專利名稱：一種復(fù)雜藥物體內(nèi)外物質(zhì)組關(guān)聯(lián)構(gòu)建代謝網(wǎng)絡(luò)的方法
技術(shù)領(lǐng)域：
本發(fā)明屬于醫(yī)藥領(lǐng)域，涉及藥物復(fù)雜組分代謝物的快速檢出、代謝途徑的分步分類廣泛預(yù)測(cè)和“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)的全面構(gòu)建。
背景技術(shù)：
中藥是中國(guó)傳統(tǒng)醫(yī)藥的瑰寶，但目前國(guó)內(nèi)外關(guān)于中藥的研究并不能系統(tǒng)的、明確的闡明中藥復(fù)雜成分及其代謝物的關(guān)系，缺乏相應(yīng)的理論與技術(shù)支撐，因此中藥體內(nèi)過程長(zhǎng)期被視為“黑箱”，是中藥現(xiàn)代化研究的主要瓶頸。生物處置知識(shí)的缺乏是為中草藥的療效和風(fēng)險(xiǎn)評(píng)估提供證據(jù)的主要障礙之一。大多數(shù)天然化合物的結(jié)構(gòu)比化學(xué)藥物更脆弱，從而更容易受到廣泛的生物代謝。更重要的是，已發(fā)現(xiàn)的天然化合物的藥效很大一部分不是來自母體化合物，而是來自其產(chǎn)生的活性代謝產(chǎn)物。此外，許多中藥成分的特點(diǎn)是口服生物利用度差，在胃腸道產(chǎn)生的代謝產(chǎn)物比中草藥成分更容易被吸收進(jìn)入流通系統(tǒng)，再進(jìn)一步發(fā)生廣泛代謝。因此，中藥成分的代謝物的檢測(cè)和鑒定，不僅是發(fā)現(xiàn)中草藥藥理活性成分的關(guān)鍵步驟，，也可能為從天然化合物發(fā)現(xiàn)新的藥物提供額外資源。然而，代謝物的檢測(cè)和鑒定絕非易事，即使是單一的化學(xué)藥物，因?yàn)樯锘|(zhì)中大量的內(nèi)源性干擾和代謝物豐度低等困難。即使采用高分辨率(如TOF和FT Obitrap)質(zhì)譜儀可實(shí)現(xiàn)對(duì)低濃度成分進(jìn)行檢測(cè)，仍要使用一些額外的軟件工具，如質(zhì)量虧損過濾器，背景扣除，和離子篩選等進(jìn)行必要的數(shù)據(jù)處理，以實(shí)現(xiàn)從龐雜的數(shù)據(jù)中分析出代謝物的相關(guān)信息。而天然化合物往往具有更特殊的代謝途徑，對(duì)天然化合物的成分檢測(cè)和代謝產(chǎn)物鑒定面臨更廣泛的干擾因素和不確定性，無疑是更艱巨的任務(wù)?，F(xiàn)有的關(guān)于中草藥代謝物的研究絕大多數(shù)局限于已分離得到的單一化合物，少數(shù)關(guān)于中藥復(fù)雜代謝成分的研究也極大程度地依賴于已知代謝途徑的幾種確證成分，即依賴于標(biāo)準(zhǔn)品的靶向性研究。針對(duì)中草藥多成分、多靶點(diǎn)的作用機(jī)制和體內(nèi)濃度低、在生物基質(zhì)中檢測(cè)困難、數(shù)據(jù)量巨大、有效目的成分不明確、缺乏標(biāo)準(zhǔn)品的特性，亟需開發(fā)高效、快速、自動(dòng)化的分析方法，對(duì)復(fù)雜生物基質(zhì)中的非靶向性成分進(jìn)行不依賴標(biāo)準(zhǔn)品的全局檢測(cè)和鑒定
發(fā)明內(nèi)容

技術(shù)問題本發(fā)明針對(duì)中草藥復(fù)雜成分在生物基質(zhì)中濃度低、干擾大、缺乏標(biāo)準(zhǔn)品、檢測(cè)困難、代謝途徑廣泛未知、數(shù)據(jù)龐雜、缺少有效數(shù)據(jù)分析手段的問題，首次提出了一種對(duì)復(fù)雜藥物成分在復(fù)雜基質(zhì)中的代謝物檢出、代謝途徑預(yù)測(cè)和“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)構(gòu)建的高效、快速、自動(dòng)化的分析方法。技術(shù)方案本發(fā)明的目的是提供一種復(fù)雜藥物成分在生物基質(zhì)中的代謝物檢出、代謝途徑預(yù)測(cè)和“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)構(gòu)建的通用性方法。這種方法的核心思路是原型成分和代謝產(chǎn)物在相同的質(zhì)譜條件下具有相似的碎裂方式，以此為基礎(chǔ)將原型成分譜和代謝產(chǎn)物譜中具有相似質(zhì)譜行為的物質(zhì)關(guān)聯(lián)起來，并通過兩者的多重對(duì)應(yīng)關(guān)系，構(gòu)建“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)。對(duì)于每一對(duì)相關(guān)聯(lián)的原型成分和代謝產(chǎn)物，利用兩者之間的精確分子量差值和碎片離子比對(duì)，快捷地預(yù)測(cè)其代謝途徑。本方法可通過如下步驟完成(見

圖1)，各步驟均利用計(jì)算機(jī)編程實(shí)現(xiàn)自動(dòng)化運(yùn)行。1、應(yīng)用LC-MS分析藥物制劑提取物樣品和給藥后生物樣品并通過設(shè)置一定的標(biāo)準(zhǔn)輸出樣品的色譜、質(zhì)譜數(shù)據(jù)根據(jù)藥物制劑和給藥生物樣品的特性選擇色譜(包括色譜柱的長(zhǎng)度、內(nèi)徑、填料，柱溫，流速，流動(dòng)相組成，洗脫梯度等)、質(zhì)譜條件(儀器的各設(shè)定參數(shù))，應(yīng)用LC-MS掃描樣品得到其總離子流色譜圖。根據(jù)實(shí)際情況，從總離子流中提取、檢出、確定具有較高強(qiáng)度和適宜保留時(shí)間的離子作為主要目標(biāo)成分。采集各主要成分的分子離子和碎片離子的精確分子量等多級(jí)質(zhì)譜信息，且每級(jí)質(zhì)譜圖都能同時(shí)做到高分辨率、高質(zhì)量精確度和高靈敏度。2、扣除內(nèi)源性干擾，構(gòu)建藥源性成分?jǐn)?shù)據(jù)矩陣比對(duì)給藥生物樣品譜和空白生物樣品譜，將給藥樣品譜中的內(nèi)源性成分扣除。3、剝離原型成分，獲得代謝產(chǎn)物譜將藥源性成分?jǐn)?shù)據(jù)矩陣再與藥物制劑樣品的數(shù)據(jù)比較，將體內(nèi)測(cè)到的原型成分剝離，獲得體內(nèi)代謝產(chǎn)物譜。4、原型成分與代謝產(chǎn)物關(guān)聯(lián)分析鑒定代謝物和代謝途徑1)原型成分與代謝產(chǎn)物關(guān)聯(lián)分析將原型成分譜和代謝產(chǎn)物譜中具有相似質(zhì)譜行為(具有相同的碎片或碎片的中性丟失)的物質(zhì)關(guān)聯(lián)起來，通過MR (Match Ratio)參數(shù)來衡量二者結(jié)構(gòu)上的關(guān)聯(lián)程度，MR值越大，表示該對(duì)化合物的關(guān)聯(lián)越緊密，MR值計(jì)算公式如下MR = VIN*2/(PFN+MFN)，其中VIN表示在一對(duì)原型成分和代謝產(chǎn)物之間，相同或一致的碎片數(shù)，PFN和MFN分別表示原型成分和代謝產(chǎn)物的質(zhì)譜碎片總數(shù)。再通過全部原型成分和代謝產(chǎn)物的多重對(duì)應(yīng)關(guān)系，構(gòu)建整方“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)。2)代謝途徑預(yù)測(cè)和代謝物鑒定對(duì)于每一對(duì)相關(guān)聯(lián)的原型成分和代謝產(chǎn)物，將其分子離子的精確質(zhì)量數(shù)差值與歸納的常規(guī)代謝反應(yīng)庫(kù)進(jìn)行匹配，逐步預(yù)測(cè)該對(duì)化合物之間的代謝關(guān)系，包括降解反應(yīng)、I相代謝、II相代謝和混合反應(yīng)類型的一步、二步、多步代謝反應(yīng)，并通過比對(duì)碎片來驗(yàn)證該候選途徑的可靠性。驗(yàn)證得可靠代謝途徑的代謝物可從譜中剔除，以簡(jiǎn)化下一步對(duì)剩余未知代謝物進(jìn)行的預(yù)測(cè)，避免產(chǎn)生過多的候選途徑。若已知原型成分的結(jié)構(gòu)，更可以通過比對(duì)原型成分和代謝產(chǎn)物的碎片來鑒定代謝產(chǎn)物的結(jié)構(gòu)。有益效果I.本發(fā)明針對(duì)中草藥多成分、多靶點(diǎn)的作用機(jī)制，建立“原型成分-代謝產(chǎn)物”的關(guān)聯(lián)網(wǎng)絡(luò)，對(duì)于全部或指定藥物成分的代謝途徑和代謝產(chǎn)物進(jìn)行準(zhǔn)確、快捷的預(yù)測(cè)和鑒定，全面闡釋了藥物成分在體內(nèi)的處置過程和相互影響，解決了傳統(tǒng)中藥復(fù)方體內(nèi)研究?jī)H限于幾種單體的研究，不足以表征復(fù)方制劑整體特征的問題。2.本發(fā)明針對(duì)天然產(chǎn)物體內(nèi)濃度低、有效目的成分不明確、代謝途徑廣泛未知、代謝物缺少標(biāo)準(zhǔn)品的問題，利用化合物的代謝規(guī)律和質(zhì)譜碎裂特征，進(jìn)行不依賴標(biāo)準(zhǔn)品的非目標(biāo)性代謝物搜尋，實(shí)現(xiàn)了從傳統(tǒng)的已知代謝物分析向未知代謝物分析的轉(zhuǎn)變，解決了中藥在體內(nèi)“非目標(biāo)性”全成分分析和藥效物質(zhì)基礎(chǔ)解釋的技術(shù)難題。3.本發(fā)明能清楚地預(yù)測(cè)出原型成分和代謝產(chǎn)物之間的代謝過程，包括降解反應(yīng)、I相代謝、II相代謝和混合反應(yīng)類型的一步、二步、多步代謝反應(yīng)。通過分步、分類的操作步驟，能預(yù)測(cè)得數(shù)量最少、正確率最高的候選途徑集，并能將已驗(yàn)證得可靠代謝途徑的代謝物從代謝產(chǎn)物譜中剔除，以簡(jiǎn)化下一步的搜尋。本發(fā)明更可以通過已知的原型成分結(jié)構(gòu)推測(cè)出代謝產(chǎn)物的結(jié)構(gòu)。 4.本發(fā)明針對(duì)復(fù)雜藥物成分在生物基質(zhì)中濃度低、干擾大、檢測(cè)困難、數(shù)據(jù)龐雜、缺少有效數(shù)據(jù)分析手段的問題，采用多級(jí)質(zhì)譜精確質(zhì)量測(cè)定配合計(jì)算機(jī)編程的手段，實(shí)現(xiàn)全分析過程的自動(dòng)化，操作簡(jiǎn)便，比傳統(tǒng)人工解析方法在效率和正確率上實(shí)現(xiàn)了幾何級(jí)數(shù)的提高，并適用于環(huán)境科學(xué)、食品、醫(yī)學(xué)、生物化工、代謝組學(xué)等多種領(lǐng)域的復(fù)雜體系研究。
具體實(shí)施例方式實(shí)施例一大鼠尿液中脈絡(luò)寧注射液“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)分析本實(shí)施例在以本技術(shù)方案為前提下進(jìn)行實(shí)施，給出了詳細(xì)的實(shí)施方式和操作過程，但本發(fā)明的保護(hù)范圍不限于下述的實(shí)施例。I、動(dòng)物實(shí)驗(yàn)方案取10只SD大鼠置于代謝籠中，禁食但可自由飲水過夜，按IOmL · kg-1的劑量尾靜脈注射給予脈絡(luò)寧注射液，收集給藥前和給藥后0-4h、4-24h的尿液，_20°C保存。2、樣品前處理將各時(shí)間點(diǎn)的尿液分別與O. 5%氨水等體積混合后，先后上樣到活化好的MAX柱，然后用50%氨水、甲醇清洗，接著用90%甲醇(含5%甲酸)洗脫。待洗脫液揮干后，用90%甲醇(含5%甲酸)溶解，20000rpm高速離心后待分析。3、色譜及質(zhì)譜參數(shù)儀器LC/MS-IT-T0F(島津，日本)。色譜參數(shù)柱型，SynergiC18 Hydro-RP 80A, 250mm X 4. 6mm i. d.,4 μ m(Phenomenex, USA);柱溫，35°C ;進(jìn)樣量,5 μ I ;流速，O. 8ml/min ;流動(dòng)相組成，水(含O. 025 % 甲酸，A)和甲醇(B);洗脫梯度，0-15min 8-12 % (B)、15_40min : 12-60 % (B)、40-50min 60% (B)、50_55min :60_8% (B)、55_65min :8% (B)。柱后分流后，O. 2ml/min 的流動(dòng)相進(jìn)入質(zhì)譜檢測(cè)器。質(zhì)譜參數(shù)離子化模式(Ionization mode), ESI (-);噴霧氣體流速(Nebulizinggasflow rate), I. 5L · min_l ;干燥氣壓力(Drying gas pressure), 0. IMpa ;應(yīng)用電壓(Appliedvoltage), -3. 5KV ；CDL 電壓(CDL voltage), Constant mode ;CDL 溫度(CDL temperature), 200 V ;分析模式(Analysis mode), MS measurement ;測(cè)定范圍(Measurement range) ,Auto MS/MS mode (MS m/z 100-1500 ；MSn m/z 50-1500);離子累積時(shí)間(Ion accumulationtime), 30ms ;轟擊能量(Energy), 30%, 80%150% 在以上條件下，全掃描得到脈絡(luò)寧注射液給藥后0_4h在大鼠尿液中的總離子流色譜圖，見圖2。3、從總離子流色譜圖中確定主要成分并扣除內(nèi)源性干擾在圖2中，確定了 527個(gè)強(qiáng)度大于1，000，000、保留時(shí)間在3_52min之間的離子峰作為脈絡(luò)寧注射液給藥后0-4h在大鼠尿液中的主要成分。然后比對(duì)大鼠空白尿液的成分譜，將樣品與空白對(duì)照中都具有的成分(質(zhì)量誤差不大于IOmDa,保留時(shí)間漂移不大于O.2min)扣除，得到180個(gè)藥源性成分，其保留時(shí)間、實(shí)測(cè)精確分子量([Μ_Η]_)和質(zhì)譜碎片見表I。表I :脈絡(luò)寧注射液給藥后0_4h在大鼠尿液中的主要藥源性成分
Table I. Retention time, accurate mass,formula, mass error and fragment ions of metabolome detected by LC-IT-TOF/MS
N0· RTMeas.m/z Formula Error (ppm) Fragment ions
I* 25.0400 137.0271 C7H6035,11 109.0357
236.0185 163.0401 C9H8033.07 134.0352,119.0568315.3450 164.0349C8H7N036.10120.0496
414.9225168.0282C7H7N04-1.79124.0348,68.0008
530.1000 177.0193C9H6040.56147.0210,133.0312,89.0472
6 30.9950 179.0315C9H8043.35135.0441
7101r7H190.nft7173.0489,171.0284,155.0436,127.0397,109.0331,97.0221,
73.8550191.0544C7H12060.0293.0331,85.0400
830.0250192.0662 ClOHl 1N03-8.3385.0314,74.0253
93.4380193.0329 C6H1007-2.07132.0401,113.0229,103.0452,85.0182
1030.4985193.0489 C10H10044.66179.0276,150.0552,134.0388
1132.9415194.0451C9H9N04-1.03150.0563,134.0390,93.0348
1213.3600198.0401C8H9N051.51154.0469,141.0419,124.0386,109.0158
1312.9500210.0375C9H9N05-1.90141.0192,109.0357,100.0024
1430.8950210.0772C10H13N04-2.38165.0158,124.0396,94.0421
1523.8370215.0552C9H12064.65197.0442,171.0659,127.0671
1622.3285215.9954C7H7N05S-1.39188.0067,136.0411,108.0463,96.9595
174.9250 217.0343 C8H1007-5.99 179.0276，150.0552,134.0388
1850.6985 222.1104 C12H17N03 -13.51 208.0811
198.4680 234.0047 C7H9N06S2.14 153.0770,136.0392
2029.6070 239.0546 C11H1206-7.53 193.0657,179.0332,177.0515,149.0614,133.0701
2121.5150 243.0444 C10H120724.27 210.0547,200.1047 165.0649
2242.6065 243.1202 C12H2005-3.29 225.1159,207.1025,181.1571,163.1121,161.0914,99.0117
2310.7305 248.0210 C12H11N053.63 140.0349,106.9828
2437.8335 262.0693 C13H13N05-7.98 ^44.0594,225.1171,218.0856,200.0709, 156.0816,
146.062
257.2025 263.0232 C9H1207S-8.39 203.0045,190.8837,183.0535,165.0527,150.0322,121.0294
2643.0865 263.1242 C15H2004-1.52 219.1426,201.1247,195.0602,153.0939,138.0652,111.0482
2735.7685 267.0870 C13H16061.87 223.0454,193.0491,179.0338, 149.0619
2845.6840 267.1437 C13H20N204 -11.60 249.1103,222.1198,108.0612
2918.1520 277.0370 C10H1407S-1.08 197.0783,159.0506
3024.5680 278.0714 C13H13N069.71 234.9898,215.0782,138.9639,124.0056,106.9824
260.1057,217.1548,205.1185,187.1125,139.0765,128.0373
3136.7835 279.1164 C15H2005-1.43 ,
123.8386
3240.2265 279.1315 C15H200518.63 237.1223,235.0922,191.0749,108.0453,91.2493
3313.6470 283.0652 C9H16010-OJl 235.9858,151.0255
3432.6665 283.0825 C13H1607-2.83 265.0674,175.0255,113.0265,103.0064,95.0174
3523.5195 285.0593 C12H1408-7.75 175.0268,113.0273,109.0344
363.5400 291.0067 C10H1208S -21.99 280.8380,218.0278,193.0392,113.0283,96.9667
277.0440,266.0504,250.0788,249.0500,221.0613,121.0399
3735.9685 295.0795 C14H1607-7.79 ,
113.0233
3844.8415 295.1130 C15H2006-17.62 205.1178
3934.4535 295.1269 C15H20069.15 253.1168,251.1330,170.0749,124.0451
4046.8915 295.1458 C18H20N202 3.73 267.0321,250.1508,130.0866
4115.0750 297.0518 C13H1408-13.80 252.1020,171.0284
282.0229,279.0892,253.1184,175.0253,167.0521,137.0466
4239.1615 297.1018 C14H1807-1.68 ,121.0661,
113.0271,103.0035
4346.1165 299.1773 C16H2805-13.70 258.0317,228.0480,219.1708
4420.1200 307.0483 C14H1208-10.75 238.9864,153.0180
4523.7020 307.0508 C14H12081.95 293.0279,201.0103，175.0209,106.9765
4651.3415 307.1138 C16H2006-14.65 159.1047,127.0348
279.0813,230.0559,220.0633,178.0136,175.0209,153.0298
4724.5285 308.0885 C14H15N0713.63 ,132.0316，
124.0085,106.9833
295.0590,281.0780,265.0627,238.0872,219.0296,202.0536
4835.8185 309.0634 C14H1408-4.53 ,193.0392,
189.0945,185.0606,175.0413,162.0802,109.0303

4939.5265309.1243C15H22N205-22.00177.0216
5016.5905313.0535C13H1409-2.24243.9774,179.0422,175.0209,137.0254
5114.4225315.0684C13H160 92.22165.0188,163.0420,152.0129,135.0322,121.0357,108.0247
5218.5750324.0871C15H19N05S-10.80296.0923,277.9981,187.0467,164.0541
5338.2985 324.1078 C15H19N07-6.48
5437.7335 325.0857 C15H1808-17.84 282.1832,236.0591,179.0593,175.0272,163.0233,113.0283
5544.9415 325.1172 C18H18N204 -6.77 306.1277,280.1027,223.1309,130.0866,124.0192
5626.2475 326.0859 C14H17N08 -10.43 175.0266,150.0571,113.0249
5723.1450 327.0731 C14H1609-3.97 175.0234,151.0409,113.0283
5839.0765 327.1045 C15H2008-13.45 283.1157,220.0195,183.0308,139.0416
5911.3250 328.0445 C13H15N07S -17.38 175.0260,153.0191,134.0486,113.0246
6017.5425 329.0494 C13H14010-2.74 175.0266,153.0211,113.0251,109.0338
311.2182,293.2113,285.0389,249.2157,247.2008,229.1426
,211.1333,
6150.3075 329.2295 C17H32N0514.88 209.1179,193.1258,185.1192,183.1382,171.1029,167.1435
,165.1301，
155.1432,125.0959
6228.5735 330.0959 C17H17N069,39 311.0908,241.1159,201.0585,162.0195
311.2210,309.2082,293.2095,291.1782,285.1814,267.1725
,229.1388,
6351.7400 330.2380 C17H33N056.36 227.1265,213.1190,211.1345,209.1150,197.1185,185.1255
,171.1027,
125.0880
6444.3815 333.0889 C17H1807-21.62 316.1278,298.1059,297.1091,171.0909,152.0396,138.0204
318.0313,209.0460,199.0051,191.0354,177.0187,173.0262
6521.4700 335.0350 C15H1209-16.71 ,169.0123,
163.0477,159.0199,151.0409,137.0255,136.0197,109.4148
6629.6510 336.0733 C15H15N08-3.27 252.0566,221.0400,175.0263,160.0390,113.0234
6732.3415 337.0895 C16H1808-3.56 191.0559,173.0469,163.0390,137.0270
6831.1690 338.0862 C15H17N08 -14.20 249.0350,175.0247,162.0560,137.0186,113.0281
6925.9450 339.0682 C15H1609-3.83 249.0350,175.0247,162.0560,137.0186,113.0281
7034.1550 339.0717 C15H1609-4.72 251.0725,208.9882,176.0325,163.0411,119.0529
7141.1765 339.1086 C16H2008-2.65 321.1710,243.1655,223.1309,209.0903,207.0773,175.0209
7218.3125 341.0504 C14H14010-4.98 175.0252,165.0208,121.0294
7326.3900 341.0828 C15H1809-12.02 323.1411,312.0511,268.0625,177.0178,175.0262,124.0039
323.1507,297.1346,279.1769,242.0505,221.1110,217.0986
7443.4515 342.1224 C15H21N0811.98 ,212.0861,
209.1150,197.0423,165.0281,130.0322,121.0294
7524.5230 343.0687 C14H160104.96 275.0459,175.0209,167.0327,152.0161
333.0820,326.0645,311.0908,269.0830,238.1166，232.1308
7640.7265 343.1128 C19H200 69.03 ,229.1159,
162.0012
7747.3865 343.2087 C18H3206-12.53 250.0805,229.1408,209.1116,171.1050
7832.2915 344.1199 C18H19N0617.15 215.0733,181.0821,171.0859
7940.9265 345.1575 C16H2608-15.65 327.1339,300.0791,259.1061,195.0735,175.0272
8041.0265 347.1728 C16H28082.59 329.1649,302.1328,161.0429
8142.5015 349.1788 C16H30081.72 332.1789^06.1193,259.0600,210.0624,193.0525,187.1350
8223.9590 353.0784 C16H1809-1.13 191.0579,179.0360,177.0202,173.0460,135.0460
8324.7985 355.0640 C15H16010-4.51 191.0354,179.0358,175.0227,153.0135,135.0472
8427.9565 356.0971 C15H19N098.99 180.0690,175.0187,135.0538,118.0345
339.0829,324.0612,312.1186,295.0932,279.0617,267.0990
,251.0719,
8535.0535 357.0973 C19H1807-3.36 241.0714,226.0671,201.0509,193.0454,189.0485,187.0439
,173.0493，
158.0386,143.0512,134.0352
8637.5335 357.1503 C17H26G8-15.96
8721.0650 359.0738 CI8HI6O82.00 255.0811
8840.9765 359.1649 C17H2808-18.38 91.0573,173.0566

8942.2265 359.2171 C18H3207-16.7 238.1314,201.1129,173.0440,171.1033,124.0086
298.1148,287.1593,260.0595,217.1548,214.0855,198.0577
9037.2835 360.1584 C16H27N08-5.00 ,185.1195,
175.0209,173.0566
9136.3585 361.1463 C16H2609-11.91 175.0202,157.0153
9213.3390 362.0533 C13H17N09S -3.59 220.0198,198.0398,190.0143
9331.8915 366.0967 C16H17N09 -14.48 193.0517,177*0597,173.0477
9427.8100 368.0827 C16H19N07S 4.35 280.0947,193.0486,175.0237,171.8282
9531.3430 369.0857 C16H18010-1.633_713’30_42’m0503’m02"H’m_0261
,134.0448
9637.9835 369.1121 C19H18N206 13.00 294.1891,254.0843,224.1444
9724.2870 371.1041 C16H200105.66
353.1236,265.1072,253.1007,241.1159,235.0922,195.0711
9831.5675 371.1125 C16H2001024.52 ,175.0272，
157.0142
9925.9000 372.0870 C15H19N010 -3.49 196.0615,175.0209,148.0455
355.1119,343.1144,273.0550,199.0879,193.0392,191.0565
10040.3015 373.1374 C20H2207-4.29 ,175.0966,
173.0484
10146.4540 373.1473 C17H26C9-4.82 ^.^9^97.1159，193.0351’Π9.1082，175·0
10243.7315373.1836 C25H2603-3.75354.1518,328.0841,223.0739,160.0029,145.0303,125.0311
10328.47403 75.1227 C16H24010-4.53261.0456,217.0448,199.0933,181.0894,175.0209,169.0799
10420.4770378.1304C18H21N0830.94359.0724,291.0972,273.0849,188.0721
10527.2435379.0868C17H1601022.42178.0519,134.0626
333.1966,315.1951,245.1539,207.1398,202.1215,162.0196
10639.8765 379.2053 C17H320913.71 ,153.0534,
124.0086
10720.0520 380.0593 C16H15NOIO -10.26 204.0290,160.0409
10826.7915 381.0762 C17H18010-8.4 @0^272.腸，23 議49’道隱,！則^
10938.1985 383.0943 C17H20010-7.05 320.1455,241.1011,232.1744,192.0400,175.0248
11034.9035 385.0859 C20H1808-5.97 341.0992,326.0722,282.0870,239.1045,203.0317,158.0291
11143.8815 387.1953 C19H3208-17.56 369.2002,269.1770,173.0440,157.0108,139.1102
374.0605,308.1517,245.0296,215.0280,200.0083,193.0520
11230.4485 389.0899 C19H18099.51 ,178.0320，
173.0440
372.2006,321.1710,303.1573,271.2128,227.0977,218.0700
11348.7440 389.1967 C18H300923.12 ,203.0997,
157.0149,128.0265
1143.7650 391.0981 C15H20012-2.70 ^^nYo2/11'0936'259-0607'217-0518'1990427'191 0588
,173.0493
11538.5975 395.0941 C18H20010-13.16 219.0402,193.0504,134.0352
364.0816,351.2147,339.0837,329.2335,317.1808,315.1951 57 I RQI
11649.6285 3972123 C17H3401019,89 251.1246,249.1103,233.1151,231.0922,197.1319,196.1014
,181.0830
384.1174,375.0631,371.0910,342.0606,322.1649,242.0802
11735.3035 403.1270 C17H24011-1.74 ,223.0571,
193.0260,187.1047,179.0523,176.0578,143.0326
11838.6375 417.1176 C17H2201223.97
11931.7915 417.1232 C18H2609S-5.75 296.0583,259.0600,208.0652
12018.7230 421.0724 C19H1801110.69 375.0695
12150.8140 427.1924 C20H30N0917.79 251.1651,207.1821,191.1415,175.0270
12237.1835 429.0717 C17H1801322.37 253.0548,175.0152
12329.5100 429.1053 C18H22012-0.93 337.0501,253.0710,179.0356,175.0257,161.0238,135.0462
12452.1085 429.1716 C16H30013-3.50 199.0933
12543.2365 429.2074 C 21H3409-16.54 311.1076,251.1784,193.1586
12645.4515 429.2123 C17H340126.52 271.1028,265.1948

12741.6765 429.2170 C21H3409-9.09 412.1871,279.0813,253.1868,174.9956
12835.7185 431.0924 C17H2001322.27 387.0899,356.0583,255.0636,175.0309,149.0242
12942.3265 433.1085 C17H2201323.78 388.1422,330.1446,288.0820,257.0817,199.0664,175.0268
13021.6170 43 8.0926 C19H21N011 -8.45 230.0389,225.0455
13110.6630 441.0653 C18H18013-1.13 285.0594,248.0268,175.0262
373.1525,327.2375,282.9373,252.0717,236.1471,195.9811
13246.5765 441.1201 C16H260141.36 ,193.0314,
175.0209,169.0123,147.7859
382.1241,341.0551,269.0458,262.1262,241.0863,223.1452
13336.9835 445.0836 C17H1801413.03 ,213.0533,
180.0697,175.0272
13438.4985 447.0865 C21H20011-3.36 285.0393,199.0387,175.0403
13540.7765 447.1172 C19H28010S -13.2 402.2095,359.0905,256.0730,249.1103,231.0995,187.1047
13627.0700 449.1100 C21H220U-LU %^^255.()659’231獅4’229.()87()’2丨5遍2’21 丨.0796
13734.5535 459.0912 C18H2001418.3 328.0841,283.0586,268.0340,175.0241,158.0410
13850.4100 459.1770 C19H28N2011 33.75 237.1015,229.9980
13939.6015 473.1421 C20H28N012 -23.46 297.1097,253.1195,251.1069,189.0617,175.0250
14032.8915 478.1322 C19H29N011S -16.52 283.0808,194.0475,175.0254,150.0571
14144.5815 481.1754 C19H3001416.63 436.2268,391.1189,348.0888,315.0680,297.1131
14229.8000 487.1085 C20H240142.26 311.0748，175.0276
351.0707,313.0729,295.0574,268.0313,215.0564,195.0269
14337.0335 487.1198 C24H24011-4.31 ,191.0555,
179.0390,177.0047,173.0491
14410.0970 489.0967 C19H22015-2.25 313.0539
,45 50.7635 495.1686 C20H3201411.51切.！職’仍麗切2.1626’251.1635
14641.9265 497.1196 C25H22011-3.62 409.1022,335.0756,323.0038,255.0659,179.0349,161.0255
342.1352,324.1253^14.1379,280.1346,272.0933,254.0826
14745.8365 504.1861 C25H31N010 -1.98 ,244.0963,
228.1025,210.1020
353.0857,335.0732^99.0546,255.0648,227.0705,203.0346
14839.2265 515.1142 C25H24012-4.85 ,191.0553,
179.0357,175.0446,173.0470
14932.6415 515.1651 C21H28N2013 23.49 340.0511,283.0805,250.0150
15010.5280 521.1413 C23H26N2012 -5.37 365.1315,245.0906,190.2907
15136.1835 527.0800 C25H20013-4.74 496.5809,399.0756,328.1014,191.0573,181.0188
15243.1865 527.2560 C26H4001I13.66 341.5665,263.1243,219.1284
367.0993,353.0857,335.0810,255.0659,211.0657,203.0384
15317.9920 529.1324 C26H260122.27 ,193.0483，
191.0565,179.0334,173.0452,161.0270
15435.6685 533.1365 C25H26G1311.25
15537.2335 537.1038 C27H220128.19 375.0674,201.0184,179.0341
15623.9720 539.1339 C27H24012-15.21 422.6096，269.0640
525.1195,367.1012,349.0919,313.0729,269.0801,228.0769
15742.0015 543.1444 C27H28012-14.73 ,210.0683，
193.0520,191.0557,175.0398,173.0484
15824.8550 550.1038 C24H25NG14 -17.45
533.0768,377.0464,375.0663,359.0390,333.0600,315.0460
15922.4200 551.1021 C24H24015-2.18 ,271.0971,
201.0164,191.0552,187.0781,179.0350
tjrAΛnnn 463.1329,377.0487,375.0678,297.0281,273.0392,201.0188
16034.0465 551.1435 C25H28014-2.00 179 0382
16127.6635558.1262C22H25N01617.56369.0803,188.0376
16226.4635565.1119C25H26015-7.26391.0640,374.0611,351.1048,215.0304,193.0469
16333.3415583.1625C26H32015-14.92 472.6297,367.1038,215.0531
16426.9350586.1090C26H25N30115.97411.9643,389.0265,381.0826,353.0928,219.0529
16529.1735593.1364C27H30015-13.32 239.0553

16632.0915 593.1399 C23H3001816.02 417.1175,353.0569,241.0863
419.1731,404.1466,390.1304,389.1206,373.1303,359.1086
16730.7450 595.1890 C28H36014 -16.97 ,251.0808,
233.0838,218.063016838.9765 617.1775 C30H34014-11.99 499.2005,441.1546,297.0958,178.9955
16932.7415 621.1764 C25H3401816.42 353.0749,337.0877,283.0748,191.0552
17032.3915 621.2545 C27H42016-0.32 295.1296,253.1230
17134.8035 649.1765 C26H3401918.33 474.1041,389.0829,367.1043,303.0460,281.0692
17234.4035 677.1695 C27H340202.36 367.1004,354.0799,338.1002
17338.8265 715.2225 C30H40N2018 -4.05 417.1177
701.1647,655.7241,507.1006,543.1419,525.1272,452.6352
,367.0972,
17437.8835 719.1738 C33H360183.48 349.0882,335.0883,331.0818,313.0636,287.0946,269.0793
,254.0532,
243.0911,127.1532
17526.8865 719.1844 C29H3602111.68 381.0795,353.0826,337.1006,219.0484
17628.9235 721.1828 C29H3802I1.94 367.1034,353.0876
17738.4485 729.1847 C31H38020-5.62 417.1171,385.0457,311.0567,267.0643,250.0452
17833.1415 757.1853 C30H38N4017 -5.94 563.1489,389.0848,374.0629,367.1010,349.0868,314.3992
17939.3765 783.2696 C36H48G19-7.92
18038.5485 835.2502 C35H48023-2.27 417.1164
*Bold components were detected in both biological sample and injection sample_4、剝離體內(nèi)原型成分，獲得代謝產(chǎn)物譜將藥源性成分譜再與原注射液樣品的數(shù)據(jù)比較，得出體內(nèi)測(cè)到的原型成分(與注射液成分精確質(zhì)量誤差不大于15mDa，保留時(shí)間漂移不大于0. 5min)共19個(gè)，剔除體內(nèi)原型成分后，獲得體內(nèi)代謝產(chǎn)物譜。5、原型成分與代謝產(chǎn)物關(guān)聯(lián)分析鑒定代謝物和代謝途徑分步一步、二步、三步、多步反應(yīng)進(jìn)行“原型成分-代謝產(chǎn)物”關(guān)聯(lián)和代謝途徑預(yù)測(cè)。對(duì)于每一步分析，先依據(jù)相似質(zhì)譜的行為(具有相同的碎片或碎片的中性丟失)將“原型成分-代謝產(chǎn)物”關(guān)聯(lián)起來，為提高關(guān)聯(lián)的準(zhǔn)確性，各步搜尋的MR參數(shù)下限設(shè)為0. 04-0. 4不等。然后將關(guān)聯(lián)的每對(duì)化合物分子離子的精確質(zhì)量數(shù)差值與常規(guī)代謝反應(yīng)庫(kù)進(jìn)行匹配，預(yù)測(cè)“原型成分-代謝產(chǎn)物”之間不同種類的代謝關(guān)系，包括降解反應(yīng)、II相代謝、I相代謝、混合類型，并通過比對(duì)碎片特征進(jìn)行驗(yàn)證。更可以利用原型成分的結(jié)構(gòu)和二者碎片的關(guān)聯(lián)性來鑒定代謝產(chǎn)物的結(jié)構(gòu)對(duì)于脈絡(luò)寧注射液給藥后0_4h在大鼠尿液中的代謝成分，共鑒定出降解產(chǎn)物12個(gè)，一步I相代謝產(chǎn)物32個(gè)，一步II相代謝產(chǎn)物21個(gè)，二步I相代謝產(chǎn)物26個(gè)，二步II相代謝產(chǎn)物15個(gè)，二步混合類型代謝產(chǎn)物32個(gè)，三步代謝產(chǎn)物38個(gè)和多步代謝產(chǎn)物3個(gè)，各類型代謝物之間有交叉，全部代謝物的預(yù)測(cè)代謝途徑列于表2。全部原型成分-代謝產(chǎn)物的關(guān)聯(lián)網(wǎng)絡(luò)見圖3。部分化合物的代謝關(guān)系和結(jié)構(gòu)鑒定如下
權(quán)利要求
1.一種復(fù)雜代謝物檢出、代謝途徑預(yù)測(cè)和體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)構(gòu)建的方法，包括制劑和生物樣品制備；多級(jí)質(zhì)譜檢測(cè)；選取主要成分，排除干擾；確證體內(nèi)的原型成分；對(duì)全部或選定的原型成分和代謝產(chǎn)物進(jìn)行關(guān)聯(lián)分析，構(gòu)建體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)；預(yù)測(cè)和確證有關(guān)聯(lián)的原型成分和代謝產(chǎn)物之間的代謝途徑，并利用原型成分的結(jié)構(gòu)鑒定代謝產(chǎn)物的結(jié)構(gòu)。
2.根據(jù)權(quán)利要求I所述的一種復(fù)雜代謝物檢出、代謝途徑預(yù)測(cè)和體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)構(gòu)建的方法，其特征在于能夠?qū)τ袠?biāo)準(zhǔn)對(duì)照品和無標(biāo)準(zhǔn)對(duì)照品、有文獻(xiàn)資料報(bào)道(化合物質(zhì)譜、色譜、理化性質(zhì)、結(jié)構(gòu)等信息)和無文獻(xiàn)資料報(bào)道的原型成分和代謝物進(jìn)行分析。
3.根據(jù)權(quán)利要求1、2所述的一種復(fù)雜代謝物檢出、代謝途徑預(yù)測(cè)和體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)構(gòu)建的方法，其特征在于能夠?qū)σ阎臀粗?、目?biāo)和非目標(biāo)的代謝物進(jìn)行分析，能夠?qū)θ亢筒糠值脑统煞诌M(jìn)行代謝分析。
4.根據(jù)權(quán)利要求I所述的原型成分和代謝產(chǎn)物關(guān)聯(lián)分析的方法，其特征在于利用化合物質(zhì)譜行為的相似程度來衡量其關(guān)聯(lián)的緊密程度，并通過原型成分和代謝產(chǎn)物的多重對(duì)應(yīng)關(guān)系，構(gòu)建體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)。
5.根據(jù)權(quán)利要求I所述的預(yù)測(cè)和確證代謝途徑的方法，其特征在于分步、分類地預(yù)測(cè)原型成分和代謝產(chǎn)物之間的代謝關(guān)系，包括降解反應(yīng)、I相代謝、II相代謝和混合反應(yīng)類型的一步、二步、多步代謝反應(yīng)，并通過比對(duì)碎片來驗(yàn)證候選途徑的可靠性。
全文摘要
本發(fā)明針對(duì)復(fù)雜成分在生物基質(zhì)中濃度低、干擾大、代謝廣泛、缺少標(biāo)準(zhǔn)品、數(shù)據(jù)龐雜、分析困難的問題，提供了一種復(fù)雜成分代謝物的快速檢出、代謝途徑的分步分類廣泛預(yù)測(cè)和“原型成分-代謝產(chǎn)物”關(guān)聯(lián)網(wǎng)絡(luò)的全面構(gòu)建的方法。方法步驟如下1、制劑和生物樣品制備；2、多級(jí)質(zhì)譜檢測(cè)；3、選取主要成分，排除干擾；4、確證體內(nèi)的原型成分；5、構(gòu)建體內(nèi)外物質(zhì)組關(guān)聯(lián)網(wǎng)絡(luò)；6、預(yù)測(cè)和確證代謝途徑，鑒定代謝物的結(jié)構(gòu)。各步驟均能利用計(jì)算機(jī)編程實(shí)現(xiàn)自動(dòng)化運(yùn)行，操作簡(jiǎn)便、準(zhǔn)確高效。本發(fā)明不依賴標(biāo)準(zhǔn)對(duì)照品，不依賴原型成分結(jié)構(gòu)，解決了單一或復(fù)雜藥物在體內(nèi)“非目標(biāo)性”全成分分析和藥效物質(zhì)基礎(chǔ)解釋的技術(shù)難題，并適用于環(huán)境、醫(yī)工、食品等諸多領(lǐng)域的研究。
文檔編號(hào)G06F19/12GK102622532SQ201210046410
公開日2012年8月1日 申請(qǐng)日期2012年2月27日 優(yōu)先權(quán)日2012年2月27日
發(fā)明者孫世頃, 張勇, 王廣基, 郝海平, 龔平 申請(qǐng)人:中國(guó)藥科大學(xué)