Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection

Abstract

An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.

Keywords: charge surface hybrid column; ion-mobility/high-resolution mass spectrometry; structural elucidation; ultraperformance liquid chromatography.

Fig. 1.

Base peak intensity chromatogram of 66 lipids (see Table 1) on CSH C₁₈ acquired in positive ion mode in the 20 min gradient.

Fig. 2.

Base peak intensity chromatogram for human plasma for positive 20 min gradient (A), negative 20 min gradient (B), positive 10 min gradient (C), and negative 10 min gradient (D) polarity. All chromatograms are acquired in HDMS^E mode and thus are from a single injection; both low- and elevated-energy data available.

Fig. 3.

Separation of PG 18:1 (9Z)/18:1 (9Z) (1) and PG 18:1 (9E)/18:1 (9E) (3), as well as PG 18:0/18:2 (9Z, 12Z) (2) (A), and separation of PC 18:1 (9Z)/18:1 (9Z) (1) and PC 18:1 (6Z)/18:1 (6Z) (2) (B) using the 20 min gradient.

Fig. 4.

Separation of phosphatidylinositol lipids in human plasma according to their equivalent carbon number (ECN). XIC for every PI was made on [M-H] with 0.05 Da extraction window.

Fig. 5.

XIC for a human plasma sample for PC 36:3 ([M+H]⁺ m/z 784.585 with 0.05 Da extraction window) on either HSS T3, CSH 20 min method, or CSH 10 min method (A) and XIC for PE 36:2 ([M-H] m/z 742.5392 with 0.05 Da extraction window) on either HSS T3, CSH 20 min method, or CSH 10 min method (B).

Fig. 6.

Comparison of Rt of bovine liver (A), brain (B), and heart (C) extracts for PC 34:2 [M+H]⁺ m/z 758.5695 (with 0.05 Da extraction window) using the 20 min chromatographic method.

Fig. 7.

XIC of a human plasma sample of TG 54:6 ([M+NH₄]⁺ m/z 896.7702 with 0.05 Da extraction window) (A) with the associated low-energy (B) and elevated-energy (C) spectra without mobility data used for the peak at 15.41 min (± 0.08 min) and the same low-energy (D) and elevated-energy (E) spectra using the mobility data making structural elucidation possible.