Unraveling the Complexity of Lipidomes by Multiple Heart-Cutting Q-TOF LC/MS with the Agilent 1290 Infinity 2D-LC Solution

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1 Unraveling the Complexity of Lipidomes by Multiple Heart-Cutting Q-TOF LC/MS with the gilent 9 Infinity D-LC Solution pplication Note iotherapeutics and iosimilars uthors Gerd Vanhoenacker, Ruben t Kindt,, Frank David, Pat Sandra,, and Koen Sandra, Research Institute for Chromatography President Kennedypark -8 Kortrijk elgium Metablys President Kennedypark -8 Kortrijk elgium bstract The gilent 9 Infi nity D-LC solution was used to study the lipid composition of blood plasma and induced sputum samples. pplying multiple heart-cutting enabled a fully automated HILIC-based lipid (sub-)class type fractionation in the fi rst dimension and online transfer of the fractions onto a high-resolution reversed-phase UHPLC column in the second dimension for separation of group members according to hydrophobicity. Detection and identifi cation of the lipids was carried out with the gilent ccurate-mass Q-TOF LC/MS System. The main advantage of this approach is a signifi cant reduction of the number of lipids that enter the mass spectrometer at a given time. This reduces the risk of misidentifi cation and erratic quantifi cation in complex lipid samples due to interfering isobaric lipids or isotopes of lipids. Udo Huber gilent Technologies, Inc. Waldbronn, Germany

2 Introduction The lipidome can be described as the complete lipid content in a given sample. Lipids exist in different classes with different physicochemical and biological properties. Each class contains many members that differ in the length of the fatty acid side-chain and the number of double bonds, among others. Lipid extracts generally are complex mixtures with a significant structural diversity and a large dynamic range. s a result, the measurement of the total lipid content is an analytical challenge and requires a high performance separation step followed by high-resolution mass spectrometry. Several approaches are available for this type of analysis, and state-of-the-art UHPLC on high performance LC columns enables the high-resolution separation required for detailed profiling of lipids in complex biological matrixes. We have recently reported on lipidomic analyses in blood plasma and induced sputum. These studies have shown that a better chromatographic separation is beneficial for MS data analysis and facilitates lipid identification and quantification. Several options are available for improving the chromatographic separation. Multidimensional chromatographic solutions are an efficient way to drastically increase peak capacity. In two-dimensional LC (D-LC), comprehensive LCxLC is known to substantially increase the chromatographic resolution as long as the two dimensions are orthogonal and the separation obtained in the first dimension is maintained upon transfer to the second dimension. In the comprehensive LCxLC mode, each fraction collected from the first dimension column is transferred to a second dimension separation for analysis. less complex approach in D-LC is heart-cutting (LC-LC), where only one or a few fractions are transferred to the second dimension. The advantage of this multidimensional mode is that the second dimension analysis time is not limited by the D-LC modulation time. Therefore, a better separation of the transferred fractions can be achieved. The application of D-LC for complex lipid samples is not new, and several column combinations have been reported. The most obvious combinations are a normal phase or HILIC separation in the first dimension and a reversed-phase (RPLC) mechanism in the second dimension. Such combinations have proven to result in good orthogonality for lipid samples. The first dimension provides a separation based on the polar head-group of the lipids, resulting in a lipid (sub-)class separation (for example, phospholipids, ceramides, acylglycerols). Here, neutral lipids will elute first before more polar and charged lipids. RPLC, conversely, separates the lipids based on hydrophobicity, that is, the carbon chain length and unsaturation (number of double bonds) of the fatty acid side chains. The application of RPLC in the second dimension is also beneficial for coupling with the mass spectrometer. The fractionation of lipids into different classes and the chromatographic analysis of the members of each class are attractive. The combination of HILIC and RPLC has been performed offline, or online using a special interface for solvent evaporation 7,8. In this pplication Note, a fully automated multiple heart-cutting Q-TOF LC/MS analysis of complex lipid extracts with the gilent 9 Infinity D-LC solution is demonstrated. Experimental Samples and sample preparation The lipid extraction protocol has been described in detail in reference. riefly, of plasma or µl of lung sputum. fter vortex mixing, ml of tert-butyl methyl ether (MTE) was added, and the samples were incubated for hour at of water induced a phase separation, generating a lower hydrophilic and an upper lipophilic layer with a protein layer at the bottom. -ml amount of the upper phase was removed and dried in a centrifugal vacuum evaporator. The dried phase was reconstituted in of MTE/isopropanol / (v/v), respectively. Instrumentation n gilent 9 Infinity D-LC solution connected to an gilent Q-TOF mass spectrometer was used for the experiments. gilent 9 Infinity inary Pump (first dimension) (G) gilent 9 Infinity inary Pump (second dimension) (G) gilent 9 Infinity utosampler (G) gilent 9 Infinity Thermostat (G) gilent 9 Infinity Thermostatted Column Compartment (GC) gilent 9 Infinity valve drive (x) (G7) gilent D-LC hardware solution kit (G), including loop kit for D-LC (option #), capillary kit for D-LC (option #), and multiple heart-cutting upgrade kit (option #7) gilent ccurate-mass Q-TOF LC/MS (G) The Jet Weaver mixer was removed in the first dimension pump to reduce the delay volume. To obtain sufficient backpressure on the first dimension separation, a calibration capillary (G-7) was installed between the pump and the autosampler.

3 Software gilent OpenL CDS ChemStation Edition (revision C..7) with gilent 9 Infinity D-LC Software (revision..) gilent MassHunter for instrument control (revision..) gilent MassHunter for data analysis (revision..) Method First dimension Column Solvent Solvent Flow rate Gradient Temperature C Second dimension RPLC HILIC gilent RX-SIL,. mm,. µm (custom made). % formic acid in acetonitrile mm ammonium formate in acetonitrile/methanol/water, //, v/v µl/min minutes: %, µl/min minutes: 7 %, µl/min minutes: 7 %, µl/min minutes: %, µl/min minutes: %, µl/min minutes: %, µl/min Column gilent ZORX Eclipse Plus C8 RRHD,. mm,.8 µm (p/n ) Solvent Solvent Flow rate Idle flow rate mm ammonium formate/. % formic acid in methanol/water, /9, v/v mm ammonium formate/. % formic acid in acetonitrile/methanol/ isopropanol, //, v/v. ml/min. ml/min Gradient. minutes: %. 8. minutes: % 8. minutes: % Temperature C Loop filling Time segments Injection Fraction :..8 minutes (Loop 8 µl) Fraction :.9.8 minutes Fraction :.. minutes Fraction : minutes Fraction : 9.. minutes Volume µl Sample temperature C Needle wash MS detection gilent JetStream technology source seconds flush port (MTE/isopropanol, /7, v/v) Positive ionization, negative ionization Drying gas temperature C Drying gas flow 8 L/min Nebulizer pressure psig Sheath gas temperature C Sheath gas flow 8 L/min Capillary voltage, V Nozzle voltage, V Fragmentor V Centroid data spectra/s Mass range m/z,7 Extended dynamic range GHz Resolution, for m/z,

4 Results and Discussion When lipid samples are analyzed with the described HILIC method, they elute in five to six groups in order of polarity, from neutral lipids to more polar and charged lipids. The separation of the individual lipids within each class is limited, and typically broad peaks are noted in real samples. This is shown for a plasma sample in Figure. The lipid classes selected in this pplication Note together with the peak codes and MHC fraction are summarized in Table. pplying the same sample to RPLC will give a different elution pattern where the separation is based on hydrophobicity and the selectivity for the individual lipids within a class is significant (Figure ). It is evident that the separation modes provide good orthogonality in this multidimensional LC setup Positive ionization Negative ionization H,G,H I,J I,J F igure. Total ion chromatogram (positive and negative ionization) for the analysis of a plasma sample with the first dimension HILIC mode. K,L K,L M,N M,N O O P P Table. Lipids, peak codes in figures, and HILIC fractions. Lipid class (abbreviation) Code MHC fraction Free fatty acids (FF) Monoacylglycerols (MG) Diacylglycerols (DG) C Triacylglycerols (TG) D Cholesterol (Chol) E Cholesterol esters (CE) F Ceramides (CER) G Glycoceramides (Hex-CER) H Glyconceramides (Hex n -CER) I Phosphatidylinositols (PI) J Phosphatidylethanolamines (PE) K Glycosphingolipids (GSL) L Phosphatidylserines (PS) M Phosphatidylcholines (PC) N Sphingomyelins (SM) O Lysophospholipids (LysoPL) P Positive ionization,p P,E C,G O G O. Negative ionization Fi gure. Total ion chromatogram (positive and negative ionization) for the analysis of a plasma sample with the second dimension RPLC mode (flow:. ml/min, gradient: to minutes: to 8 %, to 7 minutes: 8 to %). D,F

5 Using the gilent 9 Infinity multiple heart-cutting D-LC solution, a fully automated transfer of the HILIC fractions to RPLC and the subsequent analysis can be realized. The collected fractions are stored in loops installed on the -position/-port duo-valve and a single multiple heart-cutting valve, and these fractions are analyzed sequentially. Filling and emptying of the loops is performed in a countercurrent fashion to reduce carryover. The order, timing, and location of storage and analysis of the fractions can be fully controlled by the D-LC add-on software for the gilent OpenL CDS ChemStation Edition. scheme of the valve configuration is shown in Figure. Deck with six -µl loops Fractions to Waste D-Column IN OUT The MS signals for the collected fractions of the plasma and the sputum sample can be seen in Figure (for positive ionization) and Figure (for negative ionization). 8-µL loop Fraction 7 8 D-Pump D-Column Fig ure. Configuration of the -position/-port duo-valve for the multiple heart-cutting method. The six loops are installed on the multiple heart-cutting valve HILIC plasma Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction RPLC plasma C RPLC sputum Figu re. Total ion chromatogram (positive ionization) for the analysis of a plasma sample with the first dimension HILIC mode and PC (+) of the multiple heart-cutting analyses of plasma and induced sputum.

6 The advantage of performing a group-type separation before separation of individual lipids is demonstrated in Figure. In the one-dimensional RPLC separation, there is (partial) overlap of a nearly isobaric PE and PC (m/z difference is. Da), complicating data analysis. With the 9 Infinity multiple heart-cutting D-LC solution, PCs are efficiently separated from PEs in the first dimension, and both isobaric compounds can be analyzed in separate fractions. This is a clear demonstration of the benefit of the multiple heart-cutting approach for identification and quantification of individual lipids in complex mixtures.... Fraction Fraction Fraction Fraction Fraction HILIC plasma Fraction Fraction Fraction Fraction Fraction RPLC plasma C RPLC sputum Figur e. Total ion chromatogram (negative ionization) for the analysis of a plasma sample with the first dimension HILIC mode and PC ( ) of the multiple heart-cutting analyses of plasma and induced sputum. PC(p-8:/:) m/z 7.9 PC(p-:/8:) m/z 7.9 PE(8:/8:) m/z 7.8 Fraction (Phosphatidylcholines) m/z 7.9 PC(p-8:/:) PC(p-:/8:) C Fraction (Phosphatidylethanolamines) m/z 7.8 PE(8:/8:) Figure. Extracted ion chromatogram (positive ionization) for selected PEs and PCs in a plasma sample. Top trace shows the one-dimensional RPLC separation, bottom traces show the results for the multiple heart-cutting analyses where PEs and PCs are separated in the first dimension HILIC separation.

7 To further illustrate the performance of multiple heart-cutting D-LC and the usefulness of this technique in lipidome studies, some specific structure elucidations are presented in Figure 7 though Figure. The nomenclature proposed by the International Lipid Classification and Nomenclature Committee (ILCNC) is used PC(+) Fraction Fraction Conclusion The gilent 9 Infinity D-LC solution was used for fully automated multiple-heart-cutting analyses of the blood plasma and induced sputum lipidome. The lipids were fractionated in HILIC mode, and the fractions were then transferred to and analyzed with RPLC. Detection with the gilent ccurate-mass Q-TOF LC/MS System facilitated identification of the individual lipids in each fraction. Selected data clearly demonstrate the power of the system in lipidomics and might be extended to other metabolomic applications Feature Formula t R C H O N TG(:)NH C H O N TG(:)NH C 7 H 79 NO CE(:)NH C H O N TG(:)NH C H 79 NO.8. CE(8:)NH C H 7 O N TG(:)NH 7 C H 8 NO. 7.7 CE(8:)NH Figure 7. Fraction, positive ionization, triglycerides and cholesterol esters in plasma. TG and CE represent the most apolar lipids. oth groups are detected as ammonia adducts in the positive ESI mode. 7

8 . PC( ) Fraction Extracted glycoceramides Extracted ceramides glycophingolipids C ceramides Feature (glycophingolipids) Formula t R C H 97 NO HexHexHexCER(NS)() C H 87 NO HexHexCER(NS)() C H 77 NO HexCER(NS)() C H 7 NO. 7.9 CER(NS)() Feature (ceramides) Formula t R C H NO CER(NS)() C H 7 NO. 7.9 CER(NS)() C H 7 NO..9 CER(NS)() C 8 H 7 NO CER(NS)(8) C H 79 NO.9.99 CER(NS)() C H 8 NO CER(NS)() Figure 8. Fraction, negative ionization, sphingolipids in sputum. Ceramides and smaller glycosphingolipids are preferentially detected in negative ESI mode. Elution order is based on the attached sugar moiety and the fatty acid side chain. 8

9 PC( ) Fraction Extract Feature Formula t R C H 97 NO HexHexHexCER(NS)() C H 8 O P PI(8:/:) C 7 H 8 O P PI(8:/:) C 7 H 8 O P PI(8:/:) C H 8 O P PI(8:/8:) Figure 9. Fraction, negative ionization, phosphatidylinositols in sputum. Phosphatidylinositols encompass about % of detected glycerophospholipids in sputum. HexHexHexCer(NS)() is present in both Fractions and. 9

10 .. PC( ) Fraction Extract References. Sandra, K., Sandra, P., Lipidomics from an analytical perspective, Curr. Opin. Chem. iol.,, 7, Sandra, K., et al., Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry, J. Chromatogr.,, 7, Feature Formula t R C 7 H N O 9..7 NeucHexHexCER(d8:/:) C H N O 9..7 HexNcHexHexHexCER(d8:/:) C 7 H 7 NO 8 P PE(:) C 9 H 7 NO 8 P PE(:/8:) C H 7 NO 7 P PE(p:/:) C H 7 NO 7 P PE(p:/:) 7 C 9 H 7 NO 7 P. 7.9 PE(p:/8:) 8 C H 78 NO 7 P. 7. PE(p8:/:) 9 C H 8 NO 7 P PE(p8:/8:) C H 8 NO 8 P. 7. PE(8:/8:) Figure. Fraction, negative ionization, phosphatidylethanolamines, and glycosphingolipids in sputum. Plasmalogen phosphatidylethanolamines (PEp) are highly abundant in lung sputum. Complex glycosphingolipids containing N-acetylneuraminic acid (Neuc) elute in this fraction.. Telenga, E., et al., Untargeted lipidomic analysis in chronic obstructive pulmonary disease, m. J. Respir. Crit. Care Med.,, 9,. Francois, I., Sandra, K., Sandra, P., Comprehensive liquid chromatography: fundamental aspects and practical considerations a review, nal Chim cta, 9,,. Lisa, M., Cifkova, E., Holcapek, M., Lipidomic profiling of biological tissues using off-line two-dimensional highperformance liquid chromatographymass spectrometry, J. Chromatogr.,, 8, -. Cifkova, E., Holcapek, M., Lisa, M., Nontargeted lipidomic characterization of porcine organs using hydrophilic interaction liquid chromatography and offline two-dimensional liquid chromatography-electrospray ionization mass spectrometry, Lipids,, 8, Nie, H., et al., Lipid profiling of rat peritoneal surface layers by normaland reversed-phase D LC QTOF-MS, J. Lipid Res.,,, Li, M., et al., Lipid profiling of human plasma from peritoneal dialysis patients using an improved D (NP/RP) LC-QTOF MS method, nal. ioanal. Chem.,,, Fahy, E., et al., Lipid classification, structures and tools, iochim. iophys. cta.,, 8, 7 7

11 .. PC( ) Fraction (plasma) C PC( ) Fraction (sputum) (plasma) D (sputum) Feature Formula t R C 9 H 78 NO P PC(:/:)formate C 7 H 8 NO P PC(:/:)formate C H 8 NO P PC(:/:)formate C H 8 NO P PC(:/:)formate C H 8 NO P PC(:)formate C H 8 NO P PC(8:/8:)formate 7 C H 8 NO P PC(:/:)formate 8 C H 8 NO P PC(:/8:)formate 9 C 7 H 8 NO P PC(8:/:)formate C H 8 NO P PC(:)formate C H 88 NO P.7 8. PC(8:/8:)formate Figure. Fraction, negative ionization, phosphatidylcholines (PC) in sputum and plasma. Phosphatidylcholines are highly abundant in both samples. oth samples show different PC patterns. PCs are detected as formate adducts in negative ESI mode.

12 . PC( ) Fraction sn- elutes before sn- on RPLC 8. a b b a b a Feature Formula t R a C 7 H NO 9 P PC(:/8:)formate b C 7 H NO 9 P PC(8:/:)formate a C 7 H NO 9 P PC(:/8:)formate b C 7 H NO 9 P PC(8:/:)formate a C 7 H NO 9 P PC(:/8:)formate b C 7 H NO 9 P PC(8:/:)formate C 8 H 77 N O 8 P SM(d8:/:)formate C H 8 N O 8 P SM(d8:/:)formate C H 8 N O 8 P SM(d8:/8:)formate 7 C 8 H 9 N O 8 P SM(d8:/:)formate 8 C 8 H 9 N O 8 P SM(d8:/:)formate Figure. Fraction, negative ionization, lysophosphatidylcholines and sphingomyelins in sputum. Sn- lysophospholipids elute before sn- lysophospholipids in RPLC. oth lipid subclasses are detected as formate adducts in negative ESI mode. This information is subject to change without notice. gilent Technologies, Inc., Published in the US, 99-EN For Research Use Only. Not for use in diagnostic procedures.