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Journal of Chromatography A, 1250 (2012) 212–219
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Metabolic profiling of lipids by supercritical fluid chromatography/mass spectrometry Takeshi Bamba ∗ , Jae Won Lee, Atsuki Matsubara, Eiichiro Fukusaki Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Available online 28 May 2012 Keywords: Supercritical fluid chromatography Mass spectrometry Lipid Lipidomics
a b s t r a c t This review describes the usefulness of supercritical fluid chromatography (SFC) for the metabolic profiling of lipids. First, non-targeted lipid profiling by SFC/MS is described. The use of SFC/MS allows for high-throughput, exhaustive analysis of diverse lipids, and hence, this technique finds potential applications in lipidomics. Development of a polar lipid profiling method with trimethylsilyl (TMS) derivatization widens the scope of applicability of SFC/MS. SFC is a high-resolution technique that is suitable for nontargeted profiling aimed at the simultaneous analysis of many components. Next, targeted lipid profiling by SFC/MS is described. SFC is useful for the separation of lipids, such as carotenoids and triacylglycerols, which have numerous analogs with similar structures. In addition, SFC/MS shows the maximum efficiency for the target analysis of lipids in a biological sample that includes many matrices. Finally, a high-resolution, high-throughput analytical system based on SFC/MS is stated to be suitable for lipidomics because it is useful not only for the screening of lipid mixtures (as a fingerprint method) but also for the detailed profiling of individual components. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydrophobic metabolites, which are non-polar and show an affinity for other neutral and non-polar molecules, play an important role in many biochemical processes such as energy storage, cellular signaling, and cell–cell interactions. Furthermore, they are structural components of the cell membrane components [1,2]. These hydrophobic metabolites constitute a broad group of naturally occurring molecules, including fats, waxes, sterols, fatsoluble vitamins (A, D, E, and K), monoacylglycerols, diacylglycerols (DAGs), triacylglycerols (TAGs), phospholipids, sphingolipids, and others. Lipids are a class of representative hydrophobic metabolites in a biological system. The crucial roles of lipids in cell, tissue, and organ physiology have been demonstrated by a large number of genetic studies and many human diseases that involve the disruption of lipid metabolic enzymes and pathways [3]. However, the molecular composition of the cellular lipidome is complex and poorly understood. Lipidomics, defined as the systems-level analysis and characterization of lipids and their interacting moieties, is an emerging field of research. Lipidomics involves not only studies based on analytical techniques to obtain a “lipid profile”, which contains information on the composition and abundance of individual lipids,
∗ Corresponding author. Tel.: +81 06 6879 7418; fax: +81 06 6879 7418. E-mail address: [email protected] (T. Bamba). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.068
but also studies for gaining a comprehensive understanding of the various functions of lipids in a biological sample [4,5]. Lipids have high diversity because of the complex combination of hydrophobic acyl chains and a wide range of polarities resulting from the different types of attached hydrophilic molecules (Fig. 1). Therefore, it is challenging to analyze complex lipids in a mixture for the phenotype of biological samples. Hence, many previous studies have focused on the development of an effective lipid profiling system based on several chromatographic techniques such as TLC [6], GC [7,8], HPLC [9–11], ultra-high performance LC (UHPLC) [12], and supercritical fluid chromatography (SFC) [13–15]. SFC is a chromatographic technique in which a supercritical fluid (SF), which has low viscosity and high diffusivity, is used as the mobile phase. SFC, a fully mature technique, is a hybrid of GC and HPLC that incorporates many features of these two techniques [16,17]. Because of the low height equivalent to a theoretical plate (HETP) at relatively high flow rates, SFC facilitates high-throughput, high-resolution analysis [18]. Moreover, in SFC, a wide range of separation modes can be obtained through pressure and temperature manipulation [19]. Carbon dioxide (CO2 ) is the most frequently used mobile phase in SFC because it is chemically inert, non-inflammable, relatively less toxic, easy to handle, and inexpensive. Supercritical carbon dioxide (SCCO2 ) has low polarity and is hence effective for the analysis of hydrophobic compounds [20,21]. In addition, the polarity of the mobile phase in SFC can be changed considerably by adding a polar organic solvent such as
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Fig. 1. Molecular structures of various lipids: 1, phosphatidic acid (PA); 2, phosphatidylcholine (PC); 3, phosphatidylethanolamine (PE); 4, phosphatidylserine (PS); 5, phosphatidylglycerol (PG); 6, phosphatidylinositol (PI); 7, lysophosphatidic acid (LPA); 8, lysophosphatidylcholine (LPC); 9, lysophosphatidylethanolamine (LPE); 10, lysophosphatidylglycerol (LPG); 11, lysophosphatidylinositol (LPI); 12, diacylglycerols (DAG); 13, triacylglycerols (TAG); 14, monogalactosyldiacylglycerol (MGDG); 15, sphingomyeline (SM); 16, digalactosyldiacylglycerol (DGDG); 17, cerebrosides (CB); 18, ceramide (Cer); 19, lycopene; 20, -carotene; 21, lutein; 22, zeaxanthin; 23, antheraxanthin; 24, neoxanthin; and 25, violaxanthin.
methanol as a modifier. Therefore, SCCO2 is useful for the analysis of various lipids with a wide range of polarities. There are two main types of SFC: open tubular column SFC (OTSFC) and packed column SFC (PC-SFC) [22,23]. OT-SFC, which is similar to GC, can provide high-resolution separation when the sample is very complex and a large number of theoretical plates are mandatory. On the other hand, PC-SFC, similar to LC, provides high analysis speed and large sample capacity for the analysis of minor components and for preparative isolation. PC-SFC has been successfully used to analyze carotenoids, tocopherols, sterols, and squalene [24–26]. Complicated geometrical isomers and polymers extracted from plants have also been separated successfully by SFC [27,28]. Furthermore, the effectiveness of SFC in the analysis of various lipids has been demonstrated [29–31]. Mass spectrometry (MS) is a popular method to identify various lipids by using a combination of m/z and retention time data [32,33]. MS is used along with chromatography for avoiding ion suppression, a critical problem encountered in the identification of metabolites. Tandem MS (MS/MS) has also been used in lipidomics [34,35]. Highly selective quantification by MS/MS is advantageous for the profiling of various lipids in biological mixtures. In this review, we focus on the application of SFC/MS to lipidomics. Although many studies have reported on the use SFC/MS for the analysis of various compounds other than lipids, there are very few studies on the metabolic profiling of lipids by SFC/MS. Our research group has developed an effective lipidomics system based on SFC/MS, which offers several advantages such as
high throughput and high resolution and allows for the analysis of compounds with a wide range of polarities as well as the preparative isolation of lipids. Finally, we reviewed previous studies, including those published by our research group, for describing the usefulness of SFC/MS for the metabolic profiling of various lipids in a biological sample. 2. Non-targeted lipid profiling by SFC 2.1. Simultaneous analysis of diverse lipids 2.1.1. Introduction Although a lipid is generally considered to be hydrophobic, its polarity can be increased in association with highly hydrophilic molecules such as phosphoric acid and sugars. Lipids have high diversity owing to the complex combination of hydrophobic acyl chain molecular species. They also have a wide range of polarities depending on the different types of attached hydrophilic molecules. Therefore, analyzing complex lipids in a mixture is a challenging task. Simultaneous analysis of diverse lipids is too difficult with regard to their separation, detection, and identification. The first priority in lipidomics research, therefore, is the development of a practical high-throughput, high-resolution lipid profiling method. 2.1.2. Development of lipidomics system by SFC/MS In a previous study, SFC/MS was applied to develop an analytical system that enables simultaneous rapid analysis of lipids
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with varied structures and polarities [36]. The separation conditions for SFC (column type, modifier, back pressure, etc.) and the detection conditions for MS (ionization method, parameters, etc.) were investigated to develop a method for the simultaneous analysis of lipid mixtures. First, ionization conditions such as cone voltage and capillary voltage were examined by direct infusion analysis without SFC. Various phospholipids, glycolipids, neutral lipids, and sphingolipids were detected with high sensitivity by electrospray ionization (ESI). In addition, no difficulties were encountered in the ionization of non-polar lipids such as DAG and TAG by ESI in the positive-ion mode. Therefore, ESI was selected as the ionization method in this experiment. Various normal-phase and RP silica-gel-packed columns, including an unmodified silica column, an aminopropyl-modified silica column (amino silica column), a cyanopropyl-modified silica column (cyano silica column), a phenyl-modified column (phenyl silica column), an octyl-modified silica column (C8 column), and an octadodecyl (C18)-modified silica column (ODS column), were tested. When SCCO2 -containing methanol was used as the mobile phase, phosphatidylcholine (PC) was retained on each type of column. Separation based on the fatty acid (FA) side chain species was observed on the reversed-phase (RP) columns (phenyl, C8, ODS, and cyano columns) but not on the normal-phase columns (unmodified silica and amino silica columns). When the cyano column was used for the separation, a total of 14 types of lipids, including phospholipids PC, phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylglycerol [PG], phosphatidylserine [PS], lysophosphatidylcholine [LPC], and phosphatidic acid [PA]), glycolipids (monogalactosyldiacylglycerol [MGDG] and digalactosyldiacylglycerol [DGDG]), neutral lipids (DAG and TAG), and sphingolipids (sphingomyeline [SM], ceramide [Cer], and cerebrosides [CB]) were successfully detected in less than 15 min. All the lipids were detected in the positive-ion mode. TAG and DAG were detected only as positive ions, while PA and PI were more strongly detected as negative ions [37]. The use of an ODS column for the analysis of a lipid mixture resulted in separation that was dependent on the differences in the unsaturation of the FA side chains and isomer separation. As a result, the highest resolution in lipid analysis was achieved when using the ODS column, and the analysis time was less than 15 min. On the basis of these results, it can be concluded that the cyano column is the most suitable for the exhaustive analysis of lipids and that the ODS column is suitable for the analysis of the composition of the FA side chains in the lipids. Finally, SFC/MS was applied to a biological sample. The lipid derived from the leaf of Catharanthus roseus was analyzed using the cyano column. TAG, DAG, MGDG, DGDG, and PC were identified in the positive-ion mode, while only PA was detected in the negative-ion mode. By using the developed method, a crude unpurified sample was analyzed directly. In conclusion, it was demonstrated that SFC/MS is useful for the analysis of lipids in a biological sample containing various types of complex compounds. 2.2. Polar lipid profiling 2.2.1. Introduction The previous methods based on SFC/MS have certain demerits resulting from peak tailing and low detection sensitivity for polar lipids (Fig. 2). In order to solve these problems, a more advanced polar lipid profiling method was developed on the basis of SFC/MS with trimethylsilyl (TMS) derivatization [37]. Previously, a chemical derivatization method that with TMS, which has high chemical reactivity was used in the GC analysis of non-volatile compounds [38–40]. This derivatization was also applied to improve the peak shape and increase the detection sensitivity [41,42]. Upon TMS derivatization, the hydroxyl groups of the compounds are replaced
by trimethylsiloxy groups, and this can change the characteristics of the polar lipids in PC-SFC and improve the peak shapes and sensitivity. Therefore, TMS derivatization was used to establish an analytical system based on SFC/MS for the detection of different low-abundance polar lipids. In addition, the developed method was applied to analyze diverse polar lipids from biological samples. 2.2.2. Development of a method for profiling polar lipids by SFC/MS TMS derivatization was used for the analysis of 10 polar lipids: PG, PA, PI, LPC, lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), SM, and sphingosine-1-phosphate (S1P). In TMS derivatization, the extracted lipid and standard lipid samples were desolvated by N2 gas, then added to 60 L Ntrimethylsilylimidazole (TMSI) and 40 L of pyridine, and allowed to react at 37 ◦ C for 30 min. The derivatized lipids were subjected to SFC/MS analysis. The number of adducted TMS groups was related to the number of hydroxyl groups in each polar lipid: PG, 2; PI, 5; PA, 1; LPC, 1; LPE, 1; LPG, 3; LPA, 2; LPI, 5; SM, 1; and S1P, 2. In derivatization repeatability tests, the RSD (%) of the derivatized polar lipids was as follows: PG, 9.6%; PI, 3.9%; PA, 6.8%; LPC, 2.1%; LPE, 7.5%; LPG, 8.8%; LPI, 9.2%; LPA, 9%; SM, 3.5%; and S1P, 2.7%. All the RSDs were under 10%, indicating that the repeatability was sufficient for polar lipid profiling. Triple-quadrupole (QqQ) MS was used to detect several polar lipids. Multiple reaction monitoring (MRM) is a non-scanning technique that is generally performed in QqQMS, in which selectivity is enhanced by fragmentation. In MRM, two mass analyzers are used as static mass filters to monitor a particular fragment ion of a selected precursor ion [43]. The MRM conditions, including MRM transitions (precursor m/z > fragment m/z), cone voltage (CV), and MS/MS collision energy (CE), were optimized for the analysis of non-derivatized and TMS-derivatized polar lipids. By TMS derivatization, 10 polar lipids were detected as [M+H]+ ions in the positive-ion mode. Further, each polar lipid showed a specific pattern of fragmentation by collision-induced dissociation (CID). The efficiency of the cyano column, C8 column, and several ODS columns for the separation of polar lipids was investigated. Finally, an Inertsil ODS-EP column (250 mm × 4.6 mm I.D.; 5 m, GL Sciences) was chosen because sharp peaks and excellent resolution are typically obtained when using this column. This column is suitable for the separation of polar compounds because of the polar functional groups embedded between the silica surface and the ODS groups. MRM data pertaining to 20 lipids—10 non-derivatized lipids and 10 TMS-derivatized lipids—were used to compare the nonderivatization analysis with the TMS derivatization analysis (Fig. 3). The non-derivatized polar lipids were analyzed using a modifier gradient, from 15% to 30% (v/v) over 15 min. Peak tailing was observed in the analysis of PA, PI, LPE, LPA and LPI. Furthermore, it was difficult to identify the peak due to S1P because of the continuous elution after 7 min (Fig. 3A). However, pronounced peak tailing was observed in the analysis of PA, PI, LPA, LPI, and S1P by TMS derivatization (Fig. 3B). The derivatized polar lipids were analyzed with a gradient modifier from 15% to 20% (v/v) for 10 min. Furthermore, the limit of detection (LOD) of PG, PI, LPA, LPI, and S1P was enhanced 12-, 40-, 510-, 39-, and 1490-fold, respectively. Next, the developed method was applied to the analysis of sheep plasma. In comparison with lipid profiling without derivatization, that with TMS derivatization allowed for the additional detection of 20 minor species of PI, LPC, LPE, and SM, and seven molecular species of LPA, LPI, and S1P. The relative ratio of the molecular species in each polar lipid was also found by quantification. Finally, the simultaneous and detailed profiling of 10 polar lipids was successfully performed by SFC/MS with TMS derivatization.
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Fig. 2. Base peak ion (BPI) chromatograms and 2D displays of SFC/MS data obtained from the lipid mixtures by using a cyano column in the positive-ion mode (A) and negative-ion mode (B), reproduced from Ref. [36] with some alteration. 1, TAG; 2, DAG; 3, MGDG; 4, DGDG; 5, PA; 6, PC; 7, PE; 8, PG; 9, PI; 10, PS; 11, LPC; 12, SM; 13, Cer; and 14, CB.
This developed method is suitable for lipidomics, especially for targeting polar lipids. 3. Targeted lipid profiling by SFC 3.1. Carotenoids 3.1.1. Introduction Carotenoids play an important role in vision [44] and they are fat-soluble antioxidants that have a protective effect on membrane lipids [45]. Metabolic profiling of carotenoids is important for understanding their physiological functions, but a more advanced technique is required for the analysis of biological samples containing a mixture of structurally similar carotenoids. Recently, RP-HPLC has been used for the separation of carotenoids [46,47]. It is difficult to separate structurally similar carotenoids using ODS columns, which are generally employed in RP-HPLC. Columns packed with triacontyl-bonded silica (C30), whose hydrophobic sites are larger than those of ODS columns, are often used for carotenoid analysis, but the analysis time is very long [48]. 3.1.2. Carotenoid profiling by SFC Schmitz et al. employed an OT-SFC coupled with a UV/vis detector for analysis of the cis/trans isomers of carotenes [49]. They used a 7-m-long capillary column with a cross-linked stationary phase consisting of 25% cyanopropyl and 75% polymethylsiloxane. They showed the effectiveness of SFC in the analysis of thermolabile compounds, which could not be separated by LC, the commonly
used separation technique at that time. In addition, Ibanes and co-workers tried to develop a carotenoid analysis method using neat CO2 without any modifiers, with the vision to online-coupling with supercritical fluid extraction [50]. High-speed separation (in 10 min) of -carotene and lycopene was achieved using a 50-cmlong packed capillary column with highly pressurized (300 atm) CO2 . Lesellier et al. investigated various parameters such as temperature, pressure, modifier, and column stationary phase for the effective separation of carotenoids by PC-SFC [24,51–53]. One of the achievements of their study was the excellent separation of a mixture of 11 carotenoids including structural isomers. Subsequently, Lesellier et al. extended their research and made use of carotenoid analysis for column characterization [54–58]. They tried to estimate various important column characteristics such as bonding density, hydrophobicity, and polar site accessibility on the basis of the separation capacity of cis -carotenes and zeaxanthin. These achievements clearly show the advantages of SFC for carotenoid separation. 3.1.3. Carotenoid profiling by SFC/MS Co-elution of various metabolites in biological samples often interferes with the accurate identification and quantification of target compounds. The low sensitivity of photometric detection, which is widely used for the detection of carotenoids, limits the applicable sample volume. This restriction often obstructs the application of photometric detection to clinical samples. The use of MS helps overcome the shortcomings of photometric detection and allows for the detection of small amounts of carotenoids. Hence, we tried to apply SFC/MS to carotenoid analysis [15].
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Fig. 3. MRM data of the standard PG (C34:1), PA (C34:1), PI (C38:4), LPC (C18:0), LPE (C18:0), LPG (C14:0), LPA (C17:0), LPI (C18:0), SM (C18:0), and S1P (C18:1) by (A) non-derivatization and (B) TMS derivatization. Reproduced from Ref. [37] with some alteration.
Carotenes (-carotene and lycopene) and xanthophylls (zeaxanthin, lutein, antheraxanthin, neoxanthin, and violaxanthin) were analyzed as models for the separation of carotenoids. Since most of the carotenoids were not retained on normal-phase packed columns, we used the ODS column. After testing ODS columns with various packing materials, we found that seven carotenoids could be separated within 15 min on the Hibar Purospher STAR RP-18e (Merck) column. In addition, monolithic columns manufactured by a new sol–gel process [59–61], which include a continuous 3D silica network with macropores (ca. 2 m, corresponding to the pore spaces in particle-packed columns) and mesopores (ca. 13 nm, corresponding to the fine pores in particle-packed columns) [62]. The pressure drop in monolithic columns is lower than that in particlepacked columns, owing to the relatively wider flow channels in the former case [62]. Seven carotenoids were successfully separated in 10 min on such a monolithic column (Fig. 4A). Furthermore, separation performed at a flow rate of 9 mL/min yielded satisfactory results (the resolution of separation of neoxanthin and violaxanthin was 2.62), and the analysis time was reduced to 4 min (Fig. 4B). For the analysis of biological samples, three monolithic columns were connected in series, which helped improve the efficiency of separation of the target carotenoid from contaminants. 3.1.4. Profiling of carotenoids and their derivatives by SFC/MS/MS 3.1.4.1. Xanthophyll esters. We also tried to apply SFC with MS/MS for carotenoid analysis [63]. MS/MS is a powerful method for the acquisition of information about metabolites in biological samples. Partial structure information can be obtained by product ion scan, which is one of the MS/MS detection modes. Highly sensitive and selective detection of target metabolites can be performed by MRM. SFC/MS/MS was applied to the analysis of xanthophyll esters. In physiological tissues, hydroxylated xanthophylls exist not only in free form but also in FA ester form [64]. These esters have high hydrophobicity and include various structurally similar compounds having different FA chains. The esterified form of a
xanthophyll has higher thermal stability and bioavailability than does its free-form counterpart [65,66]. However, the in vivo kinetics and physiological functions of xanthophyll FA esters are not yet well understood. Conventional HPLC methods are time-consuming, requiring more than 40 min for the analysis of xanthophyll FA esters [67–69]. Thus, a high-throughput, high-sensitivity analysis system is required to obtain information about xanthophyll FA esters. In the previous study, the analysis of -cryptoxanthin (CX) was focused because recent epidemiological studies reported that CX has higher ability to prevent cancer and diabetes. CX is classified as a xanthophyll, and it exists not only in a free form, but also in a fatty-acid-esterified form in physiological tissues. First, investigations were conducted to identify the appropriate ionization conditions for -cryptoxanthin fatty acid esters (CXFAs) by infusion analysis. The molecular ions of carotenoids were observed as radical cations by ESI in the positive-ion mode. Common product ions, [M−92]+ , generated by the elimination of toluene from the polyene chain, were observed in the product ion scan. In the following experiments, MRM analysis based on this information was employed for the highly sensitive detection of CXFAs. Next, separation of a standard mixture of CXFAs was attempted using various columns under the aforementioned conditions, with the aim of identifying a suitable column for CXFA analysis. All the CXFAs were successfully separated within 20 min when Inertsil ODS-P (250 mm × 4.6 mm I.D., 5 m; GL Sciences), which is a polymeric ODS column (Fig. 5), was used. The LOD was 540 fmol for the free forms of the CXFAs and 32–130 fmol for the esterified forms. In our research, we used QqQMS, which offers higher selectivity and sensitivity than MS, since it enabled the detection of CX and CXFAs even at femtomolar concentrations. 3.1.4.2. Carotenoids and their epoxides. SFC/MS/MS has also been applied to carotenoids and their epoxides [70]. Epoxy carotenoids, which are the oxidation products of carotenoids, are potential oxidative stress markers. Several studies suggested that these
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Fig. 5. MRM data of free form, laurate ester, myristate ester, palmitate ester, stearate ester, oleate ester, linoleate ester, linolenate ester, EPA ester, DHA ester of -cryptoxanthin obtained from standard mixtures analysis by SFC/MS/MS using Inertsil ODS-P column. Reproduced from Ref. [59] with permission.
Fig. 4. Mass chromatograms of carotenoids using a monolithic ODS column (100 mm × 4.6 mm I.D.), reproduced from Ref. [15] with permission. Analysis conditions: modifier, methanol with 0.1% ammonium formate 1–5% 8 min; flow rate, (A) 3 mL/min and (B) 9 mL/min. 1, lycopene; 2, -carotene; 3, lutein; 4, zeaxanthin; 5, antheraxanthin; 6, neoxanthin; and 7, violaxanthin.
oxidation products contribute to the pro-carcinogenic effect of carotenoids in subjects suffering from oxidative stress [71,72]. Therefore, to understand the role of carotenoids in the human body, it is necessary to analyze not only carotenoids but also their oxidation products. However, it is difficult to profile epoxy carotenoids because they are present in very small amounts. Furthermore, epoxy carotenoids are isomers of hydroxyl carotenoids, which include predominant xanthophylls such as -cryptoxanthin; therefore, precise separation and identification of these carotenoids is difficult. SFC/MS/MS is the solution to these problems. First, the ionization conditions in MS/MS were investigated. The epoxidation product of -carotene was analyzed by infusion analysis. Since epoxy -carotene is an isomer of -cryptoxanthin (hydroxyl carotenoid), the m/z value for both these molecules was the same, but the results of product ion scan were different. The product ion spectrum of epoxy -carotene showed an [M−80]+ ion peak in addition to the peaks observed in the product ion spectrum of CX. Thus, these results suggested that highly sensitive structural characterization of carotenoids is possible by targeting
such structure-specific neutral losses. Under the optimized separation conditions, predominant human carotenoids and their epoxy products were efficiently separated within 20 min using an ODS column. The combination of isomer separation by SFC with highly selective detection by MS/MS is a powerful strategy for the accurate analysis of carotenoids. In addition, sub-femtomolar levels of carotenoids could be detected; limit of detection for b-carotene was 0.093 fmol. The high sensitivity of the constructed system helped in reducing the sample volume; merely 0.1 mL of human serum was required for the detection of five major carotenoids and six epoxy carotenoids. These results clearly show the advantage of SFC/MS/MS for a large number of small-scale analyses such as small-scale intervention studies. 3.2. Triacylglycerols 3.2.1. Introduction TAGs are natural compounds produced by the esterification of glycerol with FAs. In humans, TAGs serve as a source of energy stored in fat tissues and form a thermal and mechanical protective layer around important organs; further, they are the source of essential FAs (linoleic and linolenic acids), fat-soluble vitamins, and other non-polar compounds. TAG imbalance can lead to several diseases such as coronary heart disease, dyslipidaemia, and obesity or inborn errors of metabolism. The main sources of TAGs in the human diet are vegetable oils and animal fats [73,74]. TAGs have very high diversity because of the wide variety of possible FAs, esterified to three stereospecific positions of the glycerol backbone.
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Thus, separation and identification of individual TAGs in a mixture is very difficult [75]. 3.2.2. Triacylglycerol profiling by SFC Manninen et al. reported the separation of ␥- and ␣-linolenic acids containing TAGs with identical acyl carbon numbers and degrees of unsaturation by capillary SFC using a 25% cyanopropane–75% methylpolysiloxane stationary phase [76]. The resolution of 1,3-dioleoyl-2-␥-linolenoyl-sn-glycerol and 1,3dioleoyl-2-␣-linolenoyl-sn-glycerol was 1.35 on a 10 m × 50 m I.D. column, and the resolution was enhanced to 1.66 by combining two 10-m columns in series. The difference in the positions of the double bonds in one linolenic acid residue of the TAGs resulted in two series of peaks during the separation of alpine currant (Ribes alpinum) and black currant (R. nigrum) seed oils. The use of the 10-m column was found to be appropriate for screening the TAG profile in both seed oils. 3.2.3. Triacylglycerol profiling by SFC/MS Sandra et al. reported the characterization of TAGs in vegetable oils by using a silver-ion packed-column (SI-PC) SFC/MS [77]. TAGs were mainly detected as [M+Ag]+ ions by using a coordination ion spray (CIS)–electrospray ionization (ESI)-MS. In a SI-PC, TAGs are separated according to the number of double bonds. Within each group, an additional separation is observed according to the carbon number (CN), i.e. the number of carbon atoms of the three fatty acids bonded to the glycerol backbone. Solutes characterized by the same CN elute according to the number of unsaturated fatty acids, e.g. TAG (SLL) elutes before TAG (OOL). In the use of atmospheric pressure chemical ionization (APCI)-MS, TAGs were detected as [M+H]+ ions. Approximately 90 min were required to separate divers TAGs in a mixture. In our previous study, SFC/MS was also applied for the analysis of soybean lipids [78]. Diverse lipids were analyzed simultaneously by using a cyano column with a gradient modifier from 10% to 30% (v/v) for 20 min. TAG and PC were mainly detected as ammonium adduct ions ([M+NH4 ]+ ) by ESI in the positive-ion mode. Furthermore, diverse TAGs were analyzed successfully when an ODS column was used for 15 min with the gradient modifier ranged from 20% to 35% (v/v). Next, a high-throughput, high-resolution TAG profiling method was developed on the basis of SFC/MS. After testing several columns, it was found that three Chromolith Performance RP-18e columns connected in series would be the most effective for TAG profiling. Monolith columns have many advantages such as high resolution, high throughput, and low backpressure. The total column length can be increased by connecting three or more of these columns in series, and thus, the resolution can be improved. SFC/MS with three tandem monolith ODS columns was used to analyze the TAGs in a mixture of soybean lipids. Individual TAG molecules were separated effectively within 8 min on the 2D map according to the retention time and m/z (Fig. 6). For TAG identification, programmed cone voltage fragmentation in single quadrupole (Q)-MS was used. Previously, it was reported that certain structural isomers could be distinguished and identified by this method and that the sensitivity and selectivity of this method were high [79]. By fragmentation, TAG was mainly found when the cone voltage was 35 V, and two types of fragmented ions, DAG and MAG, were confirmed when the cone voltage was 50 and 90 V, respectively. The molecular structure of the TAGs was identified from fragmented ions that had a specific value of m/z. In particular, the structure of the TAGs was identified by the type and peak intensity of the fragmented ions (DAG). For example, in the case of TAG (SOL) ([M+NH4 ]+ ; m/z 902.8), three fragmented ions, DAG (SO) (m/z 605.5), DAG (SL) (m/z 603.5), and DAG (OL) (m/z 601.4), were detected. Their peak intensities were in the order SL > SO > OL. Theoretically, the stereospecific numbering
Fig. 6. Identified TAGs in 2D map by SFC/MS using three tandem monolith ODS column, reproduced from Ref. [78] with some alteration. S, stearic acid (C18); O, oleic acid (C18:1); L, linoleic acid (C18:2); Ln, linolenic acid (C18:3); P, palmitic acid (C16).
(sn)-2 position of an FA is fragmented more easily than are the sn1, 3 positions. If the peak intensity of SL were the highest, it would imply that TAG has O at the sn-2 position and that S and L are at the sn-1 and sn-3 positions respectively. Additionally, fragmentation of unsaturated FAs is easier than that of saturated FAs. Therefore, it is reasonable that the peak intensity of SO is higher than the peak intensity of OL. Finally, it was demonstrated that this method is reliable and accurate even when using single Q-MS. 4. Conclusion This review demonstrates the usefulness of SFC/MS for the metabolic profiling of hydrophobic compounds in a biological sample. Owing to the advantages of an SF as the mobile phase, highthroughput, high-resolution analysis becomes possible. SFC is also effective for the separation of diverse lipids with a wide range of polarities. In addition, while UHPLC shows excellent performance only in lipid profiling, SFC is suitable for use in preparative isolation and analysis of a high-volume lipid mixture that includes minor components as well as major ones. The particle size in the packed column used in SFC (3 m or 5 m) is larger than that in UHPLC columns (sub-2 m). Furthermore, the diameter of the SFC column (4.6 mm) is greater than that of UHPLC columns (2.1 mm). Therefore, SFC is an effective analytical method for targeting low volumes of unknown minor lipids in a biological sample. In lipidomics, SFC/MS can be effectively used for the targeted and non-targeted lipid profiling of several biological samples. In the future, various types of columns and detection methods would be used to develop lipid-profiling methods based on SFC/MS. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
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Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Metabolic profiling of lipids by supercritical fluid chromatography/mass spectrometry Takeshi Bamba ∗ , Jae Won Lee, Atsuki Matsubara, Eiichiro Fukusaki Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Available online 28 May 2012 Keywords: Supercritical fluid chromatography Mass spectrometry Lipid Lipidomics
a b s t r a c t This review describes the usefulness of supercritical fluid chromatography (SFC) for the metabolic profiling of lipids. First, non-targeted lipid profiling by SFC/MS is described. The use of SFC/MS allows for high-throughput, exhaustive analysis of diverse lipids, and hence, this technique finds potential applications in lipidomics. Development of a polar lipid profiling method with trimethylsilyl (TMS) derivatization widens the scope of applicability of SFC/MS. SFC is a high-resolution technique that is suitable for nontargeted profiling aimed at the simultaneous analysis of many components. Next, targeted lipid profiling by SFC/MS is described. SFC is useful for the separation of lipids, such as carotenoids and triacylglycerols, which have numerous analogs with similar structures. In addition, SFC/MS shows the maximum efficiency for the target analysis of lipids in a biological sample that includes many matrices. Finally, a high-resolution, high-throughput analytical system based on SFC/MS is stated to be suitable for lipidomics because it is useful not only for the screening of lipid mixtures (as a fingerprint method) but also for the detailed profiling of individual components. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydrophobic metabolites, which are non-polar and show an affinity for other neutral and non-polar molecules, play an important role in many biochemical processes such as energy storage, cellular signaling, and cell–cell interactions. Furthermore, they are structural components of the cell membrane components [1,2]. These hydrophobic metabolites constitute a broad group of naturally occurring molecules, including fats, waxes, sterols, fatsoluble vitamins (A, D, E, and K), monoacylglycerols, diacylglycerols (DAGs), triacylglycerols (TAGs), phospholipids, sphingolipids, and others. Lipids are a class of representative hydrophobic metabolites in a biological system. The crucial roles of lipids in cell, tissue, and organ physiology have been demonstrated by a large number of genetic studies and many human diseases that involve the disruption of lipid metabolic enzymes and pathways [3]. However, the molecular composition of the cellular lipidome is complex and poorly understood. Lipidomics, defined as the systems-level analysis and characterization of lipids and their interacting moieties, is an emerging field of research. Lipidomics involves not only studies based on analytical techniques to obtain a “lipid profile”, which contains information on the composition and abundance of individual lipids,
∗ Corresponding author. Tel.: +81 06 6879 7418; fax: +81 06 6879 7418. E-mail address: [email protected] (T. Bamba). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.068
but also studies for gaining a comprehensive understanding of the various functions of lipids in a biological sample [4,5]. Lipids have high diversity because of the complex combination of hydrophobic acyl chains and a wide range of polarities resulting from the different types of attached hydrophilic molecules (Fig. 1). Therefore, it is challenging to analyze complex lipids in a mixture for the phenotype of biological samples. Hence, many previous studies have focused on the development of an effective lipid profiling system based on several chromatographic techniques such as TLC [6], GC [7,8], HPLC [9–11], ultra-high performance LC (UHPLC) [12], and supercritical fluid chromatography (SFC) [13–15]. SFC is a chromatographic technique in which a supercritical fluid (SF), which has low viscosity and high diffusivity, is used as the mobile phase. SFC, a fully mature technique, is a hybrid of GC and HPLC that incorporates many features of these two techniques [16,17]. Because of the low height equivalent to a theoretical plate (HETP) at relatively high flow rates, SFC facilitates high-throughput, high-resolution analysis [18]. Moreover, in SFC, a wide range of separation modes can be obtained through pressure and temperature manipulation [19]. Carbon dioxide (CO2 ) is the most frequently used mobile phase in SFC because it is chemically inert, non-inflammable, relatively less toxic, easy to handle, and inexpensive. Supercritical carbon dioxide (SCCO2 ) has low polarity and is hence effective for the analysis of hydrophobic compounds [20,21]. In addition, the polarity of the mobile phase in SFC can be changed considerably by adding a polar organic solvent such as
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Fig. 1. Molecular structures of various lipids: 1, phosphatidic acid (PA); 2, phosphatidylcholine (PC); 3, phosphatidylethanolamine (PE); 4, phosphatidylserine (PS); 5, phosphatidylglycerol (PG); 6, phosphatidylinositol (PI); 7, lysophosphatidic acid (LPA); 8, lysophosphatidylcholine (LPC); 9, lysophosphatidylethanolamine (LPE); 10, lysophosphatidylglycerol (LPG); 11, lysophosphatidylinositol (LPI); 12, diacylglycerols (DAG); 13, triacylglycerols (TAG); 14, monogalactosyldiacylglycerol (MGDG); 15, sphingomyeline (SM); 16, digalactosyldiacylglycerol (DGDG); 17, cerebrosides (CB); 18, ceramide (Cer); 19, lycopene; 20, -carotene; 21, lutein; 22, zeaxanthin; 23, antheraxanthin; 24, neoxanthin; and 25, violaxanthin.
methanol as a modifier. Therefore, SCCO2 is useful for the analysis of various lipids with a wide range of polarities. There are two main types of SFC: open tubular column SFC (OTSFC) and packed column SFC (PC-SFC) [22,23]. OT-SFC, which is similar to GC, can provide high-resolution separation when the sample is very complex and a large number of theoretical plates are mandatory. On the other hand, PC-SFC, similar to LC, provides high analysis speed and large sample capacity for the analysis of minor components and for preparative isolation. PC-SFC has been successfully used to analyze carotenoids, tocopherols, sterols, and squalene [24–26]. Complicated geometrical isomers and polymers extracted from plants have also been separated successfully by SFC [27,28]. Furthermore, the effectiveness of SFC in the analysis of various lipids has been demonstrated [29–31]. Mass spectrometry (MS) is a popular method to identify various lipids by using a combination of m/z and retention time data [32,33]. MS is used along with chromatography for avoiding ion suppression, a critical problem encountered in the identification of metabolites. Tandem MS (MS/MS) has also been used in lipidomics [34,35]. Highly selective quantification by MS/MS is advantageous for the profiling of various lipids in biological mixtures. In this review, we focus on the application of SFC/MS to lipidomics. Although many studies have reported on the use SFC/MS for the analysis of various compounds other than lipids, there are very few studies on the metabolic profiling of lipids by SFC/MS. Our research group has developed an effective lipidomics system based on SFC/MS, which offers several advantages such as
high throughput and high resolution and allows for the analysis of compounds with a wide range of polarities as well as the preparative isolation of lipids. Finally, we reviewed previous studies, including those published by our research group, for describing the usefulness of SFC/MS for the metabolic profiling of various lipids in a biological sample. 2. Non-targeted lipid profiling by SFC 2.1. Simultaneous analysis of diverse lipids 2.1.1. Introduction Although a lipid is generally considered to be hydrophobic, its polarity can be increased in association with highly hydrophilic molecules such as phosphoric acid and sugars. Lipids have high diversity owing to the complex combination of hydrophobic acyl chain molecular species. They also have a wide range of polarities depending on the different types of attached hydrophilic molecules. Therefore, analyzing complex lipids in a mixture is a challenging task. Simultaneous analysis of diverse lipids is too difficult with regard to their separation, detection, and identification. The first priority in lipidomics research, therefore, is the development of a practical high-throughput, high-resolution lipid profiling method. 2.1.2. Development of lipidomics system by SFC/MS In a previous study, SFC/MS was applied to develop an analytical system that enables simultaneous rapid analysis of lipids
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with varied structures and polarities [36]. The separation conditions for SFC (column type, modifier, back pressure, etc.) and the detection conditions for MS (ionization method, parameters, etc.) were investigated to develop a method for the simultaneous analysis of lipid mixtures. First, ionization conditions such as cone voltage and capillary voltage were examined by direct infusion analysis without SFC. Various phospholipids, glycolipids, neutral lipids, and sphingolipids were detected with high sensitivity by electrospray ionization (ESI). In addition, no difficulties were encountered in the ionization of non-polar lipids such as DAG and TAG by ESI in the positive-ion mode. Therefore, ESI was selected as the ionization method in this experiment. Various normal-phase and RP silica-gel-packed columns, including an unmodified silica column, an aminopropyl-modified silica column (amino silica column), a cyanopropyl-modified silica column (cyano silica column), a phenyl-modified column (phenyl silica column), an octyl-modified silica column (C8 column), and an octadodecyl (C18)-modified silica column (ODS column), were tested. When SCCO2 -containing methanol was used as the mobile phase, phosphatidylcholine (PC) was retained on each type of column. Separation based on the fatty acid (FA) side chain species was observed on the reversed-phase (RP) columns (phenyl, C8, ODS, and cyano columns) but not on the normal-phase columns (unmodified silica and amino silica columns). When the cyano column was used for the separation, a total of 14 types of lipids, including phospholipids PC, phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylglycerol [PG], phosphatidylserine [PS], lysophosphatidylcholine [LPC], and phosphatidic acid [PA]), glycolipids (monogalactosyldiacylglycerol [MGDG] and digalactosyldiacylglycerol [DGDG]), neutral lipids (DAG and TAG), and sphingolipids (sphingomyeline [SM], ceramide [Cer], and cerebrosides [CB]) were successfully detected in less than 15 min. All the lipids were detected in the positive-ion mode. TAG and DAG were detected only as positive ions, while PA and PI were more strongly detected as negative ions [37]. The use of an ODS column for the analysis of a lipid mixture resulted in separation that was dependent on the differences in the unsaturation of the FA side chains and isomer separation. As a result, the highest resolution in lipid analysis was achieved when using the ODS column, and the analysis time was less than 15 min. On the basis of these results, it can be concluded that the cyano column is the most suitable for the exhaustive analysis of lipids and that the ODS column is suitable for the analysis of the composition of the FA side chains in the lipids. Finally, SFC/MS was applied to a biological sample. The lipid derived from the leaf of Catharanthus roseus was analyzed using the cyano column. TAG, DAG, MGDG, DGDG, and PC were identified in the positive-ion mode, while only PA was detected in the negative-ion mode. By using the developed method, a crude unpurified sample was analyzed directly. In conclusion, it was demonstrated that SFC/MS is useful for the analysis of lipids in a biological sample containing various types of complex compounds. 2.2. Polar lipid profiling 2.2.1. Introduction The previous methods based on SFC/MS have certain demerits resulting from peak tailing and low detection sensitivity for polar lipids (Fig. 2). In order to solve these problems, a more advanced polar lipid profiling method was developed on the basis of SFC/MS with trimethylsilyl (TMS) derivatization [37]. Previously, a chemical derivatization method that with TMS, which has high chemical reactivity was used in the GC analysis of non-volatile compounds [38–40]. This derivatization was also applied to improve the peak shape and increase the detection sensitivity [41,42]. Upon TMS derivatization, the hydroxyl groups of the compounds are replaced
by trimethylsiloxy groups, and this can change the characteristics of the polar lipids in PC-SFC and improve the peak shapes and sensitivity. Therefore, TMS derivatization was used to establish an analytical system based on SFC/MS for the detection of different low-abundance polar lipids. In addition, the developed method was applied to analyze diverse polar lipids from biological samples. 2.2.2. Development of a method for profiling polar lipids by SFC/MS TMS derivatization was used for the analysis of 10 polar lipids: PG, PA, PI, LPC, lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), SM, and sphingosine-1-phosphate (S1P). In TMS derivatization, the extracted lipid and standard lipid samples were desolvated by N2 gas, then added to 60 L Ntrimethylsilylimidazole (TMSI) and 40 L of pyridine, and allowed to react at 37 ◦ C for 30 min. The derivatized lipids were subjected to SFC/MS analysis. The number of adducted TMS groups was related to the number of hydroxyl groups in each polar lipid: PG, 2; PI, 5; PA, 1; LPC, 1; LPE, 1; LPG, 3; LPA, 2; LPI, 5; SM, 1; and S1P, 2. In derivatization repeatability tests, the RSD (%) of the derivatized polar lipids was as follows: PG, 9.6%; PI, 3.9%; PA, 6.8%; LPC, 2.1%; LPE, 7.5%; LPG, 8.8%; LPI, 9.2%; LPA, 9%; SM, 3.5%; and S1P, 2.7%. All the RSDs were under 10%, indicating that the repeatability was sufficient for polar lipid profiling. Triple-quadrupole (QqQ) MS was used to detect several polar lipids. Multiple reaction monitoring (MRM) is a non-scanning technique that is generally performed in QqQMS, in which selectivity is enhanced by fragmentation. In MRM, two mass analyzers are used as static mass filters to monitor a particular fragment ion of a selected precursor ion [43]. The MRM conditions, including MRM transitions (precursor m/z > fragment m/z), cone voltage (CV), and MS/MS collision energy (CE), were optimized for the analysis of non-derivatized and TMS-derivatized polar lipids. By TMS derivatization, 10 polar lipids were detected as [M+H]+ ions in the positive-ion mode. Further, each polar lipid showed a specific pattern of fragmentation by collision-induced dissociation (CID). The efficiency of the cyano column, C8 column, and several ODS columns for the separation of polar lipids was investigated. Finally, an Inertsil ODS-EP column (250 mm × 4.6 mm I.D.; 5 m, GL Sciences) was chosen because sharp peaks and excellent resolution are typically obtained when using this column. This column is suitable for the separation of polar compounds because of the polar functional groups embedded between the silica surface and the ODS groups. MRM data pertaining to 20 lipids—10 non-derivatized lipids and 10 TMS-derivatized lipids—were used to compare the nonderivatization analysis with the TMS derivatization analysis (Fig. 3). The non-derivatized polar lipids were analyzed using a modifier gradient, from 15% to 30% (v/v) over 15 min. Peak tailing was observed in the analysis of PA, PI, LPE, LPA and LPI. Furthermore, it was difficult to identify the peak due to S1P because of the continuous elution after 7 min (Fig. 3A). However, pronounced peak tailing was observed in the analysis of PA, PI, LPA, LPI, and S1P by TMS derivatization (Fig. 3B). The derivatized polar lipids were analyzed with a gradient modifier from 15% to 20% (v/v) for 10 min. Furthermore, the limit of detection (LOD) of PG, PI, LPA, LPI, and S1P was enhanced 12-, 40-, 510-, 39-, and 1490-fold, respectively. Next, the developed method was applied to the analysis of sheep plasma. In comparison with lipid profiling without derivatization, that with TMS derivatization allowed for the additional detection of 20 minor species of PI, LPC, LPE, and SM, and seven molecular species of LPA, LPI, and S1P. The relative ratio of the molecular species in each polar lipid was also found by quantification. Finally, the simultaneous and detailed profiling of 10 polar lipids was successfully performed by SFC/MS with TMS derivatization.
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Fig. 2. Base peak ion (BPI) chromatograms and 2D displays of SFC/MS data obtained from the lipid mixtures by using a cyano column in the positive-ion mode (A) and negative-ion mode (B), reproduced from Ref. [36] with some alteration. 1, TAG; 2, DAG; 3, MGDG; 4, DGDG; 5, PA; 6, PC; 7, PE; 8, PG; 9, PI; 10, PS; 11, LPC; 12, SM; 13, Cer; and 14, CB.
This developed method is suitable for lipidomics, especially for targeting polar lipids. 3. Targeted lipid profiling by SFC 3.1. Carotenoids 3.1.1. Introduction Carotenoids play an important role in vision [44] and they are fat-soluble antioxidants that have a protective effect on membrane lipids [45]. Metabolic profiling of carotenoids is important for understanding their physiological functions, but a more advanced technique is required for the analysis of biological samples containing a mixture of structurally similar carotenoids. Recently, RP-HPLC has been used for the separation of carotenoids [46,47]. It is difficult to separate structurally similar carotenoids using ODS columns, which are generally employed in RP-HPLC. Columns packed with triacontyl-bonded silica (C30), whose hydrophobic sites are larger than those of ODS columns, are often used for carotenoid analysis, but the analysis time is very long [48]. 3.1.2. Carotenoid profiling by SFC Schmitz et al. employed an OT-SFC coupled with a UV/vis detector for analysis of the cis/trans isomers of carotenes [49]. They used a 7-m-long capillary column with a cross-linked stationary phase consisting of 25% cyanopropyl and 75% polymethylsiloxane. They showed the effectiveness of SFC in the analysis of thermolabile compounds, which could not be separated by LC, the commonly
used separation technique at that time. In addition, Ibanes and co-workers tried to develop a carotenoid analysis method using neat CO2 without any modifiers, with the vision to online-coupling with supercritical fluid extraction [50]. High-speed separation (in 10 min) of -carotene and lycopene was achieved using a 50-cmlong packed capillary column with highly pressurized (300 atm) CO2 . Lesellier et al. investigated various parameters such as temperature, pressure, modifier, and column stationary phase for the effective separation of carotenoids by PC-SFC [24,51–53]. One of the achievements of their study was the excellent separation of a mixture of 11 carotenoids including structural isomers. Subsequently, Lesellier et al. extended their research and made use of carotenoid analysis for column characterization [54–58]. They tried to estimate various important column characteristics such as bonding density, hydrophobicity, and polar site accessibility on the basis of the separation capacity of cis -carotenes and zeaxanthin. These achievements clearly show the advantages of SFC for carotenoid separation. 3.1.3. Carotenoid profiling by SFC/MS Co-elution of various metabolites in biological samples often interferes with the accurate identification and quantification of target compounds. The low sensitivity of photometric detection, which is widely used for the detection of carotenoids, limits the applicable sample volume. This restriction often obstructs the application of photometric detection to clinical samples. The use of MS helps overcome the shortcomings of photometric detection and allows for the detection of small amounts of carotenoids. Hence, we tried to apply SFC/MS to carotenoid analysis [15].
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Fig. 3. MRM data of the standard PG (C34:1), PA (C34:1), PI (C38:4), LPC (C18:0), LPE (C18:0), LPG (C14:0), LPA (C17:0), LPI (C18:0), SM (C18:0), and S1P (C18:1) by (A) non-derivatization and (B) TMS derivatization. Reproduced from Ref. [37] with some alteration.
Carotenes (-carotene and lycopene) and xanthophylls (zeaxanthin, lutein, antheraxanthin, neoxanthin, and violaxanthin) were analyzed as models for the separation of carotenoids. Since most of the carotenoids were not retained on normal-phase packed columns, we used the ODS column. After testing ODS columns with various packing materials, we found that seven carotenoids could be separated within 15 min on the Hibar Purospher STAR RP-18e (Merck) column. In addition, monolithic columns manufactured by a new sol–gel process [59–61], which include a continuous 3D silica network with macropores (ca. 2 m, corresponding to the pore spaces in particle-packed columns) and mesopores (ca. 13 nm, corresponding to the fine pores in particle-packed columns) [62]. The pressure drop in monolithic columns is lower than that in particlepacked columns, owing to the relatively wider flow channels in the former case [62]. Seven carotenoids were successfully separated in 10 min on such a monolithic column (Fig. 4A). Furthermore, separation performed at a flow rate of 9 mL/min yielded satisfactory results (the resolution of separation of neoxanthin and violaxanthin was 2.62), and the analysis time was reduced to 4 min (Fig. 4B). For the analysis of biological samples, three monolithic columns were connected in series, which helped improve the efficiency of separation of the target carotenoid from contaminants. 3.1.4. Profiling of carotenoids and their derivatives by SFC/MS/MS 3.1.4.1. Xanthophyll esters. We also tried to apply SFC with MS/MS for carotenoid analysis [63]. MS/MS is a powerful method for the acquisition of information about metabolites in biological samples. Partial structure information can be obtained by product ion scan, which is one of the MS/MS detection modes. Highly sensitive and selective detection of target metabolites can be performed by MRM. SFC/MS/MS was applied to the analysis of xanthophyll esters. In physiological tissues, hydroxylated xanthophylls exist not only in free form but also in FA ester form [64]. These esters have high hydrophobicity and include various structurally similar compounds having different FA chains. The esterified form of a
xanthophyll has higher thermal stability and bioavailability than does its free-form counterpart [65,66]. However, the in vivo kinetics and physiological functions of xanthophyll FA esters are not yet well understood. Conventional HPLC methods are time-consuming, requiring more than 40 min for the analysis of xanthophyll FA esters [67–69]. Thus, a high-throughput, high-sensitivity analysis system is required to obtain information about xanthophyll FA esters. In the previous study, the analysis of -cryptoxanthin (CX) was focused because recent epidemiological studies reported that CX has higher ability to prevent cancer and diabetes. CX is classified as a xanthophyll, and it exists not only in a free form, but also in a fatty-acid-esterified form in physiological tissues. First, investigations were conducted to identify the appropriate ionization conditions for -cryptoxanthin fatty acid esters (CXFAs) by infusion analysis. The molecular ions of carotenoids were observed as radical cations by ESI in the positive-ion mode. Common product ions, [M−92]+ , generated by the elimination of toluene from the polyene chain, were observed in the product ion scan. In the following experiments, MRM analysis based on this information was employed for the highly sensitive detection of CXFAs. Next, separation of a standard mixture of CXFAs was attempted using various columns under the aforementioned conditions, with the aim of identifying a suitable column for CXFA analysis. All the CXFAs were successfully separated within 20 min when Inertsil ODS-P (250 mm × 4.6 mm I.D., 5 m; GL Sciences), which is a polymeric ODS column (Fig. 5), was used. The LOD was 540 fmol for the free forms of the CXFAs and 32–130 fmol for the esterified forms. In our research, we used QqQMS, which offers higher selectivity and sensitivity than MS, since it enabled the detection of CX and CXFAs even at femtomolar concentrations. 3.1.4.2. Carotenoids and their epoxides. SFC/MS/MS has also been applied to carotenoids and their epoxides [70]. Epoxy carotenoids, which are the oxidation products of carotenoids, are potential oxidative stress markers. Several studies suggested that these
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Fig. 5. MRM data of free form, laurate ester, myristate ester, palmitate ester, stearate ester, oleate ester, linoleate ester, linolenate ester, EPA ester, DHA ester of -cryptoxanthin obtained from standard mixtures analysis by SFC/MS/MS using Inertsil ODS-P column. Reproduced from Ref. [59] with permission.
Fig. 4. Mass chromatograms of carotenoids using a monolithic ODS column (100 mm × 4.6 mm I.D.), reproduced from Ref. [15] with permission. Analysis conditions: modifier, methanol with 0.1% ammonium formate 1–5% 8 min; flow rate, (A) 3 mL/min and (B) 9 mL/min. 1, lycopene; 2, -carotene; 3, lutein; 4, zeaxanthin; 5, antheraxanthin; 6, neoxanthin; and 7, violaxanthin.
oxidation products contribute to the pro-carcinogenic effect of carotenoids in subjects suffering from oxidative stress [71,72]. Therefore, to understand the role of carotenoids in the human body, it is necessary to analyze not only carotenoids but also their oxidation products. However, it is difficult to profile epoxy carotenoids because they are present in very small amounts. Furthermore, epoxy carotenoids are isomers of hydroxyl carotenoids, which include predominant xanthophylls such as -cryptoxanthin; therefore, precise separation and identification of these carotenoids is difficult. SFC/MS/MS is the solution to these problems. First, the ionization conditions in MS/MS were investigated. The epoxidation product of -carotene was analyzed by infusion analysis. Since epoxy -carotene is an isomer of -cryptoxanthin (hydroxyl carotenoid), the m/z value for both these molecules was the same, but the results of product ion scan were different. The product ion spectrum of epoxy -carotene showed an [M−80]+ ion peak in addition to the peaks observed in the product ion spectrum of CX. Thus, these results suggested that highly sensitive structural characterization of carotenoids is possible by targeting
such structure-specific neutral losses. Under the optimized separation conditions, predominant human carotenoids and their epoxy products were efficiently separated within 20 min using an ODS column. The combination of isomer separation by SFC with highly selective detection by MS/MS is a powerful strategy for the accurate analysis of carotenoids. In addition, sub-femtomolar levels of carotenoids could be detected; limit of detection for b-carotene was 0.093 fmol. The high sensitivity of the constructed system helped in reducing the sample volume; merely 0.1 mL of human serum was required for the detection of five major carotenoids and six epoxy carotenoids. These results clearly show the advantage of SFC/MS/MS for a large number of small-scale analyses such as small-scale intervention studies. 3.2. Triacylglycerols 3.2.1. Introduction TAGs are natural compounds produced by the esterification of glycerol with FAs. In humans, TAGs serve as a source of energy stored in fat tissues and form a thermal and mechanical protective layer around important organs; further, they are the source of essential FAs (linoleic and linolenic acids), fat-soluble vitamins, and other non-polar compounds. TAG imbalance can lead to several diseases such as coronary heart disease, dyslipidaemia, and obesity or inborn errors of metabolism. The main sources of TAGs in the human diet are vegetable oils and animal fats [73,74]. TAGs have very high diversity because of the wide variety of possible FAs, esterified to three stereospecific positions of the glycerol backbone.
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Thus, separation and identification of individual TAGs in a mixture is very difficult [75]. 3.2.2. Triacylglycerol profiling by SFC Manninen et al. reported the separation of ␥- and ␣-linolenic acids containing TAGs with identical acyl carbon numbers and degrees of unsaturation by capillary SFC using a 25% cyanopropane–75% methylpolysiloxane stationary phase [76]. The resolution of 1,3-dioleoyl-2-␥-linolenoyl-sn-glycerol and 1,3dioleoyl-2-␣-linolenoyl-sn-glycerol was 1.35 on a 10 m × 50 m I.D. column, and the resolution was enhanced to 1.66 by combining two 10-m columns in series. The difference in the positions of the double bonds in one linolenic acid residue of the TAGs resulted in two series of peaks during the separation of alpine currant (Ribes alpinum) and black currant (R. nigrum) seed oils. The use of the 10-m column was found to be appropriate for screening the TAG profile in both seed oils. 3.2.3. Triacylglycerol profiling by SFC/MS Sandra et al. reported the characterization of TAGs in vegetable oils by using a silver-ion packed-column (SI-PC) SFC/MS [77]. TAGs were mainly detected as [M+Ag]+ ions by using a coordination ion spray (CIS)–electrospray ionization (ESI)-MS. In a SI-PC, TAGs are separated according to the number of double bonds. Within each group, an additional separation is observed according to the carbon number (CN), i.e. the number of carbon atoms of the three fatty acids bonded to the glycerol backbone. Solutes characterized by the same CN elute according to the number of unsaturated fatty acids, e.g. TAG (SLL) elutes before TAG (OOL). In the use of atmospheric pressure chemical ionization (APCI)-MS, TAGs were detected as [M+H]+ ions. Approximately 90 min were required to separate divers TAGs in a mixture. In our previous study, SFC/MS was also applied for the analysis of soybean lipids [78]. Diverse lipids were analyzed simultaneously by using a cyano column with a gradient modifier from 10% to 30% (v/v) for 20 min. TAG and PC were mainly detected as ammonium adduct ions ([M+NH4 ]+ ) by ESI in the positive-ion mode. Furthermore, diverse TAGs were analyzed successfully when an ODS column was used for 15 min with the gradient modifier ranged from 20% to 35% (v/v). Next, a high-throughput, high-resolution TAG profiling method was developed on the basis of SFC/MS. After testing several columns, it was found that three Chromolith Performance RP-18e columns connected in series would be the most effective for TAG profiling. Monolith columns have many advantages such as high resolution, high throughput, and low backpressure. The total column length can be increased by connecting three or more of these columns in series, and thus, the resolution can be improved. SFC/MS with three tandem monolith ODS columns was used to analyze the TAGs in a mixture of soybean lipids. Individual TAG molecules were separated effectively within 8 min on the 2D map according to the retention time and m/z (Fig. 6). For TAG identification, programmed cone voltage fragmentation in single quadrupole (Q)-MS was used. Previously, it was reported that certain structural isomers could be distinguished and identified by this method and that the sensitivity and selectivity of this method were high [79]. By fragmentation, TAG was mainly found when the cone voltage was 35 V, and two types of fragmented ions, DAG and MAG, were confirmed when the cone voltage was 50 and 90 V, respectively. The molecular structure of the TAGs was identified from fragmented ions that had a specific value of m/z. In particular, the structure of the TAGs was identified by the type and peak intensity of the fragmented ions (DAG). For example, in the case of TAG (SOL) ([M+NH4 ]+ ; m/z 902.8), three fragmented ions, DAG (SO) (m/z 605.5), DAG (SL) (m/z 603.5), and DAG (OL) (m/z 601.4), were detected. Their peak intensities were in the order SL > SO > OL. Theoretically, the stereospecific numbering
Fig. 6. Identified TAGs in 2D map by SFC/MS using three tandem monolith ODS column, reproduced from Ref. [78] with some alteration. S, stearic acid (C18); O, oleic acid (C18:1); L, linoleic acid (C18:2); Ln, linolenic acid (C18:3); P, palmitic acid (C16).
(sn)-2 position of an FA is fragmented more easily than are the sn1, 3 positions. If the peak intensity of SL were the highest, it would imply that TAG has O at the sn-2 position and that S and L are at the sn-1 and sn-3 positions respectively. Additionally, fragmentation of unsaturated FAs is easier than that of saturated FAs. Therefore, it is reasonable that the peak intensity of SO is higher than the peak intensity of OL. Finally, it was demonstrated that this method is reliable and accurate even when using single Q-MS. 4. Conclusion This review demonstrates the usefulness of SFC/MS for the metabolic profiling of hydrophobic compounds in a biological sample. Owing to the advantages of an SF as the mobile phase, highthroughput, high-resolution analysis becomes possible. SFC is also effective for the separation of diverse lipids with a wide range of polarities. In addition, while UHPLC shows excellent performance only in lipid profiling, SFC is suitable for use in preparative isolation and analysis of a high-volume lipid mixture that includes minor components as well as major ones. The particle size in the packed column used in SFC (3 m or 5 m) is larger than that in UHPLC columns (sub-2 m). Furthermore, the diameter of the SFC column (4.6 mm) is greater than that of UHPLC columns (2.1 mm). Therefore, SFC is an effective analytical method for targeting low volumes of unknown minor lipids in a biological sample. In lipidomics, SFC/MS can be effectively used for the targeted and non-targeted lipid profiling of several biological samples. In the future, various types of columns and detection methods would be used to develop lipid-profiling methods based on SFC/MS. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
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