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1 49 3 Vol.49, No OCEANOLOGIA ET LIMNOLOGIA SINICA May, * 1, , 3 2 ( ; ; ) (Dinophysis acuminata), (HPLC) -2(Pectenotoxin-2, PTX2) PTX2 HPLC,, 99.2% 81.5% ( ) (Solid Phase Extraction, SPE) (Oasis HLB SPE, Phenomenex Strata-X SPE, Sep-Pak C18 SPE) PTX2, -, 78.2% HPLC PTX2, ph, ( 6.7mmol/L, ph=10.94), PTX2, Phenomenex Kinetex C18(4.8mm 250mm, 5μm),, 93.2% PTX2 PTX2 HPLC,, PTX2-2; ; ; ; O doi: /hyhz ,,, (Hallegraeff et al, 2003; FAO, 2004; Reguera et al, 2014) (Pectenotoxins, PTXs),,, (Okadaic acid, OA) (Dinophysistoxin, DTXs) (Suzuki et al, 2009; Reguera et al, 2014;, 2016) 20 PTXs, -2(Pectenotoxin-2, PTX2) (Reguera et al, 2014; García et al, 2016)( 1) 1984 PTXs Patinopecten yessoensis, (Hallegraeff et al, 2003; Miles et al, 2004; Suzuki et al, 2006; Reguera et al, 2012; Li et al, 2017) PTXs (Miles et al, 2004; Kim et al, 2008), PTXs, OA DTXs 160μg/kg, (ARfD), 400g PTXs 120μg/kg (Alexander et al, 2009) PTXs, PTXs, *, 2016YFC ;, 2016GSF ;, ,, motaxue@163.com :,,, mengmengtong@zju.edu.cn;,,, tanzj@ysfri.ac.cn : , :

2 3 : , PTX2, (Dinophysis spp.), (Dinophysis fortii) (D. acuminata) (D. acuta) (D. caudata) (D. miles) (Suzuki et al, 2009; Reguera et al, 2012; Nielsen et al, 2013; Tong et al, 2015), [ (Mesodinium rubrum) (Geminigera cryophila)] (Park et al, 2006), PTX2, PTX2,,, (Gao et al, 2017), (DAYS01-E6 ), PTX2, HPLC PTX2, - (HPLC-MS/MS), PTX2 1 PTX2 (James et al, 1999) Fig.1 The chemical structure of PTX2 (James et al, 1999) (DAYS01-E6 ) 2014 (Gao et al, 2017), (JAMR) (JATA) (Nagai et al, 2008), 15 C, (~50μmol photons/m 2 s), 14h.. 10h 30mL(~4000cells/mL) 2mL (~200000cells/mL), 30mL f/6-si,, DAYS01-E6 800mL f/2-si, 2 ~8000cells/mL,, 15μm, f/2-si 15mL, 80 C , : (a) : 100 C 10min; (b) :,, 80 C, 3 ; (c) : 15min( 300W, 40kHz); (d) :, ( 325W 25kHz / 5s/10s 20min, 20 C, 120min); (e) : (b) (d),,, C 0, C 1, L=(1 C 1 /C 0 ) 100%, 1.2.2

3 PTX2, : (1) : 80%, (d) 3min (8000r/ min),, (2) :, (d) 3min (8000r/min),, (3) - :,,,,, C, (Miles et al, 2004), : Waters Oasis HLB SPE (60mg/3mL, Waters ), Phenomenex Strata-X SPE (60mg/3mL, Phenomenex ), Waters Sep-Pak C18 SPE (60mg/ 3mL, Waters ), : (4) SPE : SPE 3mL 3mL Milli-Q, 10mL, 1mL,, 0.5mL,, 0.22μm, SPE, PTX2 T 0,,, PTX2, PTX2 T 1, T 2, R=(T 2 T 1 )/T PTX2 Sephadex LH20,, (60cm 1.2cm) (3mL),,, 10mL 1mL HPLC-MS/MS, PTX2 ( mL), 1.3 Empower3 Waters e2695 : Phenomenex Kinetex C18 (4.8mm 250mm, 5μm); : 40 C; : 235nm; : 1mL/min; A: ( 6.7mmol/L ); B: / ( , v/v, 6.7mmol/L ); : min, 20% B; min, 20% 80% B; min, 80% 20% B; min, 20% B PTX2 235nm, 26min, 15 17min, 40 C,, HPLC-MS/MS PTX QTRAP - (ESI ) : Phenomenex XB-C 18 (2.1mm 100mm, 2.6µm); : C 18 (2.1mm 50mm, 5µm); : 40 C; : 0.350mL/min; : 5µL; : A ( 50mmol/L 2mmol/L ), B 95% ( 50mmol/L 2mmol/L ); : 0 7.0min, 20% B; min, 20% 90% B; min, 90% 20% B; min, 20% B : (ESI), (MRM), ; : 20psi; : 5.5kV 4.5kV; : 550 C; 1: 345kPa; 2: 345kPa; 1 PTX2 OA DTX2 DTX1 YTX GYM SPX1, (10 200ng/mL) ; (S/N 3) (LOD), 10 (S/N 10) (LOQ), PTX2 6ng/mL, 19.8ng/mL (HPLC- MS/MS),, PTXs OA DTXs PTXs

4 3 : Tab.1 表 1 11 种脂溶性毒素的质谱分析参数 Transitions and mass spectrometry parameters for 11 marine lipophilic toxins (m/z) (ev) (ev) (v) (okadaic acid, OA) -2 (dinophysistoxin-2, DTX2) -1 (dinophysistoxin-1, DTX1) (yessotoxin, YTX) -2sa (pectenotoxin-2sa, PTX2sa) -2 (pectenotoxin-2, PTX2) -11 (pectenotoxin-11, PTX11) -12 (pectenotoxin-12, PTX12) -1 (pectenotoxin-1, PTX1) (gymnodimine, GYM) (spirolides-1, SPX1) [M H] 803.5/ / [M H] 803.5/ / [M H] 817.5/ / [M H] / / [M H] 875.5/ / [M+NH 4 ] / / [M+NH 4 ] / / [M+NH 4 ] / / [M+NH 4 ] / / [M+H] / / [M+H] / / (Wu et al, 2014), PTX2 (Suzuki et al, 2006; Ito et al, 2008) : QTRAP MIM( ), MIM-ER-EPI, (Turbo Ion Spray),, Q3 Q1, [M+NH 4 ] +, (m/z) Q2, 5V 1ms, 1.3s, m/z 876.5( 2b) 892.5( 2c) ( 2d)(Miles et al, 2004, 2006; Gerssen et al, 2009),, PTX12(874.5) PTX2(876.5) PTX11 PTX1 PTX12sa (892.5) PTX2sa(894.5), m/z 906.5, PTX6 PTX7( 2e) PTXs, IDA-EPI, + PTXs, PTX2sa PTX11 PTX12 PTX1 MRM ( 1);, EPI,,, (DAYS01-E6 ) OA DTX2 DTX1, PTXs PTX2 PTX11, PTX2 ( 5), PTXs (Cuellar-Bermudez et al, 2015), PTX2 3,, 81.51% (n=5), (78.24%) (60.23%) (45.27%)( 3) (21.45%), PTXs (García et al, 2016), PTX2,,

5 MIM (a) (b, c, d, e) Fig.2 Chromatography in MIM scanning mode on total ion (a) and selected ion (b, c, d, e) : b: m/z 876.5; c: m/z 892.5; d: m/z 874.5; e: m/z 906.5, (22.30%), (, 2014), (Halim et al, 2012),, 90.24% 94.24%,, PTX2, (81.51%>78.24%), 99.2% 81.5%, (LPE) (SPE) PTX2, ( 4) 4, PTX2, PTX2, (Miles et al, 2004, 2006; Matsushima et al, 2015; Li et al, 2017), -, 78.2% (n=5), (51.6%), (46.6%),,, PTX2, HPLC-MS/ MS, SPE,,, SPE, (Wu et al, 2014) Phenomenex Strata-X SPE Sep-Pak C18 SPE PTX2 (44%), Oasis HLB SPE (40%),,

3 : -2 573,, -,, PTX2 3 (n=5) Fig.3 Responses of different cell disruption methods on cell disruption rate and toxin extraction efficiency (n=5) : A: ; B: ; C: ; D: ; E: 4 PTX2 (n=5) Fig.

3 HPLC,, (Gerssen et al, 2009), ph OA DTXs,, PTXs, PTX2,,, (ph=2.74): A ( 50mmol/L 2mmol/L ), B 90% ( 50mmol/L 2mmol/L ); (ph 6.07): A ( 2mmol/L ), B 90% ( 2mmol/L ); (ph 10.94): A ( 6.

6 3 : ,, -,, PTX2 3 (n=5) Fig.3 Responses of different cell disruption methods on cell disruption rate and toxin extraction efficiency (n=5) : A: ; B: ; C: ; D: ; E: 4 PTX2 (n=5) Fig.4 PTX2 toxin extraction efficiency from Dinophysis spp by different extraction methods (n=5) : A: ; B: ; C: - ; D-1: Waters Oasis HLB; D-2: Phenomenex Strata-X; D-3: Waters Sep-Pak C HPLC,, (Gerssen et al, 2009), ph OA DTXs,, PTXs, PTX2,,, (ph=2.74): A ( 50mmol/L 2mmol/L ), B 90% ( 50mmol/L 2mmol/L ); (ph 6.07): A ( 2mmol/L ), B 90% ( 2mmol/L ); (ph 10.94): A ( 6.7mmol/L ), B 90% ( 6.7mmol/L ), ( 5a), PTX2, ; ( 5b), PTX2(t R =15.09min) DTX1(t R =15.11min) (, ) PTX2 DTX1, HPLC-MS/MS,, DTX1, PTX2, HPLC-MS/MS,,, DTX1 PTX2, PTX2 DTX1, OA DTXs (t R <7min), PTX2 (t R =15.77min), PTX11 (t R =14.57min) ( 5c), PTX2, (OA+DTX2 DTX1 PTX2 PTX11), PTXs (Gerssen et al, 2009), (García-Altares et al, 2013),, Gerssen (2009) (6.7mmol/L 591mmol/L), PTX2 (Gerssen et al, 2009),, 90% PTX2, (6.7mmol/L) PTX2,, PTX2 ( 6) 2.4 PTX2 PTX2 PTX2

7 HPLC-MS/MS Fig.5 TIC and XIC chromatograms of toxin extractions from Dinophsis.spp. by HPLC-MS/MS in three types of mobile phase : TIC: total ion chromatography, ; XIC: extracted ion chromatography,, ( ) PTX2 ( ) HPLC, PTX2,, 78.5%, - (78.2%), 61.4%±5.5% HPLC-MS/MS PTX2

8 3 : A, HPLC PTX2 (PTX2 ) B, PTX2 HPLC 93.2% (n=5), PTX2 6 HPLC PTX2 Fig.6 HPLC chromatogram of PTX2 2.5 HPLC-MS/MS OA DTX2 DTX1 YTX GYM SPX1, PTX2 PTX2,, PTX2 0.6μg/kg, 1.98μg/kg (n=3) (n=3), DTX2 PTX2 PTX2sa GYM SPX1, PTX2 PTX2, PTX μg/kg μg/kg, 20.19% PTX2 (P<0.05), PTX2 3 PTX2,,, PTX2,, ( C18 ), 93.2% PTX2,, PTX2,,, , 44(3): ,,, , 35(14): Alexander J, Benford D, Boobis A et al, Marine biotoxins in shellfish Summary on regulated marine biotoxins1 Scientific Opinion of the Panel on Contaminants in the Food Chain. The EFSA Journal, 1306(2009), 6 23 Cuellar-Bermudez S P, Aguilar-Hernandez I, Cardenas-Chavez D L et al, Extraction and purification of high-value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins. Microbial Biotechnology, 8(2): FAO (Food and agriculture organization of the United Nations), Marine biotoxins. Rome: FAO, 14 Gao H, An X L, Liu L et al, Characterization of Dinophysis acuminata from the Yellow Sea, China, and its response to different temperatures and Mesodinium prey. Oceanological and Hydrobiological Studies, 46(4): García C, Oyaneder-Terrazas J, Contreras C et al, Determination of the toxic variability of lipophilic biotoxins in marine bivalve and gastropod tissues treated with an industrial canning process. Food Additives & Contaminants: Part A, 33(11): García-Altares M, Diogène J, De La Iglesia P, The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: single-laboratory validation under four chromatographic conditions. Journal of Chromatography A, 1275: Gerssen A, Mulder P P J, McElhinney M A et al, Liquid chromatography tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions. Journal of Chromatography A, 1216(9): Halim R, Harun R, Danquah M K et al, Microalgal cell disruption for biofuel development. Applied Energy, 91(1): Hallegraeff G M, Anderson D M, Cembella A D et al, Manual on Harmful Marine Microalgae. In: Hallegraeff G M, Anderson D M, Cembella A D eds. Monographs on Oceanographic Methodology. Paris: UNESCO, Ito E, Suzuki T, Oshima Y et al, Studies of diarrhetic activity on pectenotoxin-6 in the mouse and rat. Toxicon, 51(4): James K J, Bishop A G, Draisci R et al, Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. Journal of Chromatography A, 844(1 2): Kim M O, Moon D O, Kang S H et al, Pectenotoxin-2

9 represses telomerase activity in human leukemia cells through suppression of htert gene expression and Akt-dependent htert phosphorylation. FEBS Letters, 582(23 24): Li M H, Sun G, Qiu J B et al, Occurrence and variation of lipophilic shellfish toxins in phytoplankton, shellfish and seawater samples from the aquaculture zone in the Yellow Sea, China. Toxicon, 127: 1 10 Matsushima R, Uchida H, Nagai S et al, Assimilation, accumulation, and metabolism of dinophysistoxins (DTXs) and pectenotoxins (PTXs) in the several tissues of Japanese scallop Patinopecten yessoensis. Toxins, 7(12): Miles C O, Wilkins A L, Hawkes A D et al, Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand. Toxicon, 48(2): Miles C O, Wilkins A L, Munday R et al, Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities. Toxicon, 43(1): 1 9 Nagai S, Nitshitani G, Tomaru Y et al, Predation by the toxic dinoflagellate Dinophysis fortii on the ciliate Myrionecta rubra and observation of sequestration of ciliate chloroplasts. Journal of Phycology, 44(4): Nielsen L T, Krock B, Hansen P J, Production and excretion of okadaic acid, pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta effects of light, food availability and growth phase. Harmful Algae, 23: Park M G, Kim S, Kim H S et al, First successful culture of the marine dinoflagellate Dinophysis acuminata. Aquatic Microbial Ecology, 45: Reguera B, Riobó P, Rodríguez F et al, Dinophysis toxins: causative organisms, distribution and fate in shellfish. Marine Drugs, 12(1): Reguera B, Velo-Suárez L, Raine R et al, Harmful Dinophysis species: a review. Harmful Algae, 14: Suzuki T, Miyazono A, Baba K et al, LC MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido, Japan. Harmful Algae, 8(2): Suzuki T, Walter J A, LeBlanc P et al, Identification of pectenotoxin-11 as 34S-hydroxypectenotoxin-2, a new pectenotoxin analogue in the toxic Dinoflagellate Dinophysis acuta from New Zealand. Chemical Research in Toxicology, 19(2): Tong M M, Smith J L, Richlen M et al, Characterization and comparison of toxin-producing isolates of Dinophysis acuminata from New England and Canada. Journal of Phycology, 51(1): Wu H Y, Guo M M, Tan Z J et al, Liquid chromatography quadrupole linear ion trap mass spectrometry for multiclass screening and identification of lipophilic marine biotoxins in bivalve mollusks. Journal of Chromatography A, 1358: PREPARATION OF PECTENOTOXIN-2 BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WANG Yu 1, 2, PENG Ji-Xing 2, WU Hai-Yan 2, GUO Meng-Meng 2, TONG Meng-Meng 1,3, TAN Zhi-Jun 2 (1. Institute of Marine Biology, Ocean College, Zhejiang University, Zhoushan , China; 2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China; 3. Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture, Zhoushan , China) Abstract A protocol for preparing pectenotoxin-2 (PTX2) from a large-scale culture of Dinophsis acuminata was conducted in high-performance liquid chromatography (HPLC). Methods of cell disruption, toxin extraction, and HPLC purification were selected and optimized. The results indicate that the most efficient cell disruption and toxin extraction were achieved in the combination of freezing + thawing + ultrasound, resulting in 99.2% and 81.5%, respectively. PTX2 was extracted using liquid-liquid extraction (methanol, acetone, diethyl ether, and dichloromethane) and Solid Phase Extraction columns (Oasis HLB SPE, Phenomenex Strata-X SPE, Sep-Pak C18 SPE). PTX2 extraction efficiency reached maximum (78.19%) with diethyl ether and dichloromethane. Studies on the optimization conditions of HPLC showed that the ph value of the mobile phase had an impact on the chromatographic reaction. When mobile phase was in alkaline condition (containing 6.7mmol/L ammonium hydroxide in acetonitrile/water, ph=10.94), PTX2 could be better chromatographically separated by Phenomenex Kinetex C18 (4.8mm 250mm, 5μm). The final PTX2 reached 93.2% in purity. This method was rapid, effective, and comprehensive for extraction, separation, and purification of PTX2 from D. acuminata cells with HPLC. Key words pectenotoxin-2; Dinophysis acuminata; high-performance liquid chromatography; extraction; preparation

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI