-UHPLC/MS

Authors: Peterka Ondřej | Jirásko Robert | Vaňková Zuzana | Chocholoušková Michaela | Wolrab Denise | Kulhánek Jiří | Bureš Filip | Holčapek Michal

Year: 2021

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Benzoylation of Nonpolar and Polar Lipid Classes in Human Plasma Characterized by RP-UHPLC/MS	The character of functional groups in lipids has a significantly effect on the extraction efficiency, chromatographic behavior, and sensitivity, which can be changed by chemical derivatization. Benzoyl chloride is a nonhazardous, commonly available, cheap derivatization agent with a high reactivity for several functional groups. Derivatization reaction, retention and fragmentation behavior were investigated for 4 nonpolar and 8 polar lipid classes. The products were characterized by high-resolution mass spectrometry and MS/MS. The derivatization reaction was thoroughly optimized using spiked pooled plasma, and the molar ratio 4:1 of pyridine with benzoyl chloride reacting for 60 min at ambient temperature provided the best yield. The repeatability and reproducibility were investigated by one operator, resp. two operators, reporting RSD lower than 20 %. The stability of the derivatives was determined at least one month and five freeze/thaw cycles stored at -80 °C by periodical measurements. The comparison of the derivatization and nonderivatization approaches were investigated using calibration curves of 22 internal standards representing 12 lipid classes. The sensitivity expressed by the ratio of calibration slopes was increased by 2 to 10-fold for almost all investigated lipid classes and even more than 100-fold for monoacylglycerols. The limit of detection was decreased 9-fold for monoacylglycerols (MG), 6.5-fold for sphingoid base (SPB), and 3-fold for diacylglycerols (DG). In total, 169 benzoylated lipid species from 11 lipid classes were identified in human plasma using the high confidence level of identification (mass accuracy, MS/MS spectra, and retention time dependences). The derivatization method enables detection of more lipid species for MG, DG, and SPB compared with nonderivatized RP-UHPLC/MS methods.