22 13 2010 Toshihiro HKURA, Minako MCHIZUKI, Michiyo HNISHI, Yuzou KA, Mitsunori DI An analytical method using liquid chromatography tandem mass spectrometry (LC/MS/MS) and liquid chromatography Time-of-Flight mass spectrometry (LC/TF-MS) equipped with electrospray ionization (ESI) was demonstrated for the determination of amount of polyphenols, such as anthocyanins (5 compounds), flavanones (5 compounds), flavones (10 compounds), flavonols (6 compounds), isoflavones (6 compounds) and anthraquinones (3 compounds), catechins(4 compounds), and furanocoumarins (4 compounds) in so-called health-promoting food. The HPLC eluted analytes within 32 min, and analytical cycle was 50 min by a sample. The sample was crushed, powdered with mortar, extracted with methanol and subjected to LC/MS/MS and LC/TF-MS without hydrolysis. The limit of quantification of developed method was 0.01-0.56μg/g(S/N=10) in samples. Amount of individual compound of polyphenols and furanocoumarins (43 compounds) was determined by analyzing each of 39 health-promoting food items which sell in the high-ranking of all obtained in Japan and the daily intake of individual compounds was estimated by the amount of compound and the daily intake of individual item. A Study on the daily intake of polyphenols and furanocoumarins through foods obtained in Ehime Prefecture was carried out by the total diet-market basket method on the basis of food classification and the data of food consumption in the Shikoku region obtained from the Japan Nutrition Survey. In several health-promoting food items, isoflavones were detected and the highest daily intake of isoflavone aglycone was estimated to be 21 mg/day. This highest daily intake of isoflavone was lower than the upper limit for safe daily extra dietary intake of soy isoflavone as a food for specified health use (30 mg/day). The dietary intake of isoflavone aglycone was estimated to be 15 mg/day. polyphenols, furanocoumarins, health-promoting food, liquid chromatography mass spectrometry, LC/MS/MS, LC/TF-MS, daily intake, food, total diet-market basket 8234 15
1) 2) 3) 4) 5) 303 4LC/MS/MS 643 LC/TF-MS 1 (LC/MS(/MS)) Alliance 2695( )Agilent 1200 SL(Agilent Technologies) Quattro micro API() micrtf II(Bruker Daltonics) 2 2 3 ChromaDex,Inc.LKT Laboratories,Inc.Alfa AesarCayman ChemicalMP Biomedicals LC/MS PTFE0.2μm Mini-UniPrep HPLC Ascentis Express RP-Amide (2.1mm i.d. 150mm, 2.7μm) (SUPELC) 秤 50100μg/mL RMilli-Q Gradient-A10 4 3 20072009 39 173 18 6) 13 16
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H H + H Anthocyanin Cyanidin-3-glucoside H H -glucose Petunidin-3-glucoside H CH3 -glucose Pelargonidin-3-glucoside H H -glucose Peonidin-3-glucoside CH3 H -glucose Malvidin-3-glucoside CH3 CH3 -glucose H Flavanone Hesperetin H CH3 H Hesperidin H CH3 -rutinose Naringenin H H H Naringin H H -neohesperidose Neohesperidin H CH3 -neohesperidose R4 R5 R4 H Flavone R4 R5 Apigenin H H H H H Vitexin H H H H glucose Cosmetin H H H -glucose H Baicalein H H H H H Baicalin H H H -glucuronic acid H Chrysin H H H H H Diosmin H CH3 H -rutinose H Luteolin H H H H H Luteolin-3,7-diglucoside -glucose H H -glucose H Rhoifolin H H H -neohesperidose H Isoflavone R4 Daidzein H H H H Genistein H H H H Glycitein H H CH3 H Daidzin H H H -glucose Genistin H H H -glucose Glycitin H H CH3 -glucose R5 H R5 R4 R4 H Flavonol R4 R5 Astragalin H H H -glucose H Hyperoside H H H -galactose H Kaempferol H H H H H Quercetin H H H H H Quercitrin H H H -rhamnose H Rutin H H H -rutinose H (-C9H15) Anthraquinone R4 R5 Purupurin H H H H H Emodin H CH3 H H H Rhein H CH H H H H H H H 7 6 H gallate H H Furanocoumarin Bergapten H CH3 Isopimpinellin CH3 CH3 Bergamottin H -C9H15 6,7-Dihydroxybergamottin H -C9H15(H)2 Catechin (-)-Epicatechin H H H (-)-Epigallocatechin H H H (-)-Epicatechin-3-gallate H -gallate H (-)-Epigallocatechin-3-gallate H -gallate H 18
5 200mg 秤 10mL flavonol 200ng/mL5.0mL 1030005 PTFE0.2μm 1μLLC/MS(/MS) 2 1 MS/MS LC/MS ESI MS/MS MS/MS 2ESI(+)43MS/MS 2 ESI(+)TF-MS (HR-EIC) MS/MS TF-MS M (M+H) HR-EICm/z 0.005 19
Intens. 1.50 1.25 1.00 0.75 0.50 0.25 0.00 22.15 min 9 24.38 min 10 12 14 16 18 20 22 24 26 Time [min] 25.63 min 11 LC/MS/MS Intens. 6000 5000 4000 3000 2000 1000 LC/TF-MS 100609006 6: MRMof 6 Channels ES+ 13.97 100 271.06 > 243.06 1.11e5 % 0 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 100609006 6: MRMof 6 Channels ES+ 13.94 100 271.06 > 153.02 6.53e5 % 0 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 100817015 7: MRMof 6 Channels ES+ 24.34 100 271.06 > 243.06 1.67e5 % 0 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 100817015 7: MRMof 6 Channels ES+ 25.63 100 271.06 > 153.02 6.90e5 % x10 4 3.94 min 6 0 genistein 271.06 -> 243.06 genistein 271.06 -> 243.06 apigenin 271.06 -> 153.02 apigenin 271.06 -> 153.02 24.34 25.64 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 apigenin 271.0600.005 genistein 271.0600.005 baicalein 271.0600.005 Time luteolin-3',7-diglucoside 611.16070.005 hesperidin 611.19700.005 8.22 min 10 10.08 min 12 11.34 min 14 C18 RP-Amide neohesperidin 611.19700.005 rutin 611.16070.005 0 0 2 4 6 8 10 12 14 16 18 Time [min] 3 LC/MS LC/MS2 HPLCRP-Amide / MS/MS 8 TF-MS 0.10.56LC 8mM- (CH3CN/H2=3/2) LCC18apigenin genisteinmrm(ms/ms) 3 RP-Amide MRM(MS/MS) 4TF-MSHR-EIC 32 50 Intens. x10 4 2.5 2.0 LC/TF-MS HR-EIC Std.mix. 100μg/L 25.47 min 21 flavonol (I.S.) 239.0700.005 20.15 min 13 1.5 18.85 min 12 30.72 min 25 20.65 min 15 1.0 20.56 min 14 26.73 min 24 25.46 min 20 24.15 min 19 0.5 22.63 min 18 13.69 min 21.42 min 10 16 22.39 min 17 31.02 min 4.36 4.72 min min 26 2.25 min 4 5 5.91 min 12.98 min 2.25 2 min 11.21 min 15.62 min 26.54 min 6 1 3.91 min 9 25.80 min 7 12.90 min 11 23 3 22 8 0.0 0 5 10 15 20 25 30 35 Time [min] 20
4 flavonol(200μg/l) (101000μg/L)flavonol 5 LC/MS/MS (r 2 )daidzein, glycitein 101000μg/L 0.997 daidzein, glycitein (r 2 )10500μg/L0.998 100μg/L 0.99.4%(n=6) (S/N=10)0.2 20μg/L LC/TF-MSdaidzin, glycitin, daidzein, glycitein, isopimpinellin, apigenin, bergamottin, emodin (r 2 ) 21
0.995 0.999 LC/TF-MS LC/MS/MS 8(r 2 ) 0.997 100μg/L 2.411.2%(n=6) (S/N=10)0.5 20μg/L (100μg/L)LC/TF-MS HR-EIC5 5 2 N,N- DMFLC/MS DMF 6 0.2μg1μg 6 LC/MS/MS0.2μg69.8 123.7%1μg60.2121.1% 0.2μg1.39.1%1μg 0.96.7% baicaleinquercetin LC/TF-MS0.2μg39.0 126.8%1μg52.2143.5% 0.2μg2.634.6%1μg 1.57.2% 0.2μg 1μgbaicalein quercetin 7 312 7 (flavonol 200μg/L) (S/N=10)0.020.56μg/g 8 413 9 1μg/ 18 2) 30mg (43 ) LC/MS/MS LC/TF-MS 1 HPLCRP-Amide / 50 2 ESI(+)flavonol 43 (S/N=10)0.2 20μg/L 3 LC/MS/MSLC/TF-MS 22
4 5 LC/MS/MS LC/TF-MS0.2μg 1μg baicaleinquercetin 6 (S/N=10) 0.010.56μg/g 7 39 43 30mg 8 43 15mg 23
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1) :, (2006.5.11) 2) :, (2006.8.23) 3) :, 2007, 454 4) :, (2007), () 5) :,11,20-28(2008) 6) :, (211) 26