UPLC/Q-TOFMS/MS as a powerful technique for rapid identification of polymethoxylated flavones in Fructus aurantii.

Polymethoxylated flavones (PMFs), as potential cancer chemopreventive agents, are widely distributed in Citrus genus. In this study, a selected ion monitoring-tandem mass (SIM-MS/MS) method for the rapid identification of PMFs in Fructus aurantii (F. aurantii) with ultra-performance liquid chromatography (UPLC) coupled to quadrupole, hybrid orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOFMS/MS) was proposed. The MS data for candidates, containing accurate mass and isotopic patterns for both precursors and their fragment ions, were acquired selectively. Based on the MS data, the mass spectrometric fingerprint (MSFP) for candidates, consisting of chemical formula and dissociation pattern, was determined. Comparing the MSFPs of the observed compounds with the diagnostic MSFP of the species, 44 PMFs were tentatively identified. The method was validated by tangeretin and sinensetin, two representative compounds of PMFs, and was considered to be suitable for the rapid screening of PMFs in crude and partially purified samples.

Characterization of polymethoxylated flavones in Fructus aurantii by off-line two-dimensional liquid chromatography/electrospray ionization-ion trap mass spectrometry.

Off-line two-dimensional reversed-phase liquid chromatography (2D-RPLC) coupled to electrospray ionization-ion trap mass spectrometry (ESI-ITMS) was operated in positive mode (PI) to characterize polymethoxylated flavonoids (PMFs) in botanical sample. The fragments of [M+H-nx15](+) produced by loss of one or more methyl group from the protonated molecules, as well as [M+H-14](+), [M+H-29](+), [M+H-33](+), [M+H-43](+), [M+H-46](+) and [M+H-61](+) fragments formed the multiple MS (MS(n)) "fingerprint" of PMFs. 42 target compounds were tentatively identified from the extract of Fructus aurantii (F. aurantii) based on this "fingerprint". Experimental outcomes indicated that the application of 2D separation method can reduce the matrix suppression of analytes caused by the coelution with interferential components and the column overloading of interferential components. 42 versus 23 target compounds were detected through 2D versus 1D method, which confirm the superiority of 2D coupled to MS in elimination of matrix effects.

Three novel terpenoids from the rhizomes of Curcuma longa.

Investigation of the EtOH extract of the rhizomes of Curcuma longa led to the isolation of two new sesquiterpenes, 2-methoxy-5-hydroxybisabola-3,10-diene-9-one (1) and 2,8-epoxy-5-hydroxybisabola-3,10-diene-9-one (2), one new monoterpene, 2-(2,5-dihydroxy-4-methylcyclohex-3-enyl)propanoic acid (3), together with five known sesquiterpenes (4-8). Among the known compounds, bisacurone A (5) and 4-methylene-5-hydroxybisabola-2,10-diene-9-one (6) were isolated from C. longa and genus Curcuma for the first time, respectively. Their structures were established on the basis of various spectroscopic analyses including HR-ESI-MS, 1D and 2D NMR, IR spectra, and by comparison of their spectral data with those of related compounds.

Two new isoflavone glycosides from Pueraria lobata.

Two new isoflavone diglycosides, formononetin 8-C-[beta-D-apiofuranosyl-(1 --> 6)]-beta-D-glucopyranoside (1) and formononetin 8-C-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranoside (2), were isolated from the roots of Pueraria lobata, together with four known compounds, 4'-methoxypuerarin (3), daidzin (4), genistin (5), and daidzein (6). The structures of these compounds were elucidated by the spectroscopic methods.

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.

Characterization of C-glycosyl quinochalcones in Carthamus tinctorius L. by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

C-Glycosyl quinochalcones are unique components in Carthamus tinctorius L. The reported C-glycosyl quinochalcones have the same quinochalcone skeleton with a hydroxyl group at the 5'-position and a glucose linked to this position with a carbon-carbon bond. In this study, the standard hydroxysafflor yellow A and water-extracted fraction of Carthamus tinctorius L. were analyzed by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOFMS) in both positive and negative ion modes. The fragmentation pathways of C-glycosyl quinochalcones were interpreted and validated by accurate mass measurement. Their fragmentation showed a special cleavage at the C-C bond except for the typical internal cleavage at the sugar moiety of other C-glycosyl flavonoids. In positive ion mode, cleavage of the 5'-glucose produced an [M+H-162](+) ion by a neutral loss, while cleavage of the 5'-glucose in negative ion mode led to an [M-H-163](-.) ion by radical cleavage. The cleavage from the carbonyl group produced fragment ions containing an A or a B ring. The fragment ions containing an A ring were common product ions of seven compounds in both ion modes, and fragment ions containing the B ring were used to judge the different substituent groups at the 3'-position. The fragmentation patterns of seven structurally related C-glycosyl quinochalcones were analyzed systematically and the formation of the fragment ions in two modes is explained in detail in this report. UPLC/Q-TOFMS is an effective tool for characterizing a complex sample, which gives higher resolution separation and generates accurate mass measurement of the product ions.

Rapid prediction and optimization of concentration conditions for preparative fractions by solid-phase extraction.

SPE is an effective tool for concentrating preparative fractions isolated from a complex sample. To guarantee high efficiency and recovery of concentration, the concentration conditions could be optimized by predicting the breakthrough volume (V(B)). In this study, a method of predicting V(B )of unknown compounds in preparative fractions at any isocratic mobile phase composition with the analytical retention parameters a and c is described. The a and c values and the relationship between half peak width (W(1/2)) and retention time of a model analyte were measured using the analytical elution mode on an SPE column, and the V(B )and retention volume (V(R)) predicted with the a and c values were validated with breakthrough experiments. However, it is impossible to measure the a and c values of multiple compounds in a complex system directly on an SPE column with a low number of theoretical plates. The correlation of the a and c values between the SPE and analytical columns was developed so that the analytical data could be transferred to the SPE column. With the calculated a and c values, we could optimize the concentration conditions on the basis of the predicted V(B )and the volume of the preparative fraction.

Polymethoxylated flavones metabolites in rat plasma after the consumption of Fructus aurantii extract: analysis by liquid chromatography/electrospray ion trap mass spectrometry.

A LC with full scan MS(n) method was developed in order to investigate the in vivo absorption and biotransformation of polymethoxylated flavones (PMFs) by analysis of plasma samples from rats after ingestion of Fructus aurantii extract. Four parent compounds and six metabolites with intact flavonoid structures were tentatively identified. The metabolites were either glucuronides of parent compounds or glucuronides of demethylated products of parent compounds.

Systematic screening and characterization of flavonoid glycosides in Carthamus tinctorius L. by liquid chromatography/UV diode-array detection/electrospray ionization tandem mass spectrometry.

The traditional Chinese medicine (TCM) is a complex system, which always consists of numerous compounds with significant difference in the content and physical and chemical properties. In this paper, a screening method based on target molecular weights was developed to characterize the flavonoid glycosides in the flower of Carthamus tinctorius L. The screening tables of aglycone and glycan were designed, respectively, in order to select and combine freely. The multiple reaction monitoring (MRM) scan mode with higher sensitivity and selectivity was adopted in the screening, which benefit the characterization for the minor components. Seventy-seven flavonoid glycosides were screened out finally, and their structures were characterized by tandem mass spectrometric method in both positive and negative ion modes. The glycosylation mode, aglycone, sequence and/or the interglycosidic linkages of the glycan portion and glycosylation position were elucidated by the fragmentation rule in the MS. Numerous compounds screened out with this method showed the structure variety in secondary plant metabolites, and the purposeful screening systemically and subsequent structure characterization offered more information about the chemical constitutions of TCM.

Simultaneous characterization of isoflavonoids and astragalosides in two Astragalus species by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry.

A method was developed for the simultaneous identification of astragalosides (AGs) and isoflavonoids (IFs) in the roots of Astragalus membranaceus and Astragalus mongholicus by HPLC coupled with atmospheric pressure chemical ionization MS/MS (HPLC-APCI-MS/MS). Diagnostic fragment ions of AGs and different group of IFs were obtained with one AG and eight IF standards analyzed by CID-MS, which were adopted as characteristic MS/MS fingerprints for further identification of these compounds in the two Astragalus species by using HPLC-APCI-MS/MS. A total of 20 IFs and 10 AGs were identified or tentatively identified. Among them, six IFs were detected in A. membranaceus for the first time and five IFs were firstly identified in A. mongholicus. The results indicate that HPLC-APCI-MS/MS is a powerful tool for the simultaneous characterization of IFs and AGs in complex matrix.

Rapid qualitative and quantitative analyses of flavanone aglycones in Fructus aurantii by HPLC ion-trap MS.

IT-MS operated in the positive mode was applied for the rapid characterization/quantification of the flavanones in extracts from Fructus aurantii. APCI-MS and CID MS/MS provide unequivocal molecular weight (MW) data of these compounds and useful information about their structures (diagnostic fragment ions). Main fragment pathways include neutral losses of H2O, C2H2O, and B-ring as well as a retro-Diels-Alder (RDA) fragment giving rise to [1,3A + H], [1,3B+H]+, and [1,4B-H2 + H]+ ions, which form the characteristic MS/MS "fingerprint" of flavanone aglycones. When screening extracts of F. aurantii for flavanone aglycones, eight target compounds were characterized using this fingerprint. Meanwhile, ESI-MS in full-scan mode was developed and validated for the quantification of the main flavanone aglycones in F. aurantii. This method is simple, accurate, fast and requires only 16 min per sample for direct detection and quantification of naringenin and hesperetin. All the results and these characteristic fragments showed that the IT-MS is a powerful tool for the structural characterization and quantitative determination offlavanone aglycones.

Characterization of polymethoxylated flavones in Fructus aurantii by liquid chromatography with atmospheric pressure chemical ionization combined with tandem mass spectrometry.

Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was operated in positive mode (PI) to characterize polymethoxylated flavonoids (PMFs) through its specific radical cations by collision-induced dissociation (CID). The fragments of [M + H - n x 15]+ produced by loss of one or more methyl group from the protonated molecule, as well as [M + H - 29]+, [M + H - 31]+, [M + H - 33]+, [M + H - 43]+, [M + H - 46]+, and [M + H - 61]+ fragment ions were detected, which were diagnostic for the polymethoxylated species, and could be adopted to form the multiple MS (MS(n)) "fingerprint" of PMFs. Based on this "fingerprint", 29 PMFs were screened out from extracts of Fructus aurantii, among which two of them were identified as sinensetin and tangeretin. It was proved that the PI was suitable for structural characterization of PMFs by APCI-MS(n).

[High performance liquid chromatography-mass spectrometry analysis of radix Angelica sciensis].

AIM: To analyze the chemical components in Danggui (the roots of Angelica sinensis (Oliv.) Diel). METHODS: HPLC-MS/MS was used to identify the main components in Danggui. Furthermore, the MS fragmentation regularity of the phthalides was proposed. The mobile phase of HPLC consisted of 0.5% acetic acid in water and 0.5% acetic acid in acetonitrile, analytical column was Hypersil ODS2 (250 mm x 4.6 mm, 5 microm), flow rate 1.0 mL x min(-1), injected volume 2 microL. The ionization source was ESI in positive ion mode. RESULTS: Ferulic acid, nine known phthalides and one unknown phthalide derivative were tentatively identified in chromatograms based on their MS data and the comparison of their UV spectra with those published in the literatures. CONCLUSION: The structural information of phthalides was obtained via HPLC-MS/MS, which provides an accurate and fast method to identify the phthalides and provides more scientific information for quality control of Danggui.