Distinguishing Radix Angelica sinensis from different regions by HS-SFME/GC-MS.

An automated headspace solvent free microextraction (HS-SFME) based gas chromatography/mass spectrometry (GC/MS) was developed for discrimination of Radix Angelica sinensis (RAS) from different cultivation regions. The MS data were subjected to principal component analysis (PCA) and hierarchical clustering analysis (HCA) to rapidly find the potential characteristic components of RAS from top-geoherb region and non top-geoherb region. Totally, fifty-one volatile organic compounds (VOCs) were identified, in which ß-ocimene, α-pinene, 3-methylbutanal, heptanes, butanal were identified as potential markers for distinguishing RAS from top-geoherb region and non top-geoherb region. Sulphur dioxide was detected in some commercial RAS samples, which implied that sulphur-fumigation might be the main reason for the quality inconsistencies of commercial RAS samples. These results suggested that RAS from top-geoherb region and non-top geoherb region could be discriminated by the method. And characteristic chemical markers found in current study can be used for ensuring consistent quality of top-geoherb of RAS.

Chemical comparison of two dosage forms of Hemp Seed Pills by UHPLC-Q-ToF-MS/MS and multivariate statistical techniques.

Hemp seed soft gel capsule (HSSGC) is a modernised dosage form that is derived from a traditional Chinese patent medicine, Hemp Seed Pills (HSP). Two dosage forms claim the same therapeutic effects; however, their chemical components and chemical equivalency are unclear. In the present study, an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-ToF-MS)-based chemical profiling approach was proposed to rapidly evaluate the chemical differences between HSP and HSSGC as model dosage forms. Samples of the two dosage forms were subjected to UHPLC-ToF-MS analysis. The datasets of retention time (TR) and mass-to-charge ratio (m/z) pairs, ion intensities and sample codes were processed with principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) to holistically compare the difference between these two dosage form samples. A clear classification trend was observed in the score plot, and a loading bi-plot was generated in which the variables are correlated with the group and the samples that were observed. The important chemical components that caused differences among the samples were explored with a Variables Importance Projection (VIP) index. Using the proposed approach, global chemical differences were found between the two dosage forms and among samples of the same dosage form. The most important components that are related to the differences were identified and most of them were attributed to Fructus Aurantii Immaturus. It is suggested that this newly established approach could be used for pre-clinical trial chemical equivalence study or the quality evaluation of the traditional medicinal products with large variations in quality.

A high-sensitivity UPLC-MS/MS method for simultaneous determination and confirmation of triptolide in zebrafish embryos.

A high-sensitivity ultra-performance liquid-chromatography (UPLC) coupled with tandem mass spectrometric method was developed for simultaneous quantification and confirmation of triptolide in both zebrafish embryos and the aqueous-exposure solution on a tandem quadrupole mass spectrometer (TQ-MS). This was achieved by performing quantification using the multiple reaction monitoring (MRM) acquisition with simultaneous characterization of the MRM peak using product ion confirmation (PIC) acquisition as it elutes from the chromatographic system. Separation was achieved on a 1.7 µm C(18) UPLC column using 0.1% formic acid water-acetonitrile mobile phase with a cycle time of 6 min. The linear range of 0.115-360 ng/mL, and lower limits of detection of 0.02 ng/mL and quantification of 0.064 ng/mL were established. This method was successfully applied to determine the time course of triptolide absorption by zebrafish embryos and the amount of triptolide remaining in the culture medium after administration of two triptolide dosages at three time points. This coupled MRM with PIC approach could provide both qualitative and quantitative results without the need for repetitive analyses. This resulted in the reduction of further confirmative experiments and analytical time, and ultimately increased laboratory productivity.

Optimisation of ultra-performance LC conditions using response surface methodology for rapid separation and quantitative determination of phenolic compounds in Artemisia minor.

A method that couples rapid, sensitive, reproducible and accurate ultra-performance LC (UPLC) with quadrupole-TOF-MS was established for the first simultaneous qualitative and quantitative analysis of phenolic compounds in Artemisia minor. Box-Behnken designs (BBDs) were applied as an effective tool to optimise major parameters that influence the resolution of UPLC, including three gradient steps and column temperature. Under optimal UPLC conditions, a total of 23 phenolic compounds in the crude methanol extracts of A. minor were well separated on a Waters Acquity UPLC BEH C(18) column (100×2.1 mm, 1.7 µm particle size) within 16.5 min, and the compounds were unequivocally or tentatively identified via comparisons with authentic standards and literature. In this study, a total of six major phenolic compounds were quantified in A. minor and the method was validated to be sensitive, precise and accurate within the LOD from 1.24 to 5.27 µg/mL, and the overall intra- and inter-day variations in detection were less than 3.76%. The recovery of the method ranged from 97.9 to 103.8% with RSDs that were less than 5.8%. These results demonstrate that this approach has the potential for quality control of A. minor and other Tibetan herbal medicines.

A new approach for simultaneous screening and quantification of toxic pyrrolizidine alkaloids in some potential pyrrolizidine alkaloid-containing plants by using ultra performance liquid chromatography-tandem quadrupole mass spectrometry.

A rapid, but sensitive and selective method for simultaneous screening and quantification of toxic pyrrolizidine alkaloids (PAs) by ultra performance liquid-chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) on a tandem quadrupole mass spectrometer (TQ-MS) is described. This was accomplished by incorporating the precursor ion scan (PIS) acquisition and multiple reaction monitoring (MRM) acquisition in the same UPLC-MS/MS run. Notably, the developed PIS approach for detecting two pairs of characteristic product ions at m/z 120/138 or 168/150, allowed specific identification of toxic retronecine and otonecine types PAs. This PIS method is highly sensitive with over 10-fold sensitivity improvement upon previously published LC-MS method. Moreover, this new approach is suitable for high sample throughput and was applied to the screening and quantifying toxic PAs in 22 samples collected from seven Parasenecio species and four Senecio species. In addition, coupling the MRM with PIS approach generated quantitative results equivalent to those obtained by conventional MRM-only approach. This coupled MRM with PIS approach could provide both qualitative and quantitative results without the need of repetitive analyses.

Qualitative and quantitative analysis of polycyclic polyprenylated acylphloroglucinols from Garcinia species using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a group of natural products isolated from different Garcinia species with a wide range of important biological activities. In this study, an ultra performance liquid chromatography (UPLC) coupled to photodiode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF) method was developed to characterize 16 PPAPs in 10 Garcinia species. In source dissociation techniques based on cone voltage fragmentation were used to fragment the deprotonated molecules and multiple mass spectrometry (MS/MS) using ramping collision energy were used to further break down the resulting product ions. The resulting characteristic fragment ions were generated by cleavage of C1-C5 bond and C7-C8 bond through concerted pericyclic reaction, which is especially valuable for differentiating three types of PPAPs isomers. As such, two new PPAPs isomers present in minor amount in the extracts of Garcinia oblongifolia were tentatively characterized by comparing their tandem mass spectra to the known ones. In addition, an UPLC-Q-TOF-MS method was validated for the quantitative determination of PPAPs. The method exhibited limits of detection from 2.7 to 21.4 ng mL(-1) and intra-day and inter-day variations were less than 3.7% and the recovery was in the range of 89-107% with RSD less than 9.0%. This UPLC-Q-TOF-MS method has successfully been applied to quantify 16 PPAPs in 32 samples of 10 Garcinia species, which were found to be a rich source of PPAPs.

Decocting-induced chemical transformations and global quality of Du-Shen-Tang, the decoction of ginseng evaluated by UPLC-Q-TOF-MS/MS based chemical profiling approach.

An UPLC-Q-TOF-MS/MS based chemical profiling method was developed to evaluate decocting-induced chemical transformations in Du-Shen-Tang, the decoction of the root of Panax ginseng. Under the optimized UPLC and Q-TOF-MS/MS conditions, over 50 peaks were separated and detected in Du-Shen-Tang within 18 min. The components were identified by comparing the mass spectra and retention time with that of reference compounds, and/or tentatively assigned by elucidating low energy CID fragment ions as well as matching empirical molecular formula with that of the published known compounds. Totally 45 major ginsenosides were identified in Du-Shen-Tang, 21 of which were determined to be newly generated during the decoction of ginseng. The mechanisms involved were further deduced to be hydrolysis, dehydration, decarboxylation and addition reactions of the original ginsenosides in white ginseng through analyzing mimic decoctions of 13 pure reference ginsenosides. Significant difference in chemical profiles between decoctions of two batches of white ginseng suggested that storage duration or other factors significantly influenced the quality consistency of not only the crude drug but also the decoction (Du-Shen-Tang) of white ginseng.

Identification and evaluation of apoptotic compounds from Garcinia paucinervis.

Four new compounds, paucinervins A-D (1-4), and 15 known ones were isolated from the leaves of Garcinia paucinervis. The structures of the new compounds were elucidated by spectroscopic evidences. All of the 19 compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to possess a fluorescent biosensor capable of detecting caspase-3 activation. Eight of them were found to activate caspase-3 in HeLa-C3 cells within 72 h at the concentration of 25 microM. Moreover, the values of IC50 were measured for all four new compounds on HeLa cells using the MTT assay. Among them, compound 2 (paucinervin B) had the lowest IC50 value of 9.5 microM, while the other three new compounds had much higher IC50 values of 29.5, 52.5, and 95.6 microM, respectively. This result shows that paucinervin B has the strongest inhibitory effect against HeLa cell growth among these four newly identified paucinervins and it may have the potential to be developed into a new anticancer candidate.

Development and validation of an ultra high-performance liquid chromatographic method for the determination of a diastereomeric impurity in (+)-pinoresinol diglucoside chemical reference substance.

(+)-Pinoresinol 4,4'-di-O-beta-D-glucopyranoside ((+)-PDG) is one of the major lignans with various pharmacological activities which could be isolated from Duzhong and other plant species. In this study, a diastereomeric impurity, (-)-pinoresinol 4,4'-di-O-beta-D-glucopyranoside ((-)-PDG), the main impurity was identified in (+)-PDG chemical reference substance (CRS) and a reliable chromatographic method for rapid purity determination of (+)-PDG CRS was firstly developed. The optimal chromatographic condition was found to be using ACN/1,4-dioxane-water (2.5:6:91.5, v/v/v) as mobile phase on a Waters Acquity UPLC HSS T3 column (2.1 mm x 100 mm, 1.8 microm) with column temperature of 37 degrees C. The method was validated and applied to determine the chromatographic purity of five (+)-PDG CRS samples. The content of (-)-PDG in four commercial (+)-PDG CRS was 8.47-20.30%, whereas no (-)-PDG was detected in our in-house prepared (+)-PDG CRS in which purity was confirmed to be 99.80%. The above results confirmed that this method is fast and highly efficient for purity determination of the (+)-PDG CRS.

Characterization of protostane triterpenoids in Alisma orientalis by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

A reliable and sensitive ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm x 2.1 mm, 1.8 microm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision-induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C-23-OH leads to dissociation of the bond between C-23 and C-24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C-23 or C-24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q-TOF-MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis.

A novel approach to rapidly explore analytical markers for quality control of Radix Salviae Miltiorrhizae extract granules by robust principal component analysis with ultra-high performance liquid chromatography-ultraviolet-quadrupole time-of-flight mass spectrometry.

In a well-controlled experiment, outliers discriminated by robust principal component analysis (RPCA) represent contents in samples which are of particular quality distinguishable from the rest of the others, therefore chemical constituents in a natural product causing discrimination between outliers and the majority of samples could be considered as analytical markers for quality control. Based on this strategy, a novel approach for rapidly exploring characteristic analytical markers was proposed for the quality control of extract granules of Radix Salviae Miltiorrhizae (EGRSM). In this study, large sizes of samples were analyzed via high-throughput ultra-high performance liquid chromatography-ultraviolet-quadrupole time-of-flight mass spectrometry (UHPLC-UV-Q-Tof MS). RPCA was first performed on the three groups of samples: RSM (the raw material), the in-house prepared aqueous extract of Radix Salviae Miltiorrhizae (AERSM) and commercial product of EGRSM, to determine the variation of specific constituents between raw material and the final products as well as the effect of manufacturing process on the overall quality. Then RPCA was performed on the commercial products of EGRSM to explore the applicability of identified characteristic markers for the quality control of EGRSM. Candidate markers were extracted by RPCA, and their molecular formulae were determined by high resolution electrospray ionization-mass spectrometric (ESI-MS) analysis. The suitability of identified markers was then evaluated by determining the relationship between quantities of the identified markers with their antioxidant activities biologically, and further confirmed in a variety of samples. In conclusion, the combination of RPCA with UHPLC-UV-Q-Tof MS is a reliable means to identify chemical markers for evaluating quality of herbal medicines.

UPLC-PDA-TOFMS based chemical profiling approach to rapidly evaluate chemical consistency between traditional and dispensing granule decoctions of traditional medicine combinatorial formulae.

In the present study, an ultra performance liquid chromatography coupled with photo-diode array detector and time-of-flight mass spectrometry (UPLC-PDA-TOFMS) based chemical profiling approach to rapidly evaluate chemical consistency between traditional and dispensing granule decoctions of traditional medicine combinatorial formulae was proposed and validated using San-Huang-Xie-Xin-Tang (SHXXT) as a model combinatorial formula. Two different kinds of decoctions, namely traditional decoction: water extract of mixed three constituent herbs of SHXXT, and dispensing granules decoction: mixed water extract of each individual herbs of SHXXT, were prepared. Batches of these two kinds of decoction samples were subjected to UPLC-PDA-TOFMS analysis, the datasets of t(R)-m/z pairs, ion intensities and sample codes were processed with supervised orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between these two kinds of decoction samples. Once a clear classification trend was found in score plot, further statistics was performed to generate S-plot, in which the variables (t(R)-m/z pair) contributing most to the difference were clearly depicted as points at the two ends of "S", and the components that correlate to these ions were regarded as the most changed components during decocting of combinatorial formula. The identities of the changed components can be identified by comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of the known compounds published in the literatures. Using the proposed approach, global chemical difference was found between traditional and dispensing granule decoctions, and berberine, palmatine, epiberberine, baicalin, wonogoside, 2-O-golloyl-1-O-cinnamoylglucose and emodin were identified as the most changed components during decocting SHXXT. It is suggested that this newly established approach could be used practically for the evaluation of chemical consistency between traditional and dispensing granule decoctions of traditional medicine combinatorial formulae.

Cytotoxic acylphloroglucinol derivatives from the twigs of Garcinia cowa.

An unusual polyprenylated acylphloroglucinol derivative unsubstituted at C-2 and C-6, garcicowin A (1), together with three other new (garcicowins B-D, 2-4) and nine known analogues, was isolated and characterized from the twigs of Garcinia cowa. The structures of 1-4 were elucidated by interpretation of their spectroscopic data. The compounds isolated were evaluated for their cytotoxicity against two cancer cell lines (HT-29 and HCT116) and against normal colon cells (CCD-18Co), and the results demonstrated their selective toxicity toward the cancer cells.

A novel strategy to rapidly explore potential chemical markers for the discrimination between raw and processed Radix Rehmanniae by UHPLC-TOFMS with multivariate statistical analysis.

In traditional Chinese medicine, raw and processed herbs are used to treat different diseases. Suitable chemical markers are crucial for the discrimination between raw and processed herbs. In this study, a novel strategy using UHPLC-TOFMS coupled with multivariate statistical analysis to rapidly explore potential chemical markers was proposed and validated. Using Radix Rehmanniae as a model herb, batches of raw and processed samples were determined by UHPLC-TOFMS. The datasets of t(R)-m/z pair, ion intensity and sample code were subjected to principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between raw and processed samples. Once a clear cluster was found, extended statistics was performed to generate S-plot, in which the variables (t(R)-m/z pair) contributing most to the difference were clearly indicated as points at the two ends of "S", and the components that correlate to these ions should be the processing-induced transformed components. These transformed components could be regarded as the potential chemical markers that can be used to distinguish between raw and processed herbs. The identity of the potential markers can be identified by comparing the mass/UV spectra and retention time with that of reference compounds and/or tentatively assigned by matching empirical molecular formula with that of the known compounds published. Using this proposed strategy, leonuride or its isomer and 5-(alpha-d-glucopyranosyl-(1-6)-alpha-d-glucopyranosyloxymethyl)-2-furancarboxaldehyde were rapidly explored as the most characteristic markers of raw and processed Radix Rehmanniae, respectively. This newly proposed strategy can not only be used to explore chemical markers but also to investigate the chemical transforming mechanisms underlying traditional herb processing.

Development and validation of a rapid capillary zone electrophoresis method for the determination of aconite alkaloids in aconite roots.

INTRODUCTION: Aconites, with aconite alkaloids as the major therapeutic and toxic components, are used for the treatment of analgesic, antirheumatic and neurological symptoms. Quantification of the aconite alkaloids is important for the quality control of aconite-containing drugs. OBJECTIVE: To establish a validated capillary zone electrophoresis (CZE) method for the simultaneous determination of six major alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in crude and processed aconite roots. METHODOLOGY: The CZE method was optimised and validated using a stability-indicating method. The optimised running buffer was a mixture of 200 mm Tris, 150 mm perchloric acid and 40% 1,4-dioxane (pH 7.8) with the capillary thermostated at 25 degrees C. RESULTS: Using the optimised method, six aconite alkaloids were well separated. The established method showed good precision, accuracy and recovery. Contents of these alkaloids in crude and processed aconites were determined and it was observed that the levels of individual alkaloids varied between samples. CONCLUSION: The developed CZE method was reliable for the quality control of aconites contained in herbal medicines. The method could also be used as an approach for toxicological studies.

Rapid optimization of dual-mode gradient high performance liquid chromatographic separation of Radix et Rhizoma Salviae Miltiorrhizae by response surface methodology.

An approach for rapid optimization of dual-mode gradient high performance liquid chromatography (HPLC) by response surface methodology (RSM) was developed for fast simultaneous separation of hydrophilic and hydrophobic components in Radix et Rhizoma Salviae Miltiorrhizae (Danshen) and its preparations. The aim of this study was to achieve a high throughput RSM optimization using a short ultra-high performance liquid chromatographic (UHPLC) column to simultaneously optimize flow rate and solvent gradient, and then transfer the optimized method to conventional HPLC for routine analytical purposes. The optimization was designed with Box Behnken design (BBD) and the global Derringer's desirability was used for describing the multicriteria response variables. Sixty-two designed experiments were performed by UHPLC with a short sub-2 microm column (2.1 mm x 50 mm, 1.7 microm) and a total running time of only 5h. The predicted gradient profile was further transferred to a long UHPLC column (2.1 mm x 100 mm, 1.7 microm) and a conventional HPLC columns (2.1 mm x 100 mm, 3.5 microm and 4 mm x 100 mm, 5 microm, respectively). Compared to the published methods, the newly developed dual-mode gradient is faster and more efficient at simultaneously separating hydrophilic and hydrophobic components in Danshen and its preparations.

An experimental design approach using response surface techniques to obtain optimal liquid chromatography and mass spectrometry conditions to determine the alkaloids in Meconopsi species.

A statistic approach using response surface methodology (RSM) for optimization of the ultra-high performance liquid chromatography (UHPLC) gradient and ionization response of electrospray ionization mass spectrometry (ESI-MS) to analyze the main alkaloids from the plant matrices of six Meconopsi species is presented. The optimization was performed with Box-Behnken designs (BBD) and the multicriteria response variables were described using global Derringer's desirability. Four parameters of UHPLC and six major parameters of ESI-MS were investigated for their contribution to analytes separation and response, leading to a total of 27 and 54 experiments being performed for each instrument, respectively. Quantitative analysis of four main alkaloids in nine samples from six Meconopsis species was employed to evaluate the statistical significance of the parameters on UHPLC-QTOF/ESI-MS analytes response. The results indicated that the optimized UHPLC-QTOF-MS method is very sensitive with the limit of detections (LODs) ranging from 0.5 to 0.1 ng/ml. The overall intra-day and the inter-day variations were less than 2.45%. The recovery of the method was in the range of 94.3-104.8% with RSD less than 4.0%. This approach has important implication in sensitivity enhancement of the ultra-trace determination of alkaloids from complex matrixes in the fields of natural products, metabolomics and pharmacokinetics.

Screening of polycyclic polyprenylated acylphloroglucinols from Garcinia species using precursor ion discovery (PID) scan and ultra performance liquid chromatography electrospray ionization Q-TOF tandem mass spectrometry.

A strategy was newly developed to rapidly screen polycyclic polyprenylated acyl-phloroglucinols (PPAPs) from the plant matrices of nine Garcinia species using ultra-performance liquid chromatography (UPLC) coupled with comprehensive mass spectrometric approaches including precursor ion discovery (PID) and tandem mass (MS/MS) scans. The PPAPs share the same diagnostic product ion at m/z 177.02 in positive MS/MS scan, which may be increased as the base peak by ramping the cone voltage from 45 to 100 V. With this ramping cone voltage PID scan, it is feasible to selectively screen the PPAPs from 29 samples of nine Garcinia species. This approach has proven to be a powerful, highly selective, and sensitive tool for rapid screening and detection of nontargeted components in natural products before the purification and structural elucidation process.

Qualitative and quantitative analysis of diterpenoids in Salvia species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

An on-line ultra-high-performance liquid chromatography (UHPLC) coupled with photodiode-array detection and quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) method has been optimized and established for the qualitative and quantitative analysis of the important diterpenoids in the methanol extracts of 12 Salvia species. Specific marker components were identified for the classification of the Salvia samples by principal component analysis. The accurate mass measurement within 3 ppm error for all the protonated molecules and subsequent fragment ions offers higher quality structural information for interpretation of fragmentation pathways of various groups of diterpenoids. Thus, a total of 21 diterpenoids from different Salvia species were separated within 10 min, and were unequivocally or tentatively identified via comparisons with authentic standards and literature. This UHPLC-QTOF-MS/MS method was validated to be sensitive, precise and accurate with the limit of detections at 3.0-16 ng/ml, and the overall intra-day and the inter-day variations less than 3%. The recovery of the method was in the range of 96.2-101.8%, with relative standard deviation (RSD) less than 3.0%. The results demonstrated that the qualitative and quantitative differences in diterpenoids were not only useful for chemotaxonomy in some Salvia species but also for the standardization and differentiation of large numbers of similar samples.

Bioassay guided discovery of apoptosis inducers from gamboge by high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

A screening system, composed of high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry, was established to find bioactive lead compound. This system succeeded in discovering apoptosis inducers from gamboge, the resin of Garcinia hanburyi. High-speed counter-current chromatography was used to provide well-separated fractions for bioassay and the resulted active fractions were rapidly identified using high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. The solvent system of n-hexane/ethyl acetate/methanol/water was optimized to the ratio of 7:3:7:3 (v/v/v/v) by a K value analysis. As a result, two active fractions were obtained. They showed apoptosis inducing effects as potent as that of taxol (500 nM) at the concentration of 1 microg/ml. Gambogenic acid (72.1%) and epimeric isogambogic acids (25.3%) were identified in one of the fractions. The other active fraction mainly contained two epimeric mixtures, gambogic acids (68.7%) and gambogoic acids (26.9%). Among them, gambogenic acid, without epimerization, has priority to be lead compound.