Anal. Chem. 2007, 79, 4188-4191
Characterization of Sphingomyelins in Lipid Extracts Using a HPLC-MS-Offline-NMR Method Jan Willmann, Kerstin Mahlstedt, and Dieter Leibfritz*
Institute of Organic Chemistry, University of Bremen, Leobener Strasse NW 2C, 28359 Bremen, Germany Manfred Spraul
Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten/Karlsruhe, Germany Herbert Thiele
Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany
Sphingomyelins were characterized using a combination of a novel isocratic reversed-phase HPLC method with electrospray time-of-flight mass spectrometric detection and optional online MS/MS. The constitution of the sphingomyelins is determined by MS/MS experiments. Baseline separation of 17 compounds of a bovine brain extract (2 main compounds and 15 minor or trace compounds) was achieved with a mobile phase consisting of methanol, 2-propanol, THF, and water on a RP-18phenyl column. In parallel, the HPLC fraction were sampled to a 600-MHz NMR spectrometer to acquire 1D and 2D NMR spectra and to elucidate the molecular structure of individual sphingomyelin components. Sphingomyelins (SMs) are ubiquitous membrane components and highly bioactive. They are particularly highly concentrated in central and peripheral neural tissue. They consist perentially of the C18 sphingoidamine base connected to varying fatty acids. Sphingomyelins are lipid signaling molecules and precursors for the second messengers free ceramide and sphingosine.1 Altered levels of phospholipids can be observed in diabetes. Furthermore sphingomyelins are involved in neurodegenerative diseases: i.e., neuronal-ceroid lipofuscinosis, where preferentially shorter fatty acids can be observed; the Niemann-Pick disease, where pathophysiologically high sphingomyelin concentrations cause cell death.2-8 Brain tissue of Morbus Alzheimer patients * To whom correspondence should be addressed. Telephone: 0049-421-2182818. Fax: 0049-421-218-4264. E-mail:
[email protected]. (1) Kolesnick, R. N. Prog. Lipid Res. 1991, 30, 1-38. (2) Hsu, F. F.; Bohrer, A.; Wohltmann, M.; Ramanadham, S.; Ma, Z.; Yarasheski, K.; Turk, J. Lipids 2000, 35, 839-854. (3) Han, X.; Abendschein, D. R.; Kelley, J. G.; Gross, R. W. Biochem. J. 2000, 352, 79-89. (4) Kolter, T.; Sandhoff, K. Angew. Chem. 1999, 111, 1632-1670. (5) Ka¨kela¨, R.; Somerharju, P.; Tyynela¨, J. J. Neurochem. 2003, 84, 1051-1065. (6) He, X.; Chen, F.; McGovern, M. M.; Schuchman, E. H. Anal. Biochem. 2002, 306, 115-123. (7) Lee, C. Y.; Lesimple, A.; Larsen, Å.; Mamer, O.; Genest, J. J. Lipid Res. 2005, 46, 1213-1228. (8) Graber, D.; Salvayre, R.; Levade, T. J. Neurochem. 1994, 63, 1060-1068.
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also contains elevated concentrations.9 Because of their biochemical and clinical relevance, several methods have been applied to analyze SMs such as fluorescence assays,6 HPLC,10,11 ESI-MS,7,12,13 HPLC-MS,5,14 MALDI-TOF MS,15,16 and NMR.17-19 Fluorescence assays have a high sensitivity and are quantitative, but the molecular structure of the measured molecules cannot be elucidated. MS-based techniques are fast, sensitive, and require only minor sample preparation.7,12-16 When coupled with a HPLC system5,14 their selectivity can be improved and extra resolution is obtained because analytes of low concentration were not suppressed during the ESI process. With NMR spectroscopy,17-19 it is possible to measure intact biomaterials nondestructively without any preceding derivatization, but its low sensitivity limits a wide application. Therefore, an efficient analytical tool is needed to identify and quantify in situ low concentrated known, but also still unidentified natural occurring species to get rapid insight into biochemical processes. A combination of HPLC separation power, MS sensitivity with accurate mass measurement of molecular and fragment ions, and NMR structure elucidation power will meet most suitably the challenge. Furthermore, the low NMR sensitivity can be compensated for by preceding concentration steps via HPLC and fraction sampling.20 (9) Han, X.; Holtzman, D. M.; McKeel, D. W., Jr.; Kelley, J.; Morris, J. C. J. Neurochem. 2002, 82, 809-818. (10) Teng, J. I.; Smith, L. L. J. Chromatogr., A 1985, 322, 240-245. (11) Ramstedt, B.; Slotte, J. P. Anal. Biochem. 2000, 282, 245-249. (12) Bru ¨ gger, B.; Erben, G.; Sandhoff, R.; Wieland, F. T.; Lehmann, W. D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2339-2344. (13) Hsu, F.-F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 437-449. (14) Karlsson, A. Å.; Miche´lsen, P.; Odham, G. J. Mass Spectrom. 1998, 33, 1192-1198. (15) Rujoi, M.; Estrada, R.; Yappert, M. C. Anal. Chem. 2004, 76, 1657-1663. (16) Jackson, S. N.; Wang, H.-Y. J.; Woods, A. S. Anal. Chem. 2005, 77, 45234527. (17) Pearce, J. M.; Komoroski, R. A. Magn. Reson. Med. 2000, 44, 215-223. (18) Tesiram, Y. A.; Saunders, D.; Towner, R. A. Biochim. Biophys. Acta 2005, 1737, 61-68. (19) Byrdwell, W. C.; Perry, R. H. J. Chromatogr., A 2006, 1133, 149-171. (20) Albert, K., Ed. On-Line LC-NMR and Related Techniques; Wiley: New York, 2002. 10.1021/ac062326h CCC: $37.00
© 2007 American Chemical Society Published on Web 05/01/2007
Figure 1. Base peak chromatogram from the bovine brain extract. For chromatographic details, see Experimental Section. Table 1. Retention Times and Peak Assignment of the Separated Sphingomyelin Molecules retention time (min)
empirical formula sphingomyelin
empirical formula phosphocholine base
empirical formula neutral loss
18.0 20.0 21.3 24.7 27.6 29.0 33.5 38.6 43.9 48.1 51.3 57.4 60.7 68.3 71.9 82.1 99.8
C39H79N2O6P C39H81N2O6P C40H78N2O6P C41H83N2O6P C41H85N2O6P C42H85N2O6P C43H87N2O6P C45H89N2O6P C46H91N2O6P C45H91N2O6P C47H93N2O6P C46H93N2O6P C48H95N2O6P C47H95N2O6P C49H97N2O6P C48H97N2O6P C49H99N2O6P
C23H49N2O5P (18:1) C23H51N2O5P (18:0) C22H47N2O5P (17:1) C23H49N2O5P (18:1) C23H51N2O5P (18:0) C24H51N2O5P (19:1) C25H53N2O5P (20:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1) C23H49N2O5P (18:1)
C18H34O (stearinic acid) C16H30O (palmic acid) C18H34O (stearinic acid) C18H34O (stearinic acid) C18H34O (stearinic acid) C18H34O (stearinic acid) C18H34O (stearinic acid) C22H40O (docosenic acid) C23H42O (tricosenic acid) C22H42O (docosanic acid) C24H44O (nervonic acid) C23H44O (tricosanic acid) C25H46O (pentacosenoic acid) C24H46O (tetracosanic acid) C26H48O (hexacosenoic acid) C25H48O (pentacosanic acid) C26H50O (hexacosanoic acid)
EXPERIMENTAL SECTION Materials. All solvents, the formic acid, and the sphingomyelin extract from bovine brain were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). High-Performance Liquid Chromatography. A HP 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) was used. A mixed-mode phenyl/C18 reversed-phase HPLC column, 250 mm × 3 mm i.d. × 5 µm particle size (Nucleodur Sphinx, Macherey-Nagel, Du¨ren, Germany) was operated under isocratic conditions at 0.6 mL/min flow for screening with a mobile phase consisting of water with 0.1% formic acid, 2-propanol, THF, and methanol (35:30:20:15), alternatively, a 250 mm × 8 mm i.d. HPLC column at 4 mL/min flow was used to collect low
concentrated compounds for offline NMR (Gilson 215 Liquid handler; Gilson International B.V., Bad Camberg, Germany). The column temperature was kept at 40 °C. The collected samples were evaporated to dryness and redissolved in chloroform/ methanol (2:1; v/v). Mass Spectrometry. A micrOTOF-Q (Bruker Daltonik GmbH, Bremen, Germany) was used by flow splitting in both cases (screening and offline NMR) for mass spectrometric detection. The capillary voltage was set to 4500 V and the end plate offset to -500 V in negative ion mode. The nebulizer gas was set to 0.4 bar; dry gas and dry heat were set to 4 L/min and 200 °C, respectively. For MS/MS experiments, the collision energy of the Analytical Chemistry, Vol. 79, No. 11, June 1, 2007
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Figure 3. Molecular structure of the sphingomyelin at 51.3 min.
Figure 2. HSQC-NMR spectrum of the chromatographic peak at 51.3 min.
quadrupole was -42 eV/z. The molecular formula was generated by matching high mass accuracy and isotopic pattern (SigmaFit, Bruker Daltonik GmbH). Nuclear Magnetic Resonance. The NMR spectra were acquired on an Avance 600-MHz NMR spectrometer equipped with CryoFit with a 30-µL NMR active volume and a 13C/1H dual CryoProbe (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Germany). In case of 2D NMR spectra, 4k data points were recorded in f2 and 1k in f1. The number of scans was varied with respect to the concentration of the sample, but 16 scans were recorded at least. RESULTS High-Performance Liquid Chromatography. With the method described in the Experimental Section, baseline separation of 2 sphingomyelins as main components and 15 minor or trace compounds of a bovine brain extract was achieved (Figure 1). Four compounds were detected that presumably have an oddTable 2. 1H and assignment
13C
numbered fatty acid and two an odd-numbered, long-chain base. The eluation order depends on the number of carbon atoms (shortchain sphingomyelins come first). The peak correlation is shown in Table 1. Mass Spectrometry. From the TOF-MS results, we obtained the precision mass to determine the molecular formula and the degree of unsaturation. The precision in mass measurement was ∼2 ppm or better. Using the online MS/MS mode, we got structural information on the sphingomyelin. Depending on the skimmer voltage, sphingomyelin plus formiate (low voltage) or sphingomyelin minus methyl formiate (high voltage) in the negative ion mode was observed. Starting from sphingomyelin minus methyl formiate in the negative MS/MS mode, the polar head group (m/z 168.049) was cleaved off and neutral loss of the fatty acid as a ketene was observed. The results were also shown in Table 1. Nuclear Magnetic Resonance. To obtain further information about the constitution of the sampled sphingomyelins, they were transferred to the NMR spectrometer and 1D and 2D NMR spectra were recorded. As an example, Figure 2 shows the HSQC of the peak at 51.3 min and Table 2 and Figure 3 represent the CH correlations. The chemical shifts of the sphingo base double bond (trans configurated) were 130/5.24 and 134/5.5 ppm and the fatty acids (cis configurated) ones were 129/5.1 ppm. All spectra were calibrated to the methyl groups of phosphocholine (3.0 ppm). DISCUSSION High-Performance Liquid Chromatography. The separation of intact hydrophobic molecules such as sphingomyelins is one of the challenging tasks in reversed-phase HPLC, especially in hyphenated techniques, where baseline separation should be
Peak Assignment of the Sphingomyelin Molecule at 51.3 min 13C chemical shift δ (ppm)
1H chemical shift δ (ppm)
assignment
D-erythro-Sphingosine
1 2 3 4 5 6 7 16/22′ 17/23′ 18/24′
64.2 53.71 71.5 130.2 134.19 32.01 28.84 32.0 22.26 13.51
59.5 66.12 53.73
1H chemical shift δ (ppm)
Fatty Acid 3.89/3.75 3.71 3.82 5.24 5.5 1.81 1.38 1.06 1.09 0.68
Phosphocholine 2′′ 3′′ 4′′
13C chemical shift δ (ppm)
4.02 3.37 3.00
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1′ 2′ 3′ 7′/12′ 8′/11′ 9′/10′ 16/22′ 17/23′ 18/24′
174.12 36.09 25.60 29.35 26.79 129.91 32.0 22.26 13.51
1.96 1.37 1.142 1.81 5.13 1.06 1.09 0.68
achieved for the best results. The numbers of compositions for the mobile phases were limited by MS detection, where phosphate buffer solutions should be avoided. Furthermore, extreme pH conditions should be avoided in order to prevent hydrolysis during the sampling process. Mass Spectrometry. Using a TOF instrument with optional MS/MS mode has the main advantage of high mass precision in mother ions and fragment ions. Combination of these data allows identification of the lipid class, reconstruction of the lipid structure, and location of a double bond position in the long-chain base or the fatty acid. Furthermore, it is possible to notice possible oxidation of the lipids. The risk of ion suppression of unknown low concentrated sphingomyelin species during the ESI process is minimized by coupling the mass spectrometer to the HPLC system. Nuclear Magnetic Resonance. With the help of NMR, we were able to determine the constitution of the sphingomyelins and locate the double bond either within the fatty acid or within the long-chain base (Table 2).
The HPLC and MS results were confirmed by NMR spectroscopy, especially the position and configuration of the double bond site. The risk of peak overlapping in the NMR spectra was avoided either by recording 2D NMR spectra or by prior sampling the sphingomyelins by HPLC. CONCLUSION We developed a novel RP-HPLC screening method for separation of sphingomyelins to get rapid insight into the composition of lipids using MS sensitivity and NMR structure elucidation potential for characterization of individual compounds without interference from compounds with the starting material. It is possible to elucidate the structure of different sphingomyelin species in different tissues to generate MS and NMR databases.
Received for review December 8, 2006. Accepted March 29, 2007. AC062326H
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