Application of Hybrid Surface Technology for Improving Sensitivity and Peak Shape of Phosphorylated and Carboxylate Lipids

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Giorgis Isaac, Ian Wilson and Robert S. Plumb
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The comprehensive lipidomic analysis of various biological tissues is a challenging task due to the extreme complexity of individual lipid classes varying in their structure, attached functional groups, polarity, dissociation and ionization behavior. Phosphorylated and carboxylate lipid species are metal sensitive and can readily absorb to stainless-steel surfaces within the flow path of LC systems. This process can lead to poor peak shape, low recovery, and reduction in sensitivity. Here we show that the Premier System with Premier CSH™ C18 column can significantly improve sensitivity, peak shape and recovery of phosphorylated and carboxylate lipids compared to standard stainless steel surface ACQUITY™ UPLC™ I-Class and CSH C18 column.


Chromatographic separation was performed using two separate chromatography systems. Either a conventional ACQUITY UPLC I-Class system/CSH C18 column (100x2.1mm) or an ACQUITY Premier system/Premier CSH C18 column (100x2.1mm)  constructed with hybrid surface technology. Both systems were equipped with a binary solvent delivery system and autosampler. The mobile phase consists of (A) ACN-Water-1M aqueous ammonium formate (600:390:10, v/v/v) in 0.1% formic acid and (B) IPA-ACN-1M aqueous ammonium formate (900:90:10, v/v/v) in 0.1% formic acid with a flow rate of 0.4 mL/min. Detection was performed using a SYNAPT™-XS mass spectrometer operated in negative ionization mode using the following instrument settings: capillary 2.5 kV, desolvation temperature 500 °C, desolvation gas 800 L/h, cone voltage 30 V, and source temperature 120 °C.

Preliminary data

The phosphate group of the phosphatidic acids (PA and LPA) and the carboxylate group of the phosphatidylserines (PS and LPS) bind to metal ions to form chelation complexes that causes peak tailing and reduced intensity. To address the issue, we have developed the use of a hybrid organic/inorganic barrier surface applied to the metal substrates in the ACQUITY Premier System and column. A serial dilution of LPA, PA, LPS and PS was analyzed using standard column with stainless steel surface ACQUITY UPLC system and Premier System. The Premier System significantly improved the peak tailing and sensitivity of both phosphorylated and carboxylate lipids compared to standard system. A 25-30 times increase in signal intensity was observed for all investigated phosphorylated and carboxylate lipid classes. It also improved peak shape and reduced tailing by minimizing analyte-surface interaction. Premier System reduced peak tailing by 65-80%  and increased lipidomics coverage by simultaneous analysis of phosphorylated and carboxylate lipids in addition to other lipid classes. This new methodology was applied to the analysis of egg (chicken) PA and brain (porcine) PS extracts and compared to that obtained with the conventional system/column. The results clearly show that the PA lipid species at m/z 671.47 (16:1_18:1 and 16:0_18:2) and 673.48 (16:0_18:1) eluted with very broad tailing peaks using the conventional system and column.  This can be compared to the data acquired with the Premier System, where the peaks are significantly improved in shape and intensity. Similarly, the method was applied for the analysis of brain porcine PS extract. The Premier System not only provided reduced peak tailing but also allowed the visualization of three additional “new” peaks. These additional peaks were identified as PS(38:2) m/z 814.55, PS(40:3) m/z 840.577 and PS(44:2) m/z 898.654. In contrast, these minor PS species were not detected using the conventional system.

Novel aspect

Hybrid surface technology improves the sensitivity and peak shape of phosphorylated and carboxylate lipid classes.

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