Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Sophora flavescens Ait., a well-known Chinese herbal medicine, is widely used in clinical practice for the treatment of viral hepatitis, cancer, gastrointestinal hemorrhage, and skin diseases. This paper is the first report on a method based on the combined use of high-performance liquid chromatography, photodiode array detection, and electrospray ionization tandem mass spectrometry for the comprehensive and systematic separation and characterization of bioactive alkaloids in Sophora flavescens Ait. A total of 22 constituents were identified on the basis of the extracted ion chromatograms for different [M+H](+) ions of the alkaloids present in S. flavescens Ait. Among these, 5 constituents were unambiguously identified by comparing the experimental data on their retention times and MS(n) spectra with those of the authentic compounds, and 17 other constituents were tentatively identified on the basis of their MS(n) fragmentation behaviors and/or molecular weight information from literatures. Furthermore, some characteristic fragmentation pathways of the alkaloids in S. flavescens Ait. were detected and examined. This information may be useful for characterizing the bioactive alkaloids present in S. flavescens Ait. and for possible applications in formulations.     

Alkaloid profiling of herbal drugs using high resolution mass spectrometry

Herbal infusions are consumed worldwide thanks to their “natural” beneficial effects, also due to the presence of alkaloids, although these compounds can have poisonous effects. A method combining online solid‐phase purification with high resolution mass spectrometry was used to define the alkaloid profiles of 117 herbs and 7 commercial blends. Forty‐one alkaloids were quantified in reference to analytical standards, while the presence of a further 116 was confirmed based on accurate mass, retention time, and fragmentation profile. The targeted study showed that 52% of herbs and 42% of commercial blends contained at least one alkaloid. Pyrrolizidines were the most commonly present (26% of samples), with concentrations generally ranging from the quantification limit to roughly 100 μg kg^?1. Moreover, a homemade infusion was studied, finding on average 45% and 6% lower extraction for pyrrolizidine and steroidal alkaloids, respectively. Nevertheless, the migration of pyrrolizidines was confirmed. The study confirmed the frequent presence, natural or accidental, of alkaloids in commercial infusion herbs, highlighting the urgent need for routine and accurate controls.     

The characterisation of selected drugs with amine-containing side chains using electrospray ionisation and ion trap mass spectrometry and their determination by HPLC-ESI-MS

The electrospray ionisation-ion-trap mass spectrometry (ESI-MS(n)) of selected drug compounds with amine-containing side chains has been investigated. Certain characteristic in-source fragmentations have been observed for these molecules. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M + H](+) ions and their predominant fragment ions. These MS(n) experiments also show certain characteristic fragmentations with respect to the amine-containing side chains. QTOF-MS/MS has been used to support the identity of the proposed fragments. The data presented in this paper therefore provides useful information on the structure of these compounds with amine-containing side chains and can be used in the characterisation of such drugs, their structurally related metabolites and unknown molecules of pharmaceutical significance extracted from animal and plant sources, for example. Amphetamine, clenbuterol, flurazepam and methadone can be identified and determined in mixtures at low ng/ml concentrations by the application of HPLC-ESI-MS which can also be used for their analysis in saliva samples.     

电喷雾离子阱质谱检测亮菌甲素琥珀酸单酯的负离子裂解途径

目的采用电喷雾离子阱（ESI-MS）质谱技术研究亮菌甲素琥珀酸单酯（ArAAE）的结构和裂解途径。方法采用蠕动注射泵直接进样,ESI-MS负离子模式检查,流速为0.3 mL/h,毛细管电压为4 500 V,雾化器压力、干燥器流速和干燥器温度分别为10 psi、5 L/min和350℃,质量扫描范围m/z 100～500。结果采用ESI-MS获得了m/z 333[M-H]-,采用ESI-MS2获得了m/z 233碎片离子,而采用ESI-MS3主要获得了m/z 215,203,189,177,171,163,147和135等碎片离子。ESI-MS和ESI-MS2主要有一种裂解途径,而ESI-MS3主要有3种裂解途径,主要是酯键的裂解,并对m/z 203,189和163特征碎片离子进行ESI-MS4质谱研究,归属了其主要特征碎片离子,分析和讨论了该化合物的结构和质谱特征。结论ESI-MS负离子模式下适用于检测亮菌甲素琥珀酸单酯各级碎片裂解,归属了主要的碎片裂解途径,其方法快速、简便,为进一步研究ArAAE的体内代谢过程与结构修饰提供试验依据。     

FAB-MS/MS法研究4(20)-双键5/7/6型紫杉烷类二萜化合物的质谱裂解特征

目的　研究4(20)-双键5/7/6型紫杉烷类二萜化合物的质谱裂解特征,以及取代基种类及位置对质谱裂解的影响,探讨该类化合物的质谱裂解规律。方法　利用FAB技术测定该类型3种化合物的［M+H］⁺和［M+Na］⁺等不同加合离子,以及由其产生的特征碎片离子的CID-MS/MS谱,并对有关特征离子进行了高分辨测定。结果　C-10位为BzO取代的化合物1和2主要以［M+Na］⁺离子形式存在,该离子主要进行失去HOBz的裂解反应,而C-10位为OH基的化合物3主要以［M+H］⁺离子形式存在,该离子以失去1个和2个H₂O的裂解反应为主导。另外,化合物1和3最终裂解产生m/z 237离子,而化合物2产生m/z 253离子。结论　MS/MS技术可以有效地进行结构差异甚微的相关化合物的结构解析。     

Application of <Superscript>1</Superscript>H-NMR spectroscopy to validation of berberine alkaloid reagents and to chemical evaluation of Coptidis Rhizoma

Berberine, palmatine, and coptisine are major pharmacologically active protoberberine alkaloids in Coptidis Rhizoma, and have been used as indices for chemical evaluation of the crude drug. ¹H-NMR spectroscopy was applied to determination of purities of commercial reagents of protoberberine alkaloids. The purities of the alkaloids were calculated from the ratios of the intensities of the H-13 singlet signal at about δ 8.7 ppm of target protoberberine alkaloids to integration of a hexamethyldisilane (HMD) signal at 0 ppm. The concentration of HMD was corrected with SI traceability using potassium hydrogen phthalate of certified reference material (CRM) grade. The purity of the reagent estimated by the ¹H-NMR was, in general, lower than that claimed by the manufacturer, leading to over-estimation of the alkaloid contents of Coptidis Rhizoma when determined by HPLC. The present quantitative ¹H-NMR method was also applicable to direct determination of protoberberine alkaloid contents in Coptidis Rhizoma.     

6种罕见核苷糖电喷雾质谱裂解规律的初步研究

本文在负离子模式下, 应用电喷雾电离多级串联质谱技术对6种罕见胸苷二磷酸单糖的裂解规律进行了初步研究。结果表明: 二级串联质谱中观察到6种罕见胸苷二磷酸单糖的高丰度碎片离子源于磷酸二酯键部分的裂解, 出现一系列特征的m/z 321、383和401碎片离子, 对应 [TDP−H]⁻ 及该碎片离子分别失去水分子和磷酸基得到的碎片离子; 罕见糖环上4位取代基的改变显著影响两个重要特征碎片离子 [glycosyl-1'-PO₃]⁻ 和[glycosyl-1'-P₂O₆]⁻ 的稳定性。     

Studies on structural elucidation of Aconitum diterpenoid alkaloid by LC-APCI-MS and effects of Aconitum diterpenoid alkaloid on cutaneous blood flow

The chemical constituents of Aconitum yesoense var. macroyesoense and Aconitum japonicum were examined using high-resolution spectral analysis. Twelve novel alkaloids were isolated from A. yesoense var. macroyesoense together with 20 known alkaloids. Eight novel alkaloids were isolated from A. japonicum together with 15 known alkaloids. An HPLC-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method was useful for the simultaneous determination of 21 Aconitum alkaloids found in A. yesoense var. macroyesoense and A. japonicum. These compounds were fairly stable under the conditions used, and the protonated molecules or fragment ions characteristic of the molecule appeared as base peaks in the mass spectra and were used for selected ion monitoring. HPLC-APCI-MS is a very promising approach for structural investigations of positional isomers and stereoisomers. This method was applied successfully to stereoisomeric Aconitum alkaloids differing in configuration at C-1, -6, or -12. Comparison of the APCI spectra showed that the abundance of fragment ions was significantly higher for the C-1, -6, or -12 beta-form alkaloid than for C-1, -6, or -12 alpha-form alkaloid. The main alkaloid constituents in the root of A. yesoense var. macroyesoense, Aconitum alkaloids of the C20-diterpenoid type, kobusine and pseudokobusine, and their acyl derivatives were examined for their peripheral vasoactivities by measuring laser-flowmetrically the cutaneous blood flow in the hind foot of mice after intravenous administration. It is thought that the hydroxyl groups of alkaloids, especially a free OH group of pseudokobusine at C-6, were important for action on the peripheral vasculature leading to dilatation, and the results indicated that esterification of the hydroxyl group at C-15 with either anisoate, veratroate, or p-nitroben-zoate may contribute to enhancement of the activity of the parent alkaloids.     

Alkaloid profiling of the traditional Chinese medicine Rhizoma corydalis using high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry

Since alkaloids are the major active constituents of Rhizoma corydalis (RC), a convenient and accurate analytical method is needed for their identification and characterization. Here we report a method to profile the alkaloids in RC based on liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (LC–Q-TOF-MS/MS). A total of 16 alkaloids belonging to four different classes were identified by comparison with authentic standards. The fragmentation pathway of each class of alkaloid was clarified and their differences were elucidated. Furthermore, based on an analysis of fragmentation pathways and alkaloid profiling, a rapid and accurate method for the identification of unknown alkaloids in RC is proposed. The method could also be useful for the quality control of RC.KEY WORDS: LC–Q-TOF-MS/MS, Alkaloid, Fragmentation pathway, Rhizoma corydalis     

In‐source collision‐induced dissociation (IS‐CID): Applications,issues and structure elucidation with single‐stage mass analyzers

A discussion of the definition, advantages, and issues with the formation of ions in the transition region between an electrospray ionization (ESI) source and the ion optics of a mass analyzer is presented. The various types of ions formed in the so‐called in‐source collision‐induced dissociation (IS‐CID) process are illustrated. Applications of IS‐CID with single‐stage mass analyzers, such as structure elucidation and quantitation, are demonstrated. The discussion is illustrated by examples of the in‐source fragmentation of ginkgolides, which are marker compounds found only in Ginkgo biloba. Supercritical fluid chromatography (SFC) with non‐aqueous eluents was used to achieve a fast resolution of the ginkgolides without the hydrolysis reactions possible with aqueous high‐performance liquid chromatography (HPLC) eluents. In‐source ion generation occurs at relatively high pressures (ca. 1–3 torr) compared to the low pressure normally observed in collision chambers of tandem mass spectrometry (MS/MS). As a result, the fragmentation process is complex and often generates ions other than the fragments observed with classic CID or the same ions at different intensities. The objective of the current tutorial is to illustrate the conditions under which single‐stage, quadrupole or time‐of‐flight mass analyzers with electrospray or in‐air (direct analysis in real time; DART) ionization can be used for quantitation and structure elucidation in a manner similar to that observed with MS/MS. While the low m/z (≤ [M±H]^±) ions formed in‐source often duplicate the ions observed in MS/MS systems, it is the focus of this discussion to illustrate the utility of in‐source generated fragment ions that may not be observed or observed at different intensities than in the collision cells of MS/MS instruments.     

防己诺林碱电喷雾质谱裂解的机制

目的对防己诺林碱的电喷雾质谱裂解机制进行研究。方法采用量子化学计算结合电喷雾质谱法预测化合物的质谱裂解机制。结果防己诺林碱电喷雾质谱分析发现,主要脱掉甲醇、羟基自由基和甲烷中性分子,以及发生逆Diels-Alder反应(RDA反应),经量子化学计算确定了脱掉-OCH3的顺序:C17-O40>C6-O43>C33-O34。结论质谱与量子化学有机结合可以预测质谱裂解机制。     

基于UPLC-Q-TOF-MS技术对黄连须生物碱类成分及其裂解规律的分析

目的 应用超高效液相色谱-飞行时间质谱联用技术(UPLC-Q-TOF-MS)分析黄连须中生物碱类成分,并对其中主要类型生物碱的裂解规律进行解析。方法 采用Waters Acquity UPLC BEH C18色谱柱,以0.1%甲酸水(A)-0.1%甲酸乙腈(B)为流动相梯度洗脱,质谱采用电喷雾(ESI)离子源,正负离子模式下采集数据,在Peakview 2.0/masterview1.0软件中依据自建的黄连化学成分数据库筛查化合物,通过精确质量数及比对Natural products HR-MS/MS Spectral Library 1.0 software所含的1000余中中草药对照品二级谱图进行裂解规律分析。结果 鉴定并推断出黄连须中19个生物碱类成分,并对其中原小檗碱型、氧化小檗碱型及普罗托品型生物碱的裂解规律进行了分析。结论 该法为黄连须成分研究提供了依据,证明了黄连须的利用价值,并对其进一步开发利用奠定了基础。     

The characterization of isobaric product ions of fentanyl using multi‐stage mass spectrometry,high‐resolution mass spectrometry and isotopic labeling

This study uses a combination of multi‐stage mass spectrometry (MSⁿ), accurate mass measurements – with high‐resolution mass spectrometry (HRMS) – and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4‐ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4‐ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.     

大鼠口服泻心汤及其配伍组方中原小檗碱类成分尿排泄动力学

目的研究大鼠灌服泻心汤及其配伍组方中3种原小檗碱类生物碱(小檗碱、巴马汀、黄连碱)尿排泄动力学的变化。方法取SD大鼠20只随机分成黄连、大黄黄连、黄芩黄连、泻心汤4组,采集灌胃给药前及给药后不同时间段尿液,用HPLC法测定尿药浓度,由尿排泄-时间数据计算尿排泄动力学参数,采用方差分析比较各组中3种原小檗碱类生物碱尿排泄动力学参数的差异。结果3种原小檗碱类生物碱排泄速率常数相近,排泄半衰期为9~16h。与黄连组比较,泻心汤组、大黄黄连组、黄芩黄连组中黄连碱尿排泄量占给药量比值减少,其中泻心汤组与之比较差异具有统计学意义(P<0.05)。结论3种原小檗碱类生物碱具有类似的尿排泄特征,大黄、黄芩和黄连配伍能减少黄连碱的尿排泄量。     

Palmatine, a protoberberine alkaloid, inhibits both Ca(2+)- and cAMP-activated Cl(-) secretion in isolated rat distal colon

BACKGROUND AND PURPOSE: The protoberberine alkaloid berberine has been reported to inhibit colonic Cl(-) secretion. However, it is not known if other protoberberine alkaloids share these effects. We have therefore selected another protoberberine alkaloid, palmatine, to assess its effects on active ion transport across rat colonic epithelium. EXPERIMENTAL APPROACH: Rat colonic mucosa was mounted in Ussing chambers and short circuit current (I (SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cAMP content was determined by an enzyme immunoassay. Intracellular Ca(2+) concentration was measured with Fura-2 AM. KEY RESULTS: Palmatine inhibited carbachol-induced Ca(2+)-activated Cl(-) secretion and the carbachol-induced increase of intracellular Ca(2+) concentration. Palmatine also inhibited cAMP-activated Cl(-) secretion induced by prostaglandin E(2) (PGE(2)) or forskolin. Palmatine prevented the elevation of intracellular cAMP by forskolin. Determination of apical Cl(-) currents showed that palmatine suppressed the forskolin-stimulated, apical cAMP-activated Cl(-) current but not the carbachol-stimulated apical Ca(2+)-activated Cl(-) current. Following permeabilization of apical membranes with nystatin, we found that palmatine inhibited a carbachol-stimulated basolateral K(+) current that was sensitive to charybdotoxin and resistant to chromanol 293B. However, the forskolin-stimulated basolateral K(+) current inhibited by palmatine was specifically blocked by chromanol 293B and not by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: Palmatine attenuated Ca(2+)-activated Cl(-) secretion through inhibiting basolateral charybdotoxin-sensitive, SK4 K(+) channels, whereas it inhibited cAMP-activated Cl(-) secretion by inhibiting apical CFTR Cl(-) channels and basolateral chromanol 293B-sensitive, KvLQT1 K(+) channels.     

Tandem MS analysis of model peptide adducts from reactive metabolites of the hepatotoxin 1,1-dichloroethylene

Dichloroethylene (DCE) is a hepatotoxin that undergoes cytochrome P450-catalyzed bioactivation in hepatocytes to form 2-chloroacetyl chloride and 1,1-dichloroethylene oxide. 2-Chloroacetyl chloride reacts with nucleophilic residues and with N-terminal amines to produce 2-chloroacetylated residues and with glutathione to form the reactive electrophile S-(2-chloroacetyl)glutathione (ClCH(2)COSG), which, in turn, is capable of sulfhydryl alkylation. 1,1-DCE oxide can bind to cysteine sulfhydryl groups and subsequently hydrolyze to form an S-carboxymethylated cysteine residue. S-Carboxymethylated, 2-chloroacetylated, and GSCOCH(2)-S-Cys-peptide adducts of model cysteine-containing peptides were synthesized, and their fragmentation patterns were characterized by electrospray tandem mass spectrometry. Synthesis of GSCOCH(2)-S-Cys-peptide adducts was achieved via a novel tert-butoxycarbonyl (tBOC) derivative of ClCH(2)COSG. CID of GSCOCH(2)-S-Cys-peptide adducts resulted in product ions and neutral losses indicative of the GSCOCH(2)-S-Cys moiety as well fragment ion pairs in the b- and y-ion series corresponding to the modified cysteine residue. S-Carboxymethylated peptides exhibited only a characteristic b- or y-series ion pair separated by 161 Da, corresponding to cysteine + CH(2)COOH. CID of 2-chloroacetylated peptides showed neutral losses of 36 (HCl), 78 (HCOCH(2)Cl), 96 (HCOCH(2)Cl + H(2)O), and 114 Da (HCOCH(2)Cl + 2H(2)O). Combinations of characteristic fragment ions, neutral losses, and ion pairs thus are characteristic for DCE-derived adducts. These features can be used in an MS/MS data reduction algorithm for the selective identification of protein targets of DCE metabolites.     

高效液相色谱-单四极杆质谱仪结合同位素峰形校正检索技术快速确定头孢呋辛水溶液降解杂质

目的:探索一种利用同位素峰形校正检索技术(CLIPS),在低分辨率单四极杆液质联用仪上,实现对未知杂质元素组成的分析,进而快速推断降解杂质结构的方法。方法:头孢呋辛对照品经水浴降解获得未知杂质混合样品,用HPLC-MS进行分离,并获得降解产物的准分子离子和碎片离子的同位素峰形;用元素组成已知的同位素峰做校正,对未知降解杂质进行CLIPS校正分析,确定元素组成,再根据质谱谱图信息初步确定其结构式。结果:水浴降解溶液中7个降解杂质得到了有效分离。用CLIPS确定了降解杂质的准分子离子和部分碎片离子的元素组成,并初步推测出其中4个降解杂质的结构。结论:同位素峰形校正检索技术结合高效液相色谱-单四极杆质谱联用仪可以确定降解杂质的元素组成,进而帮助降解杂质的结构解析和确证。     

Lactam ergot alkaloids (ergopeptams) as predominant alkaloids in sclerotia of Claviceps purpurea from Norwegian wild grasses

Four major alkaloids in the extracts from sclerotia of Claviceps purpurea, picked from wild grasses, have been identified as lactam (non-cyclol) ergot alkaloids. The structural information was obtained from ion trap MS and NMR spectroscopy. The data for one of the lactam ergot alkaloids were coinciding with ergocristam [N-(lysergyl-valyl)-cyclo(phenylalanyl-prolyl)]. The structural information of two further lactam alkaloids was suggestive of either alpha- or beta-ergocryptam [N-(lysergyl-valyl)-cyclo(leucyl-prolyl) or N-(lysergyl-valyl)-cyclo(isoleucyl-prolyl)] and ergoannam [N-(lysergyl-leucyl)-cyclo(leucyl-prolyl) or N-(lysergyl-isoleucyl)-cyclo(isoleucyl-prolyl)]. The constitution of the fourth lactam ergot alkaloid corresponded to N-(lysergyl-isoleucyl)-cyclo(phenylalanyl-prolyl), a new ergopeptam, which has not been described before. Additionally, the cyclol-analogue of the new ergopeptam was detected in the extracts and has been identified on the basis of its product ion spectrum from fragmentation of [M+H](+). The study described in this paper shows that lactam ergot alkaloids may not only be minor products of ergopeptine biosynthesis, as has been suggested hitherto, but may be major biosynthetic endproducts for some ergot strains. This is also the first report demonstrating the production of an ergot alkaloid that contains isoleucine as the second amino acid, i.e. the N-(lysergyl-isoleucyl)-moiety, by parasitic, naturally growing C. purpurea. This unusual type of ergot alkaloid has so far only been found in saprophytic cultures of C. purpurea.     

ACYLPOLYAMINES: MASS SPECTROMETRIC ANALYTICAL METHODS FOR Araneidae SPIDER ACYLPOLYAMINES

A new class of compounds, acylpolyamine has been isolated from spider venom constituents. Recent advances in highly sensitive mass spectrometric techniques have been applied successfully to characterize these acylpolyamines even with the use of a single venom gland. This has been achieved, in part, by improvements in fast atom bombardment mass spectrometry (FAB), continuous flow (FRIT) FAB-MS combined with reversed-phase high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) and high energy collision induced dissociation (CID) tandem MS/MS. Crude venom analysis without chromatographic separation can be realized directly by MALDI-MS. A charge-remote fragmentation method has provided abundant structure-related product ions and have reduced the quantity of required venom for the structure analysis of acylpolyamines. These mass spectrometric methods were proved to be useful for the analysis of complex constituents of spiders and other arthropod venom glands.