超高效液相色譜-四級(jí)桿飛行時(shí)間質(zhì)譜結(jié)合Progenesis QI 分析不同品種橙的指紋圖譜及其差異性代謝物

趙希娟，趙無疾，許華超

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超高效液相色譜-四級(jí)桿飛行時(shí)間質(zhì)譜結(jié)合Progenesis QI 分析不同品種橙的指紋圖譜及其差異性代謝物

趙希娟，趙無疾，許華超

（西南大學(xué)園藝園林學(xué)院/南方山地園藝學(xué)教育部重點(diǎn)實(shí)驗(yàn)室，重慶 400715）

【目的】建立分析不同品種橙之間差異性代謝物的方法，找出各品種橙的特征代謝物，服務(wù)于橙的代謝組學(xué)研究，為橙果實(shí)和橙汁的鑒別提供參考?！痉椒ā窟x取相同產(chǎn)地8個(gè)不同的橙品種，取其果皮的甲醇提取物，使用超高效液相色譜-四級(jí)桿飛行時(shí)間質(zhì)譜（UPLC-QToF-MS）得到對(duì)應(yīng)的指紋圖譜，結(jié)合組學(xué)軟件Progenesis QI，通過主成分分析、偏最小二乘判別分析和變量變化趨勢(shì)圖等進(jìn)行綜合評(píng)價(jià)，篩選出各品種間的差異性代謝物。根據(jù)精確分子量、二級(jí)碎片、標(biāo)準(zhǔn)品以及數(shù)據(jù)庫和相關(guān)文獻(xiàn)信息，對(duì)差異性代謝物進(jìn)行鑒定。【結(jié)果】通過UPLC-QToF-MS指紋圖譜得出8個(gè)橙品種的代謝物存在明顯差異，使用Progenesis QI建立的流程篩選得到各品種正、負(fù)離子模式下的差異性代謝物，包含保留時(shí)間、分子式、二級(jí)碎片等信息。鑒定出17種差異代謝物，其中3-羥基-5,7,3',4'-四甲氧基黃酮和木犀草素-7-O-新橙皮糖苷是血橙區(qū)別于其他7種橙的特征代謝物，橙皮苷是S26錦橙的代謝標(biāo)志物，蕓香柚皮苷-4'-O-葡萄糖苷和異橙黃酮可以作為8045甜橙的特征代謝物?！窘Y(jié)論】基于UPLC-QToF-MS和Progenesis QI建立的方法可以篩選出8個(gè)品種橙果實(shí)的差異性代謝物，鑒定了其中的17種物質(zhì)，分別對(duì)應(yīng)不同品種。該研究方法和技術(shù)路線具有通用性，適用于不同水果、不同產(chǎn)地、不同成熟度等因素引起的樣品差異分析。

超高效液相色譜-四級(jí)桿飛行時(shí)間質(zhì)譜；橙；Progenesis QI；代謝物

0 引言

【研究意義】橙屬于代表性的柑橘，富含多種代謝活性物質(zhì)[1]，如類黃酮、香豆素和類胡蘿卜素等，具有抗氧化、抗炎等生物活性[2]。因其集藥、食兩用于一身，成為大眾喜愛的水果之一，通常以鮮食或者果汁形式攝入。橙品種眾多，每種橙的品質(zhì)不同，含有的代謝成分也不同。研究表明，橙果實(shí)的營(yíng)養(yǎng)功能成分受品種、產(chǎn)地、成熟度、氣候和土壤等多種因素影響[3]。市場(chǎng)上橙的銷售容易弄虛作假，將不同品種的橙果實(shí)混合一起出售，或者將不同產(chǎn)地品質(zhì)差異大的同一品種混合，包括用非地理標(biāo)志產(chǎn)品代替地理標(biāo)志產(chǎn)品，因外觀非常接近，消費(fèi)者不容易區(qū)分而花高價(jià)錢買到品質(zhì)差的橙子；同樣，橙汁的摻假也涉及不同品種或者不同產(chǎn)地相同品種但品質(zhì)差異大的果汁混合，比如在橙汁中添加橘子汁或者其他品種的橙汁[4]。橙果實(shí)和橙汁的產(chǎn)地溯源與真實(shí)性驗(yàn)證是一項(xiàng)非常復(fù)雜的工作，因此，開發(fā)一種新的有效的方法來分析判別各品種橙代謝組分的差異以此找到其代謝標(biāo)志物，對(duì)于橙果實(shí)和橙汁的真?zhèn)舞b別具有重要意義?！厩叭搜芯窟M(jìn)展】常見的果品真?zhèn)舞b別、品質(zhì)分析和品種區(qū)分方法通常是基于其次生代謝產(chǎn)物的代謝輪廓或者定量分析，如有機(jī)酸、多酚化合物等[5-7]。實(shí)際上，定性定量分析果品中的活性成分不僅對(duì)真?zhèn)舞b別具有重要作用，而且能夠評(píng)估果品的品質(zhì)，對(duì)人們的消費(fèi)提供指導(dǎo)。多種技術(shù)可以用于果品次生代謝產(chǎn)物的分析檢測(cè)，如高效液相色譜（HPLC）[8-9]、液質(zhì)聯(lián)用（LC-MS）[10-13]和氣質(zhì)聯(lián)用（GC-MS）[14]等。隨著技術(shù)的不斷發(fā)展，超高效液相色譜（UPLC）和高分辨質(zhì)譜如四級(jí)桿飛行時(shí)間質(zhì)譜（QTof-MS）由于其分離速度快、進(jìn)樣體積小、準(zhǔn)確度高等優(yōu)點(diǎn)得到廣泛應(yīng)用[15-16]，尤其在指紋圖譜構(gòu)建和差異性代謝產(chǎn)物的分析鑒定方面得到研究者的關(guān)注[17]。其中，基于LC-MS指紋圖譜的非靶標(biāo)代謝組學(xué)可以同時(shí)分析不同產(chǎn)地或者不同品種的樣品中上百種代謝物，并比較其相似點(diǎn)以及差異之處[18-20]。Progenesis QI是新一代的代謝組學(xué)數(shù)據(jù)處理軟件，用于代謝物鑒定和生物標(biāo)志物發(fā)現(xiàn)，可以同時(shí)分析大樣本量的多組分，從色譜數(shù)據(jù)的對(duì)齊到鑒定出具有明顯差異的代謝物，處理速度快[21-22]?！颈狙芯壳腥朦c(diǎn)】目前Progenesis QI在動(dòng)物和藥物的代謝組學(xué)領(lǐng)域應(yīng)用較多，而國(guó)內(nèi)未見應(yīng)用于水果代謝組學(xué)的研究；此外，與UPLC-QToF-MS結(jié)合分析鑒定不同品種橙的差異性代謝物尚未見報(bào)道?！緮M解決的關(guān)鍵問題】基于UPLC-QTof-MS和Progenesis QI建立不同品種橙果實(shí)的指紋圖譜并同時(shí)分析不同品種間的差異性代謝物，篩選出各品種對(duì)應(yīng)的代謝標(biāo)志物，并對(duì)差異性代謝物進(jìn)行鑒定。

1 材料與方法

柑橘材料于2015年采自于中國(guó)農(nóng)業(yè)科學(xué)院柑橘研究所國(guó)家果樹種質(zhì)資源（重慶）柑橘圃，試驗(yàn)在西南大學(xué)園藝園林學(xué)院進(jìn)行。

1.1 樣品信息

為了減少環(huán)境因素對(duì)試驗(yàn)結(jié)果的影響，所有樣品（表1）保持產(chǎn)地相同，選取我國(guó)特有柑橘資源地方品種以及引進(jìn)種植的特色品種8個(gè)，每個(gè)品種均采摘于果實(shí)成熟期，從3株長(zhǎng)勢(shì)一致、健康的果樹上，于樹冠中部外圍隨機(jī)均勻采樣混勻。

表1 試驗(yàn)材料具體信息

1.2 試劑

乙腈（色譜純，美國(guó)sigma公司），甲酸（質(zhì)譜級(jí)，美國(guó)sigma公司），試驗(yàn)用水為Millipore超純水，甲醇（分析純，成都市科龍化工試劑廠）。標(biāo)準(zhǔn)品地奧司明（純度≥98.0%），橙皮苷（純度≥98.0%）和5,6,7,8,3′,4′-六甲氧基黃酮（純度≥98.0%）購買自成都克洛瑪生物科技有限公司（成都，中國(guó)）。5,6,7,4′-四甲氧基黃酮（純度≥98.0%），5,7,8,3′,4′-無甲氧基黃酮（純度≥98.0%）和橘皮素（純度≥98.0%）購自成都思天德生物科技有限公司（成都，中國(guó)）。

1.3 儀器

超聲波清洗儀KQ5200DE（昆山市超聲儀器有限公司）；超純水系統(tǒng)Milli-Q AdvantageA10（美國(guó)Millipore公司）；離心機(jī)TDL-5A（上海菲恰爾分析儀器有限公司）；UPLC-QTof-MS/MS Xevo G2-S超高效液相色譜-四級(jí)桿串聯(lián)飛行時(shí)間高分辨質(zhì)譜儀（美國(guó)Waters公司），配備電噴霧離子源，二元溶劑管理器，自動(dòng)進(jìn)樣系統(tǒng)，柱溫箱，PDA紫外檢測(cè)器；ACQUITY UPLC BEH C18色譜柱（美國(guó)Waters公司）。

1.4 樣品準(zhǔn)備

樣品采摘后，果實(shí)表面用去離子水洗凈、晾干，并分為果皮和果肉兩個(gè)部分，棄去果肉部分。果皮置于40℃鼓風(fēng)干燥箱中，待果皮烘干，約48 h后取出，經(jīng)粉碎機(jī)粉碎，過60目篩，粉末密封于密封袋中，儲(chǔ)存于玻璃干燥器中備用。

精確稱量樣品粉末0.5 g，置于10 mL離心管中，加入7 mL甲醇，渦旋搖勻后，室溫條件下300 W超聲30 min，5 000 r/min下離心15 min，取上清液于25 mL容量瓶中。殘?jiān)偌? mL甲醇重復(fù)提取兩次，合并上清液，最后用甲醇溶液定容至刻度線。提取液用初始流動(dòng)相稀釋10倍，搖勻后過0.22 μm PTFE針式濾器，前3滴棄去，續(xù)濾液置于進(jìn)樣小瓶中以待上機(jī)檢測(cè)。

1.5 液相條件

色譜柱：ACQUITY UPLC BEH C18分析柱（2.1 mm×100 mm，1.7 μm）；柱溫：40℃；進(jìn)樣量2.0 μL；流速設(shè)置為0.4 mL·min-1；流動(dòng)相的組成包括A相和B相，分別試驗(yàn)了含0.01%甲酸的水和含0.01%甲酸的乙腈、含0.01%甲酸的水和含0.01%甲酸的甲醇、含0.1%甲酸的水和含0.1%甲酸的乙腈、含0.1%甲酸的水和含0.1%甲酸的甲醇，最終優(yōu)化為A相：0.01%甲酸的水，B相：含0.01%甲酸的乙腈。采用梯度洗脫，保持A相和B相配比總和為10%：0—3.0 min，5%—15% B；1.0—8.0 min，15%—25% B；8.0—9.0 min，25%—35% B；9.0—13.0 min，35%—45% B；13.0—15.0 min，45%—60% B；15.0—16.0 min，60%—90% B；16.0—18.0 min，90%—90% B；18.0—20.0 min，90%—5% B。檢測(cè)波長(zhǎng)為283 nm和330 nm，波長(zhǎng)掃描范圍200—400 nm。

1.6 質(zhì)譜條件

UPLC-QTof-MS系統(tǒng)使用ESI離子源，在正負(fù)兩種離子模式下全掃描采集數(shù)據(jù)。質(zhì)譜掃描范圍/100—1 000，掃描時(shí)間0.2 s，參數(shù)設(shè)置與文獻(xiàn)一致[15]。

1.7 數(shù)據(jù)分析

使用Unifi軟件采集數(shù)據(jù)并對(duì)潛在標(biāo)志物進(jìn)行分子式匹配。使用Progenesis QI軟件對(duì)采集的數(shù)據(jù)進(jìn)行代謝組學(xué)部分的分析，包括峰對(duì)齊、峰提取、去卷積化、歸一化、多元統(tǒng)計(jì)分析等過程。本研究采用LC-MS技術(shù)建立8種橙果實(shí)代謝輪廓指紋圖譜，并結(jié)合主成分分析（PCA）、偏最小二乘法判別分析（PLS-DA）、變量重要性投影（VIP）等化學(xué)計(jì)量學(xué)方法尋找8個(gè)品種橙果實(shí)之間的代謝差異物，即橙類的潛在分類標(biāo)志物。

2 結(jié)果

2.1 橙的指紋圖譜

使用UPLC-QTof-MS/MS系統(tǒng)對(duì)8個(gè)品種橙果皮的甲醇提取物進(jìn)行分析，優(yōu)化了流動(dòng)相的組成和洗脫梯度。在最優(yōu)的條件下，得到了8種橙果皮的代謝輪廓指紋圖譜。如圖1-A所示，負(fù)離子模式下，8種橙的代謝成分存在差異，如血橙2—3 min出現(xiàn)的峰對(duì)應(yīng)的物質(zhì)種類和相對(duì)含量與其他橙明顯不同；長(zhǎng)葉橙可見兩個(gè)峰分別在12.36 min和11.80 min處，而另外7種橙則沒有。同樣的，正離子模式下（圖1-B），8種橙的代謝指紋圖譜差異明顯，尤其是北碚447錦橙和血橙。北碚447錦橙在保留時(shí)間2—10 min的差異性峰最明顯，血橙在5—6 min和8—9 min之間有非常明顯區(qū)別于其他橙的峰出現(xiàn)。從圖1的指紋圖譜肉眼可見8種橙的代謝組分存在明顯差異，為接下來QI軟件分析差異性代謝物奠定了基礎(chǔ)。

A：負(fù)離子模式；B：正離子模式。a：XUEC，血橙；b：XIAC，夏橙；c：XF，先鋒橙；d：S26，S26錦橙；e：EG，鵝蛋柑；f：CY，長(zhǎng)葉橙；g：BB，北碚447錦橙；h：8045，8045甜橙。*代表已鑒定的差異代謝物。下同

2.2 Progenesis QI分析

在進(jìn)行QI組學(xué)分析之前首先要考察試驗(yàn)數(shù)據(jù)的可靠性。試驗(yàn)樣品一共分為9組，包括一組QC（quality control）樣品和8組橙果實(shí)樣品（分別對(duì)應(yīng)8種橙），每組6個(gè)樣品。正離子模式下所得數(shù)據(jù)使用QI軟件處理得分析變量為13 926，即所得數(shù)據(jù)矩陣為54×13926。從圖2-A和圖2-C可見，QC樣本分布集中，且RSD<15%的變量占56.36%，RSD<30%的變量占81.32%。負(fù)離子模式下數(shù)據(jù)矩陣為54× 12015，QC樣品聚集程度相對(duì)良好，且RSD<15%的化合物占64.80%，RSD<30%的化合物占88.42%（圖2-B、2-D）?？傮w來說，本方法在整個(gè)過程中穩(wěn)定，重復(fù)性良好，適合用于橙類的代謝組學(xué)分析，所得數(shù)據(jù)穩(wěn)定可靠[23-24]。

從圖3-A可以看出，正離子模式下各品種各自聚集在一起，其中血橙、長(zhǎng)葉橙和8045甜橙距離較遠(yuǎn)，說明3種橙果實(shí)的代謝差異較大，而鵝蛋柑、先鋒橙與8045甜橙距離較近，說明這3種橙果實(shí)代謝相似。

A：正離子模式下的PCA圖；B：負(fù)離子模式下的PCA圖；C：正離子模式下RSD值分布；D：負(fù)離子模式下RSD值分布

A：正離子模式下PCA得分圖，其中第一主成分占46.83%，第二主成分占19.39%；B：負(fù)離子模式下PCA得分圖，其中第一主成分占45.83%，第二主成分占20.31%

同樣，北碚447錦橙與夏橙兩種距離相近，說明二者代謝相似。從圖3-B可以看出，各品種在負(fù)離子模式下更為集中，并且得出的聚類結(jié)果與正離子模式下相似：血橙、長(zhǎng)葉橙和8045甜橙距離較遠(yuǎn)，鵝蛋柑、先鋒橙與8045甜橙距離較近，北碚447錦橙與夏橙距離相近。

2.3 差異性代謝物的篩選

以鵝蛋柑為例分析其區(qū)別于其他7種橙的代謝物。先將UPLC-MS/MS數(shù)據(jù)導(dǎo)入Progenesis QI軟件中，以離子強(qiáng)度圖進(jìn)行圖形對(duì)齊數(shù)據(jù)，再進(jìn)行峰提取，保證數(shù)據(jù)的零丟失，自動(dòng)歸屬同一化合物產(chǎn)生的不同加合信號(hào)，再提取MSE數(shù)據(jù)，軟件自動(dòng)辨別和瀏覽離子淌度分離結(jié)果，提取所有滿足條件的組分信號(hào)，得到相應(yīng)的變量信息，數(shù)據(jù)歸一化；再將鵝蛋柑與其他品種分為兩組得OPLS-DA散點(diǎn)圖（圖4-A），鵝蛋柑在第一主成分軸上明顯分開，說明鵝蛋柑有明顯區(qū)別于其他7種橙的代謝成分。模型驗(yàn)證參數(shù)2Y=99%，2Q=98%，說明該模型的可靠性及預(yù)測(cè)性均良好。得到對(duì)應(yīng)的Loadings圖，如圖4-B所示，正軸表示鵝蛋柑，負(fù)軸表示其他品種，一個(gè)點(diǎn)代表一個(gè)變量，離原點(diǎn)越遠(yuǎn)表示該變量在該組含量越高。圖4-C為S-Plot圖，與Loadings圖類似，越是在圖的兩端代表該化合物在該組的含量越高，重復(fù)性越好。VIP圖，是重要變量性圖，通常認(rèn)為VIP值大于1的變量具有統(tǒng)計(jì)學(xué)意義。再參照變量變化趨勢(shì)圖來篩選潛在代謝標(biāo)志物，選擇在鵝蛋柑中含量高，在其他品種中含量低或者為零的變量。如圖4-D所示，該物質(zhì)只在鵝蛋柑中存在，在其他橙果實(shí)中的含量為零。最后將篩選出的變量借助高分辨質(zhì)譜的優(yōu)勢(shì)用Unifi軟件匹配可得較為準(zhǔn)確的分子式。使用同樣的流程篩選找到其他橙正負(fù)離子模式下各自的代謝標(biāo)志物（附表1—16）。

A：EG VS Group的偏最小二乘法判別分析圖，R2Y=99%，R2Q=98%；B：載荷圖；C：S-Plot圖；D：變量變化趨勢(shì)圖

2.4 差異性代謝物的鑒定

根據(jù)保留時(shí)間、精確分子量、二級(jí)碎片、標(biāo)準(zhǔn)品以及數(shù)據(jù)庫和相關(guān)文獻(xiàn)信息，對(duì)上述篩選出的差異性代謝物進(jìn)行鑒定。表2所示的是已經(jīng)鑒定出的部分代謝物分別對(duì)應(yīng)于各個(gè)橙品種?；衔?（RT=13.25 min），化合物7（RT=6.41 min），化合物11（RT=6.54 min），化合物14（RT=12.15 min），化合物15（RT=11.37 min）和化合物17（RT=10.54 min）的鑒定結(jié)果經(jīng)過標(biāo)準(zhǔn)品比對(duì)確認(rèn)（附圖1—6）。其余11種化合物包括了類黃酮-C-糖苷、類黃酮-O-糖苷、多甲氧基黃酮和其他類型的3種化合物（對(duì)應(yīng)的MS和MS/MS信息見附圖7—17）。

據(jù)報(bào)道，類黃酮-C-糖苷中C-糖基化僅發(fā)生在類黃酮骨架中的C-6和C-8位[25]，且黃酮-6-C-糖苷比-8-C-糖苷更容易失水產(chǎn)生碎片[M-H-18]-，該碎片通?？勺鳛殍b定糖基化位點(diǎn)的依據(jù)。此外，類黃酮-C-糖苷容易優(yōu)先發(fā)生糖基內(nèi)部的斷裂，產(chǎn)生[M+H–nH2O]+和[M+H–CH2O–2H2O]+等常見的碎片。鵝蛋柑在保留時(shí)間為5.18 min處的代謝物[M-H]-為461.10986，特征碎片離子341.0666（[(M–H)-C4H8O4]-）說明該化合物為C-糖基-類黃酮，碎片離子299.0537（Y0-）說明該苷元為含有3個(gè)羥基和1個(gè)甲氧基的黃酮。C環(huán)裂解產(chǎn)生的碎片209.0423（[0,4A--C2H4O2]）和167.0332（[0,4A--C3H6O3]）證明該黃酮的A環(huán)上帶有兩個(gè)羥基，而B+C環(huán)上帶有一個(gè)羥基和一個(gè)甲氧基。通過搜索數(shù)據(jù)庫SciFinder和ChemSpider查到兩個(gè)化合物符合上述裂解規(guī)律，分別為金雀花素和葒草素-4-甲基醚，因此該化合物暫定為金雀花素或葒草素-4-甲基醚，但目前的信息不能完全確定其結(jié)構(gòu)，這兩種化合物在柑橘中都有報(bào)道[26]。同理，將先鋒橙在3.68 min出峰的化合物鑒定為香葉木素6,8-二-C-葡萄糖苷。

與類黃酮-C-糖苷不同的是，類黃酮-O-糖苷易發(fā)生糖基的丟失，據(jù)此可判斷糖基的類型，如中性丟失308 Da是典型的O-二糖苷，而中性丟失470 Da則說明該化合物是O-三糖苷。以夏橙在6.55 min處出峰的化合物為例介紹類黃酮-O-糖苷的鑒定。該化合物在正離子模式下的準(zhǔn)分子離子[M+H]+為465.1384，產(chǎn)生的碎片離子如下：303.0862（Y0+）、153.0186（1,3A0+）、177.0548（[0,4B+-H2O]）、345.0964（0,2X+）和369.0966（[0,4X+-H2O]）。其中苷元離子303.0862是丟失一個(gè)己糖162 Da產(chǎn)生的，說明該化合物是O-糖基黃烷酮，帶有3個(gè)羥基和1個(gè)甲氧基。C環(huán)裂解產(chǎn)生的碎片153.0186和177.0548證明A環(huán)上有兩個(gè)羥基取代，B環(huán)上有1個(gè)羥基1個(gè)甲氧基。據(jù)此，該化合物被鑒定為橙皮素-7-O-葡萄糖苷，在Marc.果實(shí)染病的果皮中也有檢出[27]。同理，化合物2、4、16被鑒定為類黃酮-O-糖苷，如表2所示。

表2 部分差異性代謝物的鑒定信息

a保留時(shí)間 Retention time；b經(jīng)過標(biāo)準(zhǔn)品確認(rèn) Confirmed by standard

多甲氧基黃酮通常會(huì)優(yōu)先發(fā)生中性丟失，如nCH3，然后是C環(huán)的斷裂，取代基的分布可從C環(huán)裂解得到的碎片來判斷。正離子模式下，先鋒橙中保留時(shí)間為12.92 min出現(xiàn)的化合物，準(zhǔn)分子離子峰[M+H]+為359.1129，產(chǎn)生一系列的碎片包括326.07742（[M+ H-CH3-H2O]+）、344.08696（[M+H-CH3]+）、211.05952（1,2A+）和163.07469（1,3B+）。通過UNIFI軟件匹配出最接近的分子式為C19H18O7，減去黃酮的基本骨架結(jié)構(gòu)C15H10O2得到差值C4H8O5，說明該化合物為多甲氧基黃酮，其中甲氧基的個(gè)數(shù)等于差值中C的個(gè)數(shù)4，而羥基的個(gè)數(shù)為差值中O的個(gè)數(shù)減去C的個(gè)數(shù)，即為1。然后通過C環(huán)裂解得到的碎片來判斷甲氧基和羥基的取代位點(diǎn)，再通過搜索數(shù)據(jù)庫SciFinder和ChemSpider，將該化合物暫定為5-羥基-6,7,3',4'-四甲氧基黃酮。與之類似，將血橙保留時(shí)間為9.82 min處出峰的化合物鑒定為3-羥基-5,7,3',4'-四甲氧基黃酮。

此外，夏橙在2.47 min出峰的化合物被鑒定為苯噻胺，其特征碎片為101.0250；先鋒橙在7.2 min出峰的化合物是諾米林的同分異構(gòu)體，正離子模式下特征碎片為161.0597、303.0857、487.2322和469.2217，鑒定為7α-檸檬苦醇醋酸酯；鵝蛋柑在8.09 min出峰的化合物也是一種類檸檬苦素，易發(fā)生中性丟失葡萄糖和CO2，鑒定為奧巴叩酮17-O-β-D-葡萄糖苷，之前在柑橘種子中有檢出[28]。

3 討論

Wang等[29]基于LC-MS的非靶標(biāo)代謝組學(xué)對(duì)62份柑橘資源不同組織部位的類黃酮多樣性進(jìn)行了系統(tǒng)的研究，但是該研究只分析檢測(cè)了類黃酮這一類物質(zhì)。STANDER等[30]基于一種新的親水作用液相色譜-質(zhì)譜聯(lián)用（LC-MS）方法對(duì)南非果汁進(jìn)行了研究，其中包括了混合果汁和100%橙汁，研究表明柚皮苷和橙皮苷可以作為穩(wěn)定的標(biāo)志化合物用于果汁的摻假識(shí)別。JANDRI?等[3]基于UPLC-QToF-MS和化學(xué)計(jì)量學(xué)對(duì)柑橘果實(shí)（橙、柚、葡萄柚和寬皮柑橘）、不同來源的橙以及不同類型的果汁進(jìn)行了區(qū)分比較，該研究分析得到的化合物不限于類黃酮，初步鑒定出了對(duì)差異性有貢獻(xiàn)的8種標(biāo)志性化合物，例如檸檬苦素-17-β-D-吡喃葡糖苷、諾米林-17-β-D- 吡喃葡糖苷、蕓香柚皮苷和橙皮苷等，可以用于柑橘果實(shí)/果汁的真?zhèn)舞b別；同時(shí)，該課題組使用靶向和非靶向代謝組學(xué)對(duì)印度柑橘（寬皮柑橘、橙、葡萄柚）進(jìn)行了真?zhèn)舞b別，并對(duì)特征的標(biāo)志化合物進(jìn)行了鑒定，最具影響的標(biāo)志化合物有香風(fēng)草甙、野漆樹苷、異野漆樹苷、新橙皮苷、橙皮苷、柚皮苷、蕓香柚皮苷、檸檬苦素葡萄糖苷和維采寧-2[20]。然而目前國(guó)內(nèi)外的文獻(xiàn)并沒有基于LC-MS的非靶標(biāo)代謝組學(xué)對(duì)我國(guó)特色橙品種在相同產(chǎn)地條件下進(jìn)行差異性代謝產(chǎn)物的分析鑒定。橙品種繁多，風(fēng)味品質(zhì)和營(yíng)養(yǎng)價(jià)值各不相同。與果肉和種子相比，果皮含有更豐富的次生代謝產(chǎn)物，因此，本文選擇了相同產(chǎn)地的8個(gè)品種的橙果皮為研究對(duì)象。

從本研究結(jié)果可以看出，血橙、長(zhǎng)葉橙和8045甜橙的代謝物差異性較大，而北碚447錦橙與夏橙的代謝產(chǎn)物接近。利用本文建立的方法篩選出每種橙區(qū)別于其他7種橙的特征代謝物共幾百種（附表1—16），因柑橘代謝產(chǎn)物數(shù)據(jù)庫尚不完善，差異性代謝物的結(jié)構(gòu)尚不能完全確定。目前，本文已完成了17種差異性代謝物的鑒定。其中通過標(biāo)準(zhǔn)品比對(duì)，確定了6種，分別是夏橙在保留時(shí)間為6.41 min出峰的地奧司明和13.25 min出峰的橘皮素，S26錦橙在6.54 min出峰的橙皮苷，長(zhǎng)葉橙在12.15 min出峰的川陳皮素，北碚447錦橙在11.37 min出峰的5,6,7,4'-四甲氧基黃酮，8045甜橙在10.54 min出峰的異橙黃酮。另外的11種差異性代謝物根據(jù)保留時(shí)間、精確分子量、二級(jí)碎片以及數(shù)據(jù)庫和相關(guān)文獻(xiàn)信息進(jìn)行了鑒定。與已有文獻(xiàn)對(duì)柑橘種間果實(shí)/果汁的分析相比，本研究針對(duì)橙種內(nèi)代謝的差異物進(jìn)行了分析鑒定，對(duì)差異性有貢獻(xiàn)的特征代謝標(biāo)志物除了常規(guī)的類黃酮、檸檬苦素如橙皮苷、香葉木素6,8-二-C-葡萄糖苷和奧巴叩酮17-O-β-D-葡萄糖苷、7α-檸檬苦醇醋酸酯，還有6種多甲氧基類黃酮，包括3-羥基-5,7,3',4'-四甲氧基黃酮、川陳皮素、異橙黃酮、5-羥基-6,7,3',4'-四甲氧基黃酮、5,6,7,4'-四甲氧基黃酮和橘皮素。本文從代謝水平提供了鑒別橙果實(shí)及橙汁的依據(jù)。比如，8045甜橙和S26錦橙全果所榨橙汁的鑒別，可通過檢測(cè)二者的次生代謝產(chǎn)物中有無異橙黃酮和橙皮苷，以及這兩種差異性代謝物的相對(duì)含量來確定該橙汁是否摻假以及摻假的程度。若檢出異橙黃酮，沒檢出橙皮苷，或者二者相對(duì)含量的比值大于1，說明該橙汁是8045甜橙的果汁；反之，則證明該橙汁來自S26錦橙。

4 結(jié)論

本文通過超高效液相色譜-四級(jí)桿飛行時(shí)間質(zhì)譜（UPLC-QToF-MS）得到8種橙果皮的代謝產(chǎn)物指紋圖譜，使用Progenesis QI軟件結(jié)合主成分分析、偏最小二乘判別分析和變量變化趨勢(shì)圖等手段分析篩選出各品種對(duì)應(yīng)的差異性代謝物，據(jù)此得到了區(qū)分/鑒別不同品種橙的代謝依據(jù)。目前僅鑒定出17種代謝標(biāo)志物。其中木犀草素-7-O-新橙皮糖苷和3-羥基-5,7,3',4'-四甲氧基黃酮是血橙區(qū)別于其他7種橙的特征代謝物，橙皮苷是S26錦橙的代謝標(biāo)志物，蕓香柚皮苷-4'-葡萄糖苷和異橙黃酮可以作為8045甜橙的特征代謝物，而夏橙中鑒定出5種區(qū)別于其他橙的化合物，分別為橙皮素-7-O-葡萄糖苷、野漆樹苷-4'-O-葡萄糖苷、橘皮素、苯噻胺和地奧司明。該研究方法和技術(shù)路線具有通用性，可拓展至不同水果、不同產(chǎn)地、不同成熟度等因素引起的代謝物差異分析，為柑橘及其他水果的代謝組學(xué)研究提供一定的借鑒。

附表1 鵝蛋柑正離子模式下區(qū)別于其他橙的代謝物

Table S1 The metabolites in Edangan different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1273.07665.79C15H12O53.236.50.0496 2440.107912.81C21H17N3O8-2.236.4-0.0491 3433.14899.13C22H24O9-0.956.30.0476 4614.17659.13C32H27N3O10-0.736.20.0478 5611.15943.01C27H30O16-2.036.1-0.0466 6796.14873.86C36H25N7O150.746.10.0467 7827.660512.15C16H70N38O2-1.386.0-0.0464 8433.450512.81C29H56N2-2.585.9-0.0459 9367.15071.88C17H22N2O71.925.80.0458 11316.28514.6C18H37NO31.35.7-0.0433 12473.21746.24C26H32O80.845.7-0.0435 13419.133513.17C21H22O9-0.45.7-0.0435 14403.377612.16C24H50O4-1.435.6-0.0432 15419.133614.67C21H22O9-0.125.5-0.0418 16600.16135.79C19H29N5O17-3.065.40.0418 17668.28974.57C29H37N11O8-0.325.3-0.0407 18470.10830.85C23H19NO100.345.20.042 19906.207212.15C39H35N7O191.225.2-0.0398 20449.14396.55C22H24O10-0.725.20.0394 21388.114912.15C19H13N7O3-0.895.0-0.0382 22623.17912.16C19H18N20O6-0.225.0-0.0382 23889.219912.14C30H32N16O17-0.564.9-0.038 24864.494210.95C10H53N39O9-2.264.90.0372 25671.22938.18C29H38N2O16-0.124.8-0.0365 26859.238912.14C39H42N2O20-1.664.8-0.0367

p[1]*小于0.05代表兩組之間有統(tǒng)計(jì)學(xué)差異。下同

p[1]*<0.05 indicated that there are statistical differences between the two groups. The same as below

附表2 鵝蛋柑負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S2 The metabolites in Edangan different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1609.14464.67C27H30O16-2.425.82019-0.0494 2959.24999.66C62H40O110.15.76277-0.0494 3717.23274.49C42H38O11-2.055.69717-0.0484 4977.28137.33C59H46O14-0.145.6897-0.0489 5777.1943.86C28H42O25-0.365.593910.0477 6629.16199.13C41H26O72.125.546220.0475 7579.13414.17C26H28O15-2.565.52214-0.0475 8840.12963.86C55H23NO9-0.495.37510.0462 9678.08415.78C43H13N5O5-0.425.364870.0461 10735.21440.67C34H40O180.295.35637-0.0453 11461.10955.42C22H22O111.225.23974-0.0446 12642.16455.79C36H21N9O40.24.995880.0428 13960.32850.65C49H55NO19-1.144.940280.0411 14461.10995.18C22H22O1124.744-0.0405 15641.13463.01C27H30O18-2.084.68674-0.0399 16633.25258.09C32H42O13-4.414.611540.0393 17719.24596.2C24H48O24-0.524.5986-0.0393 18592.2070.61C21H39NO18-4.194.548760.0382 19649.24469.56C39H38O90.374.49205-0.0384 20839.21324.6C51H36O12-0.264.438050.0379 21737.22970.67C34H42O18-0.214.43357-0.0371 22749.16824.5C44H30O122.294.42497-0.039 23656.17919.13C36H27N5O80.574.403890.0378 24813.23565.31C50H38O111.824.360650.0374

附表3 血橙正離子模式下區(qū)別于其他橙的代謝物

Table S3 The metabolites in Xuecheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1595.20149.13C28H34O14-1.256.379690.0495 2745.208712.19C40H40O14-2.136.28810.0493 3465.1396.55C22H24O11-1.26.21340.0477 4757.25246.55C34H44O19-3.386.205840.0479 5343.12380.62C12H22O110.916.103570.0467 6609.18036.17C28H32O15-1.796.082790.0474 7348.05082.64C19H9NO61.555.99046-0.0458 8343.081411.23C18H14O70.415.913470.047 9359.112712.92C19H18O70.485.887920.0466 10494.25912.75C22H39NO11-0.955.83723-0.0446 11276.14520.94C12H21NO63.665.811750.0431 12596.28994.97C23H37N11O80.015.74448-0.044 13434.23885.14C20H35NO90.75.70059-0.0437 14389.122912.31C20H20O8-0.455.571510.0451 15302.305814.99C18H39NO21.615.533490.0424 16434.23834.99C20H35NO9-0.485.46848-0.0419 17325.07656.59C11H16O110.015.452350.0414 18348.0512.45C20H5N5O2-1.675.30489-0.0402 19359.11279.82C19H18O70.425.269850.0426 20595.16494.9C27H30O15-1.445.235210.0407 21706.13886.59C34H27NO162.135.181720.0396 22642.18062.64C31H31NO14-1.735.15193-0.04 23208.11920.59C9H13N5O-0.425.07335-0.039 24434.23855.43C20H35NO90.035.05729-0.0386 25437.193914.6C26H28O6-4.444.974250.0377 26738.14940.65C31H31NO20-2.434.949210.0374 27580.29584.39C26H45NO13-0.984.92815-0.0379 28628.17523.68C28H32O16-1.864.908440.0383 29750.2556.52C33H45NO17-4.054.908440.0376 30434.23875.26C20H35NO90.654.90625-0.0375

附表4 血橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S4 The metabolites in Xuecheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1563.14014.54C26H28O14-0.995.772880.0477 2533.17170.66C19H34O17-1.155.770190.0483 3677.16546.51C38H30O12-1.625.764430.0494 4647.15625.79C37H28O110.425.686880.0495 5503.16220.61C18H32O160.955.546560.0465 6251.11791.03C16H16N2O-4.315.45523-0.0467 7615.16629.13C37H28O90.35.303450.0464 8845.26910.62C44H46O173.435.285540.0447 9609.23632.43C33H38O113.555.03086-0.043 10563.144.68C26H28O14-1.084.941390.0409 11593.14946.58C27H30O15-3.044.893650.0424 12377.08930.66C18H18O93.984.835280.0426 13629.20445.23C35H34O112.554.811370.0414 14465.19927.37C21H30N4O80.224.78248-0.0409 15577.1555.86C27H30O14-2.254.759290.0417 16507.20857.97C22H36O130.44.52032-0.0385 17633.337613.44C33H50N2O10-2.624.51162-0.0386 18785.2229.87C45H38O13-2.514.49310.0378 19529.21354.42C21H38O15-0.484.48159-0.0381 20707.14536.6C31H32O19-1.774.475770.0385

附表5 夏橙正離子模式下區(qū)別于其他橙的代謝物

Table S5 The metabolites in Xiacheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1274.274812.72C16H35NO22.616.969310.0458 2318.300512.91C18H39NO30.66.606570.0443 3757.25156.55C34H44O19-4.576.507680.0419 4465.13846.55C22H24O11-1.536.403190.0428 5488.19143.82C25H29NO9-0.36.37004-0.0494 6349.0221.43C16H4N4O64.716.35074-0.0492 7533.08252.7C38H12O43.056.18916-0.0472 8371.15310.84C14H26O11-4.676.16312-0.0472 9936.20071.43C18H25N29O180.26.14902-0.0484 10409.19732.39C20H28N2O70.926.09424-0.0486 11581.18655.79C27H32O14-0.026.087350.0381 12228.0770.85C12H9N3O21.096.07341-0.048 13470.1080.82C23H19NO10-0.375.92974-0.0466 14630.17056.52C14H15N25O6-0.785.923980.0408 15675.26036.24C29H42N2O16-0.565.91328-0.0432 16449.17895.38C20H20N10O3-0.855.786680.0468 17344.13450.82C15H21NO81.365.706310.0432 18414.04540.6C19H11NO10-0.465.685990.0408 19332.12410.85C16H17N3O50.15.68472-0.0454 20574.19822.24C21H35NO170.795.67981-0.0443 21268.10330.61C10H13N5O4-2.925.67642-0.0451 22741.2213.78C33H40O19-3.595.59691-0.043 23393.10322.7C15H20O121.145.59395-0.0427 24378.03882.42C23H7NO5-2.415.5455-0.0431 25473.20234.69C22H32O111.235.464730.0433 26373.128413.25C20H20O70.545.4455-0.0294 27490.20785.98C25H31NO91.275.43257-0.0426 28298.06320.61C6H11N5O90.665.386350.0393 29375.09232.7C15H18O110.255.34517-0.0407

附表6 夏橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S6 The metabolites in Xiacheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1593.15014.91C27H30O15-1.846.685020.0499 2563.14044.54C26H28O14-0.495.917070.047 3798.27960.64C36H49NO19-3.775.861950.0489 4443.19422.47C21H32O104.325.860480.0498 5754.25470.6C31H37N11O12-0.445.77027-0.0488 6983.31313.83C55H52O17-0.065.73028-0.0478 7339.203515.04C15H32O83.085.724710.0473 8627.17493.36C24H36O19-4.685.70632-0.0481 9521.17220.62C18H34O17-0.225.67730.0461 10607.16576.41C28H32O15-1.955.652370.0392 11915.26775.66C42H48N2O2105.637510.0489 12536.04662.67C38H7N3O20.045.62954-0.0475 13791.25424.62C41H44O16-1.85.62789-0.048 14669.27255.23C27H46N2O170.255.6263-0.0479 15907.31139.15C57H48O11-1.155.42851-0.046 16770.24950.6C47H37N3O8-1.675.40896-0.0448 17917.26127.48C57H42O120.975.40768-0.0444 18831.32044.45C40H52N2O171.35.32064-0.0464 19779.19576.89C49H32O104.335.31993-0.046 20565.19215.04C27H34O13-1.035.16505-0.044 21993.3694.59C42H58N8O20-0.455.14148-0.0436 22672.17476.59C37H23N9O5-0.435.114580.0425 23597.18085.2C27H34O15-2.835.091980.0415 24885.25559.54C53H42O130.225.0656-0.0456 25623.15993.68C28H32O16-2.955.064310.0334 26563.13994.68C26H28O14-1.315.032430.0395

附表7 先鋒橙正離子模式下區(qū)別于其他橙的代謝物

Table S7 The metabolites in Xianfengcheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1310.12931.37C15H19NO62.586.47085-0.0494 2815.22236C36H38N4O18-3.796.3635-0.0494 3316.285214.6C18H37NO31.846.21688-0.0476 4412.14632.55C16H21N5O8-0.076.189-0.0481 5417.17555.17C19H28O100.046.11487-0.0471 6359.112912.92C19H18O71.016.02298-0.0467 7515.22757.2C28H34O9-0.15.923410.0474 8668.28954.57C29H37N11O8-0.725.869810.0461 9630.1716.52C16H27N11O1605.74865-0.0449 10302.30614.99C18H39NO22.145.73061-0.0451 11475.17630.62C28H26O72.515.6603-0.0429 12565.15514.69C26H28O14-0.185.58823-0.0422 13280.13980.68C11H21NO72.625.52591-0.0428 14704.17474.57C39H29NO12-2.245.401940.0427 15419.133714.67C21H22O90.035.33505-0.0408 16797.21014.32C31H36N6O19-0.895.32334-0.0408 17423.16292.9C21H26O9-4.835.31733-0.0411 18473.20174.69C22H32O11-0.15.31567-0.0417 19845.23386.13C37H40N4O19-2.555.25196-0.0404 20377.19241.21C16H28N2O81.355.24788-0.0404 21258.13460.94C12H19NO53.975.15397-0.0397 22625.17573.68C28H32O16-0.955.10405-0.0384 23307.17723.19C15H22N4O32.494.951130.0379 24419.13365.78C21H22O9-0.164.947990.0371 25388.114912.15C19H13N7O3-1.044.94553-0.0376 26313.071212.22C17H12O61.684.91494-0.0387 27314.09280.88C19H11N3O21.24.8526-0.0371

附表8 先鋒橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S8 The metabolites in Xianfengcheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1563.14034.68C26H28O14-0.675.74521-0.0482 2461.09870.83C24H18N2O8-0.715.68999-0.0484 3765.1814.19C41H34O15-1.945.57051-0.0485 4651.26326.2C32H44O14-3.995.443480.041 5947.27047.69C58H44O13-0.555.24041-0.0457 6863.23050.82C57H36O92.135.151010.0464 7761.25677.18C26H50O25-0.175.044080.0436 8282.08890.88C15H13N3O31.644.93064-0.0411 9471.1884.65C22H32O111.754.81185-0.039 10605.25754.51C31H42O12-0.724.800820.0412 11739.276210.48C42H44O120.224.726660.0417 12615.14615.79C40H24O71.974.696260.0437 13604.17190.66C34H27N3O8-1.14.66582-0.0402 14843.21015.73C50H36O132.14.58595-0.0408 15843.21065.83C50H36O132.754.57123-0.0396 16737.23020.67C34H42O180.534.5415-0.0369 17609.14393.01C27H30O16-3.554.52018-0.0376 18815.26860.62C47H44O13-2.794.46388-0.0368 19631.16156.51C37H28O100.874.45593-0.0399 20607.16546.41C28H32O15-2.344.44172-0.0363 21997.34295.78C60H54O14-1.234.43110.0404 22513.12553.77C22H26O140.974.36806-0.0383 23498.0822.9C33H13N3O31.774.35709-0.0364 24647.15615.79C37H28O110.334.350060.038

附表9 S26錦橙正離子模式下區(qū)別于其他橙的代謝物

Table S9 The metabolites in S26 Jincheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1449.10415.33C16H20N2O13-1.046.58198-0.0489 2575.26490.51C34H38O81.686.4651-0.0494 3644.32663.63C31H49NO13-1.646.27628-0.0479 4316.284914.6C18H37NO30.766.273780.0472 5504.20761.21C22H33NO120.146.16917-0.0461 6570.21782.16C26H35NO13-0.576.13673-0.0466 7581.18523.86C27H32O14-2.236.11955-0.0448 8345.09686.56C18H16O7-0.326.04895-0.0461 9728.39659.72C35H57N3O130.126.03694-0.0443 10413.12236.55C22H20O8-2.056.01068-0.0458 11310.12941.37C15H19NO62.745.952710.0463 12917.28845.65C33H40N16O160.335.94233-0.0436 13290.07670.61C13H11N3O5-1.515.84237-0.0449 14883.25620.63C30H38N14O180.125.8321-0.0457 15518.324915.63C28H43N3O64.655.79944-0.0449 16474.358415.26C29H47NO41.385.73263-0.0439 17790.27384.6C30H43N7O180.065.72193-0.0426 18330.15530.67C15H23NO71.865.71534-0.0432 19611.55096.55C25H66N14O3-0.965.57709-0.0425 20262.12940.68C11H19NO63.365.541040.0435 21470.10870.85C24H15N5O6-1.75.513420.0434 22298.06340.61C6H11N5O91.645.48281-0.0427 23611.19646.54C28H34O15-0.985.43932-0.0422 24406.10364.6C6H11N15O7-0.555.36619-0.0394 25396.0350.66C19H9NO9-0.045.31618-0.041 26342.140.58C12H23NO101.665.29799-0.0405

附表10 S26錦橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S10 The metabolites in S26 Jincheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1807.20374.6C29H44O26-1.385.90536-0.05 2879.20695.31C53H36O13-1.575.89934-0.0488 3777.19423.86C28H42O25-0.125.87804-0.0488 4661.17139.13C38H30O11-0.425.65283-0.0471 5840.1293.86C56H19N5O5-2.765.61629-0.0465 6821.2634.32C49H42O123.255.6064-0.0467 7565.19195.04C27H34O13-1.35.4034-0.0443 8647.15595.79C37H28O110.045.3153-0.0439 9871.17273.86C43H36O200.014.9828-0.0412 10429.17854.66C20H30O104.464.91269-0.0422 11443.1942.47C21H32O103.924.875-0.042 12855.17784.61C43H36O1904.84272-0.0408 13521.17290.62C18H34O171.094.79995-0.0411 14593.14996.58C27H30O15-2.144.79147-0.041 15975.25013.85C55H44O17-0.444.77005-0.0436 16871.24188.81C45H44O18-4.254.73234-0.0421 17613.21084.27C28H38O15-4.874.70006-0.0385 18739.20255.34C40H36O14-0.934.69833-0.0399 19708.09266.61C44H15N5O6-3.274.69533-0.0415 20960.33010.65C49H55NO190.574.68043-0.044 21683.28726.48C40H44O101.494.62997-0.0394 22908.475710.02C45H71N3O16-0.464.60686-0.0384 23432.00150.82C27H3N3O4-8.384.56015-0.0389 24862.28710.66C51H45NO120.234.54462-0.0426 25505.1355.31C24H26O12-0.364.42791-0.0365

附表11 長(zhǎng)葉橙正離子模式下區(qū)別于其他橙的代謝物

Table S11 The metabolites in Changyecheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1600.16195.79C16H21N15O110.286.60931-0.0498 2676.13055.78C34H21N5O11-0.766.55517-0.0497 3325.07686.59C11H16O110.656.35936-0.0494 4343.12240.62C12H22O11-3.246.26185-0.0486 5787.24219.87C38H42O18-2.96.16386-0.0466 6218.21237.18C12H27NO23.666.12303-0.048 7415.211814.6C24H30O60.696.08933-0.0476 8246.243910.35C14H31NO24.676.03608-0.0473 9728.39579.72C33H45N17O3-1.016.0136-0.0464 10262.12890.68C11H19NO61.336.00632-0.0461 11367.15041.88C17H22N2O71.215.99204-0.0465 12433.14939.13C22H24O9-0.145.94617-0.0447 13486.19661.25C22H31NO11-0.825.88105-0.0456 14359.112712.92C19H18O70.595.866140.0454 15343.118612.22C19H18O635.825560.0477 16230.248812.81C14H31NO4.15.82435-0.0456 17330.15480.67C15H23NO70.165.80685-0.0453 18455.20637.21C26H30O7-0.375.76497-0.0437 19345.09676.56C18H16O7-0.465.63918-0.0439 20413.12246.55C22H20O8-1.615.62896-0.0439 21766.21227.43C39H23N15O4-1.025.61459-0.0426 22706.13926.59C31H19N11O100.395.51103-0.0432 23640.345215.98C32H45N7O7-0.185.48047-0.043 24403.139512.15C21H22O81.95.40725-0.0442 25714.26417.19C19H31N21O100.85.40457-0.0409 26730.32737.44C34H51NO16-1.035.34035-0.0405 27518.32415.63C29H39N7O20.295.31737-0.0417 28614.17729.13C17H23N15O11-0.455.30624-0.0397 29367.12377.2C14H22O110.635.30548-0.0403 30533.23797.19C28H36O10-0.445.28208-0.0402 31879.322810.24C38H50N6O18-3.065.27022-0.0402 32376.12977.43C9H21N5O11-3.635.24585-0.0399

附表12 長(zhǎng)葉橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S12 The metabolites in Changyecheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1901.32510.23C39H50N8O173.155.8668-0.05 2453.193712.36C26H30O74.065.85911-0.05 3671.22724.49C36H36N2O113.765.72727-0.0499 4311.172315.93C13H28O83.615.67179-0.0493 5631.16166.61C37H28O100.935.65196-0.0488 6645.15626.59C24H30N4O174.45.49901-0.0477 7663.12065.79C29H28O180.475.20666-0.045 8632.08135.78C30H15N7O100.865.18437-0.0447 9630.20050.82C36H25N9O3-0.385.06974-0.0432 10912.39286.09C36H67NO25-0.155.00598-0.0427 11623.19537.95C29H36O15-4.494.94388-0.0424 12607.27387.4C31H44O12-3.634.90629-0.0418 13977.28077.33C59H46O14-0.764.856110.0415 14672.17426.59C23H35N3O200.14.81385-0.0419 15638.328315.97C31H49N3O11-1.714.80725-0.0413 16839.2154.6C51H36O121.884.798830.0409 17807.20244.6C54H32O8-0.14.788790.0406 18645.18480.84C31H34O153.554.71028-0.0402 19711.28117.17C41H44O11-0.044.68576-0.0399 20798.28090.64C31H49N3O212.884.65335-0.0397 21693.270310.59C41H42O10-0.394.56582-0.0389 22791.23677.18C44H40O142.714.51481-0.0385 23629.1629.13C41H26O72.184.49283-0.0389 24642.16485.79C23H29N7O15-0.174.47681-0.0387 25632.20130.68C36H31N3O8-4.014.39239-0.0374 26749.24277.41C39H42O15-3.244.31848-0.0367 27937.31989.24C40H58O250.364.254370.0366 28891.21047.19C54H36O132.294.20534-0.0358

附表13 北碚447正離子模式下區(qū)別于其他橙的代謝物

Table S13 The metabolites in Beibei 447 different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1295.11390.61C10H18N2O81.016.691560.0481 2397.19722.57C19H28N2O70.596.49125-0.0494 3376.038514.47C11H5N9O70.036.39979-0.0486 4344.13410.82C15H21NO80.46.379440.0481 5467.17575.23C19H30O13-0.516.24184-0.0472 6314.160.67C15H23NO60.666.082650.0449 7630.17046.52C16H27N11O16-0.915.957910.0442 8259.0930.61C10H14N2O62.085.904460.0418 9280.13960.68C11H21NO71.735.865630.046 10250.09260.61C9H15NO71.975.673160.0436 11343.118111.37C19H18O61.425.657190.049 12343.12350.62C12H22O110.045.633470.0412 13625.17563.68C28H32O16-1.145.562010.0442 14277.10320.61C10H16N2O70.525.527670.0393 15246.243910.35C14H31NO24.635.507680.0437 16467.18882.33C19H26N6O80.735.4848-0.0412 17415.211614.6C24H30O60.115.476730.0388 18511.20184.22C21H34O14-0.615.44497-0.041 19326.12360.82C15H19NO70.445.432130.0406 20503.21019.35C17H18N201.045.42297-0.0412 21395.20386.69C18H34O90.145.34499-0.0405 22247.13365.39C15H18O33.125.34028-0.0413 23511.20123.97C21H34O14-1.855.22583-0.0391 24362.327213.07C20H43NO42.065.223640.0415 25698.17093.72C33H31NO16-0.995.20769-0.0396 26359.18181.5C16H26N2O71.515.179750.0388 27419.13385.8C21H22O90.395.12756-0.0389 28481.22928.09C21H36O122.495.06653-0.0385 29368.19197.8C15H29NO91.185.03442-0.0381

附表14 北碚447負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S14 The metabolites in Beibei 447 different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1515.193511.37C27H32O102.325.971780.0423 2403.16224.25C18H28O103.045.80393-0.0493 3672.17426.59C21H23N17O100.115.76580.05 4369.15627.57C18H26O81.785.4549-0.0465 5635.21220.61C34H36O12-1.935.302970.0486 6439.226.69C19H36O113.515.29236-0.0446 7805.34077.72C39H54N2O160.765.28646-0.045 8423.18943.38C18H32O111.215.24883-0.0435 9777.2240.84C36H42O19-0.975.22820.038 10677.16546.51C38H30O12-1.525.189540.0454 11447.15213.41C19H28O122.795.179-0.0441 12461.09860.83C24H18N2O8-0.865.132050.0446 13592.20760.61C21H39NO18-3.085.121330.045 14423.18673.16C18H32O11-1.145.1065-0.0423 15411.18824.83C17H32O112.465.09322-0.0432 16726.3789.72C30H57N5O150.225.089570.0445 17293.1030.6C15H18O6-0.285.080030.046 18912.39326.09C38H59N9O17-2.615.009130.0435 19435.18846.93C19H32O112.674.96371-0.0419 20707.14476.6C31H32O19-2.594.915340.0432 21439.21997.02C20H32N4O70.134.84335-0.0409 22737.23040.67C34H42O180.714.695190.0386 23721.28417.74C39H46O13-3.444.59857-0.0391 24607.16526.17C28H32O15-2.654.542790.0356 25591.19133.63C25H36O16-3.024.49702-0.0388 26604.17160.66C34H27N3O8-1.624.460730.0384

附表15 8045甜橙正離子模式下區(qū)別于其他橙的代謝物

Table S15 The metabolites in 8045 Tiancheng different from those in the other oranges in the positive ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1403.13911.01C21H22O80.616.73686-0.0492 2373.306911.23C20H40N2O42.166.30116-0.048 3274.27512.72C16H35NO23.356.28815-0.0487 4303.08656.55C16H14O60.556.27881-0.0469 5767.228812.19C37H38N2O16-0.786.25069-0.0487 6470.10790.85C23H19NO10-0.676.101870.0474 7230.13960.94C11H19NO43.866.04968-0.0466 8675.26066.24C29H42N2O16-0.226.009490.0455 9316.285214.6C18H37NO31.685.93551-0.0453 10379.20761.26C16H30N2O80.345.92258-0.0456 11389.123612.31C20H20O81.235.65466-0.0416 12413.21290.5C16H32N2O10-0.255.647760.0452 13409.18190.92C16H28N2O100.615.56839-0.0429 14478.337214.18C24H47NO8-0.495.41434-0.0414 15318.300912.91C18H39NO31.865.40105-0.0414 16393.22332.33C17H32N2O80.335.27559-0.0404 17409.19663.28C20H28N2O7-0.885.253640.0409 18925.25728.07C38H40N10O18-2.475.24354-0.0406 19412.14642.55C16H21N5O80.295.19921-0.0411 20351.17641.21C14H26N2O80.455.18822-0.0401 21328.15479.05C19H21NO41.095.1572-0.0395 22373.128410.54C20H20O70.595.11595-0.0389 23212.02099.63C9HN5O22.895.03473-0.0389 24339.086511.23C19H14O60.614.99968-0.0384 25704.17534.57C39H29NO12-1.314.99680.0399 26381.18660.82C15H28N2O9-0.394.96277-0.0382 27373.092511.83C19H16O81.924.95627-0.0371 28704.395310.59C33H57N3O13-1.614.929360.0363 29668.29044.57C32H45NO14-1.384.922440.0392 30609.18096.17C28H32O15-0.854.80608-0.0368

附表16 8045甜橙負(fù)離子模式下區(qū)別于其他橙的代謝物

Table S16 The metabolites in 8045 Tiancheng different from those in the other oranges in the negative ion mode

編號(hào)Num質(zhì)荷比m/z保留時(shí)間Retention time (min)分子式Formula質(zhì)量誤差Mass error(ppm)變量重要性投影VIP[1]p[1]* 1741.21863.86C40H38O14-0.326.29005-0.0473 2633.25258.09C32H42O13-4.365.714530.0465 3719.24646.2C24H48O240.125.687420.0488 4845.26850.62C44H46O170.025.62835-0.0495 5403.16254.25C18H28O103.765.433790.0489 6638.19610.93C25H37NO183.675.33017-0.0473 7813.2375.31C39H38N6O14-0.425.29398-0.0407 8585.2860.93C36H42O70.415.26986-0.0465 9485.15260.85C18H30O152.855.23309-0.0481 10429.17745.85C20H30O101.95.008690.0435 11631.22035.41C35H36O112.94.95879-0.0426 12807.20414.6C54H32O83.444.93239-0.0402 13839.21414.6C36H32N12O130.234.87234-0.0396 14205.040.68C13H6N2O44.84622-0.0419 15733.19854.52C34H38O18-0.054.842430.0432 16795.19098.58C42H36O16-2.674.77309-0.0408 17425.18375.39C21H30O94.684.754460.0431 18563.17658.94C27H32O13-0.914.7076-0.0393 19311.172115.93C13H28O83.074.68871-0.0408 20645.15666.59C29H30N2O15-1.154.55511-0.039 21777.22297.18C36H42O19-2.334.477170.0373

附圖1 正離子模式下橘皮素標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S1 The MSEspectra of tangeretin standard in the postitive ion mode: (A) MS, (B) MS/MS

附圖2 負(fù)離子模式下地奧司明標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S2 The MSEspectra of diosmin standard in the negative ion mode: (A) MS, (B) MS/MS

附圖3 負(fù)離子模式下橙皮苷標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S3 The MSEspectra of hesperidin standard in the negative ion mode: (A) MS, (B) MS/MS

附圖4 正離子模式下川陳皮素標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S4 The MSEspectra of nobiletin standard in the positive ion mode: (A) MS, (B) MS/MS

附圖5 正離子模式下5,6,7,4'-四甲氧基黃酮標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S5 The MSEspectra of 5,6,7,4'-tetramethoxyflavone standard in the positive ion mode: (A) MS, (B) MS/MS

附圖6 正離子模式下異橙黃酮標(biāo)準(zhǔn)品的MSE圖：（A）MS（B）MS/MS

Fig. S6 The MSEspectra of isosinensetin standard in the positive ion mode: (A) MS, (B) MS/MS

附圖7 正離子模式下3-羥基-5,7,3',4'-四甲氧基黃酮的MSE圖：（A）MS（B）MS/MS

Fig. S7 The MSEspectra of 3-hydroxy-5,7,3',4'-tetramethoxyflavone in the positive ion mode: (A) MS, (B) MS/MS

附圖8 負(fù)離子模式下木犀草素-7-O-新橙皮糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S8 The MSEspectra of luteolin-7-O-neohesperidoside in the negative ion mode: (A) MS, (B) MS/MS

附圖9 正離子模式下橙皮素-7-O-葡萄糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S9 The MSEspectra of hesperetin-7-O-glucoside in the positive ion mode: (A) MS, (B) MS/MS

附圖10 正離子模式下野漆樹苷-4'-O-葡萄糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S10 The MSEspectra of rhoifolin 4'-O-glucoside in the positive ion mode: (A) MS, (B) MS/MS

附圖11 負(fù)離子模式下苯噻胺的MSE圖：（A）MS（B）MS/MS

Fig. S11 The MSEspectra of penstemide in the negative ion mode: (A) MS, (B) MS/MS

附圖12 正離子模式下5-羥基-6,7,3',4'-四甲氧基黃酮的MSE圖：（A）MS（B）MS/MS

Fig. S12 The MSEspectra of 5-hydroxy-6,7,3',4'-tetramethoxyflavone in the positive ion mode: (A) MS, (B) MS/MS

附圖13 正離子模式下7α-檸檬苦醇醋酸酯的MSE圖：（A）MS（B）MS/MS

Fig. S13 The MSEspectra of 7α-limonyl acetate in the positive ion mode: (A) MS, (B) MS/MS

附圖14 正離子模式下香葉木素6,8-di二-C-葡萄糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S14 The MSEspectra of diosmetin 6,8-di-C-glucosiden the positive ion mode: (A) MS, (B) MS/MS

附圖15 正離子模式下金雀花素的MSE圖：（A）MS（B）MS/MS

Fig. S15 The MSEspectra of scoparin in the positive ion mode: (A) MS, (B) MS/MS

附圖16 負(fù)離子模式下奧巴叩酮17-O-β-D-葡萄糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S16 The MSEspectra of obacunone 17-O-β-D-glucoside in the negative ion mode: (A) MS, (B) MS/MS

附圖17 負(fù)離子模式下蕓香柚皮苷-4'-葡萄糖苷的MSE圖：（A）MS（B）MS/MS

Fig. S17 The MSEspectra of narirutin-4'-glucoside in the negative ion mode: (A) MS, (B) MS/MS

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（責(zé)任編輯 趙伶俐）

Analysis of the Fingerprints of Different Orange Varieties and Their Differential Metabolites Based on Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry and Progenesis QI

ZHAO XiJuan, ZHAO WuJi, XU HuaChao

(Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education/College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715)

【Objective】The objective of this study is to establish an efficient method to investigate the differences between different varieties of oranges, and to find their differential metabolites, which will serve the citrus metabolomics and provide reference for the identification of oranges and orange juice. 【Method】8 orange cultivars from the same area were chosen to obtain the methanol extracts of their peels, and then based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS), the LC-MS fingerprints of the 8 oranges were obtained with obvious differences. Combined with the software Progenesis QI, the differential metabolites were screened in positive and negative modes through principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) as well as loadings plots and VIP (variable importance for projection) values. Furthermore, the differential metabolites were identified based on retention times, accurate mass, MS/MS fragments, reference standards and public databases.【Result】Based on the established method, differential metabolites of the 8 orange cultivars were screened. 17 compounds had been identified and among them, and 6 compounds were confirmed by comparison with their commercial standards. 3-Hydroxy-5,7,3',4'-tetramethoxyflavone and luteolin-7-O-neohesperidoside were the characteristic metabolites of Xuecheng. Hesperidin was the marker compound of S26 Jincheng. Narirutin-4'-glucoside and Isosinensetin could act as the characteristic metabolites of 8045 Tiancheng. 【Conclusion】The method was versatile and suitable for the analysis of sample differences caused by varieties, geographical origins and maturity.

ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry; orange; Progenesis QI; metabolites

2017-12-14；

2018-03-26

中央高?；究蒲袠I(yè)務(wù)費(fèi)（XDJK2016B014，XDJK2017D093和SWU115065）

通信作者趙希娟，E-mail：xijuanzh@swu.edu.cn

10.3864/j.issn.0578-1752.2018.13.010