Karine Ndjoko

Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers.

LC-MS-MS is becoming a very important tool for the on-line identification of natural products in crude plant extracts. For an efficient use of this technique in the dereplication of natural products, a careful study of the parameters used to generate informative MS-MS spectra is needed. In this paper, the collision-induced dissociation (CID) MS-MS spectra of ubiquitous C-glycosidic flavonoids have been systematically studied using hybrid quadrupole time-of-flight and ion-trap (IT) mass analysers under various CID energy conditions. Efficient differentiation of flavonoid C-glycoside isomers was possible, based on the comparison of CID-MS-MS spectra of particular C-glycoside unit fragments. Striking differences between 6-C and 8-C flavonoid glycosides were especially observed in the product ion spectra of their 0.2X+ fragments ([M+H-120]+). Some guidelines for the on-line characterisation of C-glycosidic flavonoids by LC-MS-MS or LC-multiple-stage MS are given.

Liquid chromatography with ultraviolet absorbance–mass spectrometric detection and with nuclear magnetic resonance spectrometry: a powerful combination for the on-line structural investigation of plant metabolites

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed.

The potential of LC-NMR in phytochemical analysis

The coupling of high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high field magnets have given a new stimulus to this technique, which has emerged since the mid 1990s as a very efficient method for the on-line identification of organic molecules. LC-NMR thus represents a potentially interesting complementary technique to LC-UV-MS in phytochemical analysis for the detailed on-line structural analysis of natural products. Recent applications have fully demonstrated the usefulness of this technique. A brief review of the applications of LC-NMR in natural product chemistry is presented in this paper, and a summary of the basic principles and modes of operation of LC-NMR is provided. Selected examples of LC-NMR analyses of plant metabolites in crude extracts or in enriched fractions are outlined and used to illustrate the different strategies for employing the technique. The practical possibilities and limitations of LC-NMR in its application to the analysis of crude plant extracts are discussed by means of several examples. Analytical strategies involving LC multi-coupled (hyphenated) techniques for the chemical screening and dereplication of crude plant extracts are presented. An analysis of the future development of the technique with respect to its application in phytochemical analysis is also given.

Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography-electrospray mass spectrometry.

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 microg/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography-electrospray mass spectrometry (LC-ES-MS) has been developed. The three main phenolic acids (1-3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC-ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5-10 microg/g for compounds 1, 2 and between 0.1 and 7.5 microg/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a SIN ratio of 3 were 0.1 (3) and 0.25 microg/g (1, 2). Recoveries were around 101% at 5 microg/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 microg/g in a standardised leaf extract which is a constituent of a phytopreparation.

On-line LC/UV/MS analysis of flavonols in the three apple varieties most widely cultivated in Brazil

Este trabalho apresenta a primeira investigacao sobre a estrutura quimica dos flavonois presentes em cascas das tres variedades de macas mais cultivadas no Brasil: Gala, Golden e Fuji. As analises foram feitas por CL/UV/EM (cromatografia liquida de alta eficiencia com detector UV com arranjo de diodos acoplada a espectrometria de massas), usando adicao poscoluna de reagentes de deslocamento de UV e tambem espectrometria de massas de multiplos estagios (EM n ) com ionizacao “electrospray” no modo negativo. A identificacao “on-line” com base nos dados de CL/UV/EM demonstrou a presenca de rutina, hiperosideo, isoquercitrina, quercitrina e outros tres derivados de quercetina-3-O-pentosideo. As analises por CL/UV/EM tambem demonstraram que as tres variedades de maca tem perfis cromatograficos semelhantes. This work describes the first detailed investigation into the chemical structures of flavonols present in the three apple varieties most commonly cultivated in Brazil: Gala, Golden and Fuji. The analyses were carried out by LC/UV/MS (high-performance liquid chromatography coupled to diode array UV detection and mass spectrometry), using post-column addition of UV shift reagents, as well as multiple stage mass spectrometry (MS n ) with electrospray ionization in the negative ion mode. Rutin, hyperoside, isoquercitrin, quercitrin, quercetin and three other quercetin-3-pentoside derivatives were identified through the characterization of on-line-based LC/UV/MS data. LC/UV/MS analysis also revealed that the three apple cultivars have similar chromatographic profiles.

On-line identification of minor flavones from sugarcane juice by LC/UV/MS and post-column derivatization

Este trabalho apresenta a identificacao “on line” de flavonas minoritarias do suco da cana-deacucar (Saccharum officinarum) , por cromatografia liquida de alta eficiencia com detector UV acoplada a espectrometria de massas (CL/UV/EM) com ionizacao quimica a pressao atmosferica, dissociacao induzida por colisao (IQPA-DIC-EM/EM) e derivatizacao pos-coluna utilizando reagentes de deslocamento de UV. As analises CLAE-UV com reagentes de deslocamento forneceram informacoes sobre a posicao da substituicao no esqueleto dos flavonoides e, em combinacao com dados de EM, estas tecnicas permitiram a identificacao “on-line” de cinco flavonas da garapa: luteolina-8- C-glucosil-7-O-glucuronideo; tricina-7-O-neoesperosideo-4’O-ramnosideo; tricina-7-O-metilglucuronato-4’-O-ramnosideo; tricina-7-O-metilglucuronideo; swertisina; e outras quatro substâncias foram parcialmente identificadas como flavonas glicosiladas. Somente a swertisina (7-O-metilapigenina-6-C-glicosideo) foi anteriormente descrita no bagaco da cana-de-acucar. This work describes the on-line characterization of minor flavones from sugarcane (Saccharum officinarum ) juice by high-performance liquid chromatography coupled to diode array UV detection and mass spectrometry (LC/UV/MS) using atmospheric pressure chemical ionizationcollision-induced dissociation (APCI-CID-MS/MS) and post-column derivatization using UV shift reagents. HPLC-UV analysis with shift reagents provided information about the substitution pattern in the flavonoid skeleton and, combined with MS data, these techniques allowed for the on-line identification of five “garapa” flavones: luteolin-8- C-glucosyl-7-O-glucuronide; tricin-7-O-neohesperoside-4’-O-rhamnoside; tricin-7-O-methylglucuronate-4’-O-rhamnoside; tricin-7-O-methylglucuronide; swertisin, while four other compounds were partially identified as glycosylflavones. Only swertisin (7- O-methylapigenin-6-C-glucoside) was reported previously in sugarcane molasses.

Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

SummaryA rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C18 reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.

LC-MS/MS ANALYSIS OF SUGARCANE EXTRACTS AND DIFFERENTIATION OF MONOSACCHARIDES MOIETIES OF FLAVONE C-GLYCOSIDES

LC-MS/MS data of the flavones from extracts of nonmodified and transgenic sugarcane (“Bowman-Birk” and “Kunitz”) led to the proposition of characteristic fragmentations that can be applied to identify the monosaccharides glucose, arabinose, and rhamnose in C-glycosylflavones. Collision-induced dissociation (CID) MS/MS generated diagnostic ions for different linked sugars in C-glycosylflavone–O-glycoside and C-glycosylflavone–O-acid derivatives, and the combination of MS data with postcolumn derivatization using UV shift reagents (which provide complementary information to determine the substitution patterns in the flavone skeleton) allowed for the identification of seven flavones not previously reported in sugarcane: 6-methoxyluteolin-8-C-arabinosyl-7-O-glucoside; diosmetin-8-C-arabinosyl-7-O-arabinoside; vitexin-7-O-rhamnoside; diosmetin-8-C-rhamnosyl-7-O-glucoside; tricetin-8–C-(6‴-O-sinapoylglucoside); 7,4′ di-O-methylapigenin-8-C-arabinosyl-rhamnoside; and luteolin-8-C-rhamnosyl-7-O-rhamnoside.