Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI‐MS/MS

This study aims to qualitatively analyze protoberberine alkaloids in crude extract of Coptidis Rhizoma using HPLC with ESI‐MS/MS. Possible specific molecular weights of protoberberine alkaloids were firstly deduced according to literatures and were adopted to screen the alkaloids in the HPLC with ESI‐MS of crude extract of Coptidis Rhizoma. As a result, 21 protoberberine alkaloids were found, including compounds of very low concentration and compounds coeluted in one peak. Among these, two compounds were positively identified and verified by comparison with standards. Ten of these compounds were first reported in this study for Coptidis Rhizoma. In addition, chromatographic retention parameters a and c of all compounds were obtained using their retention times under five gradient conditions and were applied to confirm the deduction about the structures of protoberberine alkaloids by tandem mass data.     

Establishment of a search library about benzylisoquinoline alkaloids based on selective separation on the binaphthyl column and standard analysis on C18 column

A search library about benzylisoquinoline alkaloids was established based on preparation of alkaloid fractions from Rhizoma coptidis, Cortex phellodendri, and Rhizoma corydalis. In this work, two alkaloid fractions from each herbal medicine were first prepared based on selective separation on the “click” binaphthyl column. And then these alkaloid fractions were analyzed on C18 column by liquid chromatography coupled with tandem mass spectrometry. Many structure‐related compounds were included in these alkaloids fractions, which led to easy separation and good MS response in further work. Therefore, a search library of 52 benzylisoquinoline alkaloids was established, which included eight aporphine, 19 tetrahydroprotoberberine, two protopine, two benzyltetrahydroisoquinoline, and 21 protoberberine alkaloids. The information of the search library contained compound names, structures, retention times, accurate masses, fragmentation pathways of benzylisoquionline alkaloids, and their sources from three herbal medicines. Using such a library, the alkaloids, especially those trace and unknown components in some herbal medicine could be accurately and quickly identified. In addition, the distribution of benzylisoquinoline alkaloids in the herbal medicines could be also summarized by searching the source samples in the library.     

Selective separation of structure-related alkaloids in Rhizoma coptidis with "click" binaphthyl stationary phase and their structural elucidation with liquid chromatography-mass spectrometry

It is a new task to separate structure-related compounds into a fraction according to their structural characteristics in a complex traditional Chinese medicine (TCM). This method makes separation of the components of the sample simple and structural elucidation easy. In this study, selective separation of alkaloids in Rhizoma coptidis was realized on a "click" binaphthyl column possessing a planar conjugate structure. Three kinds of alkaloids, aporphine, tetrahydroprotoberberine and protoberberine in Rhizoma coptidis showed better retention than other compounds by virtue of π-π interactions with the stationary phase. Moreover, the "click" binaphthyl column could distinguish the aporphine and tetrahydroprotoberberine alkaloids possessing two benzene rings from the protoberberine alkaloids possessing three benzene rings. After separating on the "click" binaphthyl column, the fractions containing the alkaloids were collected and then analyzed with liquid chromatography-mass spectrometry (LC-MS). Totally, 23 alkaloids were identified, and among these alkaloids, three tetrahydroprotoberberine, two aporphine and seven protoberberine alkaloids were first found in Rhizoma coptidis. These newly found alkaloids are minor compounds, and they are always neglected without eliminating the interference of compounds in large amounts by pre-separation on the "click" binaphthyl column. The typical fragmentation pathways of each class of alkaloids were summarized to illustrate their structures. In the MS(2) spectrum, the loss of a molecule of dimethylamine ((CH(3))(2)NH) was observed as the characteristic loss of aporphine alkaloids. All the tetrahydroprotoberberine alkaloids would undergo the Retro-Diels-Alder (RDA) fragmentation reaction in the MS(2) fragmentation. For protoberberine alkaloids, different characteristic fragmentations were observed with different skeleton structures.     

Analysis of protoberberine alkaloids in several herbal drugs and related medicinal preparations by non-aqueous capillary electrophoresis

A simple, rapid, reproducible, and universal non-aqueous capillary electrophoresis method has been developed for the separation and determination of three major active protoberberine alkaloids including berberine, palmatine, and jatrorrhizine within 7 min. The effects of the concentrations of acetic acid and electrolyte, the ratio of organic solvent, and the applied voltage on the separation were investigated. The optimum running buffer was composed of 50 mM ammonium acetate, 0.5% (v/v) acetic acid, and 10% (v/v) acetonitrile in methanol. The applied voltage was 18 kV. The analytes were detected by UV at 214 nm. The linearities between peak areas and the concentrations of the analytes were also investigated, and they exhibit excellent linear behavior over the concentration ranges (correlation coefficients: 0.9975-0.9986). The method was successfully applied to determine the three alkaloids in several families of herbal drugs (Rhizoma Coptidis, Cortex Berberidis, Cortex Phellodendri, Herba Chelidonii, Caulis Mahoniae) and their relevant medicinal preparations for the first time, and the recoveries of the three constituents ranged between 95.6-103.2% for berberine, 97.5-103.3% for palmatine, and 96.1 -103.6% for jatrorrhizine.     

毛细管电泳结合高效液相色谱-质谱用于天然产物活性成分的筛选

发展了毛细管电泳(CE)和高效液相色谱-质谱(HPLC-MS)相结合的用于天然产物中活性成分筛选和鉴定的方法。该方法中,用HPLC半制备柱对天然产物粗提物进行分离纯化,再用CE对HPLC纯化后的组分进行活性测试。根据HPLC-MS/MS提供的二级质谱数据,即可确定活性成分的化学结构。以乙酰胆碱酯酶为实验模型,对我们发展的筛选方法进行了验证。从黄连粗提物中确定了药根碱、巴马汀等7种活性成分,并通过CE测定了它们的半抑制率(IC₅₀)值。与传统的天然产物分离纯化和活性筛选方法相比,该方法具有简单、微量、快速、准确的优点。本文建立的方法为天然产物粗提物中活性成分的筛选提供了新技术。     

Characterization of isoquinoline alkaloids, diterpenoids and steroids in the Chinese herb Jin-Guo-Lan (Tinospora sagittata and Tinospora capillipes) by high-performance liquid chromatography/electrospray ionization with multistage mass spectrometry

This study sought to determine the primary components (isoquinoline alkaloids, diterpenoids and steroids) in crude extracts of the Chinese herb Jin-Guo-Lan, prepared from the roots of Tinospora sagittata and T. capillipes, by liquid chromatography/electrospray ionization multistage mass spectrometry coupled with diode-array detection (LC-DAD/ESI-MS(n)). After separation on a reversed-phase C(18) column using gradient elution, positive and negative ESI-MS experiments were performed. In positive ion mode, the three types of compounds showed very different characteristic ions: strong [M](+) or [M+H](+) ions were observed for isoquinoline alkaloids; [M+NH(4)](+) and/or [M+H-CO(2)](+) for diterpenoids; [M+H-nH(2)O](+) (n=1-3) for steroids. These adduct ions and/or fragments were used to deduce the mass and categories of known and unknown components in crude extracts, and their structures were further confirmed by ESI-MS(n) in positive ion mode. Moreover, UV absorption peaks obtained from DAD provided useful functional group information to aid the MS(n)-based identification. As a result, 11 compounds were unambiguously identified by comparing with standard compounds and 13 compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (13-hydroxycolumbamine and 13-hydroxyjatrorrhizine) were found to be new compounds and another one (13-hydroxypalmatine) was detected for the first time as a natural product. In addition, a [M-*CH(3)-H(2)O](*+) ion in MS(2) of [M](+) after in-source collision-induced dissociation was used to differentiate positional isomers of protoberberine alkaloids, columbamine and jatrorrhizine. Although the roots of T. sagittata and T. capillipes contain almost identical compounds, the content of the compounds in them is dramatically different, suggesting the necessity for further comparison of the bioactivities of the two species.     

Comparison of four C18 columns for the liquid chromatographic determination of deoxynivalenol in naturally contaminated wheat

Three long and 1 short reversed-phase C18 columns were compared for separation of deoxynivalenol (DON) in extracts of naturally contaminated wheat samples using liquid chromatography with ultraviolet detection and liquid chromatography/mass spectrometry (LC/MS). Among the 3 long columns used, a Symmetry C18 column with an isocratic solvent mixture of water-acetonitrile-methanol (90 + 5 + 5, v/v/v) gave the best separation for DON without interferences from other compounds in the wheat extracts. The Symmetry short (75 mm) column was comparable with the long column (250 mm) in resolving DON but significantly reduced retention time (i.e., 5.8 versus 16.3 min). Increasing the column temperature from 25 to 45 degrees C resulted in a further reduction in retention time. Identity of DON in the wheat extracts and standard solutions was confirmed by LC/MS in the positive ion mode, whereby DON appeared with an (M+1)+ ion at a mass-to-charge ratio of 297 plus fragment ions associated with loss of water and/or a 30 atomic mass unit (amu) CH2O fragment. The Symmetry short column was also capable of separating a mixture of the mycotoxins DON, 15-acetyl-DON, nivalenol, and zearalenone by use of a combination of an isocratic and gradient solvent system. The overall method showed high precision, exhibiting a relative standard deviation of 4.8%, limit of detection of 50 ng/g, and limit of quantitation of 165 ng/g. It was significantly correlated with enzyme-linked immunosorbent assay analysis, indicating its appropriateness for safety and quality assurance of wheat and related grains.     

Preparation of a stationary phase with quaternary ammonium embedded group for selective separation of alkaloids based on ion‐exclusion interaction

A stationary phase (named QA C10) with quaternary ammonium embedded between a propyl and a decyl chain was synthesized by immobilization of N,N‐dimethyldecylamine on chloropropyl–silica surface. A set of representative neutral, basic, and acidic compounds was employed to evaluate its chromatographic properties. The results illustrated that QA C10 was a mixed‐mode stationary phase possessing both hydrophobic and ionic characteristics. The QA C10 stationary phase was further used for selective separation of alkaloids from Cortex phellodendri. Under acidic condition, alkaloids could be eluted in first 8 min, while other neutral and acidic fractions were retained better on QA C10 column. Then, obtained alkaloid fraction was analyzed by LC‐MS/MS and 22 alkaloids were identified. Our study confirmed the advantages and application potential of the QA C10 stationary phase for alkaloids separation.     

Qualitative and quantitative analysis of quaternary ammonium alkaloids from Rhizoma Corydalis by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry coupled with a selective precipitation reaction using Reinecke salt

A major problem hampering the use of MALDI-MS for quantitative measurements is the inhomogeneous distribution of analytes and matrices in sample preparations. In this study, an aerospray method was utilized for sample preparation method to improve sample homogeneity across stainless steel targets for quantitative analysis of quaternary ammonium alkaloids (QAAs). A selective precipitation reaction with Reinecke salt known to selectively trap QAAs was used to facilitate the separation and purification of QAAs from the complex crude plant extracts. Palmatine and berberine as the representative QAAs in commercial Rhizoma Corydalis were successfully quantified by introducing an internal standard with similar molecular properties as analytes. The LODs were found to be 0.07 fmol, for palmatine, and 0.24 fmol, respectively, for berberine. The content of QAAs of three commercial Rhizoma Corydalis was between 0.201 and 0.245% for palmatine, and 0.049-0.057% for berberine. Furthermore, MS/MS experiments based on the accurate-mass measurements were carried out by infrared multiphoton dissociation (IRMPD) for QAAs and the corresponding tertiary alkaloids, which offered additional selectivity for this quantitative analysis method. In the fragmentation of precursor ions from QAAs, only cleavage of substituted groups attached to the A- or D-ring was observed, while cleavage between B- and C-ring from tertiary alkaloids had occurred. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for QAAs, especially in complex plant extracts.     

Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triple-quadrupole and ion-trap instruments

With 15 flavonoids as test compounds, the analytical performance of four modes of LC-MS, multiple MS (MSn) and tandem MS operation (atmospheric pressure chemical ionization (APCI), electrospray ionization, positive and negative ionization) was compared for two mass spectrometers, a triple-quadrupole and an ion-trap instrument. Two organic modifiers, methanol and acetonitrile, and two buffers, ammonium acetate and ammonium formate, were used. In general, the use of APCI in the negative ion mode gave the best response, with the signal intensities and the mass-spectral characteristics not differing significantly between the two instruments. The best results were obtained when methanol-ammonium formate (pH 4.0) was used as LC eluent. Under optimum conditions full-scan limits of detection of 0.1-30 mg/l were achieved in the negative APCI mode. Here it needs to be emphasized that up to 2-order response differences were found both between analytes and between modes of ionization. This implies that one should be very cautious when interpreting data on the screening of real-life samples. The main fragmentations observed in the MSn spectra on the ion-trap, or the tandem MS spectra on the triple-quadrupole were generally the same. The advantage of the former approach is the added possibility to ascertain precursor-->product ion relationships.     

Quantification of Fumaria officinalis isoquinoline alkaloids by nonaqueous capillary electrophoresis-electrospray ion trap mass spectrometry

A capillary electrophoresis (CE) method using non-aqueous (NA) separation solutions combined with an ion trap mass spectrometer (MS and MS/MS) as detection device is presented for the separation, identification and quantification of isoquinoline alkaloids from Fumaria officinalis. The best results were obtained with a mixture of acetonitrile-methanol (9:1, v/v) containing 60mM ammonium acetate and 2.2M acetic acid as running electrolyte and an applied voltage of 30 kV. Electrospray MS measurements were performed in the positive ionization mode with isopropanol-water (1:1, v/v) as sheath liquid at a flow rate of 3 microl/min. Alkaloids were detected as [M+H](+)-ions and showed typical fragmentation patterns in MS/MS experiments. The developed assay was used for the quantification of seven isoquinoline alkaloids representing different structural subtypes in Fumariae herba extracts and F. herba containing phytopharmaceuticals.     

One-pot synthesis of magnetic sulfonated covalent organic polymer for extraction of protoberberine alkaloids in herbs and human plasma

A novel magnetic sulfonated covalent organic polymer was prepared for magnetic solid-phase extraction of protoberberine alkaloids. The magnetic sulfonated covalent organic polymer was rapidly synthesized under mild conditions. The physicochemical properties of the prepared materials were characterized by Fourier-transform infrared spectrometry, transmission electron microscopy, and X-ray photoelectron spectroscopy. Several extraction parameters were systematically investigated, including desorption time, pH of sample solution, acetonitrile content, acetic acid content in the eluent, extraction time, and sample volume. By coupling magnetic solid-phase extraction and high-performance liquid chromatography, an efficient and sensitive method for the extraction and determination of protoberberine alkaloids in complex samples was developed. The proposed method showed great linearity (r > 0.9989), low limits of detection (0.2–0.3 ng/ml), and high precision (relative standard deviations ≤ 5.74%). The proposed method was further applied to the analysis of protoberberine alkaloids in Cortex phellodendri and human plasma samples. The recoveries were 91.50%–110.31% with relative standard deviations less than 6.63% in Cortex phellodendri and 96.12%–111.20% with relative standard deviations lower than 5.56% in plasma samples.     

中药川芎超临界CO2提取液的液相色谱-电喷雾多级质谱研究

采用HPLC-MSn技术分析和鉴定了中药川芎超临界CO2提取液中的主要成分:洋川芎内酯A、Z-藁本内酯、新蛇床内酯、3-丁基苯酞和苯酞二聚体。液相色谱-离子阱质谱联用在正离子电喷雾电离、自动多级质谱模式下进行分析。样品在ECLIPSE XDB C18色谱柱上以含0.25%乙酸水溶液和含0.25%乙酸甲醇梯度洗脱分离。对藁本内酯等苯酞内酯电喷雾电离后的碰撞裂解机理进行了探讨。Z-藁本内酯的碰撞裂解的两个主要途径为:侧链断裂脱烯和开环脱水再脱羰。实验中没有观察到孤立的脱羰过程。而这些主要成分的加钠离子的碰撞裂解过程完全不同。     

Simultaneous determination of the content of isoquinoline alkaloids in Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids by high‐performance liquid chromatography with diode array detection

A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids of D. leptopodum by high‐performance liquid chromatography with diode array detection. The chromatographic conditions were optimized on a SinoChrom ODS‐BP column to obtain a good separation of the four types of alkaloid analytes, including two aporphines (isocorydine, corydine), two protopines (protopine and allocryptopine), a morphine (sinoacutine), and three quaternary protoberberine alkaloids (berberrubine, 5‐hydroxycoptisine, and berberine). The separation of these alkaloids was significantly affected by the composition of the mobile phase, and particularly by its pH value. Acetonitrile (A) and 0.2% phosphoric acid solution adjusted to pH 6.32 with triethylamine (B) were selected as the mobile phase with a gradient elution. With this method, a new quaternary protoberberine alkaloid was isolated and the two structural isomers (isocorydine and corydine) were baseline separated. The appropriate harvest period for D. leptopodum was also recommended based on our analysis. The method for the effective fraction of the alkaloids of D. leptopodum was optimized under this method with regard to the varying significant pharmacological activities of the alkaloids.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

黄连黄柏提取液中3种生物碱的原位生成离子液体微萃取及HPLC测定

通过加入水溶性离子液体[C6MIM][Br]和离子对试剂NaPF6在黄连和黄柏的提取液中,原位生成非水溶性离子液体[C6MIM][PF6]萃取其生物碱,离心分相后,用HPLC测离子液体相中的生物碱小檗碱、巴马汀和黄连碱,其线性相关系数分别为0.9998、0.9996和0.9997,线性范围为1~2000 μg/L,检出限皆为0.5 μg/L,加标回收率96.52%~104.34%。 这种方法可用于黄连和黄柏中的小檗碱、巴马汀和黄连碱的检测。     

Rapid screening for cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.     

New “hyphenated” CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals: application to xanthones from Garcinia mangostana

Centrifugal partition chromatography (CPC) coupled online with high-performance liquid chromatography (HPLC) with diode-array detection (DAD) and mass spectrometry (MS) is presented in this work. This strategy offers the possibility to obtain simultaneously CPC fractionation of natural extracts, the HPLC fingerprint of separated fractions and structural information on molecules contained in each fraction. This new approach was applied to the fractionation and purification of xanthones from Garcinia mangostana (Clusiaceae) pericarp. A biphasic solvent system of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) was used for the CPC separation of 175?mg crude ethanolic extract. The HPLC analysis was conducted with a reversed-phase monolithic column allowing fast and repeatable separation. This combined CPC-HPLC-DAD-MS method led to isolation of 33?mg α-mangostin and 6?mg γ-mangostin at 98?% and 98.5?% purity, respectively, in 140?min. Furthermore, in the same time a total of 16 other xanthones were detected in the extract, and ten of them were identified on the basis of their UV and MS spectra.     

On-line structure characterization of pyrrolizidine alkaloids in Onosma stellulatum and Emilia coccinea by liquid chromatography-ion-trap mass spectrometry

On-line structure characterization of pyrrolizidine alkaloids in two various plant species (Onosma stellulatum W.K., family Boraginaceae and Emilia coccinea Sims., family Compositae) was performed by a newly elaborated RP-HPLC ion trap MS method with atmospheric pressure chemical ionization (APCI) interface. Different PAs (N-oxides, free bases, otonecine alkaloids) isolated were separated on Waters XTerra C18 column using a gradient elution. The combination of a CE-SPE with multiple isolation and fragmentation steps for specific masses in ion trap MS detector enabled fast and sensitive analysis of various types of PAs (N-oxides and free bases). In O. stellulatum, spectra 12 various types of structures (13 different alkaloids) have been determined for the first time: leptanthine-N-oxide, lycopsamine-N-oxide, heliospathuline, lycopsamine, trachelanthamine-N-oxide, dihydroechinatine, leptanthine, heliospathuline-N-oxide, 7-acetylintermedine, uplandicine, echimidine and echimidine-N-oxide. In E. coccinea, the following types of PAs were found: platyphylline-N-oxide, platyphylline (three stereoisomers with the same MS(n) spectrum), ligularidine, neoligularidine, neosenkirkine and also previously reported senkirkine. The method elaborated can be applied in the structural analysis of PAs in newly examined plant materials or food products but further analysis is needed to determine the stereochemistry in details.     

Optimization of HPLC-APCI-MS conditions for the qualitative and quantitative determination of total solanesol in tobacco leaves

HPLC coupled online with atmospheric pressure chemical ionization MS (APCI-MS) technique was evaluated for the qualitative and quantitative determination of solanesol in extracts of tobacco leaves. The solanesol and other compounds in the extract were separated on an Alltima C(8) (4.6 mm x 250 mm) column with methanol and water (98:2 v/v) as mobile phase, with flow rate of 0.8 mL/min and UV detection wavelength of 211 nm. In the APCI(+) mode, abundant stable [M-H(2)O + H](+) ion (m/z at 613.5) was observed, with low abundance of other fragmentation ions. A comparison of APCI-MS and ESI-MS techniques showed that APCI mode is more sensitive than ESI mode, and thus better suited for solanesol analysis. When comparing UV 211 nm and APCI-MS in SIM for solanesol quantification, the former offered better precision and reproducibility, but the latter was more than 200 times sensitive in detection. The developed method has been successfully applied to the analysis and comparison of solanesol concentration in different tobacco leaf samples.