高效液相色谱-离子阱飞行时间串联质谱法快速筛查保健食品中非法添加的磷酸二酯酶-5抑制剂及其类似物

采用离子阱飞行时间串联质谱(LC-MS-IT-TOF)技术对保健食品中非法添加的西地那非、他达那非和红地那非等28种磷酸二酯酶-5抑制剂(PDE-5)及其类似物进行快速筛查和确证。建立了此28种化合物的精确分子质量数和多级质谱碎片离子数据库。样品经甲醇超声提取,以C18色谱柱(150 mm×4.6 mm,3.5μm)分离,乙腈-0.1%乙酸为流动相梯度洗脱。结果显示,28种化合物的筛查检出限为0.2~7.0μg/L,定量下限为0.04~1.18 mg/kg(胶囊样品)和0.007~0.235 mg/L(口服液)。以定量下限浓度为加标水平的平均回收率为31.0%~114.0%,相对标准偏差(RSD)为2.5%~14.9%。利用精确质量数匹配和自建标准谱库检索,实现快速筛查;结合保留时间、同位素丰度和多级特征碎片离子对目标化合物进行确证。该方法具有简便、快速、准确、灵敏度高等优点,适用于保健食品中非法添加的磷酸二酯酶-5抑制剂(PDE-5)及其类似物的高通量筛查和定性鉴定。根据裂解规律的归纳总结,还可应用于其未知衍生物及结构类似物的分子式预测和结构推导。     

减肥产品中非法添加西布曲明类似物的ESI-MS/MS检测与确证

通过分析西布曲明和减肥类健康产品中未知化合物质谱图的质谱信息,由高分辨串联质谱获得裂解碎片离子的精密质量数,质谱软件给出碎片的可能组成,推测了西布曲明和未知化合物的质谱裂解途径,进而推测出该化合物的结构。并通过合成该结构的化合物,采用液相色谱-串联质谱法对样品作确证检验,证实样品中添加了新的西布曲明类似物。该实验提供了一种可用于检测和确证健康产品中非法添加合成药物未知类似物的方法。     

基于非靶标筛查的超高效液相色谱-三重四极杆质谱法检测尿液样品中的酰基肉碱

建立了一种基于母离子扫描模式的超高效液相色谱-三重四极杆质谱检测尿液中酰基肉碱的分析方法。对酰基肉碱类化合物所共有的m/z为60、85和144的碎片离子进行选择性检测,结合化合物母离子扫描的结果及其对应的保留时间,选取一致性较好的化合物进行筛选,再利用高分辨质谱确认,最终检测到37种酰基肉碱化合物,其中有14种尚未被HMDB和LIPID MAPS数据库收录。该方法可应用于其他生物样本(如血液、组织)中酰基肉碱的定性、定量分析,可作为检测酰基肉碱化合物的新选择。     

超高效液相色谱-四极杆飞行时间质谱法非靶向快速筛查进口粮谷中未知的农药残留

利用快速提取农药(fast pesticide extraction,FaPEx)方法前处理样品,并结合超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF)技术建立了非靶向快速筛查进口粮谷中未知的多种农药残留方法。采用1%(v/v)醋酸乙腈溶液提取粮谷中未知的农药残留,FaPEx固相萃取柱净化、浓缩,UPLC-Q-TOF检测。利用目标化合物特征离子的精确质量数、同位素匹配、二级碎片信息和保留时间进行数据库匹配,筛查可疑未知农药。结果表明,该方法无需对照标准品即可快速筛查进口粮谷中的农药残留,能够应用于进口粮谷样品的实际筛查。该方法快速、准确、分析通量高,可以为进口粮谷中农药残留的快速筛查和质量控制提供重要的方法依据。     

超高效液相色谱-Orbitrap高分辨质谱用于减肥和壮阳类保健食品中32种非法添加药物的快速筛查和确证北大核心CSCD

建立了基于超高效液相色谱-Orbitrap高分辨质谱的快速筛查及确证减肥和壮阳类保健食品中32种非法添加药物的分析方法,并总结了数据库建立和应用的相关要点。研究对象聚焦于非法添加药物的衍生物,在对比正负离子模式下各化合物响应强度的基础上建立了高分辨质谱信息库,对提取溶剂、色谱柱温度等实验条件进行了详细探究,尽可能给出了较宽的标准曲线线性范围。使用Hypersil gold vanquish色谱柱(100 mm×2.1 mm,1.9μm),梯度洗脱,流量0.3 mL/min,正、负离子全扫描/数据依赖的二级扫描模式,在17 min内完成32种目标化合物的数据采集,通过TraceFinder软件进行快速定性筛查和定量。结果显示在17 min内32种化合物能得到较好分离;2种基质加标溶液中32种化合物的一级质谱离子精确质量数的实测值与理论值均在5×10^(-6)误差之内,二级碎片离子质量数的实测值与理论值均在1×10^(-5)误差之内;方法学验证结果表明,所有化合物均显示出优异的线性关系,相关系数(r^(2))均大于0.99;固体基质中除达泊西汀、羟基硫代豪莫西地那非、硫代豪莫西地那非、硫代西地那非、去甲基硫代西地那非的回收率较低外,其余27种化合物的回收率为50.5%~84.5%,相对标准偏差(RSD)为1.2%~13%,液体基质中32种化合物的回收率范围为60.4%~109.3%,RSD为0.77%~8.2%;在48份减肥及壮阳类保健食品中检出1份阳性样品,检出率2.08%。该方法操作简单,结果定性准确,可用于减肥及壮阳类保健食品中32种非法添加药物的快速筛查及确证。     

高效液相色谱-四极杆-飞行时间质谱快速筛查鉴别食品中非法添加的62种中药材北大核心CSCD

建立了高效液相色谱-四极杆-飞行时间质谱快速筛查鉴别食品中非法添加的62种中药材的方法。依据卫生部关于进一步规范保健食品原料管理的通知(卫法监发[2002]51号)中可用于保健食品的物品名单,确定食品中可能非法添加的62种中药材原料清单,再从62种中药材中筛选其特征组分,获得不同中药材对应的特征组分清单。62种对照药材经甲醇超声提取后,于Thermo Accucore aQ色谱柱(150 mm×2.1 mm,2.6μm)上分离,在电喷雾正负离子扫描模式下,分别以0.1%(v/v)甲酸水溶液-乙腈和水-乙腈为流动相梯度洗脱,进行一级质谱和二级质谱全扫描检测,采用Library View软件建立不同中药材对应的特征组分的一级精确质量数据库和二级碎片质谱库。样品同法处理后上样分析,采用Peak View软件将样品高分辨数据与自建数据库中的质谱图、精确分子离子质量数、碎片离子质量数、保留时间等相关参数进行快速筛查鉴别分析。该工作通过建立“中药材-特征组分”对应清单,构建了食品中易非法添加的62种中药材中共388种特征组分的高分辨质谱库,每种中药材包括5~10种特征组分,通过对实际食品样品配制酒、代用茶及饮料进行筛查分析,1批次配制酒样品与淫羊藿中药材的7种特征组分匹配一致,推断该配制酒样品中加入了淫羊藿中药材。该法可实现无标准品情况下中药材的定性筛查,具有高通量、准确、简便、快捷的特点,解决了食品中非法添加中药材难以识别和确证的难题,可以实现食品中非法添加中药材的快速筛查鉴别分析。     

QuEChERS前处理结合HPLC-Q-TOF/MS非靶向快速筛查凉茶中的非法添加物

建立了QuEChERS法净化,结合高效液相色谱-四极杆-飞行时间质谱法(HPLC-Q-TOF/MS)非靶向快速筛查凉茶中未知非法添加物的方法。凉茶中的非法添加物经0.5%乙酸-乙腈提取,氨丙基粉净化,C_(18)色谱柱(100 mm×2.1 mm,1.7μm)分离,HPLC-Q-TOF/MS测定。利用8种质控化合物建立的筛查方法检出限为2.0～10μg/kg,定量下限为5.0～25μg/kg。3个不同加标水平的平均回收率为75.8%～95.6%,RSD(n=6)为2.1%～6.5%。在该定量方法基础上利用目标化合物特征离子的精确质量数、同位素匹配、二级碎片信息进行数据库匹配,无标准品情况下非靶向筛查非法添加物。结果表明,该方法无需标准品即可快速筛查凉茶中的非法添加西药,能应用于凉茶样品的实际筛查。应用该方法对50份散装凉茶样品进行测定,其中10份凉茶样品检出对乙酰氨基酚,含量为4.14～2 188 mg/kg。该方法快速、准确、分析通量高,可为凉茶中非法添加物的快速筛查和质量控制提供重要依据。     

中兽药口服液中非法添加化学药物高分辨质谱筛查

建立了适用于中兽药口服液中167种非法添加化学药物的超高效液相色谱-四极杆静电场轨道阱质谱高通量筛查方法。样品采用0.5%甲酸乙腈-水进行振荡超声提取，电喷雾电离源（ESI）、正负离子同时扫描方式进行数据采集。通过与自建的167种化合物的基本信息及色谱-质谱信息数据库进行比对，对色谱-质谱条件、提取溶剂的种类和净化条件进行优化。将经前处理后的口服液样品上机测试后，采用Trace Finder筛查软件对数据结果进行分析。结果显示，50 ng/mL非法添加药物（头孢类和内酰胺类药物为100～200 ng/mL）上机时可定性检出。不同药物配方因基质影响，其非法添加药物的检出率略有差异。该方法可基本实现中兽药口服液中非法添加化学药物的高通量快速筛查，具备操作简便、快速高效的特点，为其他兽药剂型中非法添加药物的筛查奠定了基础。     

高效液相色谱-四极杆/静电场轨道阱高分辨质谱对水产品中未知污染物的非定向快速筛查与测定

建立了高效液相色谱-四极杆/静电场轨道阱高分辨质谱对水产品中污染物的非定向快速筛查与测定的方法。筛查时样品用乙腈提取、氮气浓缩吹干、甲醇-水溶液定容，采用全扫描数据依赖二级扫描模式进样分析。利用Trace Finder软件对水产样品中未知污染物的精确质量数、同位素丰度比、二级碎片离子进行数据库检索匹配。定量时样品采用优化的QuEChERS方法净化，对筛查过程确认的三环唑、咖啡因和乙氧基喹啉3种污染物进行目标离子二级扫描模式定量分析。鱼和虾中3种化合物在5~1000 μg/L范围内线性关系良好，相关系数均大于0.99；方法检出限（LOD）为1 μg/kg，定量限（LOQ）为5 μg/kg，平均回收率为70.5~90.9%，相对标准偏差为5.4%~12.8%。筛查方法具有快速、准确、高通量等优点，结合定量方法能够用于实际水产品中未知污染物的筛查与测定。     

解吸附电晕束电离质谱法快速筛选保健食品中非法添加的5型磷酸二酯酶抑制剂

建立了解吸附电晕束离子源(DCBI)结合离子阱质谱快速检测保健食品中非法添加的3种磷酸二酯酶5(PDE5)抑制剂(伐地那非、西地那非、他达拉非)的方法。采用一级质谱筛选,二级质谱确证,对样品中非法添加物进行定性鉴别;通过二级特征碎片离子进行半定量分析,并与传统高效液相色谱-紫外(HPLC-UV)定量检测法对比。对12个市售保健品进行检测,DCBI-MS定性检测结果与HPLC-UV检测结果一致,有7个样品检出他达拉非,3个样品检出西地那非,1个样品检出伐地那非。通过研究3种PDE5抑制剂的二级质谱裂解规律,推测一个样品中含有羟基豪莫西地那非。本方法快速准确,适用于大批量复杂基质样品中PDE5抑制剂的筛查。     

超高效液相色谱-四极杆-飞行时间质谱法定性筛查保健食品中西地那非及其相关功效类非法添加化合物

建立了超高效液相色谱-四极杆-飞行时间质谱（UPLC-Q-TOF-MS）定性筛查102种西地那非及其相关功效化合物的方法。甲醇超声提取样品;采用Agilent Poroshell 120 SB-C18色谱柱（75 mm×3.0 mm,2.7 μm）,以0.1%（v/v）甲酸水溶液-乙腈为流动相梯度洗脱,流速0.4 mL/min;采用电喷雾离子（ESI）源,Q-TOF-MS作检测器,正离子模式检测;与对照品保留时间、母离子及碎片离子精确相对分子质量、元素组成比对,准确定性。西地那非等100种化合物在10种基质中的检出限为0.1~50 mg/kg或0.1~50 mg/L。并分类归纳了文中涉及的98种磷酸二酯酶5抑制剂质谱碎片裂解规律,为可疑物的识别和鉴定提供参考。该方法已应用于实际样品的测定,检出19种化合物,有效打击了非法添加行为。     

Screening for unknown synthetic steroids in human urine by liquid chromatography-tandem mass spectrometry

Chemically modified steroids (designer steroids), including tetrahydrogestrinone and norbolethone, pose a threat to the integrity of the sport community. These compounds have recently been detected in urine specimens from athletes, resulting in temporary or permanent suspension from amateur and/or professional competition. Triple quadrupole mass spectrometers enable doping control laboratories to screen for unknown, anabolic, androgenic steroids utilizing precursor ion scans. On the basis of common dissociation patterns of steroids with common structural features, characteristic product ions were selected to serve as diagnostic markers for previously unidentified drugs or drug metabolites in human urine samples. An assay was established to complement standard screening procedures. Urine specimens were enzymically hydrolyzed, partitioned into ether, concentrated, and analyzed by precursor ion scanning. Spectra from samples fortified with eight standard compounds (methyltestosterone, ethyltestosterone, 1-testosterone, gestrinone, dihydrogestrinone, tetrahydrogestrinone, norbolethone, and propyltrenbolone) and one deuterium-labeled analog (d(4)-tetrahydrogestrinone) at 50 ng/ml of urine, had precursor ion peaks other than those from common endogenous steroids. Subsequent product ion scan experiments on precursor ions of peaks of unknown origin provided structural identification of the unknown compounds.     

A fragmentation study of two compounds related to 4'-demethylepipodophyllotoxin in negative ion electrospray ionization by MSn ion-trap time-of-flight mass spectrometry

High-resolution electrospray ionization multistage tandem mass spectrometry (MS 1-7) in negative ion mode was used to determine the accurate masses and fragmentation pathways of two compounds, 4'-demethylepipodophyllotoxin and 4'-demethyl-4-azido-4-deoxyepipodophyllotoxin, which are key intermediate compounds for the preparation of podophyllotoxin-type anti-cancer drugs. The deprotonated molecules [M-H]* of both compounds were readily observed in the conventional single-stage mass spectra due to the presence of the phenolic hydroxyl group in the molecules. Abundant information on the product ions was obtained from tandem mass spectra (MS 2-7) in negative ion mode. Based on the exact masses acquired from 14 different tandem mass spectra, a similar MSn fragmentation pathway was proposed for both compounds. A characteristic product ion produced in the MS 2-4 product ion scan experiments is the cyclohexylenetrione anion [M-H-2Me-RH]* or [M-H-RH-2Me]* at m/z 351 (C19H11O7) formed by the consecutive losses of two CH3 radicals at the 3'- and 5'-positions and the neutral loss of RH, where R = a 4-substituted group (-OH or -N3), from the [M-H]* ion. This anion may be considered as diagnostic for the presence of this type of compound. The other common cleavages are the neutral losses of CO at least two times in the MS 6,7 product ion spectra. The results of this work could serve as an effective tool for the detection or determination of other derivatives of 4'-demethyl-4beta-substituted podophyllotoxin, which are widely used as intermediates for the preparation of anti-tumor drugs.     

Mass-spectrometric studies of new 6-nitroquipazines—serotonin transporter inhibitors

Six synthesized 6-nitroquipazine derivatives were examined by electron ionization (EI) and electrospray ionization (ESI) mass spectrometry in positive and negative ion mode. The compounds exhibit high affinity for the serotonin transporter (SERT) and belong to a new class of SERT inhibitors. The EI mass spectra registered in negative ion mode showed prominent molecular ions for all the compounds studied. All EI mass spectra and all ESI mass spectra showed similar fragmentation pathways of molecular ions, but the pathways differed between EI and ESI. The differences were explained with the aid of theoretical evaluation of the stability of the respective radical ions (EI MS) and protonated ions (ESI MS).     

Specificity enhancement by electrospray ionization multistage mass spectrometry – a valuable tool for differentiation and identification of ‘V’‐type chemical warfare agents

The use of chemical warfare agents has become an issue of emerging concern. One of the challenges in analytical monitoring of the extremely toxic ‘V’‐type chemical weapons [O‐alkyl S‐(2‐dialkylamino)ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. MS analysis of these compounds reveals mostly fragment/product ions representing the amine‐containing residue. Hence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical EI/MS and electrospray ionization‐MS, leading to unavoidable ambiguity in the identification of the phosphonate moiety. A set of five ‘V’‐type agents, including O‐ethyl S‐(2‐diisopropylamino)ethyl methylphosphonothiolate (VX), O‐isobutyl S‐(2‐diethylamino)ethyl methylphosphonothiolate (RVX) and O‐ethyl S‐(2‐diethylamino)ethyl methylphosphonothiolate (VM) were studied by liquid chromatography/electrospray ionization/MS, utilizing a QTRAP mass detector. MS/MS enhanced product ion scans and multistage MS³ experiments were carried out. Based on the results, possible fragmentation pathways were proposed, and a method for the differentiation and identification of structural isomers and derivatives of ‘V’‐type chemical warfare agents was obtained. MS/MS enhanced product ion scans at various collision energies provided information‐rich spectra, although many of the product ions obtained were at low abundance. Employing MS³ experiments enhanced the selectivity for those low abundance product ions and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups, for example, methylphosphonothiolates versus ethylphosphonothiolates. The new group‐specific ions elucidated in this work are also useful for screening unknown ‘V’‐type agents and related compounds, utilizing precursor ion scan experiments. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of caffeic acid derivatives in Actea racemosa (Cimicifuga racemosa,black cohosh) by liquid chromatography/tandem mass spectrometry

Caffeic acid derivatives occurring in black cohosh [Cimicifuga racemosa (L.) Nutt., Actaea racemosa (Ranunculaceae)], some of which may have pharmacological activity, were analyzed using high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in a complex plant matrix. Based on these studies, structurally characteristic product ions and neutral molecule losses were identified, which were then used during LC/MS/MS with product ion scanning, precursor scanning and constant neutral loss scanning to detect caffeic acid derivatives in a crude extract of black cohosh. Several caffeic acid derivatives were detected, and the identification of six of them were confirmed by comparison with authentic standards including caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, and cimicifugic acid B. Four other compounds were detected that appeared to be caffeic acid derivatives based on LC/MS/MS retention times, molecular weights, and fragmentation patterns during MS/MS. Since standards were unavailable for these four compounds, they were tentatively identified using LC/MS/MS as cimicifugic acid E, cimicifugic acid F, dehydrocimicifugic acid A, and dehydrocimicifugic acid B. Dehydrocimicifugic acid A and dehydrocimicifugic acid B have not been reported previously to be constituents of black cohosh.     

Analysis of aliphatic and aromatic carbonyl compounds in ambient air by LC/MS/MS.

A sensitive liquid chromatograph/tandem mass spectrometric technique (LC/MS/MS) was applied to determine aliphatic and aromatic carbonyl compounds in ambient air. Traces of the carbonyl compounds were sampled by passing through a Sep-Pak DNPH-silica cartridge. Their derivatives were thus eluted with acetonitrile, separated by reversed-phase liquid chromatography and determined by quadrupole tandem mass spectrometry in an atmospheric pressure chemical ionization (APCI) mode with multiple reaction monitoring (MRM). The detection limits (DL) of the carbonyl compounds were 0.8 - 15 ng/m3. A number of the carbonyl compounds were detected at n.d.- 14 microg/m3 levels. The precursor ion scanning analysis was applied to identify the unknown compounds.     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Cone voltage and collision cell collision-induced dissociation study of triphenylethylenes of pharmaceutical interest.

Fragmentations of three triphenylethylene compounds (toremifene and its two metabolites) with different functional side-chain groups (alcohol, acid and amine) were studied. The compounds were dissociated by collision-induced dissociation (CID) in the interface region of an electrospray ionization source (ESI(+)) and in the collision cell of a triple quadrupole mass spectrometer. Fragmentation pathways for these molecules are proposed, based on accurate mass measurements of in-source fragment ions and MS/MS experiments using product and precursor ion scanning. The side-chain functional groups were found to strongly affect the fragmentations of the molecular ions. The fragmentation pathways of the protonated molecule and sodium ion adduct were quite similar, but the subsequent stabilities of certain common fragments were surprisingly different.     

Strategies for ascertaining the interference of phase II metabolites co‐eluting with parent compounds using LC–MS/MS

LC‐MS/MS is currently the most selective and efficient tool for the quantitative analysis of drugs and metabolites in the pharmaceutical industry and in clinical assays. However, phase II metabolites sometimes negatively affect the selectivity and efficiency of the LC‐MS/MS method, especially for the metabolites that possess similar physicochemical characteristics and generate the same precursor ions as their parent compounds due to the in‐source collision‐induced dissociation during the ionization process. This paper proposes some strategies for examining co‐eluting metabolites existing in real samples, and further assuring whether these metabolites could affect the selectivity and accuracy of the analytical methods. Strategies using precursor‐ion scans and product‐ion scans were applied in this study. An example drug, namely, caffeic acid phenethyl ester, which can generate many endogenous phase II metabolites, was selected to conduct this work. These metabolites, generated during the in vivo metabolic processes, can be in‐source‐dissociated to the precursor ions of their parent compounds. If these metabolites are not separated from their parent compounds, the quantification of the target analytes (parent compounds) would be influenced. Some metabolites were eluted closely to caffeic acid phenethyl ester on LC columns, although long columns and relatively long elution programs were used. The strategies can be utilized in quantitative methodologies that apply LC‐MS/MS to assure the performance of selectivity, thus enhancing the reliability of the experimental data.