Review
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Supercritical fluid chromatography/mass spectrometry in metabolite analysis
Supercritical fluid chromatography (SFC) owes many of its advantages to the properties of supercritical CO2, which possesses benefits as mobile phase. SFC has recently gained attention as a separation technique because it can be utilized for not only non-polar but also polar compound analysis. In addition, MS is widely adopted for SFC, and the options for MS are equivalent to liquid chromatography. Sensitive and selective detection is crucial in metabolite analysis. The SFC/MS system can be an alternative approach to liquid chromatography, as can metabolite analysis using packed-column SFC in biosamples. In this review we cover the fundamentals of SFC in combination with MS, and discuss the results of metabolite analysis using SFC/MS.
Background Supercritical fluid chromatography (SFC) is a complimentary separation tech nique of both gas chromatography (GC) and liquid chromatography (LC). This separation technique has been gaining attention and has been applied to metabolite analysis. In order to get an insight into the situation, we will present the advantages of SFC and summarize the past studies of SFC combined with mass spectro metry and metabolite analysis using packedcolumn SFC. Lastly, we will explore the future of SFC by examining recent work and how advances in SFC are possible that will make it a better method for metabolite analysis. Nature of SFC Before talking about application of supercriti cal fluid chromatography (SFC) for metabo lite analysis, inevitably a question could be asked; what is supercritical fluid? Simply stated it is ‘highly compressed gas’ such as car bon dioxide, chlorofluorocarbons or ammo nia that has been used for the development of SFC [1–5] . CO2 is preferred due to the low critical parameters, as well as other ‘easy-touse’ properties such as non-inflammability, non-toxicity and low price. Nowadays, it is rare to use neat CO2 only in SFC due to the
10.4155/BIO.14.120 © 2014 Future Science Ltd
Kaori Taguchi1, Eiichiro Fukusaki1 & Takeshi Bamba*,1 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565–0871, Japan *Author for correspondence: Tel.: +81 6 6879 7418 [email protected]
fact that organic solvents are added in order to increase the solvating power of mobile phase and deactivate the residual silanol groups on the stationary phase, which results in reducing solute retention [6–10] . However, SFC is built on advantages derived from the properties of supercritical CO2 (SCCO2); therefore, we summarize the properties below. CO2 changes the phase to supercritical as temperature and pressure reach above the critical point of 31.1°C and 7.38 MPa. Typi cally, the temperature is controlled on the column through using a column heater while the pressure is controlled post-column by a module known as a backpressure regulator. Above or around the critical points, SCCO2 possesses three advantages making it the pre ferred mobile phase for SFC: physical proper ties, polarity and solubility flexibility. First of all, the viscosity of SCCO2 is similar to gas whereby its diffusivity is higher than liquid [11] , promoting throughput and resolution in the separation. Second, SCCO2 is non-polar, similar to n-hexane [12] . However, SCCO2 offers an additional advantage by chang ing the polarity, which turns out to be the most interesting. SCCO2 allows us changes, and it gives flexibility in the polarity. Even though the polarity is similar to n-hexane,
Bioanalysis (2014) 6(12), 1679–1689
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910 900
LnLLn
LLLn
890 880 m/z
LLL
SOO
SLO
SOS
A
LnLnLn LnLP
LnLnP
870
OLO
OLL
LLP
860 B
PLP
PLnP
850
OOP
OLP
SOP
POP
840 4.5
5.0
5.5
6.0
6.5 Time (min)
7.0
7.5
8.0
Figure 1. Lipid profiling performed by packed column supercritical fluid chromatography/MS. Identification of TAGs in soybean lipid (karikachi). The 2D map shows a magnified view of supercritical fluid chromatography/MS data obtained by tandem three Chromolith® Performance RP-18e columns. Small circle: peak top of each TAG. There are two types of groups that have the pattern of TAGs arrangement. Solid line arrows: (A, boxes) a group of sn-1 fatty acids changed; for example, OLP, LLP, and LnLP. Dotted line arrows: (B, circles) a group of sn-2 fatty acid changed; for example, POP, PLP and PLnP. L: Linoleic acid; Ln: Linolenic acid; O: Oleic acid; P: Palmitic acid; S: Stearic acid; TAG: Triacylglycerol. Adapted with permission from [63] .
CO2 is miscible with polar solvents, such as metha nol, acetonitrile and 2-propanol, which are modifiers in SFC. Although the polarity is also tunable by con trolling temperature and/or backpressure [13–15] , it is negligible compared with the addition of modifiers. Moreover, additives of acid, base and even water can be added with modifier into SCCO2. Due to the solubil ity flexibility allowed, SFC can offer complementary column selections from polar to non-polar stationary phase. Lesellier overviewed the retention behavior with Key terms Supercritical fluid chromatography: Chromatography technique using supercritical/subcritical carbon dioxide as the mobile phase. Supercritical CO2: A fluid state of carbon dioxide differentiated from liquid and gas above the critical temperature and pressure. Chiral analysis: Analysis of a molecule that is not superimposable on its mirror image stereoisomer. Achiral: Molecule that is superimposable on its mirror image stereoisomer and has a plane of symmetry or center of symmetry. APCI: Atmospheric pressure chemical ionization. Ionization technique that ionizes substances in the gas phase. Typically it is suitable for less polar and smaller volatile compounds.
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various columns and clarified recent understandings of the retention mechanisms in SFC [16] . His group has been intensively investigating the characteriza tion of stationary phase with retention behavior. West and Lesellier published a series of a systematic stud ies of various types of stationary phases with a single mobile phase composition: alkylsiloxane-bonded [17] , aromatic [18] , and different polar stationary phases [19] . Based on the results, they executed further compari son of the stationary phases and demonstrated that the examined columns were classified into three major groups defined as non-polar similar to C18, very polar similar to bare silica and moderately polar similar to C4 and aromatic stationary phases [20] . Their results indicated that SFC can provide the great extent of column selection from polar to non-polar stationary phase with different strength of interactions such as methylene selectivity, hydrogen bond acidity/basicity, and dipole–dipole interactions, even though the same mobile phase is used. In other words, SFC can utilize almost the full range of stationary phase with the same mobile phase. Due to this flexibility, the utilization of SCCO2 in SFC should be recognized as a suitable means for nonpolar as well as polar compound separation. The addi tion of a modifier was studied in the 1980s [10,21–23] , and Berger [24] pointed out the efficacy of SFC for
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Supercritical fluid chromatography/mass spectrometry in metabolite analysis
polar compound analysis in 1997. Unfortunately, the advantages of SFC were not properly recognized and the separation technique did not come into vogue. However, in recent years this has changed due to advances in SFC technologies and the commercializa tion of several instrument systems over the last few years. Because of the increased attention afforded to SFC, the following reviews have recently been pub lished: Saito [25] summarized the history of SFC; Tay lor introduced the separation techniques in SFC as well as the separation of polar solutes [26,27] ; West [28] presented an overview showing the analysis of polar compounds performed by SFC from the past to pres ent. As a result of these reviews, SFC finally received the proper recognition that it is amenable not only to non-polar but to polar compound analysis as pointed out by Berger, the father of modern SFC. Coupling SFC with MS Due to sensitivity and selectivity, the conjunction with MS is important for SFC [29] . SFC commonly
Review
offers superior separation to LC for isomers and enantiomers [30] . However in chiral analysis of drug discovery, MS detection is crucial to discriminate the enantiomers of interest from other achiral impurities [31] . As a result, SFC coupling to MS has been well adopted in pharmaceutical industry not only for chiral separation but for purification and analysis of a diverse set of compounds in drug discovery [32] , which lends credibility to the power of SFC in metabolite analy sis. The sensitivity and selectivity afforded by MS is crucial in metabolite analysis, therefore, we place an emphasis on MS for SFC in this review. In the past, some interfaces were studied with SFC: direct fluid introduction, moving belt interface, ther mospray interface and particle beam interface [33] . Due to several limitations; for example, sensitivity, stabil ity or analyte desorption, atmospheric pressure ion ization sources with two primary ionization methods have been widely used with SFC: atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Huang et al. reported the combination of SFC 900 880 860 840 820 SM (24:1)
800 m/z
780 760 m/z 757.6218
740
RT 4.2 min
720
SM (18:1)
700 SM (16:1)
680 660
3
4
5
6
Time (min) Figure 2. Lipid profiling performed by packed column supercritical fluid chromatography/MS. 2D map of lipids in mouse plasma analyzed in positive ion mode, showing that the SM molecular species with the same number of double bonds in the side chain are connected, illustrating the correlation between elution and carbon chain length. RT: Retention time; SM: Sphingomyelin. Adapted with permission from [44] .
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A
536.4 > 444.2 (beta-carotene) 1.39e5 2
0
5.00
15.00
10.00
5.00
10.00
15.00 552.3 > 460.2 (+O) 2.76e5
3
%
5 4
0
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15.00
10.00 6
0
4
10.00
11
15.00
0
568.3 > 488.2 (+2O [epoxy]) 1.39e3
15.00 568.3 > 476.2 (+2O) 8.15e4
8 5.00
10.00
15.00 568.3 > 488.2 (+2O [epoxy]) 4.22e3
11 10 %
%
10
552.3 > 472.2 (+O [epoxy]) 1.61e4
9 %
8
5
7
15.00
10.00
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568.3 > 476.2 (+2O) 1.14e5
9
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5.00
7 552.3 > 472.2 (+O [epoxy]) 2.29e3
10.00
5.00
0
%
6
%
10.00
5.00
%
% 0
0
0
552.3 > 460.2 (+O) 7.34e4
3
0
536.4 > 444.2 (beta-carotene) 7.65e5 2 1
%
%
1
B
5.00
10.00
15.00
0
Time (min)
5.00
10.00
15.00
Time (min)
Figure 3. Lipid profiling performed by packed column supercritical fluid chromatography/MS. Multiple reaction monitoring chromatograms obtained from supercritical fluid chromatography/MS/MS of heptane extracts of (A) human serum and (B) low-density lipoprotein. Adapted with permission from [64] .
with APCI in 1990 whereby they successfully obtained not only protonated molecular ions, but also fragment ions of steroids by means of tandem mass spectrometry (MS/MS) [34] . The APCI technique was adapted for SFC [35–37] . Sadoun and Arpino published a review on the combination of SFC with ESI in 1993 and analyzed pyridine at the pg level; they found limitations of ion ization and peak shape, and described the need for postcolumn addition of polar organic solvents [38] . Baker and Pinkston advocated the use of sheath-flow to improve the limitations, as well as pressure-regulating fluid to control backpressure and assure the flow mass transfer [39,40] . As they pointed out, we usually add methanol as make-up flow post-column to help ionization as the modifier composition is lower than 5–10%. ESI is often utilized for metabolite analysis in biosamples, so the applications are described later. In addition to ioniza tion, SFC can be coupled with various type of MS, and combination with quadropole, ion trap, orbitrap, time-
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of-flight, Fourier transform infrared spectroscopy and ion mobility have been reported [41–48] . Because of this we have almost as many MS options as LC options in SFC. Although sensitivity depends upon a compound, SFC/MS/MS with ESI offers generally four- to ten-fold better sensitivity, or in hydrophilic compound analysis it was equivalent to hydrophobic compound analysis [49] . The studies demonstrated that SFC/MS can be an alternative approach for metabolic analysis. Metabolite analysis using SFC–MS SFC is classified into two groups: open tubular column SFC (otSFC) and packed column SFC (pcSFC). otSFC is typically ‘GC-like’ because of the use of pure CO2. On the other hand, pcSFC is com monly ‘LC-like’ because of addition of a polar solvent as modifier. The addition of modifier is key for metabo lite analysis of biosamples because it allows more polar compounds in the sample to be analyzed resulting from
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518.3
520.4
522.4
536.4
542.3
544.4
546.4
548.4
550.4
568.4
570.4
572.4
580.4
608.5
LPC 18:3
LPC 18:2
LPC 18:1
LPC 19:1
LPC 20:5
LPC 20:4
LPC 20:3
LPC 20:2
LPC 20:1
LPC 22:6
LPC 22:5
LPC 22:4
LPC 22:0
LPC 24:0
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702.5
704.5
PC 30:2
PC 30:1
9.3
9.3
10.6
10.5
10.3
10.5
10.2
10.2
10.3
10.5
10.6
10.7
10.4
10.5
10.5
10.6
10.6
10.2
10.4
10.5
10.6
10.4
10.3
10.4
10.4
10.5
10.2 748.6
746.5
744.5
m/z
SM 18:2
SM 18:1
SM 18:0
SM 16:1
SM 16:0
SM 14:0
727.5
729.5
731.7
701.5
703.5
675.5
DISM 18:0 733.6
DISM 16:1 703.5
DISM 16:0 705.6
PC 34:0e
PC 33:1
PC 34:2e
PLs
9.8
9.8
9.8
9.9
9.9
9.9
9.6
9.6
9.6
9.3
9.2
9.3
PC 38:6e
PC 36:0
PC 36:1
PC 36:2
PC 36:3
PC 36:4
PC 36:5
PC 36:0e
PC 36:1e
PC 35:2
PC 35:3
PC 36:4e
PC 36:5e
PC 34:0
PC 34:1
PC 34:2
PC 34:0e
PC 34:1
PC 34:2e
PC 34:3e
PC 34:6e
PC 32:0
PC 32:1
PC 32:6
PC 31:0
PC 31:1
PC 31:2
LPC 32:0
RT (min) PLs
792.5
790.6
788.7
786.6
784.6
782.6
780.6
776.6
774.6
772.5
770.5
768.6
766.5
762.6
760.6
758.5
748.6
746.5
744.5
742.6
736.5
734.5
732.6
722.5
720.6
718.5
716.5
720.6
m/z
9.4
9.3
9.3
9.4
9.4
9.5
9.3
9.3
9.3
9.4
9.3
9.3
9.4
9.3
9.3
9.4
9.3
9.2
9.3
9.3
9.3
9.3
9.4
9.2
9.2
9.3
9.2
10.5
912.7 9.5
866.7 9.4
864.6 9.5
862.6 9.5
860.6 9.5
858.7 9.2
856.7 9.3
854.7 9.3
852.7 9.3
850.7 9.3
848.7 9.5
846.6 9.4
844.7 9.4
842.6 9.3
840.7 9.4
838.6 9.4
836.6 9.5
834.6 9.5
832.5 9.5
830.6 9.2
828.5 9.3
SM 16:0
703.5 9.9
675.5 9.9
DISM 24:6 805.6 9.5
DISM 24:1 815.7 9.6
DISM 18:0 733.6 9.6
SM 14:0
702.6
730.5
PE 39:6
PE 38:1e
PE 38:2e
PE 37:4
PE 37:5
778.5
760.7
758.7
754.5
752.5
PE 38:6e 750.6
PE 36:2e
PE 36:3e 728.5
PE 36:5e 724.5
PE 33:2
482.4
m/z
9.7
9.5
9.6
9.6
9.7
9.7
9.6
9.7
9.6
9.6
10.8
RT (min)
Neutral loss scan
LPE 18:0
RT (min) PLs
826.7 9.3
m/z
DISM 16:0 705.6 9.6
PC 45:2
PC 42:4
PC 42:5
PC 42:6
PC 42:7
PC 41:1
PC 41:2
PC 41:3
PC 41:4
PC 42:5e
PC 41:6
PC 41:7
PC 40:1
PC 40:2
PC 40:3
PC 40:4
PC 40:5
PC 40:6
PC 40:7
PC 40:1e
PC 40:9
PC 40:3e
RT (min) PLs
High collision energy precursor ion scan
List of detected PLs by online-supercritical fluid extraction–MS/MS of mouse plasma. † Typical number of carbon and double bonds of individual m/z were shown. Probable subclasses and molecular species of phospholipids for individual m/z shown in [5,6]. ‡ Ether phospholipids contain plasmalogen and alkyl ether phospholipid. DISM: Dihydrosphingomyelin; LPC: Lysophosphatidylcholine; LPE: Lysophosphatidylethanolamine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PL: Phospholipid; RT: Retention time; SM: Sphingomyelin. Adapted with permission from [65].
720.6
718.6
LPC 32:0
LPC 32:1
690.5
510.3
LPC 17:0
LPC 30:1
508.4
LPC 17:1
634.5
506.3
LPC 18:2e
692.5
496.4
LPC 16:0
LPC 26:1
494.4
LPC 30:0
482.3
10.3
480.3
LPC 16:1
‡
RT (min)
m/z
Low collision energy precursor ion scan
LPC 15:0
LPC 16:1e
PLs†
Table 1. Lipid profiling performed by packed column supercritical fluid chromatography–MS.
Supercritical fluid chromatography/mass spectrometry in metabolite analysis
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706.6
676.5
690.5
714.5
716.5
718.5
720.6
722.5
728.5
730.5
732.6
734.5
736.5
738.5
740.5
742.6
PC 30:0
PC 28:1
PC 29:1
PC 31:3
PC 31:2
PC 31:1
PC 31:0
PC 32:6
PC 32:3
PC 32:2
PC 32:1
PC 32:0
PC 34:6e
PC 34:5e
PC 34:4e
PC 34:3e
9.4
9.3
9.3
9.3
9.3
9.4
9.3
9.3
9.2
9.2
9.3
9.2
9.3
9.2
9.3
9.3
RT (min)
PLs
m/z
PC 39:4
PC 40:5e
PC 40:6e
PC 38:0
PC 39:8
PC 38:2
PC 38:3
PC 38:4
PC 38:5
PC 38:6
PC 38:7
PC 38:1e
PC 38:2e
PC 38:3e
PC 38:4e
PC 38:5e
RT (min) PLs
824.7
822.6
820.7
818.7
816.6
814.7
812.7
810.6
808.6
806.5
804.5
802.7
800.7
798.6
796.7
794.6
m/z
9.3
9.3
9.5
9.4
9.3
9.4
9.4
9.4
9.5
9.5
9.3
9.3
9.4
9.3
9.3
9.4
SM 18:1
SM 24:7
SM 24:6
SM 24:2
SM 24:1
SM 24:0
SM 22:6
SM 22:2
SM 22:1
SM 22:0
SM 20:1
SM 20:0
SM 18:2
SM 18:0
SM 16:1
RT (min) PLs
9.9
729.5 9.9
801.6 9.8
803.7 9.8
811.7
813.6 9.8
815.7 9.8
775.6 9.8
783.7 9.9
785.7 9.8
787.7 9.8
9.8
759.7 9.8
727.5 9.9
731.7 9.8
757.7
RT (min) PLs
701.5 9.9
m/z
High collision energy precursor ion scan
m/z
RT (min)
Neutral loss scan
List of detected PLs by online-supercritical fluid extraction–MS/MS of mouse plasma. † Typical number of carbon and double bonds of individual m/z were shown. Probable subclasses and molecular species of phospholipids for individual m/z shown in [5,6]. ‡ Ether phospholipids contain plasmalogen and alkyl ether phospholipid. DISM: Dihydrosphingomyelin; LPC: Lysophosphatidylcholine; LPE: Lysophosphatidylethanolamine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PL: Phospholipid; RT: Retention time; SM: Sphingomyelin. Adapted with permission from [65].
m/z
PLs†
Low collision energy precursor ion scan
Table 1. Lipid profiling performed by packed column supercritical fluid chromatography–MS (cont.).
Review Taguchi, Fukusaki & Bamba
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Supercritical fluid chromatography/mass spectrometry in metabolite analysis
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Time (min) Figure 4. Diverse polar compound analysis performed by packed column supercritical fluid chromatography/MS. MRM chromatogram of a mixture of 17 pesticides after separation on three different columns (A) COSMOSIL 5CN-MS packed column, (B) Inertsil® ODS-4 column, and (C) Inertsil® ODS-EP column. (1) Diquat dibromide, (2) fosetyl, (3) maleic hydrazide, (4) daminozide, (5) methamidophos, (6) methomyl, (7) acetamiprid, (8) carbendazim, (9) dimethirimol, (10) thifluzamide, (11) tralomethrin, (12) emamectin benzoate (B1a), (13) chlorfluazuron, (14) acequinocyl, (15) pyridaben, (16) cypermethrin, (17) etofenprox. Analytical conditions: flow rate: 3 ml/min; column temperature: 35°C; (A) and (B) modifier: 5–10% (5 min), 10–40% (2 min), 40% (10 min), 40–5% (1 min), 5% (2 min), (C) modifier: 5% (2 min), 5–10% (5 min), 10–40% (2 min), 40% (8 min). Adapted with permission from [66] .
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Key terms Open tubular column SFC: Supercritical fluid chromatography using a column having uniform distribution of the stationary phase on the interior surface of the column. This technique is not very popular compared with packed column SFC. Packed column SFC: Supercritical fluid chromatography using a column packed densely and evenly with solid-phase support.
complementary column selection and mobile phase sol vating power. Therefore, we will focus on the benefits of pcSFC in this review. There were various reports using pcSFC/UV for metabolite analysis in the 1990s. Young and Games reported the analysis of the fungi metabolite ergosterol using an amino column of a standard packed HPLC col umn [50] . The technique of pcSFC/UV was applied to the analysis of several drugs and its metabolites in biosa mples such as urine, microsomes and serum [51–57] . Hsieh et al. demonstrated rapid and simultaneous analysis of an in vitro sample containing clozapine, ondansetron, tolbutamide and primidone using pcSFC/APCI/MS/ MS for the metabolic stability test. They found a con sistent result of metabolic stability of those compounds between LC and pcSFC [58] . In addition, Xu et al. suc cessfully analyzed 15 estrogen metabolites in pcSFC/ APCI/MS and confirmed superiority in the throughput compared with HPLC/MS [59] . While another steroid analysis using ESI was reported, 25 bile acids was sepa rated simultaneously in 13 min and all the 25 bile acids were detected in rat serum samples using pcSFC/ESI/ MS/MS [60] . Bamba et al. demonstrated comprehen sive analysis of diverse lipids profiling using pcSFC/ ESI/MS/MS, and showed the effectiveness of SFC for simultaneous analysis for diverse compounds [61] . Fur thermore, his group published a review summarizing the application of pcSFC for lipidomics [62] . And recently, another application study for comprehensive metabolite analysis related to lipidomics by pcSFC/ESI/MS was published: lipid profiling in soybeans (Figure 1) [63] and mouse plasma (Figure 2) [57] , carotenoids in human serum (Figure 3) [64] , and phospholipids in mouse plasma (Table 1) [65] . These publications gave notice that we have latched onto a potential tool for metabolomics, and his group applied the metabolomics approach using SFC to not only biosamples, but also food science. Future perspective As previously mentioned, pcSFC is a powerful separa tion technique for both non-polar and polar compounds; however, we are seeing its benefits in the next stage of applications. Ishibashi et al. demonstrated simultane ous analysis of pesticides, which have a log Pow rang
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ing from -4.6 to 6.6 (Figure 4) [66] . Although this new application for pcSFC is not related to metabolite analy sis, it offers great potential allowing us to shift from a multiple method analysis to a single method analysis. In metabolite analysis, the broad coverage of constituents has been a challenging task because they are comprised of a diverse set of small molecules with various chemi cal and physical properties [67] . The solubility flexibility of SCCO2 resulting in complementary column selection advances the range of compound polarity in simultane ous analysis. To obtain the wide range of solvent polar ity, the modifier composition can range from 0 to 100% in pcSFC, however the critical point of temperature and back pressure is increased along with the increased modifier composition. In other words, the phase state of CO2 might not be supercritical any more as the modi fier gradient runs without updating temperature and backpressure. Even if CO2 changes the state from super critical to subcritical, discontinuous transition does not occur in CO2 [68] . Applying this approach to our current separation, pcSFC may enable simultaneous analysis for diverse polarity compounds that are typically analyzed not only with different methods but with different sepa ration techniques. The approach can be classified as uni fied chromatography that continuously shifts the physi cal state of the mobile phase without phase separation by controlling temperature and back pressure. For example, the phase state is changed in the order of gas, supercriti cal, subcritical and liquid [68–70] . Although there are very few publications regarding application, we think the approach will open a door to a new separation tech nique expanding the convergence regions among GC, SFC and LC for metabolite analysis. Analytical targets in metabolite analysis include highly polar compounds, but non-polar compounds such as lipids and steroids have recently emerged as important signaling molecules [71,72] . Therefore, developing a single universal method enabling simultaneous analysis for polar and non-polar compounds could be the next challenge in metabolite analysis. Unified chromatography can use the same mobile phase state as GC, SFC and LC in terms of the physical state. The benefit of the unique separation technique is to enhance the polarity range of the mobile phase from 100% CO2 (non-polar) to 100% polar sol vent (polar). The approach will enhance the compound coverage in a single run. If we can see beyond the cur rent separations, pcSFC can p rovide comprehensive information for metabolomics. Financial & competing interests disclosure This work was partially supported by MEXT KAKENHI Grant Number 23686120 Grant-in-Aid for Young Scientists (A), the Development of Systems and Technology for Advanced Measurement and Analysis Project (JST), and the Advanced
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Low Carbon Technology Research and Development Program (JST). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject mat-
Review
ter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary Nature of supercritical fluid chromatography • Supercritical fluid chromatography (SFC) owes many of its advantages to the properties of supercritical carbon dioxide (SCCO2). • SCCO2 possess many benefits as the chromatography mobile phase: low viscosity; high diffusivity and solubility flexibility resulting in better separation; faster run time; and, adoption of a wide range of column chemistry. • SFC is amenable to non-polar as well as polar compound analysis.
Coupling to MS • The sensitivity and selectivity afforded by MS is adopted in SFC. • Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the current main ionization techniques in SFC; various MS is used with SFC. • A make-up flow is sometimes required at low modifier composition for ionization.
Metabolite analysis using SFC • Packed column SFC is widely used in metabolite analysis since the addition of a modifier can enhance the solvating power of the mobile phase and the solubility of metabolites in SFC. • Analysis of a target compound and its metabolites has been carried out in various biosamples. • SFC/ESI/MS/MS is a powerful tool for lipid profiling. energy relationships. J. Chromatogr. A 1206(2), 186–195 (2008).
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Very good review that summarizes applications and techniques for polar compound analysis using supercritical fluid chromatography.
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Important example of applying supercritical fluid chromatography/MS to analyze diverse metabolites in a mixture.
61
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Informative example of coupling MS to supercritical fluid chromatography.
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Lee JW, Uchikata T, Matsubara A, Nakamura T, Fukusaki E, Bamba T. Application of supercritical fluid chromatography/mass spectrometry to lipid profiling of soybean. J. Biosci. Bioeng. 113(2), 262–268 (2012).
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Matsubara A, Uchikata T, Shinohara M et al. Highly sensitive and rapid profiling method for carotenoids and their epoxidized products using supercritical fluid chromatography coupled with electrospray ionization-triple quadrupole mass spectrometry. J. Biosci. Bioeng. 113(6), 782–787 (2012).
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Interesting paper that shows the wide coverage of compound polarity in supercritical fluid chromatography.
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Supercritical fluid chromatography/mass spectrometry in metabolite analysis
Supercritical fluid chromatography (SFC) owes many of its advantages to the properties of supercritical CO2, which possesses benefits as mobile phase. SFC has recently gained attention as a separation technique because it can be utilized for not only non-polar but also polar compound analysis. In addition, MS is widely adopted for SFC, and the options for MS are equivalent to liquid chromatography. Sensitive and selective detection is crucial in metabolite analysis. The SFC/MS system can be an alternative approach to liquid chromatography, as can metabolite analysis using packed-column SFC in biosamples. In this review we cover the fundamentals of SFC in combination with MS, and discuss the results of metabolite analysis using SFC/MS.
Background Supercritical fluid chromatography (SFC) is a complimentary separation tech nique of both gas chromatography (GC) and liquid chromatography (LC). This separation technique has been gaining attention and has been applied to metabolite analysis. In order to get an insight into the situation, we will present the advantages of SFC and summarize the past studies of SFC combined with mass spectro metry and metabolite analysis using packedcolumn SFC. Lastly, we will explore the future of SFC by examining recent work and how advances in SFC are possible that will make it a better method for metabolite analysis. Nature of SFC Before talking about application of supercriti cal fluid chromatography (SFC) for metabo lite analysis, inevitably a question could be asked; what is supercritical fluid? Simply stated it is ‘highly compressed gas’ such as car bon dioxide, chlorofluorocarbons or ammo nia that has been used for the development of SFC [1–5] . CO2 is preferred due to the low critical parameters, as well as other ‘easy-touse’ properties such as non-inflammability, non-toxicity and low price. Nowadays, it is rare to use neat CO2 only in SFC due to the
10.4155/BIO.14.120 © 2014 Future Science Ltd
Kaori Taguchi1, Eiichiro Fukusaki1 & Takeshi Bamba*,1 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565–0871, Japan *Author for correspondence: Tel.: +81 6 6879 7418 [email protected]
fact that organic solvents are added in order to increase the solvating power of mobile phase and deactivate the residual silanol groups on the stationary phase, which results in reducing solute retention [6–10] . However, SFC is built on advantages derived from the properties of supercritical CO2 (SCCO2); therefore, we summarize the properties below. CO2 changes the phase to supercritical as temperature and pressure reach above the critical point of 31.1°C and 7.38 MPa. Typi cally, the temperature is controlled on the column through using a column heater while the pressure is controlled post-column by a module known as a backpressure regulator. Above or around the critical points, SCCO2 possesses three advantages making it the pre ferred mobile phase for SFC: physical proper ties, polarity and solubility flexibility. First of all, the viscosity of SCCO2 is similar to gas whereby its diffusivity is higher than liquid [11] , promoting throughput and resolution in the separation. Second, SCCO2 is non-polar, similar to n-hexane [12] . However, SCCO2 offers an additional advantage by chang ing the polarity, which turns out to be the most interesting. SCCO2 allows us changes, and it gives flexibility in the polarity. Even though the polarity is similar to n-hexane,
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Figure 1. Lipid profiling performed by packed column supercritical fluid chromatography/MS. Identification of TAGs in soybean lipid (karikachi). The 2D map shows a magnified view of supercritical fluid chromatography/MS data obtained by tandem three Chromolith® Performance RP-18e columns. Small circle: peak top of each TAG. There are two types of groups that have the pattern of TAGs arrangement. Solid line arrows: (A, boxes) a group of sn-1 fatty acids changed; for example, OLP, LLP, and LnLP. Dotted line arrows: (B, circles) a group of sn-2 fatty acid changed; for example, POP, PLP and PLnP. L: Linoleic acid; Ln: Linolenic acid; O: Oleic acid; P: Palmitic acid; S: Stearic acid; TAG: Triacylglycerol. Adapted with permission from [63] .
CO2 is miscible with polar solvents, such as metha nol, acetonitrile and 2-propanol, which are modifiers in SFC. Although the polarity is also tunable by con trolling temperature and/or backpressure [13–15] , it is negligible compared with the addition of modifiers. Moreover, additives of acid, base and even water can be added with modifier into SCCO2. Due to the solubil ity flexibility allowed, SFC can offer complementary column selections from polar to non-polar stationary phase. Lesellier overviewed the retention behavior with Key terms Supercritical fluid chromatography: Chromatography technique using supercritical/subcritical carbon dioxide as the mobile phase. Supercritical CO2: A fluid state of carbon dioxide differentiated from liquid and gas above the critical temperature and pressure. Chiral analysis: Analysis of a molecule that is not superimposable on its mirror image stereoisomer. Achiral: Molecule that is superimposable on its mirror image stereoisomer and has a plane of symmetry or center of symmetry. APCI: Atmospheric pressure chemical ionization. Ionization technique that ionizes substances in the gas phase. Typically it is suitable for less polar and smaller volatile compounds.
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various columns and clarified recent understandings of the retention mechanisms in SFC [16] . His group has been intensively investigating the characteriza tion of stationary phase with retention behavior. West and Lesellier published a series of a systematic stud ies of various types of stationary phases with a single mobile phase composition: alkylsiloxane-bonded [17] , aromatic [18] , and different polar stationary phases [19] . Based on the results, they executed further compari son of the stationary phases and demonstrated that the examined columns were classified into three major groups defined as non-polar similar to C18, very polar similar to bare silica and moderately polar similar to C4 and aromatic stationary phases [20] . Their results indicated that SFC can provide the great extent of column selection from polar to non-polar stationary phase with different strength of interactions such as methylene selectivity, hydrogen bond acidity/basicity, and dipole–dipole interactions, even though the same mobile phase is used. In other words, SFC can utilize almost the full range of stationary phase with the same mobile phase. Due to this flexibility, the utilization of SCCO2 in SFC should be recognized as a suitable means for nonpolar as well as polar compound separation. The addi tion of a modifier was studied in the 1980s [10,21–23] , and Berger [24] pointed out the efficacy of SFC for
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polar compound analysis in 1997. Unfortunately, the advantages of SFC were not properly recognized and the separation technique did not come into vogue. However, in recent years this has changed due to advances in SFC technologies and the commercializa tion of several instrument systems over the last few years. Because of the increased attention afforded to SFC, the following reviews have recently been pub lished: Saito [25] summarized the history of SFC; Tay lor introduced the separation techniques in SFC as well as the separation of polar solutes [26,27] ; West [28] presented an overview showing the analysis of polar compounds performed by SFC from the past to pres ent. As a result of these reviews, SFC finally received the proper recognition that it is amenable not only to non-polar but to polar compound analysis as pointed out by Berger, the father of modern SFC. Coupling SFC with MS Due to sensitivity and selectivity, the conjunction with MS is important for SFC [29] . SFC commonly
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offers superior separation to LC for isomers and enantiomers [30] . However in chiral analysis of drug discovery, MS detection is crucial to discriminate the enantiomers of interest from other achiral impurities [31] . As a result, SFC coupling to MS has been well adopted in pharmaceutical industry not only for chiral separation but for purification and analysis of a diverse set of compounds in drug discovery [32] , which lends credibility to the power of SFC in metabolite analy sis. The sensitivity and selectivity afforded by MS is crucial in metabolite analysis, therefore, we place an emphasis on MS for SFC in this review. In the past, some interfaces were studied with SFC: direct fluid introduction, moving belt interface, ther mospray interface and particle beam interface [33] . Due to several limitations; for example, sensitivity, stabil ity or analyte desorption, atmospheric pressure ion ization sources with two primary ionization methods have been widely used with SFC: atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Huang et al. reported the combination of SFC 900 880 860 840 820 SM (24:1)
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Time (min) Figure 2. Lipid profiling performed by packed column supercritical fluid chromatography/MS. 2D map of lipids in mouse plasma analyzed in positive ion mode, showing that the SM molecular species with the same number of double bonds in the side chain are connected, illustrating the correlation between elution and carbon chain length. RT: Retention time; SM: Sphingomyelin. Adapted with permission from [44] .
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Figure 3. Lipid profiling performed by packed column supercritical fluid chromatography/MS. Multiple reaction monitoring chromatograms obtained from supercritical fluid chromatography/MS/MS of heptane extracts of (A) human serum and (B) low-density lipoprotein. Adapted with permission from [64] .
with APCI in 1990 whereby they successfully obtained not only protonated molecular ions, but also fragment ions of steroids by means of tandem mass spectrometry (MS/MS) [34] . The APCI technique was adapted for SFC [35–37] . Sadoun and Arpino published a review on the combination of SFC with ESI in 1993 and analyzed pyridine at the pg level; they found limitations of ion ization and peak shape, and described the need for postcolumn addition of polar organic solvents [38] . Baker and Pinkston advocated the use of sheath-flow to improve the limitations, as well as pressure-regulating fluid to control backpressure and assure the flow mass transfer [39,40] . As they pointed out, we usually add methanol as make-up flow post-column to help ionization as the modifier composition is lower than 5–10%. ESI is often utilized for metabolite analysis in biosamples, so the applications are described later. In addition to ioniza tion, SFC can be coupled with various type of MS, and combination with quadropole, ion trap, orbitrap, time-
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of-flight, Fourier transform infrared spectroscopy and ion mobility have been reported [41–48] . Because of this we have almost as many MS options as LC options in SFC. Although sensitivity depends upon a compound, SFC/MS/MS with ESI offers generally four- to ten-fold better sensitivity, or in hydrophilic compound analysis it was equivalent to hydrophobic compound analysis [49] . The studies demonstrated that SFC/MS can be an alternative approach for metabolic analysis. Metabolite analysis using SFC–MS SFC is classified into two groups: open tubular column SFC (otSFC) and packed column SFC (pcSFC). otSFC is typically ‘GC-like’ because of the use of pure CO2. On the other hand, pcSFC is com monly ‘LC-like’ because of addition of a polar solvent as modifier. The addition of modifier is key for metabo lite analysis of biosamples because it allows more polar compounds in the sample to be analyzed resulting from
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518.3
520.4
522.4
536.4
542.3
544.4
546.4
548.4
550.4
568.4
570.4
572.4
580.4
608.5
LPC 18:3
LPC 18:2
LPC 18:1
LPC 19:1
LPC 20:5
LPC 20:4
LPC 20:3
LPC 20:2
LPC 20:1
LPC 22:6
LPC 22:5
LPC 22:4
LPC 22:0
LPC 24:0
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702.5
704.5
PC 30:2
PC 30:1
9.3
9.3
10.6
10.5
10.3
10.5
10.2
10.2
10.3
10.5
10.6
10.7
10.4
10.5
10.5
10.6
10.6
10.2
10.4
10.5
10.6
10.4
10.3
10.4
10.4
10.5
10.2 748.6
746.5
744.5
m/z
SM 18:2
SM 18:1
SM 18:0
SM 16:1
SM 16:0
SM 14:0
727.5
729.5
731.7
701.5
703.5
675.5
DISM 18:0 733.6
DISM 16:1 703.5
DISM 16:0 705.6
PC 34:0e
PC 33:1
PC 34:2e
PLs
9.8
9.8
9.8
9.9
9.9
9.9
9.6
9.6
9.6
9.3
9.2
9.3
PC 38:6e
PC 36:0
PC 36:1
PC 36:2
PC 36:3
PC 36:4
PC 36:5
PC 36:0e
PC 36:1e
PC 35:2
PC 35:3
PC 36:4e
PC 36:5e
PC 34:0
PC 34:1
PC 34:2
PC 34:0e
PC 34:1
PC 34:2e
PC 34:3e
PC 34:6e
PC 32:0
PC 32:1
PC 32:6
PC 31:0
PC 31:1
PC 31:2
LPC 32:0
RT (min) PLs
792.5
790.6
788.7
786.6
784.6
782.6
780.6
776.6
774.6
772.5
770.5
768.6
766.5
762.6
760.6
758.5
748.6
746.5
744.5
742.6
736.5
734.5
732.6
722.5
720.6
718.5
716.5
720.6
m/z
9.4
9.3
9.3
9.4
9.4
9.5
9.3
9.3
9.3
9.4
9.3
9.3
9.4
9.3
9.3
9.4
9.3
9.2
9.3
9.3
9.3
9.3
9.4
9.2
9.2
9.3
9.2
10.5
912.7 9.5
866.7 9.4
864.6 9.5
862.6 9.5
860.6 9.5
858.7 9.2
856.7 9.3
854.7 9.3
852.7 9.3
850.7 9.3
848.7 9.5
846.6 9.4
844.7 9.4
842.6 9.3
840.7 9.4
838.6 9.4
836.6 9.5
834.6 9.5
832.5 9.5
830.6 9.2
828.5 9.3
SM 16:0
703.5 9.9
675.5 9.9
DISM 24:6 805.6 9.5
DISM 24:1 815.7 9.6
DISM 18:0 733.6 9.6
SM 14:0
702.6
730.5
PE 39:6
PE 38:1e
PE 38:2e
PE 37:4
PE 37:5
778.5
760.7
758.7
754.5
752.5
PE 38:6e 750.6
PE 36:2e
PE 36:3e 728.5
PE 36:5e 724.5
PE 33:2
482.4
m/z
9.7
9.5
9.6
9.6
9.7
9.7
9.6
9.7
9.6
9.6
10.8
RT (min)
Neutral loss scan
LPE 18:0
RT (min) PLs
826.7 9.3
m/z
DISM 16:0 705.6 9.6
PC 45:2
PC 42:4
PC 42:5
PC 42:6
PC 42:7
PC 41:1
PC 41:2
PC 41:3
PC 41:4
PC 42:5e
PC 41:6
PC 41:7
PC 40:1
PC 40:2
PC 40:3
PC 40:4
PC 40:5
PC 40:6
PC 40:7
PC 40:1e
PC 40:9
PC 40:3e
RT (min) PLs
High collision energy precursor ion scan
List of detected PLs by online-supercritical fluid extraction–MS/MS of mouse plasma. † Typical number of carbon and double bonds of individual m/z were shown. Probable subclasses and molecular species of phospholipids for individual m/z shown in [5,6]. ‡ Ether phospholipids contain plasmalogen and alkyl ether phospholipid. DISM: Dihydrosphingomyelin; LPC: Lysophosphatidylcholine; LPE: Lysophosphatidylethanolamine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PL: Phospholipid; RT: Retention time; SM: Sphingomyelin. Adapted with permission from [65].
720.6
718.6
LPC 32:0
LPC 32:1
690.5
510.3
LPC 17:0
LPC 30:1
508.4
LPC 17:1
634.5
506.3
LPC 18:2e
692.5
496.4
LPC 16:0
LPC 26:1
494.4
LPC 30:0
482.3
10.3
480.3
LPC 16:1
‡
RT (min)
m/z
Low collision energy precursor ion scan
LPC 15:0
LPC 16:1e
PLs†
Table 1. Lipid profiling performed by packed column supercritical fluid chromatography–MS.
Supercritical fluid chromatography/mass spectrometry in metabolite analysis
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706.6
676.5
690.5
714.5
716.5
718.5
720.6
722.5
728.5
730.5
732.6
734.5
736.5
738.5
740.5
742.6
PC 30:0
PC 28:1
PC 29:1
PC 31:3
PC 31:2
PC 31:1
PC 31:0
PC 32:6
PC 32:3
PC 32:2
PC 32:1
PC 32:0
PC 34:6e
PC 34:5e
PC 34:4e
PC 34:3e
9.4
9.3
9.3
9.3
9.3
9.4
9.3
9.3
9.2
9.2
9.3
9.2
9.3
9.2
9.3
9.3
RT (min)
PLs
m/z
PC 39:4
PC 40:5e
PC 40:6e
PC 38:0
PC 39:8
PC 38:2
PC 38:3
PC 38:4
PC 38:5
PC 38:6
PC 38:7
PC 38:1e
PC 38:2e
PC 38:3e
PC 38:4e
PC 38:5e
RT (min) PLs
824.7
822.6
820.7
818.7
816.6
814.7
812.7
810.6
808.6
806.5
804.5
802.7
800.7
798.6
796.7
794.6
m/z
9.3
9.3
9.5
9.4
9.3
9.4
9.4
9.4
9.5
9.5
9.3
9.3
9.4
9.3
9.3
9.4
SM 18:1
SM 24:7
SM 24:6
SM 24:2
SM 24:1
SM 24:0
SM 22:6
SM 22:2
SM 22:1
SM 22:0
SM 20:1
SM 20:0
SM 18:2
SM 18:0
SM 16:1
RT (min) PLs
9.9
729.5 9.9
801.6 9.8
803.7 9.8
811.7
813.6 9.8
815.7 9.8
775.6 9.8
783.7 9.9
785.7 9.8
787.7 9.8
9.8
759.7 9.8
727.5 9.9
731.7 9.8
757.7
RT (min) PLs
701.5 9.9
m/z
High collision energy precursor ion scan
m/z
RT (min)
Neutral loss scan
List of detected PLs by online-supercritical fluid extraction–MS/MS of mouse plasma. † Typical number of carbon and double bonds of individual m/z were shown. Probable subclasses and molecular species of phospholipids for individual m/z shown in [5,6]. ‡ Ether phospholipids contain plasmalogen and alkyl ether phospholipid. DISM: Dihydrosphingomyelin; LPC: Lysophosphatidylcholine; LPE: Lysophosphatidylethanolamine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PL: Phospholipid; RT: Retention time; SM: Sphingomyelin. Adapted with permission from [65].
m/z
PLs†
Low collision energy precursor ion scan
Table 1. Lipid profiling performed by packed column supercritical fluid chromatography–MS (cont.).
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Time (min) Figure 4. Diverse polar compound analysis performed by packed column supercritical fluid chromatography/MS. MRM chromatogram of a mixture of 17 pesticides after separation on three different columns (A) COSMOSIL 5CN-MS packed column, (B) Inertsil® ODS-4 column, and (C) Inertsil® ODS-EP column. (1) Diquat dibromide, (2) fosetyl, (3) maleic hydrazide, (4) daminozide, (5) methamidophos, (6) methomyl, (7) acetamiprid, (8) carbendazim, (9) dimethirimol, (10) thifluzamide, (11) tralomethrin, (12) emamectin benzoate (B1a), (13) chlorfluazuron, (14) acequinocyl, (15) pyridaben, (16) cypermethrin, (17) etofenprox. Analytical conditions: flow rate: 3 ml/min; column temperature: 35°C; (A) and (B) modifier: 5–10% (5 min), 10–40% (2 min), 40% (10 min), 40–5% (1 min), 5% (2 min), (C) modifier: 5% (2 min), 5–10% (5 min), 10–40% (2 min), 40% (8 min). Adapted with permission from [66] .
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Key terms Open tubular column SFC: Supercritical fluid chromatography using a column having uniform distribution of the stationary phase on the interior surface of the column. This technique is not very popular compared with packed column SFC. Packed column SFC: Supercritical fluid chromatography using a column packed densely and evenly with solid-phase support.
complementary column selection and mobile phase sol vating power. Therefore, we will focus on the benefits of pcSFC in this review. There were various reports using pcSFC/UV for metabolite analysis in the 1990s. Young and Games reported the analysis of the fungi metabolite ergosterol using an amino column of a standard packed HPLC col umn [50] . The technique of pcSFC/UV was applied to the analysis of several drugs and its metabolites in biosa mples such as urine, microsomes and serum [51–57] . Hsieh et al. demonstrated rapid and simultaneous analysis of an in vitro sample containing clozapine, ondansetron, tolbutamide and primidone using pcSFC/APCI/MS/ MS for the metabolic stability test. They found a con sistent result of metabolic stability of those compounds between LC and pcSFC [58] . In addition, Xu et al. suc cessfully analyzed 15 estrogen metabolites in pcSFC/ APCI/MS and confirmed superiority in the throughput compared with HPLC/MS [59] . While another steroid analysis using ESI was reported, 25 bile acids was sepa rated simultaneously in 13 min and all the 25 bile acids were detected in rat serum samples using pcSFC/ESI/ MS/MS [60] . Bamba et al. demonstrated comprehen sive analysis of diverse lipids profiling using pcSFC/ ESI/MS/MS, and showed the effectiveness of SFC for simultaneous analysis for diverse compounds [61] . Fur thermore, his group published a review summarizing the application of pcSFC for lipidomics [62] . And recently, another application study for comprehensive metabolite analysis related to lipidomics by pcSFC/ESI/MS was published: lipid profiling in soybeans (Figure 1) [63] and mouse plasma (Figure 2) [57] , carotenoids in human serum (Figure 3) [64] , and phospholipids in mouse plasma (Table 1) [65] . These publications gave notice that we have latched onto a potential tool for metabolomics, and his group applied the metabolomics approach using SFC to not only biosamples, but also food science. Future perspective As previously mentioned, pcSFC is a powerful separa tion technique for both non-polar and polar compounds; however, we are seeing its benefits in the next stage of applications. Ishibashi et al. demonstrated simultane ous analysis of pesticides, which have a log Pow rang
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ing from -4.6 to 6.6 (Figure 4) [66] . Although this new application for pcSFC is not related to metabolite analy sis, it offers great potential allowing us to shift from a multiple method analysis to a single method analysis. In metabolite analysis, the broad coverage of constituents has been a challenging task because they are comprised of a diverse set of small molecules with various chemi cal and physical properties [67] . The solubility flexibility of SCCO2 resulting in complementary column selection advances the range of compound polarity in simultane ous analysis. To obtain the wide range of solvent polar ity, the modifier composition can range from 0 to 100% in pcSFC, however the critical point of temperature and back pressure is increased along with the increased modifier composition. In other words, the phase state of CO2 might not be supercritical any more as the modi fier gradient runs without updating temperature and backpressure. Even if CO2 changes the state from super critical to subcritical, discontinuous transition does not occur in CO2 [68] . Applying this approach to our current separation, pcSFC may enable simultaneous analysis for diverse polarity compounds that are typically analyzed not only with different methods but with different sepa ration techniques. The approach can be classified as uni fied chromatography that continuously shifts the physi cal state of the mobile phase without phase separation by controlling temperature and back pressure. For example, the phase state is changed in the order of gas, supercriti cal, subcritical and liquid [68–70] . Although there are very few publications regarding application, we think the approach will open a door to a new separation tech nique expanding the convergence regions among GC, SFC and LC for metabolite analysis. Analytical targets in metabolite analysis include highly polar compounds, but non-polar compounds such as lipids and steroids have recently emerged as important signaling molecules [71,72] . Therefore, developing a single universal method enabling simultaneous analysis for polar and non-polar compounds could be the next challenge in metabolite analysis. Unified chromatography can use the same mobile phase state as GC, SFC and LC in terms of the physical state. The benefit of the unique separation technique is to enhance the polarity range of the mobile phase from 100% CO2 (non-polar) to 100% polar sol vent (polar). The approach will enhance the compound coverage in a single run. If we can see beyond the cur rent separations, pcSFC can p rovide comprehensive information for metabolomics. Financial & competing interests disclosure This work was partially supported by MEXT KAKENHI Grant Number 23686120 Grant-in-Aid for Young Scientists (A), the Development of Systems and Technology for Advanced Measurement and Analysis Project (JST), and the Advanced
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Low Carbon Technology Research and Development Program (JST). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject mat-
Review
ter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary Nature of supercritical fluid chromatography • Supercritical fluid chromatography (SFC) owes many of its advantages to the properties of supercritical carbon dioxide (SCCO2). • SCCO2 possess many benefits as the chromatography mobile phase: low viscosity; high diffusivity and solubility flexibility resulting in better separation; faster run time; and, adoption of a wide range of column chemistry. • SFC is amenable to non-polar as well as polar compound analysis.
Coupling to MS • The sensitivity and selectivity afforded by MS is adopted in SFC. • Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the current main ionization techniques in SFC; various MS is used with SFC. • A make-up flow is sometimes required at low modifier composition for ionization.
Metabolite analysis using SFC • Packed column SFC is widely used in metabolite analysis since the addition of a modifier can enhance the solvating power of the mobile phase and the solubility of metabolites in SFC. • Analysis of a target compound and its metabolites has been carried out in various biosamples. • SFC/ESI/MS/MS is a powerful tool for lipid profiling. energy relationships. J. Chromatogr. A 1206(2), 186–195 (2008).
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Supercritical fluid chromatography/mass spectrometry in metabolite analysis
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Important example of applying supercritical fluid chromatography/MS to analyze diverse metabolites in a mixture.
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Informative example of coupling MS to supercritical fluid chromatography.
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Interesting paper that shows the wide coverage of compound polarity in supercritical fluid chromatography.
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Critical review of unified chromatography.
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