CHEMICAL ENGINEERING SERIES – Supercritical Fluids Separation Processes - Volume 1 by Sciencenetwork

CHEMICAL ENGINEERING SERIES – Supercritical Fluids Separation Processes - Volume 1

Publisher: Science Network ISBN: 978-0-9869554-1-9 First published in January, 2014 Printed in Canada

A free online edition of this book is available at www.sciencenetwork.ca Additional hard copies can be obtained from reprint@sciencenetwork.ca

CHEMICAL ENGINEERING SERIES

Supercritical Fluids Volume 1

 Science Network Online Open Access Publisher

Table of contents

Team Entrepreneurship Abstract

Introduction

1- Construction of a profiling method for carotenoids using SFC/MS with monolithic columns

1-1 Introduction

1-2 Results

1-2-1 Investigation of analysis conditions

1-2-2 Detection limit

1-2-3 Analysis of biological sample

2- Metabolic profiling of b-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry

2-1 Introduction

2-2 Results

2-2-1 Construction of Î˛ CXFA analysis system by SFC-QqQMS

2-2-2 Analysis of Î˛ CXFAs in Citrus unshiu peel by SFC-QqQMS

2-2-3 Comparison between results of GC-MS and SFC-QqQMS

Conclusion

References

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F L U I D S

Metabolite profiling of carotenoids and their derivatives by supercritical fluid chromatography coupled with mass spectrometry

Atsuki Matsubara, Yusuke Wada, Eiichiro Fukusaki, Takeshi Bamba Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan E-mail :

atsuki_matsubara@bio.eng.osaka-u.ac.jp

wing to its favorable properties such as low viscosity and high diffusivity, a supercritical fluid (SF) can be used as the mobile phase in chromatography. Supercritical fluid chromatography (SFC) can provide high-speed and highresolution separation. SFC is most commonly used as a preparative method. However, SFC can also be used to perform high-precision analysis of hydrophobic metabolites, because of the low polarity of supercritical carbon dioxide (SCCO 2). With the aim of application of SFC to bioanalysis, we have established an analytical system in which mass spectrometer as a detector is coupled to SFC. This chapter will present analysis system for carotenoids and their derivatives constructed using SFC/MS. Seven carotenoids including structural isomers were separated using a monolithic column within 3 minutes at the flow rate of 9 ml/min. In addition, three monolithic columns were connected in head-to-tail fashion to indicate an expected improvement in the resolution. The system enabled sufficiently separation of the carotenoids from biological samples containing complex matrices. Next, analysis system for carotenoid fatty acid esters was constructed using triple-quadrupole mass spectrometer (QqQMS) as a detector. -cryptoxanthin (β CX) and nine β CX fatty acid esters (β CXFAs) were sufficiently separated in 20 minutes. The limit of detection was 540 fmol for the free form and 32–130 fmol for the esterified forms. These results demonstrate that both the throughput and the sensitivity of this SFC-QqQMS system are considerably higher than those of conventional methods. When this system was applied to the analysis of Citrus unshiu, β CX and five CXFAs were directly detected with much simpler sample pre-preparation. The analysis of other citrus fruits indicated that the β CXFA profiles varied considerably with their breed variety. Furthermore, using the theoretically estimated analytical parameters, it was confirmed that citrus fruits contained a small amount of β CX esterified with short-chain fatty acid such as butyric acid. These results clearly showed the usefulness of SFC/MS in bioanalysis. We hope that SFC/MS technique will be generally used in profiling method for hydrophobic compounds in various fields such as medical, pharmaceutical, and biological science.

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Introduction A substance whose temperature and pressure are above its critical point is called a supercritical fluid (SF). Owing to its favorable properties such as low viscosity and high diffusivity, an SF can be used as the mobile phase in chromatography. In most case, SFC is used for preparative isolation of chiral compounds owing to the fact that SFC facilitates rapid separation of such compounds and that it renders solvent evaporation unnecessary [1-5]. However, SFC has the potential to be a powerful analytical tool for other applications. Especially, SFC is useful for the separation of hydrophobic compounds because the polarity of supercritical carbon dioxide (SCCO2) is relatively low as that of hexane [6]. Metabolomics is the exhaustive profiling of metabolites contained in organisms. The metabolomics approach can characterize a phenotype with high resolution. Thus, recent years, metabolomics is applied to various researches in the field of life science; discovery of biomarkers, improvement of fermenting process, quality estimation, etc. However, metabolomics technology has not been well established because of the chemical diversity of the target metabolites [7-11]. Particularly, an analysis method for highly hydrophobic metabolites has still not been established. At present, mass spectrometry (MS) is the most popular detection method used in metabolomics for analysis of metabolites [7,8,12]. Through MS, it is possible to obtain information for identification, such as the molecular weight and structure. It is also possible to obtain information for quantification with a high sensitivity. MS is mostly used along with a separation method for avoiding ion suppression, which is a critical drawback of MS, and for identifying metabolites. Identification of metabolites is carried out by combining the mass-to-charge ratio (m/z) and the migration time. Thus far, metabolomics targeting low-molecular-weight metabolites in the glycolytic system and the tricarboxylic acid cycle was performed by GC/MS or capillary electrophoresis (CE)/MS [13-16]. SFC coupled with MS may be the last piece of the puzzleâ&#x20AC;&#x201D;SFC coupled with MS has the potential to be a powerful analytical tool for metabolomics targeting hydrophobic metabolites. In this chapter, we present a summary of carotenoid analysis as examples of hydrophobic metabolites by SFC/MS.

1- Construction of a profiling method for carotenoids using SFC/MS with monolithic columns [17] 1-1 Introduction Carotenoids are fat-soluble pigments that are biosynthesized by combining eight isoprene units. They play an essential role in not only the pigmentation of natural resources but also physiological functions. For example, they function as 2

light

harvesting

complexes

and

provide

photoprotection

during

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photosynthesis. Carotenoids are indispensable to animals as well as plants, because some of them are essential for vision as provitamin A [18, 19]. Carotenoids are generally classified into two molecular classes: carotenes that do not contain oxygen and xanthophylls that do contain oxygen. Their polarities are slightly different. Moreover, each of them has many structural isomers and cis-trans isomers. Metabolic profiling of carotenoids is important for understanding their physiological functions and food quality estimation, but advanced technology is required to analyze biological samples containing a mixture of structurally similar carotenoids. We applied SFC for carotenoids analysis, because as mentioned above, SFC is highly suitable for the separation of hydrophobic compounds. Furthermore, MS detection was applied instead of photometric detection, which is the conventional method for carotenoid detection. Co-elution with various metabolites in biological samples often interferes with the accurate identification and quantification of target compounds. This may be a serious problem in the case of photometric detection of carotenoids that have many analogous compounds. The MS enables the separation of carotenoids, which cannot be achieved by chromatography; therefore, more precise profiles of carotenoids can be obtained from biological samples. Furthermore, the high sensitivity of the MS allows us to target minor constituents. In addition, monolithic columns manufactured by a new sol-gel process [20– 22] were applied as analytical columns. It consists of continuous 3-D silica network and its gaps containing construction called macropore (ca. 2 μm, correspond to pore spaces of particle-packed columns) and mesopore (ca. 13 nm, correspond to fine pores of particle-packed columns) [23]. The back pressure of monolithic columns is lower than that of particle-packed columns, owing to their wider flow channels [23]. Because of this characteristic of monolithic columns, the analysis time can be reduced by increasing the flow rate and the resolution can be improved by using several columns [17–20]. In fact, it has been reported that monolithic columns are effective in separating isomers of β-carotene [24]. 3

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1-2 Results 1-2-1 Investigation of analysis conditions Carotenes (β-carotene and its structural isomer, i.e., lycopene) and xanthophylls (zeaxanthin and neoxanthin and their structural isomers, namely, lutein and violaxanthin, respectively, and antheraxanthin; their structures are shown in Fig. 1) were analyzed as a model for the separation of carotenoids. First, ionization conditions of the mass spectrometer were investigated using their standards under the following mobile phase conditions: mobile phase, supercritical carbon dioxide (SCCO2); modifier, methanol with 0.1% ammonium formate 10%; flow rate, 3 ml/min. Generally, atmospheric pressure chemical ionization (APCI) is used to analyze compounds with high hydrophobicity, such as carotenoids [25], although good ionization is obtained by electrospray ionization (ESI). Hence, we decided to adopt ESI as the ionization method and optimized the analysis conditions such as the voltage, source temperature, the flow rates in SFC and make up. Next, we examined the column and separation conditions. First, we tried to use particle-packed columns. Most of the carotenoids were not retained in the normal-phase columns; hence, we used the ODS column. After testing ODS columns with various packing materials, 7 carotenoids were separated within 15 min using Hibar Purospher STAR RP-18e (Merck). Both improved separation and improved throughput were observed compared to the separation and throughput for the previously reported method involving HPLC [26]. These results indicate that the new SFC method is effective in the highresolution and high-speed separation of hydrophobic compounds. Next, we used a monolithic silica column to carry out an analysis at a higher speed and resolution. The gradient condition was investigated by analyzing a standard mixture using methanol with 0.1% ammonium formate as the modifier. Seven carotenoids were successfully separated in 10 min (Fig. 2A) under the following conditions: the modifier gradient, outlet pressure, flow rate, and column temperature were 1–6% (10 min), 10 MPa, 3 ml/min, and 35 °C, respectively. The inlet pressure was as low as 15 MPa (outlet pressure: 10 4

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MPa, flow rate: 3 ml/min). Therefore, we tried to carry out an analysis at a high flow rate by making optimum use of the low back pressure. The conditions in the analysis of the standard mixture of carotenoids were the same as mentioned above, except for the flow rate. The inlet pressure was maintained at a relatively low level: 18 MPa for the flow rate of 5 ml/min and 21 MPa for the flow rate of 7 ml/min (approximately the same inlet pressures were observed for the particle sizes of 5 column).

m and 3

m, respectively, in a particle-packed

Separation performed at a flow rate of 9 ml/min yielded quite

satisfactory results (the resolution of separation of neoxanthin and violaxanthin was 2.62), and the analysis time was reduced to 4 min (Fig. 2B). The synergistic effect of SFC and the monolithic column enabled us to develop a high-speed and high-resolution profiling method for carotenoids.

1-2-2 Detection limit Experiments were conducted to determine the sensitivity of the mass spectrometer for the detection of several carotenoids at a flow rate of 3.0 ml/min. The detection limit (S/N > 3) of β-carotene was 34.5 pg; lycopene, 36.2 pg; lutein, 55.0 pg; zeaxanthin, 48.4 pg; antheraxanthin, 37.5 pg; neoxanthin, 44.9 pg; and violaxanthin, 43.9 pg. These results indicated that the use of the mass spectrometer as a detector led to an improvement in not only selectivity but also sensitivity.

1-2-3 Analysis of biological sample Next, we analyzed a biological sample—acetone extracts of green algae, Chlamydomonas reinhardtii—in order to evaluate the usefulness of this system for profiling an actual biological sample. Samples were prepared without preprocessing, so the extracts clearly contained hydrophobic contaminants such as lipids and chlorophylls. They also contained carotenoids, which were very similar in structure to the target compounds. When such samples were analyzed, it was difficult to identify and quantify target compounds precisely, even if the authentic standards were separated successfully. In fact, when the 5

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sample was analyzed using a monolithic ODS column under the conditions mentioned earlier (flow rate: 3 ml/min), there were several peaks apart from those corresponding to the target carotenoids in the mass chromatograms, and some of these peaks overlapped with the target peaks (Fig. 3A). We therefore attempted to improve resolution by using several monolithic columns with very low back pressure. The use of three monolithic columns resulted in the successful separation of co-eluted compounds. For example, when a single monolithic column was used, the resolution of neoxanthin and the peak of retention time 7.2 min (m/z 601) was 0.9, but when three columns were used, the resolution improved to 1.8 (Fig. 3B). On the other hand, there were some peaks whose resolution was not improved (e.g., neoxanthin and violaxanthin). The characteristics of SCF, which is the mobile phase in SFC, are known to be greatly affected by pressure. The variations in the pressure gradient inside the column caused by the elongation of the column might have an effect on the characteristics of SCCO2 and the retention behavior of each compound. However, the peaks obtained with connected columns were more scattered than those obtained with a single column; hence, the separation with connected columns is suitable for metabolite identification. It is suggested that this is an extremely practical profiling method because it enables us to carry out highly accurate identification and quantification of samples containing complex matrices. Moreover, there is no need for pretreatment during sample preparation, and the analysis time is very short; hence, it is possible to perform extremely high-throughput analysis of biological samples by using this profiling method.

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2- Metabolic profiling of b-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry [27] 2-1 Introduction Hydroxylated xanthophyll exists not only in a free form but also in a fatty-acidesterified form in physiological tissues [28]. This ester has high hydrophobicity, and it contains many kinds of structurally similar compounds having different fatty acid chains. It has been reported that the esterified form of xanthophyll is more thermally stable and bioavailable than its free-form counterpart [29, 30]. However, in vivo kinetics and physiological functions of xanthophyll fatty acid esters are not yet well understood. Conventional highperformance liquid chromatography (HPLC) methods are time-consuming, requiring more than 40 min for the analysis of xanthophyll fatty acid esters [31-33]. They also require complicated sample pre-preparation processes such as multiple extractions, concentration, and purification [31-34]. Thus, there is a need for an analysis system with high throughput and high sensitivity to obtain information about xanthophyll fatty acid esters. Because SFC has unique properties and is an effective analytical tool for the analysis of hydrophobic metabolites, as mentioned above, SFC is a promising candidate for such a system. We constructed a novel analysis system for CX and b-cryptoxanthin fatty acid ester (CXFA) using SFC. Selectivity and sensitivity of detector are important for separating structurally similar compounds and detecting trace components in biological samples. Therefore, triple quadrupole mass spectrometry (QqQMS) was used as the detector. QqQ-MS is a powerful tool for metabolite analysis. For example, a product ion scan for structure elucidation and selective reaction monitoring (SRM) allows highly selective and sensitive quantification and can be performed using collision-induced dissociation. To assess the usefulness of the SFC-QqQMS system, we applied it to the analysis of CX and CXFAs in citrus fruits.

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2-2 Results 2-2-1 Construction of CXFA analysis system by SFC-QqQMS Firstly, investigations were conducted to identify appropriate ionization conditions for CXFAs (Fig. 4) by infusion analysis. The molecular ions of carotenoids were observed as radical cations using electrospray ionization in positive mode. Common product ions, [M-92]+, generated by elimination of toluene from the polyene chain were observed by product ion scan. For the following experiments, SRM analysis based on this information was employed to detect CXFAs with high selectivity. Next, to find a suitable column for CXFA analysis, the standard mixture of CXFAs was analyzed using various columns under the aforementioned conditions. Most of the CXFAs were not retained in normal-phase columns. As preferred reverse-phase column, YMC Carotenoid (250 × 4.6 mm (i.d.), 5 m; YMC) was selected because a C30 column modified with a triacontyl chain is generally used to analyze xanthophyll fatty acid esters [31-34]. However, the CXFAs were not eluted within 30 min, possibly because the hydrophobic interaction between the CXFAs and the column stationary phase was strong. By contrast, all of the CXFAs were successfully separated within 20 min with the use of Inertsil ODS-P (250 × 4.6 mm (i.d.), 5 m; GL Sciences), which is a polymeric-type ODS column (Fig. 5A). The density of ODS chain in polymeric-type column is higher than that of monomeric-type and polymeric ODS column has high stereo-selectivity. This is the reason why Inertsil ODS-P column could separate CXFAs, such as oleate/linoleate ester and linolenate/EPA ester, which were not separated with monomeric-type ODS column. Next, we obtained the calibration curve using an Inertsil ODS-P column to validate the constructed system. The relative standard deviation (RSD) for retention time was less than 1.1% and for peak area was less than 9.2%. These results show that the SFC-QqQMS system has good repeatability. The linearity was also good since the R2 value was greater than 0.99 for every compound. In addition, the limit of detection (LOD) was also examined, and it was 540 fmol 8

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for the free form and 32–130 fmol for the esterified forms. In our research, we used QqQMS, which offers higher selectivity and sensitivity than MS; it enabled the detection of CX and CXFAs even at femtomolar concentrations. Thus, we successfully developed and validated an analysis system for CX and CXFAs by SFC-QqQMS. 2-2-2 Analysis of CXFAs in Citrus unshiu peel by SFC-QqQMS Next, we analyzed acetone extracts of Citrus unshiu to evaluate the usefulness of this system for profiling an actual biological sample. The acetone extract of a freeze-dried peel of this fruit was subjected to SFC-QqQMS analysis, and the CX free form and five different esterified forms—laurate (C12:0) ester, myristate (C14:0) ester, palmitate (C16:0) ester, stearate (C18:0) ester, and oleate (C18:1) ester—were directly detected (Fig. 5B). As mentioned above, conventional techniques such as HPLC are quite time-consuming [31-34]. The SFC-QqQMS system is free from this flaw. Therefore, the SFC-QqQMS system developed in this study is an effective analysis tool for CXFAs. In these experiments, we also targeted CXFAs for which standards are not available by theoretically estimating the analytical parameters. The structures of these CXFAs were estimated using the information on the fragment pattern and retention time. For example, the structure of CX butyric acid (C4:0) ester was estimated by the following three steps. First, the fragment ion [M-92]+, which is specific to carotenoids, was evaluated. Second, the presence of the ion of m/z 552.4, which is generated by removing the fatty acid from the molecule, was confirmed. Third, it was identified as being the butyric acid ester on the basis of the fact that shorter CXFAs have a shorter retention time. As a result, it was confirmed that citrus fruits contained a small amount of CX butyric acid ester, caproic acid (C6:0) ester, caprylic acid (C8:0) ester, and capric acid (C10:0) ester (Fig. 5C). Because previous studies have reported that capric acid ester is the shortest CXFA in citrus fruits [34, 35], this is a new discovery concerning CXFA and is attributable to the high sensitivity of QqQMS. 9

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2-2-3 Comparison between results of GC-MS and SFC-QqQMS Finally, to investigate the properties of the fatty acid used for CX esterification, the total fatty acid profile in Citrus unshiu was studied using GC-MS. The total fatty acids in Citrus unshiu were present in the following (decreasing) amounts: oleic acid (39.7% ± 1.0%), linoleic acid (26.7% ± 0.3%), palmitic acid (19.5 ± 0.3%), and linolenic acid (10.6 ± 0.6%) (Fig. 6A). In contrast, the fatty acids located in CXFA obtained by SFC/MS/MS in Citrus unshiu were present in the following (decreasing) amounts: laurate ester (33.7%), myristate ester (28.6%), palmitate ester (26.0%), and oleate ester (6.9%) (Fig. 6B). These results show that the profiles of fatty acids located in CXFA were distinct from total contents. According to the review article published by Antonio et al. in 2005, xanthophylls were suggested to be esterified by acyl-coenzyme A; however, further research is needed to confirm the biosynthetic pathway of xanthophyll fatty acid esters. If the CXFAs are actually formed by enzyme reactions, the results indicate that citrus fruits contain an enzyme that preferentially uses the short-chain fatty acid to esterify CX. This finding, obtained using our SFC-QqQMS system, may be helpful to clarify the mechanism of xanthophyll esterification.

Conclusion In this review, an overview of carotenoid analysis performed by SFC/MS has been provided. Metabolic profiles of carotenoids in biological samples containing complex matrices can be obtained by using both of these methods. As of now, only GC, LC, and CE are mainly used for metabolite analysis. However, it is difficult to simultaneously perform high-speed analysis of compounds with high hydrophobicity by using these technologies. SFC is suitable for the analysis of such compounds. The application of SFC can be extended by integrating SFC and a QqQ MS, high-resolution MS (time-offlight MS, FT-ICR MS), and hybrid MS, with which we can perform highly sensitive and selective quantification and structural information. In fact, we could detect CXFAs of the order of femtomoles using QqQMS for detection. 10

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Supercritical fluid extraction (SFE) is another separation application of SF. Online coupling of SFC with supercritical fluid extraction (SFE), which is based on the fact that SFs are efficient solvents, has already been reported [3637]. This method is useful for obtaining a high-throughput screening. In addition, it is expected that this method could be effective for the analysis of compounds that are easily decomposed during organic solvent extraction. In other words, precise information of metabolites present in a target cell can be obtained by online SFE-SFC analysis. In conclusion, although bioanalysis technologies based on SF are currently not well established, there is great potential for development. In future, it is expected that these technologies will be extensively used for metabolite analysis.

References

[1]

Abbott

pharmaceutical

E.,

Veenstra

applications

T.D., of

Issaq

H.J..(2008),

packed-column

Clinical

supercritical

and fluid

chromatography. J. Sep. Sci. 31, 1223-1230. [2]

Toribio L., del Nozal M.J., Bernal J.L., Alonso C., JimĂŠnez J.J. (2007)

Enantiomeric separation of several antimycotic azole drugs using supercritical fluid chromatography. J. Chromatogr. A, 1144, 255-261. [3]

Su .B, Bao Z., Xing H., Yang Y., Ren Q. (2009), Enantioseparation of

paroxetine intermediate on an amylose-derived chiral stationary phase by supercritical fluid chromatography. J. Chromatogr. A, 1216, 5140-5146. [4]

Toribio L., Alonso C., del Nozal M.J., Bernal J.L., MartĂn M.T. (2006)

Semipreparative enantiomeric separation of omeprazole by supercritical fluid chromatography. J. Chromatogr. A, 1137, 30-35 [5]

Yang Y., Su B., Yan Q., Ren Q. (2005), Separation of naproxen

enantiomers by supercritical/subcritical fluid chromatography. J. Pharm. Biomed. Anal. 39, 815-818.

S U P E R C R I T I C A L

[6]

F L U I D S

Ikushima Y., Saito N., Arai M., Arai K. (1991), Solvent polarity

parameters of supercritical carbon-dioxide as measured by infraredspectroscopy. Bull. Chem. Soc. Japan, 64, 2224-2229. [7]

Fukusaki E., Kobayashi A.(2005), Plant metabolomics: Potential for

practical operation. J. Biosci. Bioeng. 100, 347-354. [8]

Dunn W.B., Bailey N.J.C., Johnson H.E. (2005), Measuring the

metabolome: current analytical technologies. Analyst, 130, 606-625. [9]

Bamba T., Fukusaki E. (2006), Technical problems and practical

operations in plant metabolomics. J. Pest. Sci. 31, 300-304. [10]

Hollywood K., Brison D.R., Goodacre R. (2006), Metabolomics:

Current technologies and future trends. Proteomics, 6, 4716-4723. [11]

Wu Z.M., Huang Z.Q., Lehmann R., Zhao C.X., Xu G.W. (2009), The

Application

Chromatography-Mass

Spectrometry:

Methods

Metabonomics. Chromatographia, 69, 23-32. [12] Bedair M., Sumner L.W. (2008), Current and emerging mass-spectrometry technologies for metabolomics. Trac-Trends in Anal. Chem. 27, 238-250 [13] Pasikanti K.K., Ho P.C., Chan E.C.Y. (2008), Gas chromatography/mass spectrometry in metabolic profiling of biological fluids. J. Chromatogr. B, 871, 202-211. [14]

Monton M.R.N., Soga T. (2007) Metabolome analysis by capillary

electrophoresis-mass spectrometry. J. Chromatogr. A, 1168, 237-246 [15]

Ramautar R., Somsen G.W., de Jong G.J. (2009) CE-MS in

metabolomics. Electrophoresis, 30, 276-291. [16]

Harada K,. Fukusaki E. (2009), Profiling of primary metabolite by

means of capillary electrophoresis-mass spectrometry and its application for plant science. Plant Biotech. 26, 47-52. [17]

Matsubara A., Bamba T., Ishida H., Fukusaki E., Hirata K.(2009),

Highly sensitive and accurate profiling of carotenoids by supercritical fluid chromatography coupled with mass spectrometry., J. Sep. Sci. 32, 1459-1464. [18]

Rao, A.V., Rao, L.G. (2007), Carotenoids and human health,

Pharmacol. Res., 55, 207-216. 12

S U P E R C R I T I C A L

[19]

F L U I D S

Noziere, P., Graulet, B., Lucas, A., Martin, B., Grolier, P., Doreau, M.

(2006), Carotenoids for ruminants: From forages to dairy products, Anim. Feed Sci. Technol., 131, 418-450. [20]

Nakanishi, K., Soga, N. (1991), Phase Separation in Gelling Silicaâ&#x20AC;&#x201C;

Organic Polymer Solution: Systems Containing Poly(sodium styrenesulfonate), J. Am. Ceram. Soc., 74, 2518-2530. [21]

Nakanishi, K., Soga, N. (1992), Phase separation in silica sol-gel

system containing polyacrylic acid I. Gel formaation behavior and effect of solvent composition, J. Non Cryst. Solids., 139, 1-13. [22]

Nakanishi, K., Soga, N. (1992), Phase separation in silica sol-gel

system containing polyacrylic acid II. Effects of molecular weight and temperature, J. Non Cryst. Solids., 139, 14-24. [23]

Cabrera, K., Lubda, D., Eggenweiler, H.M., Minakuchi, H., Nakanishi,

K. (2000), A New Monolithic-Type HPLC Column For Fast Separations, J. High Resol. Chromatogr., 23, 93-99. [24]

Lesellier, E., West, C., Tchapla, A. (2003), Advantages of the use of

monolithic stationary phases for modelling the retention in sub/supercritical chromatography: Application to cis/trans-Î˛-carotene separation, J. Chromatogr. A, 1018, 225-232. [25]

Rodriguez-Bernaldo de Quiros, A., Costa, H. S. (2006), Analysis of

carotenoids in vegetable and plasma samples: A review, J. Food Compos. Anal., 19, 97-111. [26]

Bunea, A., Andjelkovic, M., Socaciu, C., Bobis, O., Neacsu, M., Verhe,

R., Camp, J. V. (2008), Total and individual carotenoids and phenolic acids content in fresh, refrigerated and processed spinach (Spinacia oleracea L.), Food Chem., 108, 649-656. [27]

Wada Y., Matsubara A., Uchikata T., Iwasaki Y., Morimoto S., Kan K.,

Okura T., Fukusaki E., Bamba T., Metabolic profiling of b-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry, J. Sep. Sci., submitted

S U P E R C R I T I C A L

[28]

F L U I D S

Wingerath, T., Stahl, W., Sies, H.(1995), ß-Cryptoxanthin selectively

increases in human chylomicrons upon ingestion of tangerine concentrate rich in beta-cryptoxanthin esters, Arch. Biochem. Biophys., 324, 385-390. [29]

Fu, H., Xie, B., Fan, G., Ma, S., Zhu, X., Pan, S. (2010), Effect of

esterification with fatty acid of ß-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method, Food Chem., 122, 602609. [30]

Bowen, P. E,, Herbst-espinosa, S. M,, Hussain, E. A,, Stacewicz-

sapuntzakis, M. (2002), Esterification does not impair lutein bioavailability in humans, J. Nutr., 132, 3668-3673. [31]

Dugo, P., Herrero, M., Giuffrida, D., Ragonese, C., Dugo, G.,

Mondello, L. (2008), Analysis of native carotenoid composition in orange juice using C30 columns in tandem, J. Sep. Sci., 31, 2151-2160. [32]

Inbaraj, B. S., Lu, H., Hung, C. F., Wu, W. B., Lin, C. L., Chen, B.

H.(2008), Determination of carotenoids and their esters in fruits of Lycium barbarum Linnaeus by HPLC-DAD-APCI-MS., J. Pharm. Biomed. Anal., 47, 812-818. [33]

Schlatterer, J., Breithaupt, D. E. (2005), Cryptoxanthin Structural

Isomers in Oranges, Orange Juice, and Other Fruits, J. Agric. Food Chem., 53, 6355-6361. [34]

Pérez-Gálvez, A., Mínguez-Mosquera, M. I. (2005), Esterification of

xanthophylls and its effect on chemical behavior and bioavailability of carotenoids in the human, Nutr. Res., 25, 631-640. [35]

Dugo, P., Herrero, M., Kumm, T., Giuffrida, D., Dugo, G., Mondello,

L.(2008),

Comprehensive

normal-phase

reversed-phase

liquid

chromatography coupled to photodiode array and mass spectrometry detection for the analysis of free carotenoids and carotenoid esters from mandarin, J. Chromatogr. A. 1189, 196-206. [36]

Sato K, Sasaki SS, Goda Y. (1999), Direct connection of supercritical

fluid extraction and supercritical fluid chromatography as a rapid quantitative

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method for capsaicinoids in placentas of Capsicum. J. Agric. Food Chem. 47, 4665-4668. [37]

Voorhees KJ, Gharaibeh AA, Murugaverl B. (1998), Integrated

SFE/SFC/MS system for the analysis of pesticides in animal tissues. J. Agric. Food Chem. 46, 2353-2359.

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Figures

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Fig. 1 Structures of carotenoids investigated

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Fig. 2

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Mass chromatograms of carotenoids using a monolithic ODS column (100 mm × 4.6 mm ID).

Analysis conditions: mobile phase (CO2) flow, 3.0 ml/min; modifier (methanol with 0.1% w/v ammonium formate), 1-11% (20min); outlet pressure, 10.0 MPa; oven temperature, 35°C; detection, single-quadrupole mass spectrometer. (a) lycopene, (b) β-carotene, (c) lutein, (d) zeaxanthin, (e) antheraxanthin, (f) neoxanthin, and (g) violaxanthin (reproduced from ref. [17] with some alteration).

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Fig. 3

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Analysis of carotenoids in Chlamydomonas reinhardtii by SFC-MS using (i) a Chromolith

Performance RP-18e (100 x 4.6 mm ID) (ii) three connected Chromolith Performance RP-18e. (a) ď ˘-carotene, (b) lutein, (c) zeaxanthin, (d) antheraxanthin, (e) neoxanthin, and (f) violaxanthin Analysis conditions: mobile phase (CO2) flow, 3.0 ml/min; modifier (methanol with 0.1% w/v ammonium formate), 1-11% (20min); outlet pressure, 10.0 MPa; oven temperature, 35Â°C; detection, single-quadrupole mass spectrometer. (reproduced from ref. [17] with some alteration).

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Fig. 4

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Structures of ď ˘CXFAs used in this research. (A) Free form, (B) laurate (C12:0) ester, (C)

myristate (C14:0) ester, (D) palmitate (C16:0) ester, (E) stearate (C18:0) ester, (F) oleate (C18:1) ester, (G) linoleate (C18:2) ester, (H) linolenic (C18:3) ester, (I) EPA (C20:5) ester, and (J) DHA (C22:6) ester.

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Fig. 5

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Analysis of ď ˘CXFAs in Citrus unshiu. Mass chromatograms (m/z 552.4 > 460.4, free

form; m/z 734.7 > 642.7, laurate ester; m/z 762.7 > 670.7, myristate ester; m/z 790.7 > 698.7, palmitate ester; m/z 818.7 > 726.7, stearate ester; m/z 816.7 > 724.7, oleate ester; m/z 814.7 > 722.7, linoleate ester; m/z 812.7 > 720.7, linolenic ester; m/z 836.7 > 744.7, EPA ester; m/z 862.7 > 770.7, DHA ester) obtained by SRM mode analysis using Inertsil ODS-P column. (A) Standard mixtures and (B) Citrus unshiu peel. ď ˘CX

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Fig. 5 (C) ď ˘CXFA compositions of Citrus unshiu obtained by SFC-QqQMS

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Fig. 6

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Comparison between (A) fatty acid composition obtained by GC-MS and (B) ď ˘CXFA composition obtained by SFC-QqQMS.