Pre-column derivatization and gas chromatographic determination of alkaloids in bulbs of Fritillaria

A method of precolumn derivatization GC with FID detection was developed for a simultaneous analysis of five major steroidal alkaloids of Fritillaria species, namely ebeiedine, ebeiedinone, verticine, verticinone and imperialine. Derivatization was carried out by trimethylsilylation of the hydroxyl-containing Fritillaria alkaloids to the corresponding trimethylsilylates with trimethylsilylimidazole. Reaction conditions were optimised and the alkaloids derivatives were characterised by on-line GC-MS. The validated GC method demonstrated a good linearity at the sampling ranges used. This analytical method is simple, convenient and reproducible. The developed assay was successfully applied to the determination of the major pharmacologically active alkaloids in three commonly used antitussive Fritillaria species: F. cirrhosa, F. thunbergii and F. pallidiflora.     

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.     

Characterizing distribution of steroidal alkaloids in Fritillaria spp. and related compound formulas by liquid chromatography-mass spectrometry combined with hierarchial cluster analysis

Bulbus Fritillariae (BF), referred to the bulbs of several Fritillaria species (Liliaceae), is a commonly used antitussive and expectorant herb in traditional Chinese medicine (TCM). Due to the complexity of BF botanical origin in the herbal markets, it is urgently needed to develop a reliable method for species identification. Previous studies based on morphological and histological as well as molecular biological techniques have respective limitations. For the purpose of finding a possible discriminant method among Fritillaria species, 27 steroidal alkaloids in 17 Fritillaria species and 12 BF-containing compound formulas were identified and characterized by a high-performance liquid chromatography with mass spectrometry (HPLC-MS) method. The estimated relative composition of steroidal alkaloids was used to carry out a chemotaxonomical study on Fritillaria species by means of hierarchial cluster analysis. In addition, the characteristic occurring patterns of the examined bases were compared in an effort to distinguish the botanical origin of BF-containing compound formulas. The results demonstrated that the qualitative and quantitative differences in steroidal alkaloids were useful not only for chemotaxonomy in some medicinal Fritillaria species but also for species identification in compound formulas. The described method has important implications in quality control of BF-containing TCM preparations, allowing for the prevention of BF confusion, and also revealing the possible presence of adulteration.     

Determination of the major isosteroidal alkaloids in bulbs of Fritillaria by high-performance liquid chromatography coupled with evaporative light scattering detection

A new direct HPLC analytical method using evaporative light scattering detection coupled with a low-temperature adapter for the simultaneous determination of the major biologically active isosteroidal alkaloids in Bulbus Fritillariae, a commonly used antitussive traditional Chinese medicinal (TCM) herb, has been developed. The simultaneous separation of eight Fritillaria alkaloids was achieved on a reversed-phase C8 column with an isocratic mobile phase system consisting of acetonitrile-methanol-water (66.5:3.5:30, v/v) containing 0.006% triethylamine. This method provides good reproducibility and sensitivity for the quantification of six major isosteroidal alkaloids, namely peimissine, verticine, verticinone, imperialine, isoverticine and ebeiedine in different Fritillaria species with overall intra- and inter-day precision and accuracy of less than 11% and higher than 90%, respectively. The assay was successfully utilized to quantify the major biologically active alkaloids in five Fritillaria species. The results demonstrate that this method is simple, selective, and suitable for the quality control of this commonly used antitussive TCM herb, Bulbus Fritillariae. reserved.     

Simultaneous determination of seven major isosteroidal alkaloids in bulbs of Fritillaria by gas chromatography

The present paper describes the development of a most simple, sensitive, and specific gas chromatographic method to date, for the direct determination of seven major bioactive isosteroidal alkaloids, namely ebeiedine, ebeiedinone, ebeienine, hupehenine, isoverticine, verticine, verticinone and imperialine, in Fritillaria species, a commonly used antitussive traditional Chinese medicinal (TCM) herb. In the present study, a commercially available Supelco SAC-5 capillary column (30 m x 0.25 mm, 0.25 microm) specifically designed for the analysis of steroids was utilized for the direct determination of Fritillaria alkaloids. Calibration curves were obtained by spiking authentic compounds and the internal standard (solanidine) into herbal samples prior to extraction. Extraction was conducted simply by shaking the pre-alkalized diethyl ether solution (5.0 ml) containing dried herb (0.1 g) for 2 h. All calibration curves showed good linear regressions (r2>0.995) within test ranges. The assay was reproducible and accurate with the overall intra- and inter-day variation and accuracy of less than 10% and more than 90%, respectively. The developed GC method was successfully utilized to analyze seven major bioactive alkaloids in seven Fritillaria species, and the results demonstrate that this direct GC analytical method is suitable for the quality control of this commonly used antitussive TCM herb.     

Global detection and semi‐quantification of Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus by a non‐targeted multiple reaction monitoring approach

Methods based on triple quadrupole tandem mass spectrometry have been widely used and reported as highly selective and sensitive methods for quantifying substances of herbal medicines. However, most of them were limited to targeted components, due to the difficulties to optimize the multiple reaction monitoring transitions without authentic standards. This study proposed a novel strategy for non‐targeted optimization of multiple reaction monitoring method based on the diagnostic ion guided family classifications, tandem mass spectrometry database establishment, and transitions and collision energy screening. Applying this strategy, 59 Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus have been classified, and 51 of these Fritillaria alkaloids were successfully detected by the optimal multiple reaction monitoring method. For semi‐quantification, the easy‐to‐obtain Fritillaria alkaloids of each type, such as verticinone for cevanine type and peimisine for jervine type, were used as the reference standards to calibrate the other Fritillaria alkaloids in the same type. The method was demonstrated a good linearity (R² > 0.998) with satisfactory accuracy and precision, and the lower limits of quantification of verticinone and peimisine were estimated to be 0.076 and 0.216 pg, respectively. In addition, the results suggested that the proposed strategy might obtained high quality metabolomics data in discrimination of Fritillaria unibracteata and Fritillaria ussuriensis.     

Determination of the appetite suppressant P57 in Hoodia gordonii plant extracts and dietary supplements by liquid chromatography/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods

Hoodia gordonii is traditionally used in South Africa for its appetite suppressant properties. P57AS3 (P57), an oxypregnane steroidal glycoside, is the only reported active constituent from this plant as an appetite suppressant. Effective quality control of these extracts or products requires rapid methods to determine P57 content. New methods of liquid chromatography/mass spectrometry (LC/MS) and LC-UV for analysis of P57 from H. gordonii have been developed. The quantitative determination of P57 was achieved with a Phenomenex Gemini (Torrance, CA) reversed-phase column using gradient mobile phase of water and acetonitrile, both containing 0.1% acetic acid. The method was validated for linearity, repeatability, and limits of detection and quantification. Good results were obtained in terms of repeatability (relative standard deviation <5.0%) and recovery (98.5-103.5%). The developed methods were applied to the determination of P57 for H. gordonii plant samples, one related genus (Opuntia ficus-indica), and dietary supplements that claim to contain H. gordonii.     

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb.

Bulbus Fritillariae derived from plants of various Fritillaria species is the most commonly used antitussive traditional Chinese medicinal herb and is called Beimu. Herbs derived from similar and/or different species of Fritillaria are also used in Japan and Turkey as traditional or folk medicines. Isosteroidal alkaloids are the main bioactive ingredients in Fritillaria species. As the contents and structure types of these bioactive alkaloids vary in different Fritillaria species, quality control of these active principles in herbal Beimu is very important to ensure its safe and effective clinical use. This review describes the development of chromatographic analyses for the simultaneous qualitative and quantitative determination of the main bioactive Fritillaria isosteroidal alkaloids in herbal and biological samples. The recently developed direct HPLC-evaporative light scattering detection method is the most simple, selective and sensitive assay, and is readily used as a suitable quality control method for the analysis of the active principles of herbal Beimu.     

Identification of the major metabolites of tectorigenin in rat bile by liquid chromatography combined with time-of-flight and ion trap tandem mass spectrometry

A novel methodology for the identification of tetorigenin and its metabolites in rat bile has been created using liquid chromatography (LC) combined with time-of-flight (TOF) and ion trap multiple mass spectrometry (IT-MSn). As a means to discriminate amongst unknown organic compounds in complex biological matrices, the proposed methodology relies upon the combination of LC/TOF-MS to provide accurate mass measurements in generating a molecular formula while benefiting from the complementary structural information provided by the LC/IT-MSn. In this study, the combined approach has been applied to the metabolic fingerprinting chromatograms of rat bile samples before and after tectorigenin administration. All possible metabolites are investigated based on accurate mass data and isotope function using LC/TOF-MS and structural confirmation using LC/IT-MSn. Seven phase II metabolities of tectorigenin in rat bile have been successfully elucidated using this novel LC approach and are being reported for the first time.     

Determination of bitter orange alkaloids in dietary supplement Standard Reference Materials by liquid chromatography with atmospheric-pressure ionization mass spectrometry

A liquid chromatographic atmospheric-pressure ionization electrospray mass spectrometry (LC–API–ES–MS) method has been developed for the determination of five bitter orange alkaloids (synephrine, octopamine, n-methyltyramine, tyramine, and hordenine) in bitter orange-containing dietary supplement standard reference materials (SRMs). The materials represent a variety of natural, extracted, and processed sample matrices. Two extraction techniques were evaluated: pressurized-fluid extraction (PFE) and sonication extraction. The influence of different solvents, extraction temperatures, and pH were investigated for a plant material and a processed sample. The LC method uses a new approach for the separation of highly polar alkaloids. A fluorinated, silica-based stationary phase separated the five alkaloids and the internal standard terbutaline in less than 20 min. This method enabled the determination of the dominant alkaloid synephrine and other minor alkaloids in a variety of dietary supplement SRMs.     

Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry

Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.     

Identification and quantitation of pyrethroid pesticide residues in vegetables by solid-phase extraction and liquid chromatography/electrospray ionization ion trap mass spectrometry

A quantitative method consisting of solid-phase extraction (SPE) followed by liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) analysis was developed for the identification and quantitation of ten pyrethroid pesticides commonly used in vegetables. The best HPLC separation was achieved using a gradient program of methanol/water mixture. For the vegetable samples, an SPE procedure to clean up the matrices was carried out prior to LC/MS analysis. Under the optimum conditions, the limits of quantification of the pyrethroid pesticides (tetramethrin, allethrin, fenpropathrin, lambda-cyhalothrin, cypermethrin, deltamethrin, fenvalerate, bioresmethrin, permethrin and bifenthrin) ranged from 0.03 to 0.1 mg kg-1 with relative standard deviations<20%, and the mean recoveries ranged from 69.5 to 102.5%. The proposed method has been successfully applied to the determination of pyrethroids in six vegetables with satisfactory results.     

Chemistry, bioactivity and geographical diversity of steroidal alkaloids from the Liliaceae family

Plants belonging to the Liliaceae family have been the topic of research in many phytochemical and pharmacological laboratories because they contain structurally complex and biologically fascinating steroidal alkaloids. This review, citing 153 references, summarises the chemistry, bioactivity and geographical diversity of steroidal alkaloids isolated from Veratrum and Fritillaria species, so as to illustrate the chemo-diversity and biological significance of these alkaloids, along with their geographical distribution where this is discernible.     

Simultaneous Determination of Six Steroidal Alkaloids of Veratrum dahuricum by HPLC–ELSD and HPLC–MSn

A high performance liquid chromatography coupled with evaporative light scattering detection (HPLC–ELSD) and electrospray ionization multistage mass spectrometry (HPLC–ESI–MSⁿ), respectively, has been performed for the simultaneous determination of six steroidal alkaloids, including pseudojervine, veratrosine, jervine, veratramine, 3-veratroylzygadenine, 3-angeloylzygadenine, in Veratrum dahuricum collected in different seasons. The plants were soaked in methanol and extracted ultrasonically. The six steroidal alkaloids were obtained by silica gel column chromatography, eluting with gradient petroleum and acetyl acetate. The intra-day and inter-day precisions of the method were evaluated and were less than 1.4%. The content of steroidal alkaloids in the plant varied significantly from spring to autumn, confirming the necessity to control the quality of V. dahuricum during its preparation and application.     

Rapid determination of 5-hydroxymethylfurfural by DART ionization with time-of-flight mass spectrometry

DART (direct analysis in real time), a novel technique with wide potential for rapid screening analysis, coupled with high-resolution time-of-flight mass spectrometry (TOF-MS) has been used for quantitative analysis of 5-hydroxymethylfurfural (5-HMF), a typical temperature marker of food. The DART/TOF-MS method was optimised and validated. Quantification of 5-HMF was achieved by use of a stable isotope-labelled 5-HMF standard prepared from glucose. Formation of 5-HMF from saccharides, a potential source of overestimation of results, was evaluated. Forty-four real samples (honey and caramelised condensed sweetened milk) and 50 model samples of heated honey were analysed. The possibility of using DART for analysis of heated samples of honey was confirmed. HPLC and DART/TOF-MS methods for determination of 5-HMF were compared. The correlation equation between these methods was DART?=?1.0287HPLC?+?0.21340, R ²?=?0.9557. The DART/TOF-MS method has been proved to enable efficient and rapid determination of 5-HMF in a variety of food matrices, for example honey and caramel.     

Quantitative analyses of indoloquinazoline alkaloids in Fructus Evodiae by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

Fructus Evodiae (Wuzhuyu), the fruits of Evodia rutaecarpa and related varieties, is widely used in traditional Chinese medicine. The bioactive constituents include the indoloquinazoline alkaloids rutaecarpine, evodiamine and dehydroevodiamine. A new assay based on high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/UV/APCI-MS/MS) was developed for the measurement of the indoloquinazoline alkaloids in commercial Fructus Evodiae products. Initially, the MS/MS fragmentation pathways of indoloquinazoline alkaloids were investigated to identify fragment ions that might be useful for the sensitive and selective detection of trace indoloquinazoline alkaloids during LC/MS/MS. Then, quantitative MS analysis of five indoloquinazoline alkaloids in 12 commercial Fructus Evodiae products from different geographical sources was performed. Analyte recovery was in the range of 97.5-105.3% for all with relative standard deviations (RSDs) below 6%, the intra-assay and inter-assay RSDs were less than 7%, and good linear relationships were shown with correlation coefficients for the analytes exceeding 0.999. Therefore, this LC/MS/MS assay facilitated the rapid quantitative analysis of rutaecarpine, evodiamine, evodiamide, 14-formyldihydrorutaecarpine and dehydroevodiamine in 12 commercial Fructus Evodiae products with excellent recovery, repeatability, accuracy and sensitivity. This method is simple and specific and can be used for identification and quality control of this traditional Chinese remedy.     

Determination of seven benzoylphenylurea insecticides in processed fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatographic method with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the sensitive and selective determination of seven benzoylphenylurea insecticide residues (diflubenzuron, triflumuron, lufenuron, flufenoxuron, teflubenzuron, chlorfuazuron, hexaflumuron) in pear baby purée, concentrated lemon juice, and tomato pulp. The general sample extraction/partition method for our established multiresidue methods has been used. The entire procedure involves extraction of residues with acetone and partition into ethyl acetate/cyclohexane. Chromatographic determination was performed using a C18 column and isocratic elution. Fourteen MS/MS transitions of precursor ions were monitored (two for each pesticide) using negative ESI. The majority of mean recoveries at fortification levels of 0.002-0.020 and 0.020-0.200 mg/kg were in the range 77-102% with relative standard deviations between 2 and 10%. The excellent sensitivity and selectivity of this LC/MS/MS method allowed quantitation and identification at low levels in difficult matrices with a run time of 4 min.     

高效液相色谱-串联质谱法测定化妆品中10种生物碱

建立高效液相色谱-串联质谱同时测定化妆中10种生物碱的分析方法。实验优化了提取条件、净化方式和仪器条件等参数。样品经80%（v/v）甲醇水溶液超声提取，离心后过滤。采用Waters BEH C₁₈色谱柱分离，在多反应监测模式下测定，外标法定量。结果显示，在各自范围内，10种生物碱具有良好的线性关系，相关系数（R²）>0.9900，方法的检出限为5.0~12.5 μg/kg，定量限为12.5~50.0 μg/kg。在1倍、2倍和6倍定量限的加标水平下，10种生物碱的回收率为70.91%~116.75%，相对标准偏差为0.49%~9.98%（n=6）。该方法操作简单，适用于化妆品中10种有毒生物碱的快速筛查和定量分析。     

Determination of mepiquat chloride residues in cotton and soil by liquid chromatography/mass spectrometry

A liquid chromatographic/mass spectrometric (LC/MS) method is reported for the determination of the onium-type plant growth regulator mepiquat chloride in cotton and soil. The pesticide was extracted from the sample with ethanol and water containing 2% NH4Cl. The extract was cleaned up on a solid-phase extraction C18 column, and the pesticide was determined by LC/MS. The average recoveries were 85.9-93.8, 81.3-91.7, and 78.1-94.7%, with corresponding relative standard deviations of 3.4-9.6, 3.7-12.3, and 4.0-9.8%, from soil, cotton leaves, and cotton seeds, respectively. A coefficient of determination of R2 = 0.9964 was obtained for the analyte calibration graph, from 0.05 to 100 microg/mL. Decision limits, CCalpha, and detection capability, CCbeta, were calculated. Electrospray ionization LC/MS in the positive-ion mode (ESI+) was used to detect mepiquat chloride in extracts of soil, cotton leaves, and cotton seeds. The ion at m/z 114 in the mass spectrum was monitored.