Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection
2014; Elsevier BV; Volume: 55; Issue: 8 Linguagem: Inglês
10.1194/jlr.d047795
ISSN1539-7262
AutoresCarola W.N. Damen, Giorgis Isaac, James Langridge, Thomas Hankemeier, Rob J. Vreeken,
Tópico(s)Analytical Chemistry and Chromatography
ResumoAn 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. 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. Lipids are the building blocks of all cell membranes and are thus essential for various biological functions varying from membrane trafficking to signal transduction. 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Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients.J. Proteome Res. 2010; 9: 2377-2389Crossref PubMed Scopus (179) Google Scholar). Although we have extensive experience with this type of chromatography, and in separating both inter- and intralipid classes, optimal separation of different isomers has not been achieved. Recently, Bird et al. (27Bird S.S. Marur V.R. Stavrovskaya I.G. Kristal B.S. Separation of cis-trans phospholipid isomers using reversed phase LC with high resolution MS detection.Anal. Chem. 2012; 84: 5509-5517Crossref PubMed Scopus (43) Google Scholar) described the separation of cis/trans phospholipids, exploring different RP C18 columns. An Ascentis Express C18 (2.7 µm particles) column gave identical separations and peak shape for cis/trans isomers as a charged surface hybrid (CSH) C18 column (1.7 µm particles). However, due to the lack of an ultra-high-pressure LC system [ultraperformance LC (UPLC)], the CSH C18 column was run at suboptimum conditions; hence, it was suggested to increase the flow rate to optimum conditions and thus improve peak shape and separation and shorten analysis time. The CSH material contains a low-level positive surface charge, in acidic mobile phases, in order to enhance the separation, in addition to increasing the loading capacity. Herein, we demonstrate the use of CSH C18 material for the separation of different lipid molecular species and different isomers within these classes in complex biological samples, in combination with ion-mobility spectrometry coupled with high-resolution MS. Ion mobility is, besides chromatographic separation, adding an additional separation dimension located within the mass spectrometer and therefore enhancing structural elucidation of lipids. Using ion mobility, collisional cross-sections (CCSs) of ions can be calculated, and besides accurate mass, fragmentation information, and retention time (Rt), these CCS values can be added to a searchable library for a routine work flow to increase the identification confidence. It was recently demonstrated that the CCS values are highly reproducible even using different machines (35Paglia G. Williams J.P. Menikarachchi L. Thompson J.W. Tyldesley-Worster R. Halldórsson S. Rolfsson O. Moseley A. Grant D. Langridge J. et al.Ion mobility derived collision cross sections to support metabolomics applications.Anal. Chem. 2014; (In press.)Crossref Scopus (246) Google Scholar). The chromatographic separation in combination with the ion-mobility separation is of pivotal importance for structural elucidation of these lipid isomers. HPLC-grade methanol originated from Actu-All Chemicals BV (Oss, The Netherlands). HPLC-grade chloroform, ultra liquid chromatography (ULC)-MS-grade water, 2-propanol (IPA), acetonitrile (ACN), and 99% pure ULC-MS formic acid were purchased from Biosolve BV (Valkenswaard, The Netherlands). Ammonium formate, dichloromethane (DCM), leucine enkephalin (Leu-Enk), and poly-dl-alanine (product number P9003) were from Sigma Aldrich (St. Louis, MO). All lipid standards (as shown in supplementary Table I), and extracts of bovine heart, liver, and brain were purchased from Avanti Lipids (Alabaster, AL) and from Nu-Chek Prep (Elysian, MN). Human plasma with heparin as anticoagulant was obtained from healthy volunteers who had given their informed consent for use of their plasma for method development. Throughout the entire paper and to follow a common standard lipid language, the lipid nomenclature described by LIPID MAPS (http://www.lipidmaps.org) was followed (3Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-861Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar, 4Fahy E. Subramaniam S. Murphy R.C. Nishijima M. Raetz C.R. Shimizu T. Spener F. van Meer G. Wakelam M.J. Dennis E.A. Update of the LIPID MAPS comprehensive classification system for lipids.J. Lipid Res. 2009; 50: S9-S14Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar). For example, 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine is PC (17:0/17:0), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine is LPC (19:0/0:0), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine is PE (15:0/15:0), and so forth. A mixture of 66 lipids was prepared as shown in Table 1. Stock solutions of 1 mg/ml were prepared in chloroform-methanol (2:1, v/v) and stored at −20°C. Stock solutions were diluted prior to analysis in IPA-ACN-water (2:1:1, v/v/v).TABLE 1.List of mixture of 66 lipid standards with corresponding concentration and RtLipid SubclassLipid Molecular SpeciesConcentration (pmol/μl)Rt (min)Peak number (as marked in Fig. 1)FAC13:1 (12Z)41.24aPeak ID not shown in Fig. 1. Retention time value is obtained in negative ion mode only. CE, cholesterol ester; Cho, cholesterol; D5-DG, D5-diacylglycerol; LPE, lyso-phosphatidylethanolamide; LPG, lyso-phosphatidylglycerol; LPI, lyso-phosphatidylinositol; LPS, lyso-phosphatidylserine; MG, monoacylglycerol; PS, phosphatidylserine.C17:1 (10Z)42.30aPeak ID not shown in Fig. 1. Retention time value is obtained in negative ion mode only. CE, cholesterol ester; Cho, cholesterol; D5-DG, D5-diacylglycerol; LPE, lyso-phosphatidylethanolamide; LPG, lyso-phosphatidylglycerol; LPI, lyso-phosphatidylinositol; LPS, lyso-phosphatidylserine; MG, monoacylglycerol; PS, phosphatidylserine.C23:1 (14Z)45.44aPeak ID not shown in Fig. 1. Retention time value is obtained in negative ion mode only. CE, cholesterol ester; Cho, cholesterol; D5-DG, D5-diacylglycerol; LPE, lyso-phosphatidylethanolamide; LPG, lyso-phosphatidylglycerol; LPI, lyso-phosphatidylinositol; LPS, lyso-phosphatidylserine; MG, monoacylglycerol; PS, phosphatidylserine.MG14:1 (9Z)41.11517:1 (10Z)41.70919:2 (10Z, 13Z)41.8710D5-DG1,3-14:0/14:027.60271,3-15:0/15:0210.17321,3-16:0/16:0212.98361,3-17:0/17:0213.60401,3-19:0/19:0214.55461,3-20:5 (5Z, 8Z, 11Z, 14Z, 17Z)/20:5 (5Z, 8Z, 11Z, 14Z, 17Z)25.41161,3-20:4 (5Z, 8Z, 11Z, 14Z)/20:4 (5Z, 8Z, 11Z, 14Z)27.88281,3-20:2 (11Z, 14Z)/20:2 (11Z, 14Z)213.23381,3-20:0/20:0214.9549DG19:1/19:1 (10Z)413.654219:1/19:1 (10Z) 1,3 isomer413.6543D5-TG14:0/16:1 (9Z)/14:0214.995015:0/18:1 (9Z)/15:0215.655316:0/18:0/16:0216.245719:0/12:0/19:0216.245817:0/17:1 (10Z)/17:0216.085520:4 (5Z, 8Z, 11Z, 14Z)/18:2 (9Z, 12Z)/20:4 (5Z, 8Z, 11Z, 14Z)214.904820:2 (11Z, 14Z)/18:3 (6Z, 9Z, 12Z)/20:2 (11Z, 14Z)215.465120:5 (5Z, 8Z, 11Z, 14Z, 17Z)/22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)/20:5 (5Z, 8Z, 11Z, 14Z, 17Z)214.104420:0/20:1 (11Z)/20:0217.1362TG19:2 (10Z, 13Z)/19:2 (10Z, 13Z)/19:2 (10Z, 13Z)415.5752PC17:0/20:4 (5Z, 8Z, 11Z, 14Z)27.252518:1 (9Z)/18:0410.843319:0/19:0413.604121:0/22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)211.7435LPC17:0 LPC41.508PA16:0/18:147.9229PE15:0/15:046.462017:0/17:0411.383418:0/18:0413.2037LPE17:1 (10Z) LPE41.257PS16:0/18:1 (9Z)46.381917:0/20:4 (5Z, 8Z, 11Z, 14Z)35.701718:1 (9Z)/18:1 (9Z)46.542121:0/22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)29.2031LPS17:1 (10Z) LPS41.002PG14:0/14:043.811317:0/17:048.363018:1 (9Z)/18:1 (9Z)46.712218:1 (9E)/18:1 (9E)47.362618:0/18:2 (9Z, 12Z)46.9724LPG17:1 (10Z) LPG41.003LPI17:1 (10Z) LPI251.001Cardiolipin14:1 (9Z)/14:1 (9Z)/14:1 (9Z)/15:1 (10Z)413.293915:0/15:0/15:0/16:1 (9Z)414.824722:1 (13Z)/22:1 (13Z)/22:1 (13Z)/14:1 (9Z)416.325924:1 (15Z)/24:1 (15Z)/24:1 (15Z)/14:1 (9Z)416.8961Sphingolipidd17:151.084d17:051.166d18:1/12:0 SM53.4911d18:1/12:0 Cer54.7215d18:1/25:0 Cer514.4945d18:1/12:0 Gluc Cer53.8414d18:1/12:0 Lac Cer53.5612Sphingomyelind18:1/17:0 SM46.8323ChoCho45.8418CE17:0416.336018:2 (TT)415.865418:1416.165623:0417.1963a Peak ID not shown in Fig. 1. Retention time value is obtained in negative ion mode only. CE, cholesterol ester; Cho, cholesterol; D5-DG, D5-diacylglycerol; LPE, lyso-phosphatidylethanolamide; LPG, lyso-phosphatidylglycerol; LPI, lyso-phosphatidylinositol; LPS, lyso-phosphatidylserine; MG, monoacylglycerol; PS, phosphatidylserine. Open table in a new tab Lipids were extracted from human plasma with a slightly adapted protocol from Bligh and Dyer (36Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). In brief, to 25 µl of plasma 100 µl of cold (−20°C) chloroform-methanol (2:1, v/v) was added. The sample was vortex mixed for 30 s at ambient temperatures and allowed to stand for 5 min at ambient temperatures. After vortex mixing for 30 s, the sample was centrifuged at 12,000 g for 5 min. The lower organic phase was transferred to a clean tube and evaporated to dryness under a gentle stream of nitrogen. Immediately prior to analysis, the sample was reconstituted in 25 µl chloroform-methanol (1:1, v/v) and diluted to 20 and 10 times the original volume of plasma in IPA-ACN-water (2:1:1, v/v/v) for positive and negative ion mode, respectively. Five microliters was injected into the UPLC system for positive ion mode and 10 µl for negative ion mode. Separation of lipids was carried out on an Acquity™ UPLC system (Waters Corporation, Milford, MA). Mobile phase A consisted of 10 mM ammonium formate with 0.1% formic acid in water-ACN (40:60, v/v), and mobile phase B was 10 mM ammonium formate with 0.1% formic acid in ACN-IPA (10:90, v/v). As weak wash, ACN-water-IPA (30:30:40, v/v/v) was used, and the strong wash was a mixture of IPA-water-formic acid-DCM (92:5:2:1, v/v/v/v). Gradient elution was applied at a flow rate of 0.4 ml/min through a CSH C18 column [100 × 2.1 mm inner diameter, particle size 1.7 µm (Waters Corporation, Milford, MA)] and thermostatted at 55°C. Initial conditions started with 40% B, and immediately a linear gradient (curve 6) was started from 40% to 43% B in 2 min. In the following 0.1 min, the percentage of mobile phase B was increased to 50%. Over the next 9.9 min, the gradient was further ramped up to 54% B, and the amount of mobile phase B was increased to 70% in 0.1 min. In the final part of the gradient, the % B was increased to 99% in 5.9 min. The eluent composition returned to the initial conditions in 0.1 min, and the column was equilibrated at the initial conditions for 1.9 min before the next injection, leading to a total run time of 20 min. Sample injections of 5 µl of both the lipid standard mixture and human plasma samples were carried out, and the autosampler temperature was set at 10°C. For both positive and negative ion mode, the same chromatographic conditions were used. Lipidomics studies often involve hundreds of samples with multiple replicate injections, and therefore, a shorter chromatographic run of 10 min was explored as an alternative to reduce the analysis time for isomeric separation. For this short method, all conditions were the same except the gradient. Initial gradient conditions started with 40% B, and immediately a linear gradient (curve 6) was started from 40% to 43% B in 1 min. In 0.1 min, mobile phase B was increased to 50% B. Over the next 4.9 min, the gradient was further ramped to 54% B, and the amount of mobile phase B was increased to 70% in 0.1 min. In the final part of the gradient, the % B was increased to 99% in 2.9 min. The eluent composition returned to the initial conditions in 0.1 min, and the column was equilibrated at the initial conditions for 0.9 min before the next injection. The UPLC system was coupled to a traveling wave ion-mobility-enabled hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (SYNAPT G2-S HDMS, Waters Corporation, Wilmslow, United Kingdom). Detailed descriptions of this mass spectrometer can be found elsewhere (37Konijnenberg A. Butterer A. Sobott F. Native ion mobility-mass spectrometry and related methods in structural biology.Biochim. Biophys. Acta. 2013; 1834: 1239-1256Crossref PubMed Scopus (190) Google Scholar, 38Pringle S.D. Giles K. Wildgoose J.L. Williams J.P. Slade S.E. Thalassinos K. Bateman R.H. Bowers M.T. Scrivens J.H. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument.Int. J. Mass Spectrom. 2007; 261: 1-12Crossref Scopus (663) Google Scholar). Electrospray positive and negative ionization modes were used. A capillary voltage and sampling cone voltage of (±) 0.6 kV and 30 V were used respectively for both polarities of electrospray ionization. The desolvation source conditions used nitrogen gas at 700 l/h with a constant desolvation temperature of 450°C. The source temperature was set at 120°C. Data were acquired over the m/z range of 50–1,200 Da. The mass spectrometer was operated in ion-mobility (HDMSE) mode for acquisition in both polarities. During this acquisition method, the first quadrupole Q1 was operated in a wide band radio frequency (RF) mode only, allowing all ions to enter the T-wave collision cell. The "trap" T-wave was operated at 4 V causing no fragmentation of the lipids. The intact lipid ions entered the helium cell region of the ion-mobility spectrometry (IMS) cell that was operated at 180 ml/min; the main function of the helium cell was to reduce the internal energy of ions and minimize fragmentation. The lipid ions then entered the IMS cell, held under 80 ml/min flow of nitrogen, to separate species according to their charge, mass, and CCS area. As the separated ions exited the IMS cell, they entered the "transfer" T-wave where two discrete and alternating acquisition functions were used. The first function, typically set at 2 eV, collects low-energy or unfragmented data, while the second function collects elevated-energy or fragment ion data, typically operated using a collision energy ramp from 30 to 55 eV. In both instances, argon gas is used for collision induced dissociation (CID). The trap T-wave, IMS T-wave, and the transfer T-wave all carried different wave velocities; these were 314, 600, and 190 m/s, respectively. The Stepwave was operated at default settings with a wave velocity of 300 m/s and a wave height of 15.0 V. Calibration of the ion-mobility cell for CCS calculations was performed using poly-dl-alanine at a concentration of 10 mg/L in water-ACN
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