On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC × LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC × LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC × LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa.     

Isolation and Purification of Ginsenosides from Plant Extract of Panax quinquefolium L. by High Performance Centrifugal Partition Chromatography Coupled with ELSD

A high performance centrifugal partition chromatography (HPCPC) combined with evaporative light scattering detection (ELSD) was developed for the separation and purification of ginsenosides from Panax quinquefolium. Three compounds, ginsenosides Rc, Rb₁, and Re were isolated and purified by HPCPC using an optimized two-phase solvent system composed of ethyl acetate–n-butanol–water (1:1:2, v/v/v). The purities of the three ginsenosides were 96.5, 97.6, and 98.5%, respectively as determined by liquid chromatography (LC–ELSD). The CPC fractions were analyzed by LC–ELSD and electrospray ion source mass spectroscopy (ESI-MSⁿ) in negative ion mode. The identification of the ginsenosides Rc, Rb₁, and Re in the extract of P. quinquefolium was based on matching their retention times, the detection of the molecular ions, and the fragment ions of the molecular ion obtained in the CID experiments with those of the authentic standards and data reported in the literature. The results demonstrate that HPCPC coupled with ELSD is a feasible and efficient technique for systematic isolation of non-chromophoric components from traditional medicinal herbs.     

State-of-the-art separation of ginsenosides from Korean white and red ginseng by countercurrent chromatography

Ginseng (Panax ginseng C. A. Meyer) has been one of the most popular herbs used for nutritional and medicinal purposes by the people of eastern Asia for thousands of years. Ginsenosides, the mostly widely studied chemical components of ginseng, are quite different depending on the processing method used. A number of studies demonstrate the countercurrent chromatography (CCC) separation of ginsenosides from several sources; however, there is no single report demonstrating a one-step separation of all of these ginsenosides from different sources. In the present study, we have successfully developed an efficient CCC separation methodology in which the flow-rate gradient technique was coupled with a new solvent gradient dilution strategy for the isolation of ginsenosides from Korean white (peeled off dried P. ginseng) and red ginseng (steam-treated P. ginseng). The crude samples were initially prepared by extraction with butanol and were further purified with CCC using solvent gradients composed of methylene chloride–methanol–isopropanol–water (different ratios, v/v). Gas chromatography coupled with flame ionization detector was used to analyze the components of the two-phase solvent mixture. Each phase solvent mixture was prepared without presaturation, which saves time and reduces the solvent consumption. Finally, 13 ginsenosides have been purified from red ginseng with the new technique, including Rg₁, Re, Rf, Rg_2, Rb₁, Rb₂, Rc, Rd, Rg₃, Rk₁, Rg₅, Rg₆, and F₄. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg₁, Re, Rf, Rh_1, Rb₁, Rb₂, Rc, and Rd by using a single-solvent system. Thus, the present technique could be used for the purification of ginsenosides from all types’ ginseng sources. To our knowledge, this is the first report involving the separation of ginsenoside Rg₂ and Rg₆ and the one-step separation of thirteen ginsenosides from red ginseng by CCC.     

Development of Green and Efficient Extraction of Bioactive Ginsenosides from Panax ginseng with Deep Eutectic Solvents

The extraction of active constituents from natural sources in a green and efficient manner is considered an important field in the pharmaceutical industry. In recent years, deep eutectic solvents (DESs), a new type of green solvent, have attracted increasing attention. Therefore, we aimed to establish a green and high-efficiency extraction method for ginsenosides based on DESs. This study takes Panax ginseng as a model sample. Eighteen different DESs were produced to extract polar ginsenosides. Ultrasound-assisted extraction (UAE) was applied for simplicity and efficiency. A binary DES synthesized using choline chloride and urea at a proportion of 1:2 prepared by a heating stirring method is proven to be more effective than other solvents, such as the widely used 70% ethanol for the extraction of ginsenosides. Three variables that might affect the extraction, including the DES content in the extraction solvent, liquid/solid ratio, and ultrasound extraction time, were evaluated for optimization. The optimum extraction conditions for ginsenosides were determined as follows: DES water content of 20 wt%, liquid/solid ratio of 15 mL g⁻¹, and an ultrasonic extraction time of 15 min. The extraction yield for the optimized method is found to be 31% higher than that for 70% ethanol, which achieves efficient extraction. This study shows that DESs are available to extract ginsenosides for use in traditional Chinese medicine. The discovery also contributes to further research into the green extraction of ginsenosides.     

A four-dimensional separation approach by offline 2D-LC/IM-TOF-MS in combination with database-driven computational peak annotation facilitating the in-depth characterization of the multicomponents from Atractylodis Macrocephalae Rhizoma (Atractylodes macrocephala)

Plant metabolites represent complex chemical system, which renders it difficult to clarify the chemical composition by conventional liquid chromatography/mass spectrometry (LC/MS) due to the limited selectivity and peak capacity. The rhizomes of Atractylodes macrocephala have been utilized as a traditional Chinese medicine Atractylodis Macrocephalae Rhizoma (Bai-Zhu), and have been reported containing multiple categories of plant metabolites. Targeting the multicomponents from A. macrocephala, an integral approach by offline two-dimensional liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) was established and validated. By configuring an XBridge Amide column of Hydrophilic Interaction Chromatography and an Atlantis Premier BEH C18AX column of mixed ion exchange and reversed-phase modes, the established 2D-LC/IM-QTOF-MS system showed high orthogonality up to 0.91. Dimension-enhanced, data-independent high-definition MS^E (HDMS^E) in the positive ESI mode was conducted on a Vion IM-QTOF mass spectrometer, and its hyphenation to offline 2D-LC could enable the four-dimensional separation (each dimension in 2D-LC, IM, and MS). Particularly, HDMS^E facilitated the acquisition of high-definition MS¹ and MS² spectra. In-house library-driven computational peak annotation by the bioinformatics platform UNIFI could efficiently process and annotate the HDMS^E data for the structural elucidation. By integrating reference compounds comparison, we could identify or tentatively characterize 251 components from A. macrocephala (including 115 sesquiterpenoids, 90 polyacetylenes, 11 flavonoids, 9 benzoquinones, 12 coumarins, and 14 others), which indicated large improvement in identifying those minor plant components, compared with the conventional LC/MS approach. Conclusively, offline 2D-LC/IM-QTOF-HDMS^E in combination with computational data interpretation proves to be powerful facilitating the in-depth multicomponent characterization of herbal medicine.     

Separation and identification of multiple constituents in Xiao Chai Hu Decoction (Sho‐saiko‐to) by bioactivity‐guided fractionation combined with LC‐ESI‐QTOFMS/MS

Xiao Chai Hu Decoction (XCHD), named Sho‐saiko‐to in Japanese, is a well‐known traditional Chinese medicine formula used in Asia. However, the characterization methods used in the past have lacked sensitivity and the nature of the active constituents of XCHD remains unclear. This study was carried out to establish the hyphenated method of bioactivity‐guided fractionation and liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOFMS/MS) in order to identify the major bioactive constituents of XCHD. D101 macroporous resin was used to separate and enrich the material base into four fractions, XCHD‐1, XCHD‐2, XCHD‐3 and XCHD‐4. Each fraction was then evaluated for its antidepressant effect using depression‐related parameters. An LC‐ESI‐QTOFMS/MS method in both positive and negative ion mode was also applied for separation and identification of the biological active fractions of XCHD. As a result, 79 compounds including polysaccharides, flavonoids, saikosaponins, ginsenosides, licoricesaponins and gingerols were detected, 69 of them were identified or tentatively characterized. Based on our preliminary characterization investigations, polysaccharides, gingerols and flavonoids in XCHD may contribute to the antidepressant effect of XCHD. In conclusion, the hyphenated method of bioactivity‐guided fractionation and LC‐ESI‐QTOFMS/MS was meaningful for the isolation and preliminary identification of the biological active components in complex matrices of traditional Chinese medicine. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of flavanones in Citrus juices by means of one- and two-dimensional liquid chromatography

1-D and 2-D comprehensive (LC×LC) liquid chromatography methods have been developed and compared for the separation and quantification of flavanones in various Citrus juices. 1-D analyses were carried out on a superficially porous C18 column, whereas the 2-D LC approach was composed of a polyethylene glycol silica narrow-bore column packed with totally porous particles in the first dimension (D1) and a superficially porous C18 column in the second dimension (D2). Low-selectivity correlations were ensured by the complementary separation mechanisms offered by the D1 and D2 columns. Quantification was carried out both manually and by means of a software capable of detecting and quantifying each peak from the 2-D plot. Limit of detection (LOD) values as low as 0.023 μg/mL were obtained for hesperidin used as reference material for 1-D LC analyses, whereas values as high as 0.432 μg/mL were obtained by comprehensive LC. This discrepancy can be traced back to the minor sensitivity experienced in comprehensive LC due to both sample dilution in D1 and the high flow rates employed in D2. On the other hand, the separation capabilities of the LC×LC approach allowed to reduce the interferences coming from the matrix and to achieve the separation of some critical pairs, e.g. hesperidin/naringin difficult to accomplish in 1-D LC.     

Quick identification of apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor detection

Analytical high-speed counter-current chromatography (HSCCC), a unique liquid-to-liquid separation technology, has an inherent capability to provide perfect fractionation for tracking active ingredients of medicinal herbs, in a quick, efficient, and high-recovery manner. A high throughput screening (HTS) method which utilizes a novel biosensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique, was newly established and proved to be very sensitive in detecting apoptosis induced by various known anticancer drugs. The first combination of both advanced techniques formed an efficient platform for drug discovery and succeeded in quickly identifying the most potent apoptotic constituent of a Chinese herb namely Isodon eriocalyx. The system of n-hexane/ethyl acetate/methanol/water was used as the separation solvent. The solvent ratio was first set at 3:5:3:5 to check the water-soluble part of the crude extract, and then 1:1:1:1 was used to isolate the target compounds. The active fraction was tracked and purified continuously using HSCCC which was guided by the apoptosis detection at gradually decreased drug concentrations. As a result, the most potent apoptosis inducer in this herb was discovered by analytical HSCCC equipped with a 16 ml mini-coil column, using less than 50 ml diphase solvent, from about 50 mg active fraction. It was identified as eriocalyxin B, a well-known antitumor natural product, by NMR analysis of the HSCCC purified fraction.     

Offline coupling of high-speed counter-current chromatography and gas chromatography/mass spectrometry generates a two-dimensional plot of toxaphene components

High-speed counter-current chromatography (HSCCC), a separation technique based solely on the partitioning of solutes between two immiscible liquid phases, was applied for the fractionation of technical toxaphene, an organochlorine pesticide which consists of a complex mixture of structurally closely related compounds. A solvent system (n-hexane/methanol/water 34:24:1, v/v/v) was developed which allowed to separate compounds of technical toxaphene (CTTs) with excellent retention of the stationary phase (S_f = 88%). Subsequent analysis of all HSCCC fractions by gas chromatography coupled to electron-capture negative ion mass spectrometry (GC/ECNI-MS) provided a wealth of information regarding separation characteristics of HSCCC and the composition of technical toxaphene. The visualization of the large amount of data obtained from the offline two-dimensional HSCCC–GC/ECNI-MS experiment was facilitated by the creation of a two-dimensional (2D) contour plot. The contour plot not only provided an excellent overview of the HSCCC separation progress, it also illustrated the differences in selectivity between HSCCC and GC. The results of this proof-of-concept study showed that the 2D chromatographic approach involving HSCCC facilitated the separation of CTTs that coelute in unidimensional GC. Furthermore, the creation of 2D contour plots may provide a useful means of enhancing data visualization for other offline two-dimensional separations.     

Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination

In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC × LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC × LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products.     

Screening and identification of multi-component in Qingkailing injection using combination of liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

An approach for screening and identification of multi-component in complex traditional Chinese medicine systems with a combinative LC/MS (MSⁿ) technique was described in this paper. The chemical profile of Qingkailing injection, a well-known traditional Chinese formula in China, was studied using the established method as for an application. Benefit from combining the accurate mass measurement of LC/TOF-MS to generate elemental compositions and the complementary multilevel structural information provided by LC/ion trap MSⁿ, 33 components in Qingkailing injection were identified in all. The three isomers of chlorogenic acid, isochlorogenic acid and neochlorogenic acid which are derived from Flos Lonicerae, one of the medicinal materials in Qingkailing, were differentiated by verifying their MS³ fragmentation data. All the components identified were surveyed and classified according to their medicinal materials derivation. This study is expected to provide an effective and reliable pattern for comprehensive and systematic characterization of the complex traditional Chinese medicine systems.     

Hyphenated HPLC-NMR and its applications in drug discovery

Hyphenated HPLC-NMR is a fast growing technology, allowing rapid and detailed structural characterization of unknown mixtures. The technical aspects of the technology are reviewed on the basis of system configuration, operation, solvent suppression, HPLC and NMR optimization, and detection. The combined use of HPLC-NMR and HPLC-MS is also described and discussed. Various applications of HPLC-NMR and integrated HPLC-NMR-MS in drug discovery, especially in the separation and structure elucidation of drug impurities, reaction mixtures, degradation products, in vitro and in vivo metabolites, and combinatorial library samples, are illustrated.     

Polar Aprotic Modifiers for Chromatographic Separation and Back-Exchange Reduction for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.     

Pharmacokinetic study of five ginsenosides using a sensitive and rapid liquid chromatography–tandem mass spectrometry method following single and multiple oral administration of Shexiang Baoxin pills to rats

Shexiang Baoxin pills (SBP) are a traditional Chinese medicine that are used for treating coronary heart disease. Ginsenosides are the main effective components of SBP, but a comprehensive and deep pharmacokinetic study of ginsenosides in SBP, including multiple dosing and linear or nonlinear properties, is lacking. This study was designed to investigate and compare the pharmacokinetic characteristics of ginsenosides in SBP at a single dose and in multiple doses. A liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of the ginsenosides Rg1, Re, Rb3, Rc and Rb1 in rat plasma. Rats were randomly assigned to receive a single dose of 4, 8 or 12 g/kg and multiple doses (4 g/kg) of SBP for 8, 15 or 22 consecutive days. The results revealed that ginsenosides, following a single oral dose of 4 or 8 g/kg, were absorbed rapidly, with a T_max ranging from 0.250 to 1.08 h. The AUC_0–t and C_max of the ppd‐type ginsenosides Rb3, Rc and Rb1 were greater than those of the ppt‐type ginsenosides Rg1 and Re. Nondose‐dependent exposure was observed at doses of 4–12 g/kg for all of the ginsenosides. After multiple dosing, the plasma levels of the ppt‐type ginsenosides decreased, whereas those of the ppd‐type ginsenosides did not change significantly. In conclusion, the LC‐MS/MS method was successfully applied to investigate the pharmacokinetics of ginsenosides after single and multiple oral administrations of SBP. The ginsenosides did not accumulate after multiple dosing. The ppd‐type ginsenosides displayed more favorable pharmacokinetic properties compared with the ppt‐type ginsenosides. Copyright © 2014 John Wiley & Sons, Ltd.     

The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis

The microcapillary liquid chromatography (μLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the μLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of μLC systems.     

A systematic strategy for rapid identification of chlorogenic acids derivatives in Duhaldea nervosa using UHPLC-Q-Exactive Orbitrap mass spectrometry

Duhaldea nervosa (Wallich ex Candolle) A. Anderberg was widely used for food spice and folk medicine. However, it is still insufficient in the constituent’s characterization of D. nervosa. In this study, a systematic strategy for rapid detection and identification of constituents was proposed based on UHPLC-Q-Exactive Orbitrap mass spectrometry in parallel reaction monitoring mode combining anion exchange resin separation, expected compounds predicted and diagnosis fragmentation ions techniques. Finally, 149 chlorogenic acids derivatives were unanimously and tentatively characterized from D. nervosa, 102 of them were report for the first time. This results widely extended the chemical constituents of D. nervosa, which will facilitate understanding the effective substance and quality control. Meantime, it is possible for this strategy to exhibit a wide application for chemical’s characterization in different sample.     

Comparison of High-Temperature Gradient Heart-Cutting and Comprehensive LC × LC Systems for the Separation of Phenolic Antioxidants

Natural phenolic antioxidants were separated using comprehensive 2D HPLC on a Purospher Star RP-18e column in the first dimension and on two parallel Zirconia Carbon columns working in alternating cycles in the second dimension. The combination of the two columns provides great differences in separation selectivity in each dimension and an almost orthogonal 2D system. Temperature and solvent gradients were compared for the separation of the first-dimension fraction in the stop-flow heart-cutting 2D setup. Temperature gradients provide shorter separation times in comparison with solvent gradients. However, the time required for post-run column equilibration is too long for comprehensive LC × LC. High-temperature isocratic separation was employed in the second dimension of the comprehensive setup, allowing improvement of the fraction transfer frequency between the two dimensions and shorter 2D separation time in comparison to the earlier published method. The approach was applied to the analysis of beer and wine.     

Separation and purification of steroidal saponins from Paris polyphylla by microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection

A method of microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection was successfully developed for the separation and purification of steroidal saponins from Paris polyphylla. The main extraction conditions including microwave power, liquid/solid ratio, irradiation time, and extraction temperature were optimized using an orthogonal array design method. A suitable two‐phase solvent system consisting of n‐heptane/n‐butanol/acetonitrile/water (10:19:6:20, v/v/v/v) was employed in the separation and purification of the extracts of P. polyphylla. A total of 7.1 mg polyphyllin VII, 4.3 mg gracillin, 9.2 mg dioscin, and 10.2 mg polyphyllin I were obtained from 1.5 g P. polyphylla in less than 300 min, the purities of which determined by HPLC were 96.7, 97.3, 98.7, and 98.6%, respectively. The identification and characterization of these compounds were performed by LC–ESI‐MS and ¹H NMR spectroscopy. The results demonstrated that the proposed method is feasible, economical and efficient for the extraction, separation and purification of effective compounds from natural products.     

Optimization of a liquid chromatography method based on simultaneous electrospray ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides

Different reversed-phase liquid chromatography (LC) columns of conventional dimensions were coupled to an ultraviolet photodiode array detector (UV-DAD) and a magnetic sector-type spectrometer, equipped with an electrospray ionization (ESI) source, by a laboratory-made flow splitter. A mixture of three flavonoid-O-glycosides was employed to examine the effects of the solvent composition, the flow rate, the stationary phase, the pH and the organic acid added, on the chromatographic separation, the UV-DAD detection, the ESI process and the entire LC system with ESI-MS and UV-DAD detection. In the positive ion mode, methanol containing 1% acetic acid was by far the most sensitive in ESI-MS analysis, whereas an acetonitrile/water mobile phase containing 0.5% formic acid was proved to give the best sensitivity in LC/ESI-MS/UV-DAD analysis. In the negative ion mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid, while addition of bases decreased the sensititvity. The optimal flow rate was higher in negative ESI (20-50 micro L/min) than in positive ESI (5 micro L/min), and the percentage of organic phase had an influence on the sensitivity of ESI-MS detection. With regard to the selection of a suitable C(18) reversed-phase LC column, a column which is well end-capped is to be preferred, because residual silanol groups appear to impair the separation of flavonoid glycosides. The optimized LC/ESI-MS/UV-DAD method was applied to a commercial Crataegus extract, which is used in phytomedicine to treat cardiovascular problems and is known to be rich in flavonoids. It is demonstrated how UV spectra and first-order ESI mass spectra allow a fast characterization of flavonoids, even if reference compounds are not available or at hand.