郭澤琴,霍佳麗,王建華,吳志軍
(1. 重慶大學(xué)生物工程學(xué)院, 重慶 400044;2. 中國科學(xué)院成都生物研究所, 四川 成都 610041)
β-內(nèi)酰胺類抗生素質(zhì)譜裂解機理研究
郭澤琴1,霍佳麗1,王建華1,吳志軍2
(1. 重慶大學(xué)生物工程學(xué)院, 重慶 400044;2. 中國科學(xué)院成都生物研究所, 四川 成都 610041)
采用電噴霧-四極桿-飛行時間串聯(lián)質(zhì)譜(ESI-Q-TOF-MS/MS)對3種頭孢類和2種青霉素類藥物的[M+Na]+裂解規(guī)律進行研究。頭孢類藥物[M+Na]+裂解可產(chǎn)生許多互補的碎片離子,1,3-H重排和retro-Diels-Alder (RDA)是其重要的裂解方式;青霉素類藥物[M+Na]+裂解除了一些常規(guī)的中性分子丟失外,在氘代實驗的基礎(chǔ)上提出了比以往更合理的H2S丟失途徑,即1,5-H重排。本實驗豐富了β-內(nèi)酰胺類藥物的質(zhì)譜裂解途徑,對應(yīng)用質(zhì)譜技術(shù)快速鑒定復(fù)雜體系中β-內(nèi)酰胺類抗生素具有重要作用。
β-內(nèi)酰胺類抗生素;電噴霧-四極桿-飛行時間串聯(lián)質(zhì)譜(ESI-Q-TOF-MS/MS);裂解規(guī)律;氫重排
β-內(nèi)酰胺類藥物是使用最早的一類抗生素,在臨床和畜牧業(yè)中被廣泛用于疾病的預(yù)防和治療。在速成養(yǎng)殖中,β-內(nèi)酰胺類藥物作為飼料添加劑可以促進牲畜的生長[1-8],但該類藥物在食用動物組織中容易殘留。隨著抗生素的過度使用,動物體內(nèi)抗生素殘留量增加,長期食用含有β-內(nèi)酰胺類抗生素殘留的食品,可對人體造成嚴重的危害[2-4]。因此,需要一種快速、有效的方法對食品中β-內(nèi)酰胺類抗生素進行有效的檢測和監(jiān)控。
質(zhì)譜分析具有檢測速度快、靈敏度高和樣品耗費少等優(yōu)點,已成為復(fù)雜體系中痕量成分分析的一種重要方法[18-19]。質(zhì)譜鑒定工作的可靠性需要豐富的帶有結(jié)構(gòu)信息的碎片離子來保證。對質(zhì)子加合的β-內(nèi)酰胺類藥物的質(zhì)譜研究已有一些報道,其主要的裂解方式為β-內(nèi)酰胺環(huán)和碳氮鍵的斷裂[8-17],但關(guān)于該類抗生素鈉加合離子[M+Na]+的斷裂方式卻未見報道。本工作利用高分辨電噴霧-四極桿-飛行時間串聯(lián)質(zhì)譜 (ESI-Q-TOF-MS/MS)法研究5種具有代表性的β-內(nèi)酰胺類藥物(包括3種頭孢類藥物和2種青霉素類藥物) [M+Na]+的斷裂方式,其結(jié)構(gòu)式示于圖1,并且利用氘代實驗對其斷裂機理進行驗證。
注:1.頭孢克洛;2.頭孢拉啶;3.頭孢羥氨芐;4.氨芐西林;5.阿莫西林圖1 5種β-內(nèi)酰胺類抗生素的結(jié)構(gòu)式 Fig.1 The structure of five kinds of β-lactam antibiotics
1.1主要儀器和裝置
microTOF Q質(zhì)譜儀:德國Bruker公司產(chǎn)品,配有電噴霧(ESI)離子源,Compass 數(shù)據(jù)處理系統(tǒng),自動進樣器。
1.2試劑和樣品
β-內(nèi)酰胺類抗生素對照品:美國Sigma-Aldrich公司產(chǎn)品;甲醇(色譜純):美國Fisher Scientific公司產(chǎn)品;質(zhì)譜校正液:美國Agilent公司產(chǎn)品;實驗用水:自制超純水;氘代試劑D2O和CD3OD:美國CIL公司產(chǎn)品;氘代溶液配制:分別取0.1 mg頭孢克洛(1)、頭孢拉定(2)和氨芐西林(4)于2 mL D2O中,放置24 h。
1.3質(zhì)譜條件
電噴霧(ESI)離子源;ESI源電壓-500 V;毛細管電壓-4 500 V;霧化氣體:高純氮氣;霧化氣壓力30 kPa;進樣速度180 μL/h;干燥氣溫度180 ℃;干燥氣流速4.0 L/min;在正離子模式下先進行一級質(zhì)譜全掃描,m/z100~3 000,再選擇[M+Na]+離子進行二級質(zhì)譜分析。
2.1頭孢類抗生素1~3的[M+Na]+MS/MS分析及裂解規(guī)律研究
對頭孢克洛(1)[M+Na]+m/z390進行二級質(zhì)譜分析,可以得到豐富的碎片離子峰,示于圖2,其主要碎片離子組成列于表1?;パa離子對m/z213和m/z200為β-內(nèi)酰胺環(huán)斷裂產(chǎn)生的離子峰,m/z213再丟失一分子NH3生成m/z196;互補離子對m/z257和m/z156由[M+Na]+發(fā)生RDA反應(yīng)產(chǎn)生中間體m/z390經(jīng)氨交換和四元環(huán)氫重排而得到,m/z257再脫去一分子NH3得到m/z240,該中間體也可經(jīng)酯化反應(yīng)和麥?zhǔn)现嘏臶20]生成m/z231,或直接發(fā)生自由基斷裂生成m/z230;互補離子對m/z285和m/z128由[M+Na]+經(jīng)1, 3-H重排而得到,前者再經(jīng)RDA反應(yīng),進一步斷裂生成m/z124,其裂解途徑經(jīng)氘代實驗得到驗證,示于圖2b。頭孢克洛(1) [M+Na]+可能的斷裂過程示于圖3,化合物2、3[M+Na]+的裂解途徑與化合物1相似,示于圖4。
注:a.[M+Na]+二級質(zhì)譜圖;b.[M/D+Na]+ 二級質(zhì)譜圖;c.氘代試驗一級掃描圖圖2 頭孢克洛(1)[M+Na]+ m/z 390的二級質(zhì)譜圖Fig.2 Product ion scan of the selected precursor [M+Na]+ at m/z 390 for cefaclor (1)
表1 頭孢克洛(1)主要碎片離子的組成
圖3 頭孢克洛(1) [M+Na]+ 可能的斷裂過程Fig.3 Major fragmentation patterns of [M+Na]+ for cefaclor (1)
圖 4 頭孢拉啶(2) [M+Na]+ 的二級質(zhì)譜圖Fig.4 Product ion scan of the selected precursor [M+Na]+ for cefradine (2)
2.2青霉素類抗生素4~5的[M+Na]+MS/MS分析及裂解規(guī)律研究
氨芐西林4[M+Na]+m/z372的MS/MS圖示于圖5,其主要碎片離子組成列于表2。例如,互補離子對m/z267和m/z128由m/z372經(jīng)1, 3-H重排而得到。m/z372可丟失一分子CO產(chǎn)生m/z344,后者再脫去一分子CO2和H2S產(chǎn)生m/z266;還可連續(xù)脫去一分子NH3,CO2和H2S生成m/z277,其中H2S易通過1, 5-H重排而脫去,氨芐西林(4) [M+Na]+可能的斷裂過程示于圖6,該裂解途徑經(jīng)氘代實驗得以確證(圖5b),并且從氘代裂解圖中可以推斷出S元素是以DHS或D2S的形式丟失的。阿莫西林(5) [M+Na]+的裂解途徑與氨芐西林(4)相似,其二級質(zhì)譜圖示于圖7。
與[M+H]+相比,β-內(nèi)酰胺類藥物的[M+Na]+裂解途徑更豐富,可產(chǎn)生酰胺交換,四氫重排,1, 3、1, 5-H重排以及RDA反應(yīng)等裂解途徑??赡艿脑蚴琴|(zhì)子化位點不同,在質(zhì)譜中H+傾向于與氮結(jié)合發(fā)生電荷誘導(dǎo)裂解,而Na+更傾向于與氧結(jié)合。
注:a.[M+Na]+二級質(zhì)譜圖;b.[M/D+Na]+二級質(zhì)譜圖; c.氘代試驗一級掃描圖圖5 氨芐西林(4)m/z 372的二級質(zhì)譜圖 Fig.5 Product ion scan of the selected precursor [M+Na]+ at m/z 372 for ampicillin (4)
表2 氨芐西林(4)主要碎片離子的組成
本實驗采用高分辨ESI-Q-TOF-MS/MS在正離子模式下對5種β-內(nèi)酰胺類抗生素[M+Na]+二級質(zhì)譜的裂解規(guī)律進行分析研究,并在氘代實驗的基礎(chǔ)上,解釋了這類化合物的裂解過程,提出了1, 3、1, 5-H重排以及RDA反應(yīng)等裂解機理,豐富了離子化學(xué)機理,為使用質(zhì)譜技術(shù)快速鑒定復(fù)雜體系中非目標(biāo)β-內(nèi)酰胺類藥物提供了理論基礎(chǔ)。
圖6 氨芐西林(4) [M+Na]+ 可能的斷裂過程Fig.6 Major fragmentation patterns of [M+Na]+ for ampicillin (4)
圖7 阿莫西林(5) [M+Na]+ 的二級質(zhì)譜圖Fig.7 Product ion scan of the selected precursor [M+Na]+ at m/z 388 for amoxicillin (5)
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StudyontheCleavageRuleofβ-LactamAntibioticsbyElectrosprayQuadrupoleTime-of-FlightTandemMassSpectrometry
GUO Ze-qin1, HUO Jia-li1, WANG Jian-hua1, WU Zhi-jun2
(1.CollegeofBioengineering,ChongqingUniversity,Chongqing400044,China; 2.ChengduInstituteofBiology,ChineseAcademyofSciences,Chengdu610041,China)
The fragmentation of sodiated molecules [M+Na]+of five kinds ofβ-Lactam antibiotics, including three cephalosporins and two penicillins, were studied by electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS). For cephalosporins, besides the cleavage of active tetracycle, 1, 3-H rearrangement and retro-Diels-Alder reaction (RDA) played the substantial roles in the production of many informative product ions, and the D-labeling experiments supported the postulated mechanisms.The processes of sequential losses of a CO2and H2S molecule from the precursor sodiated molecules for penicillins possibly involving 1, 5-H rearrangement were elucidated and further confirmed by D-labeling experiment. This work enriches the fragmentation ofβ-lactam antibiotics and will be valuable for the rapid identification of untargetedβ-lactam antibiotics in the complicated matrix by mass spectrometry.
β-lactam antibiotics; electrospary quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS); fragmentation mechanisms; H rearrangement
2013-04-10;
:2013-07-01
郭澤琴(1989~),女(漢族),安徽人,碩士研究生,藥物分析專業(yè)。E-mail: guozq1008@163.com
王建華(1962~),男(漢族),安徽人,教授,從事藥物合成與分析和藥物制劑工程研究。E-mail: wjh@cqu.edu.cn 吳志軍(1979~),男(漢族),湖南人,青年研究員,從事藥物質(zhì)譜分析與測試研究。E-mail: wuzj@cib.ac.cn
O 657.63
:A
:1004-2997(2014)01-0045-07
10.7538/zpxb.2014.35.01.0045