16种核苷与核苷酸LC检测方法的建立及质谱裂解规律的研究

目的 本文旨在建立一个通用LC-MS法用于分离和鉴定核苷(酸)(类)化合物。方法 选用ODS-AQ柱,流动相A为缓冲液1(120 mmol/L Na₂HPO₄,120 mmol/L KH₂PO₄和3 mmol/L TBAOH),流动相B为水,流动相C为甲醇,梯度洗脱,对16种核苷和核苷酸化合物进行分离分析。采用电喷雾离子化(ESI)检测,喷雾电压4000 V(正离子模式)/3500 V(负离子模式),雾化气压力45 psi,干燥气流量10 L/min,去溶剂温度350 ℃。碎裂电压为180 V,碰撞能量设置为5~30 eV,对4种不同类型的核苷和核苷酸化合物的质谱碎片进行分析。结果 建立的LC方法对16个代表性核苷(酸)化合物达到了基线分离(R>2.0),基于质谱碎片规律,构建核苷(酸)化合物的质谱平台。结论 建立的LC-MS方法可为未知的核苷(酸)(类)化合物的分离与鉴定提供有用的指导。     

罂粟碱的体内与体外代谢物研究

利用HPLC-MSⁿ检测罂粟碱及其在大鼠体内(大鼠粪便)和体外(肝微粒体, 肠道菌)代谢物。体内与体外的代谢物经C₁₈小柱进行富集和纯化后, 直接采用优化的HPLC-ESI/ITMSⁿ方法对样品进样分析。以罂粟碱标准品为对象优化高效液相色谱/电喷雾离子阱质谱(HPLC-ESI/ITMSⁿ )实验条件, 分析总结其电喷雾质谱的一级电离规律和多级质谱裂解规律, 作为罂粟碱大鼠体内与体外代谢物结构分析的依据。代谢物的结构推导主要依据代谢物的色谱保留时间及其电喷雾离子阱质谱HPLC-ESI/ITMSⁿ电离规律。在大鼠粪便中有原药及其8种代谢产物。体外代谢物检测到原药的脱甲基和羟基化。     

真菌对人参皂苷Rb1及人参二醇系皂苷的代谢作用

目的　研究真菌对人参皂苷Rb₁(G-Rb₁)及人参二醇系皂苷(PDS)的代谢作用。方法　在恒温振荡条件下,10种真菌对其底物G-Rb₁ 及PDS分别进行代谢,不同时间采集代谢物,正丁醇萃取,用薄层色谱(TLC)、高效液相色谱(HPLC)和电喷雾质谱(ESI-MS)检测代谢成分。结果　代谢产物中存在化合物G-Rb₁,G-Rd ,G-F₂,CK和Ppd。结论　发现6种真菌对底物有不同程度的代谢作用,其代谢过程可能为G Rb₁(或PDS)→G Rd→G F₂ →CK→Ppd ,并通过控制这一过程可获得目的代谢产物CK。     

HPLC-ESI/MS分析5-羟基黄酮在大鼠体内的代谢产物

目的研究5-羟基黄酮在大鼠体内的代谢产物。方法应用高效液相色谱-电喷雾质谱(HPLC-ESI/MS)检测大鼠灌胃5-羟基黄酮后血浆、尿液、胆汁和粪便中的代谢产物。实验采用Zorbax C18色谱柱,0.05%甲酸乙腈-0.05%甲酸水二元线性梯度洗脱进行色谱分离,并与电喷雾质谱联用,根据正离子模式的分子离子峰获得化合物分子量信息,推测化合物的可能结构。结果仅在大鼠粪便中检测到原形成分,在大鼠尿液、粪便、血浆、胆汁中检测到5-羟基黄酮葡糖醛酸结合物。结论 5-羟基黄酮吸收差,在大鼠体内主要以II相代谢产物葡萄糖醛酸结合物的形式存在。     

藤黄总酸注射液中藤黄酸的高效液相色谱法测定

目的:首次建立一种高效液相法用于藤黄总酸注射液(以下简称THZS)中藤黄酸的含量测定。方法:样品经酸化后,用乙醚萃取,挥干乙醚,流动相溶解进样分析。色谱条件为:C₁₈(4.6mm×150mm,5μ)色谱柱,甲醇-0.1% H₃PO₄(9∶1)为流动相,检测波长360nm。结果:该法的专属性强,平均回收率100.3%。结论:可用于THZS质量研究。     

液相色谱-电喷雾离子肼质谱联用法在体内药物分析中的应用

在现代药物研发中,液相色谱-质谱联用法对药物的吸收、分布、代谢和消除分析是一种强有力的工具。电喷雾离子肼作为一种质谱检测模式,可产生MSn多级裂解碎片峰,进而获得丰富的结构信息,在体内药物分析时可利用这些碎片峰(MSn)建立一种强专属性和高灵敏度的高效液相色谱-质谱联用(HPLC-MS)方法。该文评述了高效液相色谱-电喷雾离子肼串联质谱(HPLC-ESI-MSn)方法在体内药物分析中的应用。     

LC/MS分析大鼠体内氧化苦参碱及其主要代谢物LC/MS分析大鼠体内氧化苦参碱及其主要代谢物

目的研究氧化苦参碱在大鼠体内的主要代谢产物。方法以氧化苦参碱和苦参碱为对象优化液相色谱/电喷雾离子阱质谱(LC/ESI-ITMSⁿ)实验条件，分析总结其电喷雾质谱的一级电离规律和二级质谱裂解规律，作为氧化苦参碱大鼠体内代谢物结构分析的依据。健康大鼠腹腔肌注40 mg·kg^-1氧化苦参碱，收集0~24 h的尿样，尿样中的代谢物经C₁₈小柱进行富集与纯化后，在优化的LC/ESI-ITMSⁿ条件下进样分析。代谢物的结构推导主要依据代谢物的色谱保留时间及其电喷雾离子阱质谱(ESI-ITMSⁿ)电离规律。结果在大鼠尿样中有原药及其6种I相氧化及还原代谢产物，且主要代谢物为苦参碱。未发现II相代谢物。结论本法不仅操作简便、快速，而且灵敏度高、专属性强。该分析技术是研究药物代谢最有效的方法之一。     

高分离度快速液相色谱-离子阱质谱分析参麦注射液化学成分

目的:通过高分离度快速液相色谱(RRLC)-离子阱质谱联用技术定性分析参麦注射液及其中间体的化学成分。方法:色谱分离采用Agilent Rapid Resolution HT Extend-C18色谱柱(2.1 mm×50 mm,1.8μm),5 mmol.L-1醋酸铵-乙腈梯度洗脱,流速0.4 mL.min-1。质谱条件为电喷雾离子源,负离子模式检测。结果:利用多级质谱信息,结合对照品对照和文献分析,确定了数十种化合物的可能结构。结论:通过RRLC-离子阱质谱联用技术,为中药注射液的化学成分研究提供了一种快速、灵敏、高效的分析方法。     

五味子酯甲在大鼠体内代谢产物的鉴定

目的 考察五味子酯甲在大鼠体内的代谢转化。方法 采用超高效液相色谱串联四级杆飞行时间质谱(UPLC-TOF-MS/MS)分析鉴定大鼠灌胃五味子酯甲后,其在尿样中的代谢产物。Phenomenex UPLC C₁₈色谱柱,流动相为乙腈-1‰甲酸水,梯度洗脱,质谱仪离子源为电喷雾离子源(ESI),正离子方式检测。结果 经代谢物软件处理后,根据MS/MS给出质谱碎片信息对五味子酯甲代谢产物进行结构推测,共检测到5种代谢产物。结论 五味子酯甲在大鼠体内的代谢途径主要为氧化反应和还原反应。     

当归化学成分的HPLC-MS/MS分析

目的分析当归中的主要化学成分。方法采用高效液相色谱-质谱联用的方法对当归中一些主要化学成分进行定性研究，并对当归中苯酞类化合物的质谱裂解规律进行了初步探讨。色谱柱为Hypersil ODS2(250 mm×4.6 mm，5 μm)，流动相为水(含甲酸0.5%)-乙腈(含甲酸0.5%)，流速为1.0 mL·min^-1，进样量为2 μL。质谱的离子源为电喷雾离子源，采用正离子模式。结果初步推测出阿魏酸、9个已知的苯酞类化合物和一个未知的苯酞二聚物的衍生物。结论通过液相色谱-质谱联用分析可获得苯酞类化合物的丰富的结构信息，为这些化合物的定性提供了一种快速有效的方法，也为当归药材的质量控制提供更多科学依据。     

藤黄中新藤黄酸的分离及其结构

藤黄系藤黄科植物(Garcinia hanburryi)所分泌的干燥树脂,有攻毒蚀疮,破血散结等功效,近年有报道藤黄制成注射液后,临床治疗恶性肿瘤有一定疗效。藤黄中含主要酸性成分为藤黄酸,对其结构和化学方面的研究工作已有百余年历史,其结构如(Ⅰ),并有报告藤黄酸为藤黄抗癌的有效成分。我们从藤黄树脂中除分离得到藤黄酸以外,还得到另一个新的酸性化合物藤黄Ⅱ号,经用UV、IR、~1HNMR、MS等测定,推论其结构为(Ⅱ),命名为新藤黄酸,该化合物对人体肝癌细胞和某些小鼠移植性肿瘤均有一定作用,研究结果将另文报道,本文主要报道新藤黄酸的分离和结构研究。     

叶下珠中新多酚化合物的分离与鉴定

目的:研究植物药叶下珠(Phylanthus urinaria L.)的化学成分。方法:用各种色谱技术进行分离纯化,经MS,¹H,¹³CNMR和HMBC光谱分析鉴定其结构。结果:分得4个多酚类化合物,其结构分别鉴定为1-O-没食子酰基 3,6-六羟基联苯二甲酰基-2,4-去羟甲基诃子酰基-吡喃葡糖(I),焦棓酚(pyrogalol,I),咖啡酸(cafeicacid,II)和(brevifolincarboxylic acid,IV)。结论:化合物I为一新的鞣花单宁(命名为phylanthusinU),I,II,IV为首次从该植物中分得。     

液相色谱 质谱联用法鉴定9种皮质激素药物

目的:建立皮质激素的液相色谱 质谱分析方法,为限制药物滥用提供检测手段。方法:采用RP-HPLC-UV-MS联用法,同时对9 种皮质激素进行色谱分离及质谱鉴定,利用质谱解析软件研究了该类化合物的质谱裂解规律,并应用本法鉴定了药物制剂、送检物及尿样中的皮质激素。结果:在正离子检测方式下,9 种皮质激素的质谱断裂方式存在共性,即对于含有氟的分子,二级质谱优先脱去HF;含有醋酸酯的分子,二级质谱易产生脱CH₃COOH的特征碎片离子。每种化合物的检测限约为6 ng。结论:本法可用于皮质激素的体外、体内定性分析。     

Identification of gentamicin impurities by liquid chromatography tandem mass spectrometry

An HPLC/MS/MS method was developed for identification of impurities in gentamicin. The HPLC was performed on a Synergy Hydro-RP column using 50 mM trifluoroacetic acid (TFA), pH 2 adjusted with ammonium solution and methanol as mobile phase. All impurities in gentamicin were separated from main gentamicin components. Atmospheric pressure chemical ionization (APCI) was used and product mass spectra of protonated molecules were acquired. Seventeen impurities were detected in gentamicin. Reference compounds: gentamicins: C_2b, B, B₁, G-418, sisomicin, garamine and gentamines: C₁, C_1a, C₂, C_2a were used for spectra interpretation and impurities identification. All MS/MS spectra were interpreted and fragmentation transitions for gentamicins and in general for aminoglycoside antibiotics (AG) were proposed. All impurities were identified. More than one isomere were proposed for three impurities.     

N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸、脯氨酸和焦谷氨酸的合成

报道11个预期有血管紧张素转化酶抑制活性的N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸(VII_1～9)、脯氨酸(VII₁₀)和焦谷氨酸(VIl₁₁)的合成和鉴定。所有上述化合物以及与VⅡ_1～9相应的叔丁酯(VI_1～9)均未见文献报道。药理初试结果显示，化合物VII₈，VII₉和VI₁₀均有明显降压活性。     

Targeting amine- and phenol-containing metabolites in urine by dansylation isotope labeling and liquid chromatography mass spectrometry for evaluation of bladder cancer biomarkers

Metabolomics is considered an effective approach for understanding metabolic responses in complex biological systems. Accordingly, it has attracted increasing attention for biomarker discovery, especially in cancer. In this study, we used a non-invasive method to evaluate four urine metabolite biomarker candidates—o-phosphoethanolamine, 3-amio-2-piperidone, uridine and 5-hydroxyindoleactic acid—for their potential as bladder cancer diagnostic biomarkers. To analyze these targeted amine- and phenol-containing metabolites, we used differential ¹²C₂-/¹³C₂-dansylation labeling coupled with liquid chromatography/tandem mass spectrometry, which has previously been demonstrated to exhibit high sensitivity and reproducibility. Specifically, we used ultra-performance liquid chromatography (UPLC) coupled with high-resolution Fourier transform ion-cyclotron resonance MS system (LC-FT/MS) and an ion trap MS with MRM function (LC-HCT/MS) for targeted quantification. The urinary metabolites of interest were well separated and quantified using this approach. To apply this approach to clinical urine specimens, we spiked samples with ¹³C₂-dansylatedsynthetic compounds, which served as standards for targeted quantification of ¹²C₂-dansylated urinary endogenous metabolites using LC-FT/MS as well as LC-HCT/MS with MRM mode. These analyses revealed significant differences in two of the four metabolites of interest—o-phosphoethanolamine and uridine—between bladder cancer and non-cancer groups. O-phosphoethanolamine was the most promising single biomarker, with an area-under-the-curve (AUC) value of 0.709 for bladder cancer diagnosis. Diagnostic performance was improved by combining uridine and o-phosphoethanolamine in a marker panel, yielding an AUC value of 0.726. This study confirmed discovery-phase features of the urine metabolome of bladder cancer patients and verified their importance for further study.     

Synthesis of [2,3,4,5,6‐2H5]phenyl glucosinolate

Starting from commercially available [2,3,4,5,6‐²H₅]benzoic acid, [2,3,4,5,6‐²H₅]phenyl glucosinolate was synthesized. Under negative‐ion electrospray‐ionization mass spectrometric conditions, this compound affords a peak at m/z 399. Since this m/z value is not known from the ions derived from natural glucosinolates, the [2,3,4,5,6‐²H₅]phenyl glucosinolate reported here is useful as an internal standard for the quantification of glucosinolates by negative‐ion mass spectrometry (MS) and liquid chromatography (LC)/MS techniques. Copyright © 2007 John Wiley & Sons, Ltd.     

HPLC-MS/MS法测定人血浆中的左西孟旦及其主要代谢物

建立快速、 灵敏、 易操作的LC-MS/MS法测定人血浆中的左西孟旦及其代谢物OR-1855和OR-1896的浓度。根据待测物的不同性质， 采用两套液相色谱系统和电离方式分别测定人血浆中的左西孟旦和代谢物OR-1855、 OR-1896。测定左西孟旦时， 用瑞舒伐他汀为内标， 血浆样品经甲醇沉淀蛋白， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(55∶45∶0.02， v/v/v)为流动相， Capcell MG III C₁₈柱(35 mm×2.0 mm ID， 3 μm)进行分离， 采用电喷雾电离源，以选择反应监测(SRM)方式进行负离子检测。测定代谢物OR-1855和OR-1896时， 用多索茶碱为内标， 血浆样品经乙酸乙酯萃取， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(65∶35∶0.1， v/v/v)为流动相， Zorbax Extend C₁₈柱(150 mm×4.6 mm ID， 5 μm)进行分离， 采用电喷雾电离源， SRM方式进行正离子检测。测定血浆中左西孟旦方法的线性范围为0.10～50.0 ng·mL^-1， 定量下限可达0.10 ng·mL^-1； 测定血浆中代谢物OR-1855和OR-1896方法的线性范围均为0.20～100 ng·mL^-1， 定量下限均可达0.20 ng·mL^-1。本方法专属性好， 准确、 快速， 适用于左西孟旦注射液的临床药代动力学研究。     

Simultaneous quantification of 19 diterpenoids in Isodon amethystoides by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method was developed to characterize and quantify 19 diterpenoid compounds in Isodon amethystoides simultaneously. By employing a Diamonsil C₁₈ column, 19 constituents were separated within 15 min using a gradient elution consisted of methanol containing 0.1% formic acid and 0.1% aqueous formic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode in a single run and quantified by a multiple-reaction monitor (MRM). All standard calibration curves showed good linearity (r² > 0.99) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.06–3.25% and 1.56–3.84%. The recovery studies for the quantified compounds were between 95.82 and 108.3% with RSD values less than 1.86%. The results indicated that the method is simple, rapid, specific and reliable. This method was successfully applied for identification and quantification of 19 diterpenoids in 11 batches of I. amethystoides. The results showed that the contents of diterpenoids in I. amethystoides from different sources were widely varied.     

Study on the aconitine‐type alkaloids of Radix Aconiti Lateralis and its processed products using HPLC‐ESI‐MSn

The type and content change of alkaloids of Radix Aconiti Lateralis (Lateral root of Aconitum carmichaeli Debx, an important and popular medicinal herb in Traditional Chinese Medicine) in processing were studied using high performance liquid chromatography‐electrospray ionization‐multi‐stage mass spectrometry (HPLC‐ESI‐MSⁿ). Extract containing alkaloids, which were known to be the main bioactive components of the herb, was prepared by 1% (v/v) hydrochloric acid solution. An HPLC method which can simultaneously separate these alkaloids was established with gradient elution mode. Forty‐eight compounds were structurally identified by employing LC‐MSⁿ techniques; the MSⁿ spectra of most alkaloids displayed a characteristic behaviour of loss of CH₃COOH (60 u), CH₃OH (32 u), C₆H₅COOH (122 u), CO (28 u) and H₂O (18 u). Among them, the fragmentation ion C6H5COOH (122 u) was reported for the first time. By comparison, 22 compounds were found both in the crude materials and the processed products; 17 alkaloids were only found in the processed products and 9 alkaloids,which existed in crude material, could not be detected after processing. In the process of identification, we found new kinds of alkaloids, with protonated molecules at m/z 452, 468, and 482. We called these compounds dehydra‐hypaconine, dehydra‐mesaconine, and dehydra‐aconine, respectively. Copyright © 2012 John Wiley & Sons, Ltd.