ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29 997 2013 © The Japan Society for Analytical Chemistry

Tentative Identification of Torulene Cis/trans Geometrical Isomers Isolated from Sporidiobolus pararoseus by High-Performance Liquid Chromatography–Diode Array Detection–Mass Spectrometry and Preparation by Column Chromatography

Qiuyu SHI,* Heya WANG,* Chao DU,* Weiguo ZHANG,** and He QIAN*†

*School of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China **Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China

The major (β-, γ-carotene, torulene, and torularhodin) were determined by high-performance liquid chromatography, with torulene present in the largest amount (167.0 μg/g), followed by torularhodin (113.4 μg/g), β-carotene (52.1 μg/g) and γ-carotene (15.4 μg/g). In addition, cis/trans torulene isomers were further identified by developing an HPLC-DAD coupled with an atmospheric-pressure chemical ionization (APCI) MS method, following isolation and purification torulene from crude pigments by column chromatography. A total of 8 torulene geometrical

isomers were resolved within 60 min by employing a YMC C30 column and a binary gradient mobile phase consisting of methanol–methyl tert–butyl ether–water, (50:47.5:2.5, v/v/v) (A) and methanol–methyl tert–butyl ether–water, (8:90:2, v/v/v) (B). Geometrical isomers behave differently with respect to bioavailability; therefore, it is of great importance to expand our knowledge on their biological roles to determine the appropriate method to separate torulene cis/trans isomers.

Keywords Carotenoids, cis/trans, geometrical isomers, Sporidiobolus pararoseus, torulene

(Received July 8, 2013; Accepted August 21, 2013; Published October 10, 2013)

pararoseus, has not been well-studied with respect to its possible Introduction bioactivity and nutritional purposes, most likely because it has so far not been found in food.12 As shown in Fig. 1, it has 13 Carotenoids are a class of natural pigments widely distributed in conjugated double bonds, and is assumed to have higher plants, fruits and several microorganisms including algae, antioxidant activity that is associated with the number of bacteria, and yeasts of the genera Rhodotorula,1,2 conjugated double bonds in contrast to β-carotene (11 conjugated Rhodosporidium,3 Sporidiobolus, Sporobolomyces4 and Phaffia,5 double bonds).13 In addition, Galano et al. reported that torulene ranging from their original evolutionary role as light-quenching possesses better free-radical scavenging activity than β-carotene pigments to antioxidants,6 precursors of vitamin A, colorants towards the OOH radical,14 exhibits even higher reactivity in and possible tumor-inhibitors7–9 for the food, pharmaceutical aqueous solutions, and has more efficient electron-transfer and chemical industries. The security and negative assessment reactions than , which has been recognized as being of synthetic food dyes by the modern consumer, have given rise one of the most reactive carotenoids towards free radicals.15,16 to a strong interest in natural coloring alternatives.10 Compared Individual carotenoids are capable of forming different with the extraction of carotenoid pigments from plants and cis/trans geometrical isomers; they predominantly occur in their vegetables, the microbial production of carotenoids can offset all-trans configuration, the thermodynamically more stable problems of seasonal and geographic variability, and is a more form.17,18 The geometrical configuration is known to effect the suitable choice for industrial applications because of the biochemistry of carotenoids in certa in situ ations. For example, economic advantages of microbial processes using natural several studies have suggested that cis-isomers of lycopene are low-cost substrates as carbohydrate sources.11 better absorbed than the all-trans form because of the shorter Sporidiobolus pararoseus belongs to the class Urediniomycetes, length of the cis-isomers, and the greater solubility of cis-isomers which can biosynthesize intracellular carotenoids in abundance. in mixed micelles. This results from the lower tendency to These contain four major pigments: β-carotene, γ-carotene, aggregate.19 It indicates that carotenoid cis-isomers may possess torulene, and torularhodin. All of these fulfill the requirement good bioavailability profiles. The appropriate methodology for for pro-vitamin A, an unsubstituted β- ring (Fig. 1). the separation and characterization of cis/trans torulene isomers 20 Torulene, one of the most principal carotenoid in Sporidiobolus is potentially of great importance. Therefore, a C30 column with gradient mobile phases that have better resolution than a † To whom correspondence should be addressed. C18 column is often adopted for the simultaneous separation of E-mail: [email protected] torulene geometrical isomers.21 998 ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29

pooled, and water was added to wash free of acetone. The crude pigments were concentrated in a rotary evaporator and stored at –20°C under a nitrogen atmosphere to remove excess lipids.

Isolation and purification of torulene After evaporating the solvent by the aforementioned protocol, the extracts were redissolved in n-hexane/acetone (10:1, v/v), and then applied in a sealed glass column (35 × 500 mm) with silica gel (150 g, 100 – 200 mesh) that was slurry-packed into n-hexane. The extract was eluted with n-hexane, which was gradually enriched with acetone. Fraction 1 was eluted with n-hexane, and further fractioned with silica-gel chromatography (200 – 300 mesh) by eluting repeatedly with light-petroleum (boiling point range, 30 – 60°C) until β-carotene, γ-carotene, and torulene were well separated from each other. Unidentified Fractions 2 and 4 were eluted with n-hexane/acetone (10:1, v/v) and acetone, respectively, while Fraction 3 (torularhodin) was obtained by elution with n-hexane/acetone (2:1, v/v).25 The primary evaluation of crude pigments was carried out by TLC Fig. 1 All-trans chemical structures of the major four pigments in with a mobile phase of a mixture of n-hexane and acetone (50:1, yeast of Sporidiobolus pararoseus. v/v). The torulene fraction was collected and flushed with nitrogen gas, after which it was eluted with light petroleum through a silica-gel column repeatedly until TLC plates yielded a single band of torulene. All of the preparative procedures The objective of this study was to develop a simple were carried out in dim lighting in order to minimize degradation HPLC-DAD-APCI-MS method to separate and identify cis/trans and isomerization. The torulene solution was concentrated torulene isomers for nutritional studies after purification and under a vacuum, dried with nitrogen gas, and stored at –20°C preparation by silica column chromatography in Sporidiobolus containing 0.1% butylated hydroxytoluene (BHT, w/v) to pararoseus. To the best of our knowledge, this paper is the first prevent oxidation for subsequent HPLC analysis. to report on the comprehensive characteristics of the elution order on a C30 column, the UV-vis spectra and the MS HPLC separation of carotenoids in Sporidiobolus pararoseus fragmentation of torulene geometrical isomers present in equipped with a C18 column microorganisms. HPLC analyses of carotenoids were carried out in a Hitachi Chromaster system (Tokyo, Japan) equipped with CM-5110 quaternary pumps coupled to a Hitachi Model CM-5430 diode Experimental array detector (DAD) and a CM-5210 autosampler. The crude pigments and purified torulene were both separated on an Materials Agilent Zorbax SB-C18 column (5 μm, 250 × 4.6 mm i.d.) under Sporidiobolus pararoseus (JD-2 CCTCC M 2010326) was different conditions. For crude pigments, a mobile-phase system obtained and characterized by our lab. HPLC-grade solvents, was used consisting of solvent A, ACN/water/formic acid including methanol (MeOH), acetonitrile (ACN), and methyl 86:10:4 (v/v/v), and B, ethyl acetate/formic acid 96:4 (v/v). tert–butyl ether (MTBE), were purchased from Tedia Ltd., Gradient elution was conducted as follows: 100% A at the (Fairfield, OH). Deionized water was obtained from Milli-Q beginning to 100% B within 25 min and then changed to 100% Water Purification Systems (Millipore, Bedford, MA). A in 5 min, as recommended by Davoli et al.26 with some Analytical-grade n-hexane, light-petroleum (boiling point range, modifications; however, ACN/THF (60:40, v/v) was used to 30 – 60°C), dimethyl sulfoxide (DMSO), acetone, ethyl acetate, separate purified torulene. The UV-visible spectrum was tetrahydrofuran (THF), and formic acid were all from Sinopharm obtained for wavelengths ranging from 200 to 600 nm. Chemical Reagent Co., Ltd. (Shanghai, China). Standards of The four major carotenoids in Sporidiobolus pararoseus were all-trans-β-carotene and lycopene were obtained from Sigma quantified by HPLC using external calibration curves of Chemicals (St. Louis, MO). β-carotene due to the unavailability of commercial standards of γ-carotene, torulene, torularhodin and similarity in structure and Preparation of crude carotenoid pigments extinction coefficient between β-carotene and γ-carotene, The cultivation of Sporidiobolus pararoseus was according to torulene and torularhodin.27 The limits of detection (LODs) and routine procedures followed by Han et al.22 All procedures quantification (LOQs) for β-carotene commerical standard was were performed in dim lighting in order to prevent analyte determined by signal-nosie ratios (S/N) of 3 and 10, respectively.6 decomposition. Four-day old cells were harvested by centrifugation (12000 rpm, 20 min, 4°C), washed with distilled HPLC analyses of torulene geometrical isomers equipped with a water, and resuspended in an equal volume of DMSO in a C30 column shaking bath to increase the extraction efficiency at room Purified torulene was put on a YMC C30 column (5 μm, temperature. The colored solution was separated by 250 × 4.6 mm i.d.) to separate its cis/trans isomers. Mobile centrifugation (8000 rpm, 10 min, 4°C) and collected. This phase A was composed of MeOH, MTBE and water with the treatment was repeated for several times.4 Acetone was then following ratio: 50:47.5:2.5 (v/v/v). Mobile phase B contained added, and the mixture was vortexed and centrifuged (8000 rpm, 8% MeOH, 90% MTBE, and 2% water (v/v/v). The gradient 10 min, 4°C). This procedure was repeated until the cells procedure was as follows: 100% A isocratic (25 min), 100 – 85% became almost discolored.23,24 The pigmented layer was then A (10 min), 85 – 45% A (20 min), 45% A isocratic (5 min), and ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29 999

12,28 45 – 100% A (2 min). The column temperature was operated at features, retention time, and TLC RF values. The four main 25°C with a flow rate of 0.8 mL/min. The HPLC separation peaks all showed typical three-peak-carotenoid profiles from efficiency was estimated based on the resolution (Rs) and peak DAD spectra (Table 1). The two neighboring less-polar yellow purities. The LODs and LOQs for all-trans-β-carotene separated carotenoids with RF values of 0.98 and 0.88 on TLC plates on the C30 column was also confirmed. All-trans-torulene was absorbed maximally at 425, 452, 478 and 430, 461, 489 nm. quantified by the standard curve of all-trans-β-carotene under This were tentatively assigned as β- and γ-carotene, respectively. the above-mentioned condition. Because commercial The reddish-pink band with an RF of 0.72, together with a red all-cis-forms of torulene were not available, the cis isomers polar pigment that was located in the origin position of the TLC were quantified based on a curve of the corresponding all-trans plates displayed three well-defined peaks at 460, 485, 518 and torulene. 471, 495, 524 nm, respectively. These were tentatively determined as torulene and torularhodin, conforming to that Mass spectrometry previously reported by Davoli et al.26 In order to unambiguously confirm the identity of the cis/trans The LODs and LOQs were 3 and 10 ng for all-trans-β-carotene. torulene isomers, the analysis of mass spectra is necessary. The obtained linear-regression equation was Y = 342407X – 106 Experiments were carried out on an LCQ Deca XP Max mass for all-trans-β-carotene (1 – 100 μg/mL, R2 = 0.9997). Because spectrometer (Finnigan, USA) equipped with a multi-mode ion of the variation of the carotenoids among different samples, and source (electrospray ionization (ESI) and APCI). The APCI due to the unavailability of an authentic reference compound source heater temperature was set to 450°C, while the capillary suitable for quantitation purposes, their contents were quantitated temperature was held at 150°C with the APCI positive-ion solely using all-trans-β-carotene, providing an overview of their mode. Other MS parameters were set as follows: current contents. Thus, the contents of four main carotenoids were corona, 6 μA; sheath flow rate, 30 (arbitrary units); sweep flow approximately quantified, as presented in Table 1. The results rate, 4 (arbitrary units). showed that torulene had significantly higher amount than the other three carotenoid pigments, followed by torularhodin, β-carotene and γ-carotene. Results and Discussion HPLC (C18) analysis of purified torulene by column chromatography Qualitative and quantitative composition of crude carotenoid Following the purification and preparation torulene by column pigments chromatography, it was identified by adding one drop of a Crude pigments obtained from Sporidiobolus pararoseus were 0.1 mol L–1 HCl solution (in methanol) to test the presence of well separated through a C18 column within 30 min. This epoxycarotenoids before further HPLC analysis. As a result, the produced a total of four major peaks, as described in Fig. 2, color of the carotenoid solution did not change, which indicated which was coherent with the behavior on TLC plates, displaying the absence of an epoxy group.29 In order to make further four distinguishable bands.23 Preliminary identifications of efforts to validate our own purified torulene by silica-gel carotenoids are mainly based on their UV-visible spectroscopic chromatography, it was contrasted to its counterparts of available commercial standards of β-carotene and lycopene due to the lack of an authentic torulene standard. The retention time and spectroscopic features of the above-mentioned three pigments were compared to each other. Spectroscopic properties were used by early chromatographers to distinguish between the pigments, and as a means to gain insight into possible structures. Torulene differed from β-carotene in the retention time (Fig. 3) and UV-vis spectrum (Table 1), but it was similar to lycopene in

retention time. However, torulene, which had λmax values of 460, 485, and 518 nm showed distinctive UV-visible characteristics from lycopene, which exhibited absorption maxima at wavelengths of 450, 475, and 507 nm. Moreover, there was an obvious cis peak of torulene around 380 nm based on the UV-vis absorption spectra (Table 1). Some of the minor HPLC peaks observed in the chromatogram (Fig. 3), except for the main peak of torulene, were believed to be cis-isomers of torulene according to the relative shifts of their absorption 30 Fig. 2 HPLC profile of total carotenoids extract from Sporidiobolus maxima, and compared with data previously reported. Thus, it pararoseus. (1) Torularhodin, (2) torulene, (3) γ-carotene, (4) is necessary to develop a valid method to verify the presence of β-carotene. cis-torulene isomers.

Table 1 Tentative identification data and contents of the main four carotenoids of Sporidiobolus pararoseus

a b –1 c Peak Assigned carotenoid tR/min λmax/nm λmax/nm Cis-peak/nm Content/μg g dry mass cell

1 Torularhodin 17.9 471, 495, 525 470, 495, 524 393 113.4 ± 8.4 2 Torulene 21.6 462, 484, 516 460, 485, 518 381 167.0 ± 9.8 3 γ-Carotene 22.2 434, 461, 490 430, 461, 489 351 15.4 ± 1.2 4 β-Carotene 23.1 426, 452, 478 425, 452, 478 338 52.1 ± 3.7 a. Obtained in the present study. b. Reported by Roland W. S. Weber et al.27 c. Average of duplicate analyses ± standard deviation. 1000 ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29

Table 2 Retention time, resolution (Rs), peak purity and estimated content of torulene geometrical isomers in Fig. 4

Retention Peak purity, Content/ Peak Compound R a time/min s % μg g–1 c

1 Torulene isomer 1 17.4 1.0 98.4 2.7 ± 0.4 2 Torulene isomer 2 18.5 1.1 (1,2)b 96.1 2.0 ± 0.2 3 Torulene isomer 3 22.9 1.4 (2,3) 88.5 8.8 ± 0.9 4 Torulene isomer 4 24.6 1.1 (3,4) 98.9 10.7 ± 1.5 5 Torulene isomer 5 27.1 1.5 (4,5) 90.6 4.4 ± 0.8 6 Torulene isomer 6 37.7 9.5 (5,6) 91.3 1.8 ± 0.5 7 Torulene isomer 7 39.1 1.0 (6,7) 93.1 26.2 ± 2.7 8 Torulene isomer 8 54.6 1.4 (7,8) 98.8 110.2 ± 3.3

a. Rs = 2 (tR2 – tR1)/(w1 + w2), whereas wn = band width of an analyte at the baseline.6 b. Numbers in parentheses represent values between two neighboring peaks. Fig. 3 HPLC chromatograms of torulene purified by a silica-gel c. Average of duplicate analyses ± standard deviation. column (1), standards of all-trans-lycopene (2) and β-carotene (3).

strength and the selectivity of the mobile phase to sample components were properly controlled. Our identifications of torulene isomers were generally based on combined information obtained from their chromatographic

behavior on a C30 column, UV-visible, and mass-spectrometric characteristics33 (Tables 2 and 3). However, it is well known that carotenoids are nonpolar compounds; thus, their ionization for mass spectrometry is rather difficult. APCI-MS was taken into account because carotenoids lack a site for protonation detection by ESI.34,35 As a result, a total of 8 torulene geometrical isomers that had the protonated molecular ion [M+H]+ at m/z

535, consistent with C40H54, and in common except for Peak 6 (not detected) among purified torulene, were tentatively confirmed by mass spectrometry. At the same time, Peaks 1, 5 and 7 showed similar mass spectra, producing a fragment ion at m/z 443 [M+H–92]+ ([M–92]+ 442) due to the loss of toluene, Fig. 4 HPLC chromatogram of cis/trans isomers of torulene while peaks 2, 3, 4, 7 and 8 were further identified by the separated on a YMC C30 column. See the text above for detailed consecutive loss of xylene at m/z 429 [M+H–106]+ ([M–106]+ chromatographic condition in the method of HPLC analysis. Peak 428) from the polyene chain, verified in Table 3, which are the identification and characterization are given in Table 3, as below. general characteristic fragments of carotenoids.32,35 Absorption spectroscopy is still the major technique to identify and quantitate carotenoids. In the UV region, an absorption peak referred to as the cis peak is apparent for cis isomers, with the Determination of torulene cis/trans isomers by HPLC intensity of this peak varying with the position and number of 20,36 (C30)-DAD-APCI-MS cis double bonds. The cis peaks of Z-torulene isomers It has been well established that polymeric C30 phases exhibit appeared at around 375 – 380 nm (Table 3), being similar to 37 superior shape selectivity in comparison to C18 reverse phases in those reported in the literature. A distinction between mono-Z separating various carotenoids and their geometrical isomers.31 and di-Z carotenoid isomers can be made by considering the Consequently, our own purified standard of torulene was carried shift in their absorption maxima relative to those of the all-E out with a C30 column for further separation. A simple HPLC isomers, which is normally around 2 – 12 nm in the case of the method that could resolve cis- and trans-α and β- in former.24,35 In this sense, peaks 1 and 2 (Fig. 4), which exhibited Taiwanese sweet potatoes, developed by Liu et al., was used to a characteristic UV-visible spectrum, with a hypsochromic shift separate the various torulene cis/trans isomers.6 Although there ranging from 12 to 20 nm, together with their “cis peaks” is resemblance in chemical structure among α-, β-carotene and appearing near the region of ultraviolet around 375 – 376 nm torulene, the process slowed the overlap of several peaks, but compared to peak 8, the one corresponding to the all-trans failed to well separate various isomers of torulene (data not configuration of torulene on the basis of the absence of cis peak shown), as a result of the strong solvent strength and weak in the near UV/vis spectrum and the location of its absorption polarity of the mobile phase. After various studies, the optimized maxima in comparison to those of the remaining isomers,12 were HPLC condition (the Experimental section) was developed to tentatively assumed to be di-cis-torulene isomers.27 Peaks 3 to separate 8 geometrical isomers of torulene within 60 min 7 were all tentatively identified as being mono-cis isomers, (Fig. 1), with the retention times ranging from 17.4 to 54.6 min, mainly considering the UV-visible spectral characteristics, the resolution (Rs) from 1.0 to 9.5 and the peak purity from 88.5 which indicated the appearance of typical cis peaks at about to 98.9% (Table 2).29,32 Most peaks of the torulene isomers 376 – 380 nm, and displayed a series of hypsochromic shifts of were adequately resolved (Fig. 4), implying that both the solvent λmax values from 3 to12 nm in contrast to the all-trans torulene. ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29 1001

Table 3 Identification data for all-trans and cis isomers of torulene in Fig. 4

Peak Assigned torulene isomer Cis-peak/nm Absorption maxima/nm DB/DII (Q-ratio) m/z found

1 di-Z-Torulene isomer 1 376 450, 473, 501 0.38 535.3 [M+H] 442.3 [M–92] 2 di-Z-Torulene isomer 2 375 450, 472, 503 0.21 535.3 [M+H] 429.1 [M+H–106] 3 mono-Z-Torulene isomer 1 378 457, 479, 511 0.37 535.3 [M+H] 429.3 [M+H–106] 4 mono-Z-Torulene isomer 2 380 454, 477, 508 0.28 535.3 [M+H] 429.2 [M+H–106] 5 mono-Z-Torulene isomer 3 377 450, 476, 506 0.30 535.3 [M+H] 443.2 [M+H–92] 6 mono-Z-Torulene isomer 4 376 4520, 477, 510 0.14 — 7 mono-Z-Torulene isomer 5 378 450, 481, 513 0.24 535.3 [M+H] 442.3 [M–92] 428.2 [M–106] 8 All-E-Torulene — 462, 486, 521 — 535.3 [M+H] 534.4 [M] 429.3 [M+H–106]

In principle, each carbon-carbon double bond in the polyene chain of carotenoids can undergo geometric isomerization. Conclusions Some double bonds are, however, sterically hindered because the cis configuration would lead to a sterical hindrance between Our study used HPLC-DAD to confirm that Sporidiobolus the hydrogen and the methyl group of the polyene chain, as is pararoseus was rich in four main carotenoids; namely, torulene, the case of the C-7, 8 and C-11, 12 double bonds. Thus, the torularhodin, γ-carotene and β-carotene, which can all act as commonly encountered cis-isomers of β-carotene are the 15-cis, vitamin A precursors. The synthesis of different natural 13-cis and 9-cis isomers. Torulene is unsymmetrical, having commercially important carotenoids, as mentioned above, by two different end-groups, but 13 conjugated double bonds; thus, Sporidiobolus pararoseus has led us to consider it for potential more torulene cis isomers are formed. It is well known that the utility of carotenoid-synthesizing applied in nutraceuticals for intensity of the cis peak is greater since the cis bond is nearer to cosmetic and pharmaceutical purposes.38 Moreover, an the centre of the molecule. For that purpose, the ratio of the HPLC-DAD-APCI-MS method was developed to separate 8 absorbance of the second absorption band in the visible region, torulene cis/trans isomers within 60 min by employing a C30 known as the Q ratio or DB/DII, was calculated as a useful tool column and gradient mobile phases. This method may be for identification. At present, no attempts to locate the position applied to analyze other carotenoids and their geometrical of the torulene cis double bonds were made due to a lack of isomers in different species of yeasts. supporting data; therefore, it is more challenging to annotate cis/trans isomers of torulene on the basis of their spectroscopic characteristics.24 We could deduce the presence of 3-cis-, 9-cis-, Acknowledgements 13′-cis-, 9′-cis-, 5′-cis-torulene according to the chemical structure of all-trans-torulene; however, the exact positions of This work was supported by grant No. 2012BAD36B02 from cis double bonds can not be further confirmed by HPLC-MS, the Technical Study and Products Development of Ecological except for NMR spectroscopy, which makes the authentic and High-value Utilization of Regional Speciality Resources identification of every configuration of carotenoids stereoisomers Program. possible.18,36 Carotenoids are susceptible to geometrical isomerization, because their double bonds are sensitive to external conditions, References such as light and heat, but the relative content of the 8 torulene geometrical isomers in Sporidiobolus pararoseus were 1. F. M. Squina and A. Z. Mercadante, Braz. J. Pharm. Sci., comparatively stable. Also, the proportion of the all-trans 2003, 39, 310. isomer among torulene geometrical isomers accounted for 2. H. Sakaki, H. Nochide, S. Komemushi, and W. Miki, J. 64.8 ± 2.3% (average of duplicate analyses ± standard Biosci. Bioeng., 2002, 93, 338. deviation), existing as the dominant configuration of torulene 3. S. Sperstad, B. F. Lutnæs, S. K. Stormo, S. L. Jensen, and stereoisomers in Sporidiobolus pararoseus during the whole B. Landfald, J. Ind. Microbiol. Biotechnol., 2006, 33, 269. process of extraction, purification and HPLC-MS analysis after 4. P. Buzzini, M. Innocenti, B. Turchetti, D. Libkind, M. V. adding BHT to minimize torulene decomposition and Broock, and N. Mulinacci, Can. J. Microbiol., 2007, 53, isomerization. The LODs and LOQs were 2.3 and 7.7 ng for 1024. the all-trans-β-carotene performed with a C30 column. The 5. A. F. Xiao, H. Ni, H. N. Cai, L. J. Li, W. J. Su, and Q. M. obtained linear-regression equation was Y = 302060X – 1.3 × 106 Yang, World J. Microbiol. Biotecnol., 2009, 25, 2029. (0.5 – 80 μg/mL, R2 = 0.9992). The estimated content of 6. S. C. Liu, J. T. Lin, and D. J. Yang, Food Chem., 2009, 116, cis-torulene isomers ranged from 1.8 to 110.2 μg/g, as presented 605. in Table 2. 7. R. G. Ziegler, Am. J. Clin. Nutr., 1991, 53, 251S. 8. S. Carpentier, M. Knaus, and M. Suh, Crit. Rev. Food Sci., 1002 ANALYTICAL SCIENCES OCTOBER 2013, VOL. 29

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