李 毅，朱騰高，王國鳳，陳煥文

(東華理工大學(xué)，江西省質(zhì)譜科學(xué)與儀器重點(diǎn)實(shí)驗(yàn)室，江西 南昌 330013)

單次掃描檢測大表面痕量賴氨酸和黑索今的質(zhì)譜分析方法

李 毅，朱騰高，王國鳳，陳煥文

(東華理工大學(xué)，江西省質(zhì)譜科學(xué)與儀器重點(diǎn)實(shí)驗(yàn)室，江西 南昌 330013)

為了進(jìn)一步提高大表面樣品的分析速度，減少給定樣品表面的掃描次數(shù)，建立了一種單次掃描即可檢測分布于大表面樣品上任意位置痕量待測物的質(zhì)譜分析方法。以人造革表面(4 cm×4 cm)添加的賴氨酸和黑索今為代表性檢測對象，用一塊無毛刺等邊三角形金屬銅電極(邊長為8.6 cm)緊貼在樣品表面，將甲醇-水(3∶7，V/V)電離試劑涂灑在金屬電極表面，讓其與樣品表面保持濕潤接觸，并使三角形電極尖端對準(zhǔn)質(zhì)譜儀的離子入口；然后在金屬電極上施加+5.8 kV高壓，在電場的驅(qū)動下，濕潤表面的待測物朝著質(zhì)譜入口移動，并在三角形電極尖端形成待測物離子進(jìn)入質(zhì)譜儀檢測。結(jié)果表明，該方法可單次掃描檢測隨機(jī)分布在皮革樣品表面上任意位置的非均勻分布的賴氨酸和黑索今，檢測限可達(dá)6.2×10-7μg/cm2，分析單個樣品表面的時間不到2 s；與采用電噴霧解吸電離質(zhì)譜等順次掃描檢測(采樣面積不到1 mm2)的方法相比，本方法的分析速度提高了1 000倍。

單次掃描；大表面樣品；非均勻分布；常壓質(zhì)譜；賴氨酸；黑索今

Abstract: Ambient ionization mass spectrometry has significantly improved the efficiency of analysis complex matrix sample due to its high sensitivity, selectivity and throughput. At present, direct ionization sources coupled with mass spectrometer including desorption electrospray ionization (DESI), real time online (DART), matrix assisted laser desorption ionization (MALDI), laser ablation electrospray ionization (LAESI), desorption atmospheric pressure chemical ionization (DAPCI), extractive electrospray ionization (EESI) etc. are applied for the direct ionization targeting molecular followed by mass spectrometer characterization. A famous ionization technique namely DESI is the most representative ambient source, which can ionization the polar compounds with bulk solid surface focused on a large number of studies on the detection methods of explosives. However, DESI in the ionization of solid explosives is needed continuous scanning and a single sampling area can not exceeding 1 cm2, resulting in time-consuming and inaccurate enough results. In order to further improve the analysis rate of the large surface sample and decrease the number of scanning for a given sample surface, a novel mass spectrometry method with a single scan was established for detecting trace substance distributed in large sample surface of any position. Using artificial leather surface (length of a side: 4 cm×4 cm) added lysine and royal demolition explosive (RDX) as representative detection object, the triangular metal copper electrode (length of a side: 8.6 cm) which was applied the ionization regent with methanol-water (3∶7,V/V) was closed to the sample surface, making the tip of the triangle metal copper electrode on the mass spectrometer entrance. The trace substance form into ions in triangle electrode tip towards mass spectrometry when applying +5.8 kV high voltage on the metal copper electrode. This experimental results show that the novel method with a single scan can detect randomly and non-uniform distributed on the surface of the leather sample of lysine and/or RDX at any position, providing the speed for a single sample analysis within 2 s, the limit of detection (LOD) of 6.2×10-7μg/cm2. compared with analyzing speeding on desorption electrospray ionization mass spectrometry (single sampling area less than 1 cm2), the analyzing speed of the established method is 1 000 times than DESI. Overall, this method can provide a promising analytical tool for advanced studies on monitoring of trace explosive, the identification of the food quality and the products in the import and export trade, and the pharmacokinetics of drug metabolism in vivo.

Keywords： single scan; large surface sample; non-uniform distribution; atmospheric pressure mass spectrometry; lysine; royal demolition explosive (RDX)

直接質(zhì)譜分析技術(shù)能夠提高分析效率，拓展應(yīng)用領(lǐng)域，是質(zhì)譜學(xué)研究的前沿和熱點(diǎn)[1]。目前，常見的直接離子化技術(shù)，如電噴霧解吸電離(DESI)[2-4]、實(shí)時在線分析(DART)[5-7]、基質(zhì)輔助激光解吸電離(MALDI)[8-9]、激光消融電噴霧電離(LAESI)[10]、電噴霧輔助激光解吸電離(ELDI)[11]、表面解吸常壓化學(xué)電離(DAPCI)[12-14]和電噴霧萃取電離(EESI)[15-17]等越來越多地應(yīng)用于復(fù)雜基體樣品的直接分析，這些方法具有樣品預(yù)處理簡單、消耗量少、響應(yīng)速度快、靈敏度高等特點(diǎn)。在諸多的直接離子化技術(shù)中，Cooks教授發(fā)明的DESI是該領(lǐng)域最具代表性的方法，其優(yōu)點(diǎn)是能夠直接檢測塊狀固體表面的極性物質(zhì)，在爆炸物檢測方面有大量的研究報道。然而，DESI在檢測固體表面分布不均的爆炸物時，需要多次連續(xù)地掃描固體表面的多個位點(diǎn)，且單次采樣面積不大于1 cm2，檢測范圍有限，對整個樣品進(jìn)行分析不僅耗時長，還可能存在漏檢、檢測結(jié)果不夠準(zhǔn)確的情況。

本研究擬建立一種單次掃描即可檢測分布于大表面樣品上任意位置痕量待測物的質(zhì)譜分析方法，單次掃描檢測隨機(jī)分布在皮革樣品表面任意位置非均勻分布的賴氨酸和黑索今。希望該方法能夠解決其他常壓電離技術(shù)單次檢測面積小、漏檢、檢測效率較低等問題，同時為行李包上痕量爆炸物的監(jiān)測、食品的品質(zhì)鑒定、進(jìn)出口貿(mào)易中商品的鑒定等提供新的研究思路。

1 實(shí)驗(yàn)部分

1.1 主要儀器與裝置

LTQ-XL型線性離子阱質(zhì)譜儀：美國Finnigan公司產(chǎn)品，配有Xcalibur2.0數(shù)據(jù)處理系統(tǒng)。

1.2 主要材料與試劑

賴氨酸標(biāo)準(zhǔn)品：上海藍(lán)季科技發(fā)展有限公司產(chǎn)品；黑索今(RDX)：由公安部第三研究所提供；甲醇(色譜純)：美國Tedia公司產(chǎn)品；超純水(電阻率18.2 MΩ·cm)：美國Thermo Scientific公司產(chǎn)品。

1.3 實(shí)驗(yàn)方法

設(shè)置LTQ-MS正離子檢測模式，放電電壓+5.8 kV，離子傳輸管溫度200 ℃，質(zhì)量掃描范圍m/z50～400。單次掃描金屬電極離子源裝置示于圖1。離子的選擇窗口為1.0 u，碰撞能量為13%～25%，其他參數(shù)由LTQ-MS系統(tǒng)自動優(yōu)化得到，實(shí)驗(yàn)過程無需對任何樣品進(jìn)行預(yù)處理，以人造革表面(4 cm×4 cm)添加的賴氨酸和黑索今為代表性檢測對象，將一塊無毛刺等邊三角形金屬銅電極(邊長為8.6 cm)緊貼在賴氨酸或黑索今表面，將甲醇-水(3∶7，V/V)電離試劑涂灑在金屬電極表面，讓其與樣品表面保持濕潤接觸，并使三角形電極尖端對準(zhǔn)質(zhì)譜儀離子入口，然后在金屬電極上施加+5.8 kV高壓。在電場的驅(qū)動下，使樣品表面的待測物朝著質(zhì)譜入口移動，形成待測物離子進(jìn)入質(zhì)譜儀檢測。所得質(zhì)譜數(shù)據(jù)扣除背景后導(dǎo)出，質(zhì)荷比精確到整數(shù)位。

圖1 實(shí)驗(yàn)裝置原理示意圖Fig.1 Schematic diagrams of the metal electrode ion source

2 結(jié)果與討論

2.1 人造革表面痕量賴氨酸和黑索今的常壓金屬電極-質(zhì)譜分析

檢測以黑索今為代表的痕量爆炸物對保障國土安全具有重要意義。黑索今不但影響人類的中樞神經(jīng)系統(tǒng)，還顯示出對腎、胃腸的毒性，被視為具有一定的致癌性，是重要的工業(yè)污染物之一[18-21]。賴氨酸是控制人體生長的抑長素中的重要成分，對人的中樞神經(jīng)和周圍的神經(jīng)系統(tǒng)起著重要的調(diào)節(jié)作用；如果缺乏賴氨酸，會造成胃液分泌不足而出現(xiàn)厭食、營養(yǎng)性貧血，致使中樞神經(jīng)受阻、發(fā)育不良[22-26]。本研究以人造革表面(4 cm×4 cm)添加的黑索今和賴氨酸為代表性檢測對象，按1.3節(jié)方法對其進(jìn)行直接質(zhì)譜分析，實(shí)驗(yàn)結(jié)果示于圖2。在正離子檢測模式下可形成質(zhì)子化準(zhǔn)分子離子[M+H]+，質(zhì)子化黑索今和賴氨酸的準(zhǔn)分子離子信號峰分別為m/z233和m/z147。

注：a.RDX(m/z 223)一級質(zhì)譜圖，插圖為MS/MS圖；b.Lys(m/z 147)一級質(zhì)譜圖，插圖為MS/MS圖圖2 正離子模式下，金屬電極-質(zhì)譜檢測人造革表面RDX和Lys的串聯(lián)質(zhì)譜圖 Fig.2 Metal electrode-mass spectrometry of leather surface of RDX and Lys samples in positive ion mode

為了排除假陽性，對黑索今m/z223進(jìn)行串聯(lián)質(zhì)譜分析，得到m/z209、207、195和177碎片離子。其中，碎片離子m/z209比m/z223少了14 u，推測為中性丟失一分子CH2；m/z207比m/z223少了16 u，推測為中性丟失一分子O；m/z177比m/z223少了46 u，推測為中性丟失一分子NO2；m/z149比m/z223少了74 u，推測為失去CH2+N+NO2。該結(jié)果與胡燕等[18]報道的一致。

本實(shí)驗(yàn)進(jìn)一步研究了賴氨酸在碰撞誘導(dǎo)解離(CID)中的裂解規(guī)律，選擇質(zhì)子化的賴氨酸m/z147進(jìn)行多級串聯(lián)質(zhì)譜分析。在二級質(zhì)譜中，母離子m/z147主要產(chǎn)生m/z129和m/z101碎片離子，以及響應(yīng)較弱的m/z130、119和105碎片離子。其中，m/z129比m/z147少了18 u，推測是由于電荷誘導(dǎo)羧基中C—O單鍵發(fā)生斷裂，中性丟失一分子H2O形成的；m/z101比m/z129少了28 u，推測為分子內(nèi)重排后中性丟失一分子CO形成的。由實(shí)驗(yàn)原理可知，單次掃描大表面金屬電極-質(zhì)譜法能使待測物與溶劑形成溶劑化分子，在電場作用下形成極化團(tuán)簇，待測物在電場及電滲流的作用下朝著質(zhì)譜入口移動，在質(zhì)譜入口形成離子進(jìn)行檢測，極大地提高了不均勻大表面單個樣品的分析速度。單次掃描大表面金屬電極-質(zhì)譜法能夠?qū)θ嗽旄锉砻?4 cm×4 cm)的黑索今和賴氨酸進(jìn)行定性分析，檢測限可達(dá)6.2×10-7～6.2×10-3μg/cm2，分析單個樣品表面的時間不到2 s，同時可對黑索今和賴氨酸的結(jié)構(gòu)進(jìn)行鑒定。

2.2 條件優(yōu)化

為了獲得穩(wěn)定的信號和較好的重現(xiàn)性，本實(shí)驗(yàn)對電極的材質(zhì)、電極尖端的角度、電壓和人造革表面濕潤情況進(jìn)行了優(yōu)化，并選取賴氨酸的二級碎片離子m/z101作為研究對象。不同電極材質(zhì)與信號強(qiáng)度的關(guān)系示于圖3a，可見，當(dāng)電極材料為銅金屬時，m/z101信號強(qiáng)度高，這可能是因?yàn)殂~電極的導(dǎo)電性優(yōu)于鋅電極和鋁電極。圖3b顯示，電極尖端的角度為60°時，信號強(qiáng)度最佳，這可能是由于施加在等邊三角形電極的高壓在等邊三角形表面產(chǎn)生的梯度電場能夠使待測物朝著電極尖端移動，極大地提高了目標(biāo)化合物的質(zhì)子化效率。圖3c顯示，施加電壓為5～5.8 kV時，m/z101信號強(qiáng)度逐漸增大，當(dāng)電壓大于5.8 kV時，信號強(qiáng)度反而下降，這是由于在電壓大于5.8 kV時，銅金屬電極尖端容易放電。圖3d表明，當(dāng)人造革表面涂撒250 μL電離試劑時，m/z101信號強(qiáng)度最佳，其他參數(shù)由系統(tǒng)自動優(yōu)化。

a.不同金屬材質(zhì)；b.施加電壓；c.電極尖端角度；d.電離試劑體積圖3 金屬電極-質(zhì)譜法對賴氨酸檢測的工作參數(shù)優(yōu)化 Fig.3 Optimization working parameters of metal electrode-mass spectrometry for Lys detection

2.3 金屬電極-質(zhì)譜法與DESI-MS法檢測RDX和Lys的結(jié)果對比

將待測物RDX和Lys均勻或非均勻的涂撒在人造革表面，分別以串聯(lián)質(zhì)譜中m/z207、101為特征離子，采用單次掃描大表面金屬電極-質(zhì)譜法和DESI-MS法檢測RDX和Lys。對比結(jié)果可知，本方法比DESI-MS法的分析速度提高了52 倍，其特征離子強(qiáng)度、檢測時間和RSD列于表1。

表1 本方法與DESI-MS法檢測黑索今和賴氨酸的結(jié)果對比Table 1 Comparison of novel method and DESI-MS for detecting RDX and Lys

3 結(jié)論

本研究建立了一種單次掃描即可檢測分布于大表面樣品上任意位置痕量黑索今和賴氨酸的質(zhì)譜分析方法。該方法無需對任何樣品進(jìn)行預(yù)處理，每個樣品在30 s時內(nèi)即可完成質(zhì)譜分析，分析速度快，可檢測樣品表面均勻、非均勻分布的待測物，獲得的二級離子信號強(qiáng)度和檢測效率均優(yōu)于DESI法。

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Mass Spectrometric Detection of Lysine and Royal Demolition Explosive on Large Surfaces

LI Yi, ZHU Teng-gao, WANG Guo-feng, CHEN Huan-wen

(JiangxiKeyLaboratoryforMassSpectrometryandInstrumentation,EastChinaInstituteofTechnology,Nanchang330013,China)

O657.63

A

1004-2997(2017)05-0515-06

10.7538/zpxb.youxian.2016.0067

2016-07-04;

2016-09-23

國家自然科學(xué)基金(21520102007)；長江學(xué)者和創(chuàng)新團(tuán)隊發(fā)展計劃項目(IRT13054)；江西省科技計劃項目(20152ACB21021)資助

李 毅(1988—)，男(漢族)，江西九江人，碩士研究生，從事質(zhì)譜技術(shù)的應(yīng)用研究。E-mail: liyi_jiujiang@sina.com

陳煥文(1973—)，男(漢族)，江西興國人，教授，從事質(zhì)譜分析研究。E-mail: chw8868@gmail.com

時間：2016-12-28；網(wǎng)絡(luò)出版地址：http:∥www.cnki.net/kcms/detail/11.2979.TH.20161228.0937.028.html