DSpace at VNU: Semi-preparative HPLC separation followed by HPLC UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed

Accepted Manuscript Title: Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed Author: Santi Kongmany Truong Thi Hoa Le Thi Ngoc Hanh Kiyoshi Imamura Yasuaki Maeda Luu Van Boi PII: DOI: Reference: S1570-0232(16)31046-7 http://dx.doi.org/doi:10.1016/j.jchromb.2016.10.016 CHROMB 20298 To appear in: Journal of Chromatography B Received date: Revised date: Accepted date: 19-6-2016 10-10-2016 14-10-2016 Please cite this article as: Santi Kongmany, Truong Thi Hoa, Le Thi Ngoc Hanh, Kiyoshi Imamura, Yasuaki Maeda, Luu Van Boi, Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.10.016 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed Santi Kongmanya, Truong Thi Hoab, Le Thi Ngoc Hanhc, Kiyoshi Imamurad,*, Yasuaki Maedad and Luu Van Boie a Department of Chemistry, Faculty of Natural Science, National University of Laos, Dongdok Campus, P.O Box 7322, Xaythany District, Vientiane, Laos b Danang Environmental Technology Center, Institute of Environmental Technology, Vietnam Academy of Science and Technology, Tran Dai Nghia Road, Ngu Hanh Son District, Danang, Vietnam c Graduate School of Engineering, Osaka Prefecture University, 1-2 Gaken-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan d Research Organization for University-Community Collaborations, Osaka Prefecture University, 1-2 Gaken-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan e Faculty of Chemistry, Vietnam National University, Hanoi, 19 Le Thanh Tong St., Hanoi, Vietnam * Corresponding author: +81-72-254-9863 Email: k_imamura@riast.osakafu-u.ac.jp Highlights  A group of phorbol esters (PEs) was extracted with MeOH from JCL seeds  More than seven isomers of phorbol esters with mw of 710 were identified  All of them were tigliane-type of diterpens with isomeric dicarboxylic moiety  Main five PEs are isolated using semi-preparative HPLC analysis  They were assigned as components of Jatropha factors as cited in the references Abstract Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components contained in the PEs The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C1, C2, C3, and C4/5, but J6 was a mixture of Jatropha factor C6 and its isomer based on the data of UV and LC-MS/MS, and J2 was identified using 1H-NMR analysis By characterization using LC-MS/MS analysis, all components of a series of PEs were elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380 Keywords: preparative HPLC, reverse phase ODS-3 column, Jatropha curcas seeds, phorbol ester isomers, tandem mass spectrometry characterization, 1H NMR spectrometry Introduction The non-edible seed oil from Jatropha curcas Linnaeus (JCL) has recently been reported as a promising feedstock for the production of biodiesel [1,2] JCL is a flowering plant belonging to the Euphorbiaceae plant family that is typically grown in tropical and sub-tropical regions, where it is cultivated in free-draining sands and loams with no water logging [3] JCL is native to Mexico and Central America, and was introduced to Africa and Asia by Portuguese sailors in the 16th century [4] JCL seeds contain about 34% (w/w) of oil, which is lower than that of sun flower seeds (50%) or rapeseed (40–48%), but higher than that of soybean (18%) [5] However, JCL seed oil is non-edible because it contains numerous toxic phytochemicals, with PEs being identified as the main toxic compounds [6] PEs have been reported to exhibit a wide range of interesting biological activities, including the hyper-activation of protein kinase C (PKC), which can lead to abnormal signal transduction and negative responses, including tumorigenesis, skin irritation, inflammation, platelet aggregation and cell differentiation [7] PEs have also been reported to exhibit molluscicidal, fungicidal and insecticidal activities, indicating that the extract of JCL seed oil could be used as a bio-control agent in agrochemical applications following its detoxification [8-10] Furthermore, a PE was used as an intermediate in the synthesis of the promising anti-HIV compound, prostratin [11] The concentrations of the different PEs in the seed kernels of JCL can vary from 0.87–3.32 mg/g, depending on the area of cultivation [6,12,13] The utilization of PEs as value-added by-products could be essential for promoting the production of biodiesel from JCL In 1988, Hirota et al [14] reported the isolation and structural characterization of a 12-deoxy-16-hydroxyphorbol diester from the seed oil of JCL, which was subsequently assigned as Jatropha factor C1, as shown in Fig In 2002, four additional components of 12-deoxy-16-hydroxyphorbol diester were isolated and named as Jatropha factors C2, C3, C4/5 (epimeric isomers) and C6 [15] From a structural perspective, PEs are essentially diterpenes consisting of a tetracyclic carbon skeleton (A, B, C and D), which are also known as tigliane-type diterpenes The carbon skeleton of PEs contains two hydroxyl groups at the C13 and C16 positions, which are combined with a dicarboxylic acid In light of the large number of potential variations of the carboxylic acid group, many different isomers could be formed, although only six components of the 12-deoxy-16-hydroxyphorbol diesters have been isolated and identified to date High performance liquid chromatography (HPLC) over a C18 (ODS) reversed-phase column with UV absorbance at 280 nm has been used for the quantitative analysis of the PEs in JCL with phorbol 12-myristate 13-acetate (TPA) as an internal reference standard [16-19, 26] The PEs found in the seeds and leaves of JCL, as well as those found in numerous animal tissues, were recently separated using a modified ODS column This method allowed for the PEs to be separated into five peaks, which were subsequently quantified by HPLC tandem mass spectrometry [12, 13] Preparative HPLC has been used for the purification of PEs using a normal phase column [19] This technique has also been applied for the purification and isolation of specific compounds from JCL extracts using sequentially by a C18 reversed phase column, followed by a normal phase silica gel column [15] The use of HPLC in conjunction with tandem mass spectrometry is a powerful and highly specific analytical tool for the identification of the individual components found in plant extracts [20, 22, 24, 25] MS/MS spectra can also provide useful information pertaining to the fragmentation of the individual components, which can be used to search for novel metabolites with similar structural features/core scaffolds This level of detail can be particularly useful for determining the presence of related components in plant materials and investigating the phytochemical degradation, metabolism and biosynthesis in a quantitative and qualitative manner [12, 13] The purpose of the present study is to elucidate the structural feature of a series of PEs extracted from Jatropha curcas oil with MeOH Using HPLC/PDA analysis, a series of PEs of MeOH extract was separated into more than seven components of PE A reversed phase semi-preparative HPLC analysis of an ODS-3 column was revised for the isolation of five peaks of components (J2, J3, J5, J6 and J7) from the extracts, and their structures was assigned on the basis of the UV, LC-MS/MS and 1H NMR analysis The characterization using by LC-MS/MS analysis was conducted to elucidate the structural specification of a series of PEs consist of more than seven components Experimental 2.1 Materials The JCL seeds used in this study were cultivated in Trang Bang, Vietnam MeOH, n-hexane, chloroform and ethyl acetate were all purchased in the analytical grade from Wako Pure Chemical Industry (Kyoto, Japan) Acetonitrile (HPLC analytical grade), anhydrous sodium sulfate (analytical grade), activated silica gel (Wakogel®C-200, grade: column chromatography analysis), and deuterated dichloromethane (NMR spectral grade) and phorbol 12-myristate 13-acetate (TPA) were also purchased from Wako Pure Chemical Industry The purified water used in the current study was produced in Osaka Prefecture University (Sakai, Japan) 2.2 Extraction and purification of PEs The JCL seeds were manually cracked using a pair of pliers and the kernels were separated from the seeds The seed kernels were subsequently homogenized using a grinder A sample of the homogenized material (116 g) was suspended in MeOH (150 mL) and the resulting mixture was extracted under ultrasonic irradiation for 30 using an ASU-10 ultrasonic apparatus (As One, Tokyo, Japan) The mixture was then allowed to stand until the MeOH layer was clearly separated from the residue and the supernatant was transferred to a L Erlenmeyer flask The residue was then extracted times with MeOH (100 mL), and the combined extracts were filtered and concentrated under vacuum at 40 °C to approximately 50 mL The volume of the MeOH extract was adjusted to 100 mL with MeOH and transferred to a 250 mL separating funnel The lower oily layer of the mixture was removed, and the remaining MeOH layer was extracted three times with 50 mL of MeOH-saturated hexane The lower MeOH layer was then evaporated to dryness under vacuum at 40 °C to give an oily residue, which was dissolved in ethyl acetate (30 mL) and transferred to a 100 mL separating funnel Water (10 mL) was then added to the separating funnel, and the resulting mixture was vigorously shaken for a few before being separated into two phases by centrifugation The ethyl acetate layer was subjected to washing process two times to give an ethyl acetate extract containing PEs, which is referred to hereafter as crude PEs 2.3 Clean-up using column chromatography The oily residue (3.1 g) obtained from the MeOH extraction process was fractionated by column chromatography The oily residue was previously coated on a powder of anhydrous Na2SO4 (4–5 g) by dissolving in MeOH and then removing the solvent using a rotary evaporator under a reduced pressure A silica gel column was prepared by packing 17 g of silica gel (C-200), which had been activated overnight at 130 °C, into a glass column (30 × 1.5 cm i.d., Pyrex glass, glass fritted at the bottom, with a Teflon stop cock) as a slurry in n-hexane The top of the column was then packed with the Na2SO4 coated with the oily residue The column was then eluted sequentially with the following solvents at a flow rate of 2.5 mL/min: 20 mL of n-hexane, 100 mL of n-hexane/chloroform (50/50, v/v), 100 mL of chloroform, 100 mL of MeOH/chloroform (5/95, v/v), 100 mL of MeOH/chloroform (35/65, v/v) and 50 mL of MeOH The eluents were fractionated into 200 drops/tube (about mL) using an SF2120 fraction collector (ADVANTEC, Tokyo, Japan) The fractionation process was initiated from chloroform (100%) and continued until MeOH (100%) The target PE compounds were collected in fractions 52–63, corresponding to the use of 5% MeOH in chloroform as the eluent The PE-containing fractions were subsequently combined and concentrated to give residues, which were purified by preparative HPLC and analyzed by LCMS/MS 2.4 Isolation of PEs using semi-preparative HPLC The individual PEs were isolated from the PE-containing fractions described above by preparative HPLC using a GL-7480 HPLC system (GL Science Inc., Tokyo, Japan) equipped with a PDA detector (GL-7452, GL Science Inc.), an auto-sampler (GL-7420, GL Science Inc.) and a column oven (GL-7430, GL Science Inc.) The HPLC system was fitted with a preparative Inertsil® ODS-3 column (modified C18, m, 7.6  250 mm, GL Science Inc.), which was operated at 40 C with a flow rate of mL/min Water (A) and acetonitrile (B) were used as the mobile phases, and the system was subjected to the following gradient elution process: 50% B (0 min), 80% B (0–10 min), 80% B (10–25 min), 100% B (25–30 min), 100% B (30–45 min), 50% B (45–50 min) and 50% B (50–65 min) An 80 l aliquot of sample was repeatedly injected onto the system The eluted compounds were monitored at wavelengths of 220, 250 and 280 nm, and the eluents themselves were collected using a CHF122SC fraction collector (ADVANTEC, Tokyo, Japan), which was operated in the timer mode (i.e., collecting the eluent in a tube every 20 seconds) The fraction collector was connected to an in-line HPLC analysis system and the fractionation process was initiated 10 after the sample injection An 80 l aliquot of PE-containing fractions (1.5 mL) described in section 2.3 was repeatedly purified by preparative HPLC and fractionated into five portions containing J2, J3, J5, J6 and J7 2.5 HPLC/PDA analysis The PE sample solutions were analyzed by high performance liquid chromatography (HPLC) on a Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with a degasser unit (DGU-20A), auto-sampler (SIL-20A), column oven (CTO-20A) and photo diode array (PDA) detector (SPD-M20A) The resulting data were acquired and analyzed using version 1.2 of the Lab Solution software (Shimadzu) Solvent A (10 mM aqueous ammonium acetate) and solvent B (acetonitrile, CH3CN) were used as the mobile phases The HPLC system was fitted with an analytical Inertsil® ODS-3 column (modified C18, 4.6 m, 5.0  150 mm, GL Science Inc., Tokyo, Japan), which was eluted at a flow rate of 0.5 mL/min with the following gradient: 20% A (0–2 min), 18% A (2–2.20 min), 13% A (2.20–13.50 min), 8% A (13.50–18 min), 0% A (18–22 min), 0% A (22–25 min), 20% A (25–25.01 min) and 20% A (32 min) The column temperature was maintained at 35 C and the eluent was scanned at wavelengths in the range of 190–400 nm The PEs eluted from the column were monitored at a wavelength of 280 nm The concentration of (purified) extract used for analysis was 40 mg/L (diluted solution) The concentration of each isolated compound used for analysis was 6.3 mg/L (J2), 15 mg/L (J3), 11 mg/L (J5), 12 mg/L (J6), 17 mg/L (J7) The injection volume was 20 µL 2.6 LC/MS/MS analysis All of the compounds were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an 8030 LCMS system (Shimadzu), which was connected to an in-line HPLC-DAD system through a valve unit (FCV-12HAH) The injection volume was 20 µL This system was used to estimate the molecular weight and structure of the individual components The LC-MS/MS experiments were conducted under the following MS conditions: ionization interface (electrospray ionization (ESI)) voltage, +4.5 kV; nebulizer gas, N2; nebulizer gas flow rate, 2.0 L/min; drying gas, N2; drying gas flow rate, 15 L/min; heat block temperature (HB), 120 C; and desolvation temperature (DL), 250 C The samples were separately analyzed in two different modes, including (i) the precursor-ion-scan mode (product ion, m/z 311; mass range, m/z 300–800; collision energy, −17 V; scan speed, 0.2 sec/scan) and (ii) the production-ion-scan mode (precursor ion, m/z 728; mass range, m/z 200–800; collision energy, −10 V; scan speed, 0.2 sec/scan) The argon gas pressure for the collision was set at 230 kPa 2.7 Characterization by NMR A sample of the J2 component of the PEs (ca mg), which was isolated from the Jatropha seed, was dissolved in 0.7 mL of deuterated dichloromethane (CD 2Cl2) and analyzed by 1H NMR (400 MHz) using a JNM-GX400 FT-NMR spectrometer (JEOL, Tokyo, Japan) Each sample was scanned for more than h and the resulting data were analyzed using the Delta NMR software (ver 5.0.4.4) (JEOL) too low to be analyzed by 1H NMR spectroscopy Furthermore, the observed values for these components in Fig were overestimated as a consequence of using TPA as an external standard during their quantification by HPLC analysis [10] 3.4 Characterization of the PEs by LC-MS/MS Analysis 3.4.1 The ESI precursor ion (m/z 293) scan analysis The purified PEs by column chromatography was subjected to the LC-MS/MS analysis The analytical parameters of the apparatus were optimized using by J2 fraction as a standard described in section 3.2.1 Tigliane-type PEs typically gives two diagnostic ions of the diterpene skeleton with m/z values of 311 and 293 [22] The precursor ion scan chromatogram (m/z 293) and the precursor ion spectra of the different peaks in Fig were analyzed in order to elucidate their quasi-molecular ions (parent ions) The precursor ion spectra of J2, of which the structure was identified by 1H-NMR spectrum was shown in Fig 5-J2 The two highly abundant mass ions, including an ammonia adducted quasi-molecular mass ion, [M+NH4]+ with m/z value of 728 and a fragment ion, [M-OH2+H]+ with m/z value of 693 were obtained, accompanying with much weaker hydrogen adducted molecular ion [M+H]+ with m/z value of 711 The other components of PEs (J1, J3, J4, J5, J6 and J7) also showed the almost similar patterns of the precursor ion spectra with that of J2 These results indicated that all components of PEs were the 12-deoxy-16-hydroxyphorbol diesters with same molecular weight of 710 3.4.2 The ESI product ion (m/z 728) scan analysis The ESI product ion (m/z 728) scan chromatogram and the product ion spectra of the different peaks identified in the chromatogram were shown in Fig The ESI product ion chromatogram contained seven peaks, which were the same as those observed in the HPLC 13 chromatogram (UV 280 nm) shown in Fig 2, except that the peak for J6 was composed of two components The product ion spectrum of J2 (in Fig 7-J2) revealed two highly abundant ions, an adducted molecular ion with m/z value of 728 and a second peak with m/z value of 693 A quasi-molecular ion with m/z value of 711 was also observed The peak with m/z value of 675 was attributed to the loss of two waters from the phorbol ester molecule This means that the phorbol moiety consist of two tertiary hydroxyl groups positioned at C4 and C9 [21] The fragment ion with m/z value of 311 was attributed to [M-R(COOH)2-OH2]+, which are produced by the proton transfer-mediated cleavage of the two carboxylic acid groups with loss of a water from the molecular ion, as shown in Fig The loss of an additional water molecule would then produce the ion of [M-R(COOH)2-2OH2]+ with m/z value of 293 A proton adducted dicarboxylic acid fragment ion with m/z value of 383 [R(CO2H)2+H]+ was observed, which are produced by the proton transfer-mediated cleavage of two carboxylic groups The corresponding water loss ion was observed with m/z value of 365, as well as a fragment ion with m/z value of 337 produced by cleavage of ketone group (CO m/z 28) The fragment responsible for the peak with m/z value of 382, which was equal to the difference in mass between m/z 693 and 311, was determined to be the molecular weight of dicarboxylic acid (C24H32O4, m/z 382) By comparing to the product ion spectra of J2 to those of J1, J3, J4, J5, J6, and J7, the same fragmentation characteristics of these compounds indicated the identical structural feature of phorbol ester Taken together with both of LC-MS/MS and HPLC/PDA analysis, more than seven PEs were observed and seven components (J1, J2, J3, J4, J5, J6, and J7) identified in the current study were the tigliane-type 12-deoxy-16-hydroxyphorbol diesters with the same molecular weight (m/z 710) that were based on different isomers of the 14 dicarboxylic acid moiety (C24H32O4, m/z 382) Conclusion Using the HPLC/PDA analysis, a series of PEs extracted from Jatropha seeds were separated into more than seven components of PE The preparative HPLC over a reversed-phase ODS-3 column was revised to isolate the five main components of J2, J3, J5, J6 and J7, which were individually isolated with chromatographic purities of more than 90% The structures of J2, J3, J5 and J7 were assigned to be Jatropha factor C1, C2, C3, and C4/5, respectively However, J6 was contaminated with the isomeric form consisted of four conjugated double bonds in dicarboxylic acid moiety The subsequent characterization of these materials by UV and LC-MS/MS revealed that they were the PE congeners of an isomeric tigliane-type diterpene consisting of an alkyl chain with two carboxylic acid groups Most notably, they all had the same molecular mass (m/z 710) Acknowledgements The authors greatly appreciate the financial support that they have received for this project from the Science and Technology Research Partnerships for Sustainable Development (SATREP) project 15 References [1] L.E Rincón, J.J Jaramillo, C.A Cardona, Comparison of feedstocks and technologies for biodiesel production : An environmental and 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sunlight and UV irradiation, Doctor thesis at Osaka Prefecture University, February (2015) 18 Legends of Figures Fig Molecular structures of six components of phorbol esters isolated from Jatropha curcas seeds (referred to [15]) (a) Jatropha factor C1, (b) Jatropha factor C2, (c) Jatropha factor C3, (d) Jatropha factor C4/5, and (e) Jatropha factor C6 19 Fig Chromatogram of PEs extracted with MeOH from JCL seed kernel in the retention range from 10 to 22 (a) UV contour view (200–400 nm) (b) HPLC chromatogram (UV 280 nm) 20 Fig HPLC chromatogram (UV 280nm) of five components of phorbol esters isolated J2: tR15.3 (6.3mg/L), J3: tR 16.5 (15 mg/L), J5: tR 17.6 (11 mg/L), J6: tR 18.6 (12 mg/L), J7: tR 19.1 (17 mg/L) The values in parentheses are the concentrations estimated by external standard of TPA 21 Fig UV absorption spectra of components of J1, J2, J3, J4, J5, J6 and J7 22 Fig ESI precursor-ion (m/z 293)-scan chromatogram (a) and the corresponding precursor-ion spectra of identified peaks (b) 23 Fig Schematic interpretation of the fragmentation of J2 molecule and the proton transfer cleavage process of carboxylic group 24 Fig ESI product-ion (m/z 728)-scan chromatogram (a) and the corresponding product-ion spectra of identified 7peaks (b) 25 Legends of Tables Table Comparison of 1H NMR spectrum of the 12-deoxy-16-hydroxyphorbol moiety of J2 with those of Jatropha factor C1 reported by Hirota et al (1988) [14] and Haas et al 2002[15]) Jatropha factor C1 (Hirota et al 1988) (CDCl3, 400 MHz) J2 At carbon Positions*1 (CD2Cl2, 400 MHz) Shift (ppm) Type Shift (ppm) Type 7.56 2.26 2.48 2.41 5.60 2.97 5.11 s s dd d d m s 19.1 19.1 4.9 - 7.59 2.26 2.50 2.44 5.62 3.00 5.24 s s dd d d m s 10 3.23 11 1.97 br s - 3.28 m - 2.00 12 2.14 dd 15.1, 7.1 1.60 dd 15.1, 11.4 13 - - - 14 1.15 d 5.0 15 - - - 16 4.28 d 3.54 d 17 1.19 18 Jatropha factor C1 (Haas et al 2002) (CD2Cl2, 500 MHz) Shift (ppm) Type 19.1 19.1 4.2 - 7.57 2.25 2.48 2.42 5.60 2.97 5.10 s s d d d o s br s - 3.24 br s - m - 1.98 m - 2.17 dd 14.6, 7.7 2.12 dd 15.1, 7.1 1.60 dd 14.6, 11.0 1.60 dd 15.1,11.4 - - - - - - 1.16 d 5.4 1.18 d 6.0 - - - - - - 11.6 4.33 d 11.7 4.30 d 11.8 11.8 3.58 d 11.7 3.57 d 11.8 s - 1.20 s - 1.19 s - 0.86 d 6.3 0.90 d 6.3 0.88 d 6.4 19 1.75 o - 1.78 d 1.6 1.75 o - 20 4.09 d 12.1 4.04 br d 12.3 4.02 d 13.1 3.99 d 8.3 3.99 br d 12.3 3.97 d 13.1 1.59 o - - 1.59 o - J (Hz) J (Hz) J (Hz) 19.2 19.2 4.7 - Note: the symbols “s, br s, d, dd, ddd, dt, t, br t, q, tq, m, o” are the multiplicity type assignment for H atoms in the molecules (s: singlet; br s: broad singlet; d: doublet, dd: doublet-doublet, ddd: doublet-doublet-doublet, t: triplet, dt: doublet-triplet; br t: broad triplet; q:quartet; tq: triplet-quartet; m: multiplet; o: overlap *1 refer to structure a in Fig 26 Table Comparison of 1H NMR spectrum of the dicarboxylic acid moiety of J2 with those of Jatropha factor C1 reported by Hirota et al 1988[14], Haas et al 2002[15] Jatropha factor C1 (Hirota et al 1988) (CDCl3, 400 MHz) J2 (CD2Cl2, 400 MHz) Position*1 1' 2' 2' 3' 4' 5' 6' 7' 8' 9' 10' 10 11' 12' 13' 14' 15' 16' 17' 18' 19' 20' 21' 22' 23' 24' Shift (ppm) Type J (Hz) 2.96 2.95 5.46 5.16 2.49 3.51 5.32 5.99 6.65 5.15 5.07 dd dd ddd dd q dt o t dt d d 17.8, 8.7 18.1, 9.3 15.2, 8.6, 6.2 15.2, 9.0 9.0 9.7., 5.3 3.16 1.76 1.83 1.69 5.66 6.31 6.13 6.16 6.08 5.70 2.06 1.39 0.88 d m m dd dd dd m o m dt q br tq t 11.0 16.7, 10.2 15.7 10.0 9.0 8.2,7.5 14.9, 7.8 14.8, 9.6, 14.5, 7.3 7.3 7.3 7.3 Jatropha factor C1 (Haas et al 2002) (CD2Cl2, 500 MHz) Shift (ppm) Type J (Hz) Shift (ppm ) Type J (Hz) 3.01 2.94 5.44 5.18 2.50 3.53 5.32 6.03 6.64 5.19 5.10 dd dd ddd dd dd td dd t td d d 18.1, 9.3 18.1, 9.3 15.2, 9.3, 5.6 15.2, 8.6 9.0, 4.9 9.7, 4.9 10.3,10.0 11.0 16.8, 10.5 16.8 10.5 3.00 2.92 5.46 5.18 2.52 3.51 5.34 6.02 6.67 5.16 5.09 dd dd ddd dd br q br dt o t dt d d 17.8, 6.1 17.8, 8.7 15.1, 8.7, 6.1 15.1, 9.0 9.0 9.8, 5.3 3.19 1.78 1.88 1.70 5.66 6.32 6.15 6.15 6.05 5.72 2.09 1.42 0.91 d m m dd dd dd m dd m ddd td tq t 9.0 9.2 9.7, 8.2 8.2, 7.5 15.1, 7.6 14.8, 9.6 3.18 1.78 1.85 1.69 5.68 6.33 6.16 6.16 6.08 5.72 2.08 1.42 0.91 d o m dd dd m o o m dt q tq t 15.0, 9.6 14.8, 7.3, 7.3 7.3, 7.6 7.3 7.3 10.4 16.8, 10.4 16.8 10.4 9.0 8.8, 7.8 15.0, 7.8 14.5, 7.3 7.3 7.3 7.3 Note: the symbols “s, br s, d, dd, ddd, dt, t, br t, q, tq, m, o” are the multiplicity type assignment for H atoms in the molecules s (singlet), br s (broad singlet), d (doublet), dd (doublet-doublet), ddd (doublet-doublet-doublet), t (triplet), dt (doublet-triplet), br t (broad triplet), q (quartet), tq (triplet-quartet), m (multiplet), and o (overlap) *1 refer to structure a in Fig 27 .. .Semi-preparative HPLC separation followed by HPLC/ UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed Santi Kongmanya, Truong Thi Hoab,... PE-containing fractions were subsequently combined and concentrated to give residues, which were purified by preparative HPLC and analyzed by LCMS/MS 2.4 Isolation of PEs using semi-preparative HPLC. .. aliquot of PE-containing fractions (1.5 mL) described in section 2.3 was repeatedly purified by preparative HPLC and fractionated into five portions containing J2, J3, J5, J6 and J7 2.5 HPLC/ PDA analysis