Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available.     

Liquid chromatography-tandem mass spectrometry analysis allows the simultaneous characterization of C-glycosyl and O-glycosyl flavonoids in stingless bee honeys

The analysis of the phytochemicals present in stingless bee honey samples has been a difficult task due to the small amounts of samples available and to the complexity of the phytochemical composition that often combines flavonoid glycosides and aglycones. Honey samples produced in Venezuela from Melipona species were analyzed using a combination of solid-phase extraction and HPLC-DAD-MSn/ESI methodologies with specific study of the fragment ions produced from flavonoid glycosides. The analyses revealed that flavonoid glycosides were the main constituents. The honey samples analyzed contained a consistent flavonoid pattern composed of flavone-C-glycosides, flavonol-O-glycosides and flavonoid aglycones. The HPLC-DAD-MSn/ESI analysis and the study of the fragment ions obtained allowed the characterization and quantification for the first time of five apigenin-di-C-glycosides, and ten quercetin, kaempferol and isorhamnetin O-glycosides (di- and tri- glycosides), and the aglycones pinobanksin, quercetin, kaempferol and isorhamnetin in the different samples. This is the first report of flavonoid-C-glycosides in honey. The results show that the content of flavonoid-glycosides (mean values of 2712 μg/100 g) in stingless bee honeys is considerably higher than the content of flavonoid aglycones (mean values of 315 μg/100 g). This differs from previous studies on Apis mellifera honeys that consistently showed much higher aglycone content and smaller flavonoid glycoside content. The occurrence of relevant amounts of flavonoid glycosides, and particularly C-glycosides, in stingless bee honeys could be associated with their putative anticataract properties.     

Analysis of scopolamine and its eighteen metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method is described for the determination of scopolamine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC–MS/MS). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of scopolamine. After extraction procedure, the pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol/ ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MS/MS system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention-times and full scan MSⁿ spectra with those of the parent drug. The results revealed that at least 18 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, hydroxyscopolamine N-oxide, p-hydroxy-m-methoxyscopolamine, trihydroxyscopolamine, dihydroxy-methoxyscopolamine, hydroxyl-dimethoxyscopolamine, glucuronide conjugates and sulfate conjugates of norscopolamine, hydroxyscopolamine and the parent drug) and the parent drug existed in urine after ingesting 55 mg/kg scopolamine to healthy rats. Hydroxyscopolamine, p-hydroxy-m-methoxyscopolamine and the parent drug were detected in rat urine for up 106 h after ingestion of scopolamine.     

Identification and partial characterization of C-glycosylflavone markers in Asian plant dyes using liquid chromatography-tandem mass spectrometry

Flavonoids in the grasses (Poaceae family), Arthraxon hispidus (Thunb.) Makino and Miscanthus tinctorius (Steudel) Hackel have long histories of use for producing yellow dyes in Japan and China, but up to now there have been no analytical procedures for characterizing the dye components in textiles dyed with these materials. LC-MS analysis of plant material and of silk dyed with extracts of these plants shows the presence, primarily, of flavonoid C-glycosides, three of which have been tentatively identified as luteolin 8-C-rhamnoside, apigenin 8-C-rhamnoside and luteolin 8-C-(4-ketorhamnoside). Two of these compounds, luteolin 8-C-rhamnoside (M=432), apigenin 8-C-rhamnoside (M=416), along with the previously known tricin (M=330) and several other flavonoids that appear in varying amounts, serve as unique markers for identifying A. hispidus and M. tinctorius as the source of yellow dyes in textiles. Using this information, we have been able to identify grass-derived dyes in Japanese textiles dated to the Nara and Heian periods. However, due to the high variability in the amounts of various flavonoid components, our goal of distinguishing between the two plant sources remains elusive.     

Metabolic profiling of Actaea species extracts using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Despite persistent questions about the safety of black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.), its products continue to be one of the most popular botanical supplements in the United States market. Black cohosh products have been associated with cases of liver toxicity, but subsequent evaluation found some products to be adulterated with other related plants from the same genus. US FDA regulations require that black cohosh products be unadulterated, and correct identification of different species of Actaea is a key first step for their good manufacturing practice. We have developed a phytochemical method to distinguish four different groups of Actaea, including: species other than A. racemosa, Asian species, A. racemosa, and North American species other than A. racemosa. Using HPLC-TOF-ESI-MS technique and principal component analysis, we identified 15 chemical markers (1-3, 5-6, 8-10, 12, 16-21). Three marker compounds were unambiguously identified using authentic standards, and 12 marker compounds were tentatively identified by comparison of fragmentation patterns with previously reported data. The presence of these marker compounds is critical for discrimination among the four groups of closely related plants. The use of metabolic profiling to distinguish black cohosh from related species of Actaea has broader implications in the identification of markers to help authenticate other important medicinal plants.     

液相色谱-串联质谱法测定毛发中的司坦唑醇

采用液相色谱-串联质谱(LC-MS/MS)建立了毛发中司坦唑醇的分析方法,并将其应用于单次给药动物实验中的动物毛样品分析。将10 mg样品碱水解后加入戊烷提取,然后进行LC-MS/MS分析,采用正离子电喷雾电离、多反应监测模式测定,方法的最低定量限为25 pg/mg。剃去豚鼠背部中央的毛,以60 mg/kg的剂量于豚鼠腹腔注射司坦唑醇,然后隔天在同一部位剃取其毛,两周内该豚鼠毛中均能检出司坦唑醇;给药后豚鼠毛中司坦唑醇的含量在第1周内保持稳定,在给药后第10天达到峰值。所建立的方法毛发取样量少,特异性强,灵敏度高,适用于毛发中司坦唑醇的分析。     

液相色谱-串联质谱法测定面粉及其制品中的联二脲

建立了面粉及其制品中联二脲的液相色谱-串联质谱（LC-MS/MS）测定及确证方法。采用纯水振荡提取样品中的联二脲,联二脲经高锰酸钾氧化后,转变为偶氮甲酰胺,再加入对甲苯亚磺酸钠使偶氮甲酰胺衍生为对甲苯磺酰氨基脲。以乙腈和2 mmol/L乙酸铵溶液（含0.2%（v/v）甲酸）为流动相进行梯度洗脱,经Shimpak XR-ODSⅡ色谱柱（150 mm×2.0 mm,2.2 μm）分离,电喷雾正离子模式电离（ESI⁺）,多反应监测（MRM）模式检测,同位素内标法定量。方法的线性范围为1~20000 μg/kg,相关系数为0.9999,定量限为5 μg/kg;当添加水平为5.0、10.0与50.0 μg/kg时,联二脲的平均回收率为78.3%~108.0%,相对标准偏差（RSDs）不大于5.73%。本方法新颖、可靠、灵敏、线性范围广,而且实用性强,可用于面粉及其多种制品中联二脲的测定和确证。     

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.     

Quantitation of free and total bisphenol A in human urine using liquid chromatography-tandem mass spectrometry

Bisphenol A (BPA) is employed in the synthesis of polycarbonate plastics and epoxy resins and is widely used in consumer products including as a coating for the inside of almost all food and beverage containers and thermal-imaging paper. Bisphenol A is considered to have important health implications because it possesses weak estrogenic activity and can leach from storage containers resulting in its consumption by both humans and animals. It is metabolized in the body and excreted into urine as a glucuronide derivative. In this report, we present an accurate, selective, sensitive, and reproducible high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the quantitation of BPA in human urine, which is not prone to exogenous contamination. BPA-glucuronide is hydrolyzed enzymatically, extracted with toluene, derivatized with dansyl chloride, and the BPA-(dansyl)(2) derivative is analyzed using reversed-phase HPLC/MS/MS. Calibration was linear to 50 ng/mL with a limit of quantitation of 50 pg/mL and a limit of detection of 5 pg/mL.     

In vivo metabolism study of rhubarb decoction in rat using high-performance liquid chromatography with UV photodiode-array and mass-spectrometric detection: a strategy for systematic analysis of metabolites from traditional Chinese medicines in biological samples

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.     

液相色谱-四极杆-飞行时间串联质谱法高通量分析美国红参的脑苷脂分子种

利用液相色谱-四极杆-飞行时间串联质谱法(LC-Q-TOF MS/MS)分析美国红参(Parastichopus californicus)脑苷脂的分子种组成。以氯仿-甲醇(2：1,v/v)溶液提取,经皂化和固相萃取柱(SPE)净化后,在正离子模式下,通过自动二级质谱方式同时获取样品脑苷脂分子的一级和二级质谱图。筛取含180 Da中性丢失碎片的分子,再结合二级质谱中的长链碱和脂肪酸碎片,确定每种脑苷脂分子的结构。通过该法一次性共分析出123种美国红参脑苷脂分子种;含有的长链碱共18种,其中植物鞘氨醇型与鞘氨醇型长链碱的相对含量比为1：2;脂肪酸为18~25碳的饱和或单不饱和脂肪酸,以24碳脂肪酸为主,2-羟基脂肪酸占58.62%,饱和与单不饱和脂肪酸的相对含量比约为1：3。LC-Q-TOF MS/MS法分析海参脑苷脂分子种的灵敏度高,准确,简便,为海参脑苷脂的构效关系研究及功能食品开发提供理论依据。     

高效液相色谱-串联质谱法同时测定辽东楤木不同部位中5种皂苷类成分的含量

建立了高效液相色谱-串联质谱法（HPLC-MS/MS）用于测定辽东楤木不同部位中的楤木皂苷Ⅱ、楤木皂苷Ⅳ、楤木皂苷Ⅴ、楤木皂苷Ⅹ和楤木叶皂苷Ⅱ的含量,并比较了楤木不同部位中上述5种皂苷的含量差异。以Alltima C₁₈柱（250 mm×4.6 mm,5 μm）为分析柱,以乙腈和0.1%（体积分数）甲酸水溶液为流动相,梯度洗脱,流速为0.8 mL/min。在电喷雾正离子多反应监测（MRM）模式下进行检测。结果表明该方法中楤木皂苷Ⅱ、楤木皂苷Ⅳ、楤木皂苷Ⅴ、楤木皂苷Ⅹ和楤木叶皂苷Ⅱ分别在0.17~108 μg/L、0.53~329 μg/L、0.77~480 μg/L、0.77~480 μg/L和0.82~510 μg/L范围内线性关系良好,相关系数（r²）均大于0.999,提取回收率为99.0%~100.2%。该方法简单、快速、灵敏,可用于测定辽东楤木药材不同部位中的皂苷类成分的含量。测定结果发现楤木不同部位中上述5种皂苷总量多少的顺序为根皮 >叶 >种子 >芽。     

液相色谱-串联质谱法检测玩具中的3种异噻唑啉酮类防腐剂

建立了液相色谱-串联质谱快速测定玩具中异噻唑啉酮类防腐剂(2-甲基-4-异噻唑啉-3-酮、5-氯-2-甲基-4-异噻唑啉-3-酮和1,2-苯并异噻唑啉-3-酮)的分析方法。样品经去离子水超声提取后进行液相色谱-串联质谱分析。色谱流动相为甲醇-水(15:85, v/v),等度洗脱,在选择反应监测(SRM)模式下定性和定量分析。3种被分析物的工作曲线线性范围均为2.0～1 000 μg/L,方法的定量限(信噪比大于10)为0.04 mg/kg,灵敏度优于欧盟玩具协调标准EN71-11-2005中推荐的方法。两种类型玩具样品中的加标回收率分别为95.9%～105.2%和94.7%～102.8%,精密度分别为3.04%～4.96%和2.36%～4.79%。应用本方法对10种玩具样品进行了测试,结果完全能满足欧盟玩具协调标准EN71-9-2005对玩具中异噻唑啉酮类防腐剂的检测要求。     

Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

纳升级反相液相色谱-串联质谱法分析锦灯笼提取物中的蛋白质

通过水提、酸沉法得到锦灯笼果实提取物,其中蛋白质含量为188 mg/g(以提取物干重计),共含有18种氨基酸,其中8种人体必需氨基酸占氨基酸总量的31%。基于鸟枪法蛋白质组学的分析方法,用纳升级反相液相色谱-串联质谱(nano-RPLC-MS/MS)系统分析锦灯笼果实提取物中蛋白质的酶解产物,结合数据库检索,共鉴定得到60种蛋白质;通过生物信息学分析,得到锦灯笼提取物中的蛋白质具有催化活性、抗氧化活性、酶调节活性、养分贮液囊活性、运输活性、结合活性六大生物活性,其中鉴定到与抗氧化相关的蛋白质有3种,为锦灯笼中蛋白质的功能性质的进一步研究奠定了基础。     

Simultaneous determination of flonicamid and its metabolites in vegetables using QuEChERS and reverse-phase liquid chromatography-tandem mass spectrometry

This paper presents a rapid analytical method for the simultaneous determination of flonicamid and its metabolites N-(4-trifluoromethylnicotinoyl) glycine (TFNG), 4-trifluoromethylnicotinic acid (TFNA), and 4-trifluoromethylnicotinamide (TFNA-AM) in vegetables using QuEChERS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were extracted with acetonitrile. The extract was purified through QuEChERS method with primary secondary amine (PSA) and graphite carbon black (GCB). Then the extract was diluted with 0.1% formic acid in water, and analyzed by LC-MS/MS on a Waters Acquity BEH C18 column with methanol/0.1% formic acid in water as mobile phase with gradient elution. The linearity of the analytical response across the studied range of concentrations (0.20-500 μg/L) was excellent, obtaining correlation coefficients higher than 0.998. No significant matrix effects were observed. Recovery studies were carried out on spiked spinach and cucumber blank samples, at four concentration levels (0.01, 0.05, 0.50 and 2.0 mg/kg) performing six replicates at each level. Mean recoveries of 81.3-94.8% with CVs of 2.4-7.0% were obtained. The method demonstrated to be suitable for the simultaneous determination of flonicamid and its metabolites in vegetables.     

Qualitative and quantitative determination of the major coumarins in Zushima by high performance liquid chromatography with diode array detector and mass spectrometry

A method, high-performance liquid chromatography coupled with diode array and electrospray tandem mass spectrometry (HPLC-DAD-ESI-MS), was developed to qualitatively identify and quantitatively determine the 10 major active coumarins of Zushima. The analysis was performed by using a ZORBAX SB-C18 analytical column (250 mm x 4.6 mm ID, 5 microm) at gradient elution of 0.5% aqueous formic acid and acetonitrile with diode array detection (325 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. The proposed method was successfully applied for the qualitative and quantitative analysis of 10 coumarins in five different species of Zushima which had great variation on the contents of investigated coumarins.     

Metabolic analysis of rhubarb extract by rat intestinal bacteria using liquid chromatography-tandem mass spectrometry

Through investigation of the metabolism of rhubarb extract by rat intestinal bacteria, a total of 14 components in rhubarb extract were found to be biotransformed. These components included aloe-emodin-O-glucosides, emodin-O-glucosides, chrysophanol-O-glucosides, physcion-O-glucosides and the corresponding aglycones. Rhein also could be biotransformed by rat intestinal bacteria. Twelve major metabolites were detected in the incubation sample. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M H](-) ions were similar to those of free anthraquinones, thus allowing the rapid identification of the metabolites formed in incubation samples. The results suggested that the proposed hydrolysis of glycoside group followed by hydrogenation in quinoid moiety and/or further acetylation was the major biotransformation pathway for these anthraquinone glycosides by rat intestinal bacteria.     

液相色谱-串联质谱法检测蜂蜜中杀虫脒及其代谢产物残留

建立了液相色谱-串联质谱分析洋槐蜜、荆条蜜、蜂巢蜜、杂花蜜、野蜂蜜中杀虫脒及其代谢产物残留的方法。样品经氢氧化钠水溶液稀释溶解后,采用Waters Oasis HLB固相萃取柱净化。样品提取液经Agilent XDB-C18色谱柱分离,以0.1%甲酸水溶液和乙腈为流动相进行梯度洗脱。以电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式检测,基质匹配标准溶液外标法定量。杀虫脒及其代谢产物(4-氯邻甲苯胺)在2.5～250 μg/L范围内呈线性相关,相关系数(r)均大于0.999;定量限(S/N＞10)为5 μg/kg,检出限(S/N＞3)为2.5 μg/kg。各种蜂蜜基质样品在5、10和20 μg/kg添加水平时,杀虫脒及其代谢产物的回收率范围分别为75.8%～113.8%和85.6%～114.3%,相对标准偏差(RSD)分别为4.8%～10.2%和4.7%～9.1%,可以满足蜂蜜中杀虫脒及其代谢产物残留量的检测需要。