Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

Abstract

Reversed-phase ultrahigh-performance liquid chromatography - mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2 micrometer particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole - time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

Simple and Reproducible Derivatization with Benzoyl Chloride: Improvement of Sensitivity for Multiple Lipid Classes in RP-UHPLC/MS

“…The retention dependences are additional parameters supporting a high confidence in identification. 32,46 Polynomial dependences of retention times on the length of the fatty acyl chain (Figure S-12) and on the double bond(s) number (Figure S-13) confirm the logical lipid series. The identification was performed on 2.5 times more concentrated plasma for a better comparison with previous publications.…”

“…The identification was performed on 2.5 times more concentrated plasma for a better comparison with previous publications. 26,32 In total, 169 derivatized lipid species belonging to 11 lipid classes have been identified by the benzoylation method, and the summary of lipids is shown in Table S-5. The lipid nomenclature follows the shorthand notation for lipid structures published by Liebisch et al 47 and the LIPID MAPS 48 classification system.…”

“…The lipid nomenclature follows the shorthand notation for lipid structures published by Liebisch et al 47 and the LIPID MAPS 48 classification system. In comparison to five papers 26,32,34,49,50 using nonderivatization methods (Table S- 6), the new derivatization method enables the identification of more lipid species for sphingoid bases, monoacylglycerols, and diacylglycerols (except for Lipid MAPS reporting 1,2-and 1,3isomers). The number of fatty acids is comparable to another two methods, but the derivatization method enables the detection of all lipids in the positive ion mode compared to other methods forced to switch to the negative ion mode.…”

“…The mobile phase gradient was set as follows: 0 min, 65% A; 8 min, 50% A; 21 min, 5% A; 21−23 min, 5% A; 24−25 min, 65% A, where phase A was ACN/H 2 O (3:2, v/v), phase B was IPA/ACN (9:1, v/v), and both phases contained 8 mM ammonium formate and 0.1% formic acid. 32 Each sample was injected once, because the relative standard deviation (RSD) between injections was less than 10% (data not shown).…”