Liquid chromatography of cardiac glycosides

Summary LC was used for the separation of several cardiac glycosides with water-alcohol eluents on a silanised silica column. The influence of temperature and composition of the eluent on the retention time, the retention volumes of the glycosides, the selectivity and the capacity factor of the column were studied. Higher temperature and a higher ethanol content in the eluent reduce the retention time, the selectivity and the capacity factor but the efficiency of the column increases. The best separation of six glycosides studied was obtained at 50°C, the ethanol content in the eluent being about 30%.     

Resolution of dianthrone glycosides and anthraquinone glycosides by thin-layer chromatography

Summary Existing TLC systems for the separation of dianthrone glycosides (sennosides) and anthraquinone glycosides were found unsatisfactory. Separation of these two groups of glycosides was achieved using a conventional TLC development tank and also in a modern VARIO-KS-Chamber at a relative humidity of 32%. The separation in a VARIO-KS-Chamber was superior.     

Droplet counter-current chromatography for the preparative isolation of various glycosides

Droplet counter-current chromatography has proven to be an efficient method for separating polar compounds in the range of a few mg to gram quantities, and in this respect is complementary to the popular high-performance liquid chromatography. This is exemplified in the following by the direct obtention of four bioactive saponins in pure form from the crude extract of Hedera helix berries, isolation of an extremely unstable catechin glycoside from Dalbergia nitidula and the separation of flavone glycosides. A rapid thin-layer chromatography method for selecting the solvent system is also described.     

Use of steroid glycosides for the affinity chromatography of cholesterol

A number of sorbents for the sorption of cholesterol has been synthesized from eight saponins of the spiro- and furostanol series immobilized on amino-Silochrome. The most effective have proved to be a sorbent containing capsicosin. Amino-Silochrome, and also sorbents containing digitonin, F-gitonin, and purpureagitoside cause hemolysis. The sorption capacity of the sorbents obtained falls on passing from saponins of the spirostanol series to saponins of the furostanol series, and also with a decrease in the length of the oligosaccharide moiety of the saponins. M. V. Lomonosov Moscow State University. 2nd Moscow State Medical Institute, Institute of Chemistry, Academy of Sciences of the Moldavian SSR, Kishenev. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 652–655, September–October, 1980.     

Extraction of iridoid glycosides and their determination by micellar electrokinetic capillary chromatography

Several methods for the extraction of two iridoid glycosides, catalpol and aucubin, from the plant matrix (Veronica longifolia leaves) were compared. Pressurized hot water extraction and hot water extraction were the most efficient isolation techniques for both. Pressurized liquid extraction and maceration with various organic solvents were also tested. Relative to the amounts extracted with hot water, ethanol extracted only 22% of catalpol and 25% of aucubin and pressurized hot water extracted 83% of catalpol and 92% of aucubin. The lowest relative standard deviations, 22% for catalpol and 8% for aucubin, were achieved with hot water extraction (13 repetitions), and the highest relative standard deviations, 76% for catalpol and 73% for aucubin, with pressurized liquid extraction (five repetitions). A fast capillary electrophoretic method was developed for the quantitative determination of catalpol and aucubin.     

One step purification of flavanone glycosides from Poncirus trifoliata by centrifugal partition chromatography

Flavanone glycosides were successfully separated from the crude extract of Poncirus trifoliata by preparative centrifugal partition chromatography with a two-phase solvent system composed of ethyl acetate-acetonitrile-water (3:2:5, v/v/v). Naringin (50.0 mg), neoponcirin (16.8 mg), and poncirin (71.9 mg) were purified from the 524 mg crude extract in only one step. The purities of the isolated compounds were determined to be over 90% by HPLC analysis and their structures were elucidated by (1)H NMR, (13)C NMR, and ESI-MS.     

Identification of carbohydrate isomers in flavonoid glycosides after hydrolysis by hydrophilic interaction chromatography

A method for carbohydrate isomers (saccharide units) identification in flavonoid glycosides after hydrolysis by an HPLC system with two detectors (diode array UV-VIS detector and evaporative light scattering detector) was set up in this work. Experimental procedure was optimized with two types of model glycosides, namely rutin and hesperidin. The model glycosides were hydrolyzed to saccharide units and aglycone parts; the aglycone was characterized by its UV-VIS spectrum and the saccharide unit was identified by its retention time and elution profile (anomeric signals). Acidic and enzymatic hydrolyses were compared in the first step of the method; in acidic hydrolysis, trifluoracetic acid was used, while glucosidase, galactosidase, and hesperidinase were used in enzymatic hydrolysis. A complete enzymatic hydrolysis was achieved with hesperidin and neohesperidin, but not with 3-O-glycosides. The method was applied for the identification of a glycone from a glycoside isolated from Polygonum lapathifolium (Polygonaceae sp.).     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

Simultaneous separation of flavanone glycosides and polymethoxylated flavones in citrus juices using liquid chromatography

We present a simultaneous liquid chromatographic method for the separation of two flavonoid compound families, flavanone glycosides (FGs) and polymethoxylated flavones (PMFs), which are usually found in citrus fruit species and varieties. This technique permits the quantitation of six FGs (narirutin, naringin, hesperidin, neohesperidin, didymin, poncirin) and six PMFs (sinensetin, hexamethoxyflavone, nobiletin, scutellarein, heptamethoxyflavone and tangeretin). This technique, to be used to characterize a citrus juice by its polyphenolic profile, has been applied to the determination of flavonoid compounds in grapefruit- and orange juice. Differentiation of orange juice varieties and mixtures containing tangor juice using polyphenolic profiles and flavonoid content has been achieved.     

Determination of quercetin glycosides and free quercetin in buckwheat by capillary micellar electrokinetic chromatography

A simple and reliable method for determination of quercetin glycosides and free quercetin in buckwheat flower, leaves, stems and achenes was developed. The method consists of flavonoid extraction from freeze‐dried homogenous material in 50% v/v methanol solution and in presence of an antioxidant, cleaning of extract and analyte isolation using SPE. Analytical step uses capillary micellar electrokinetic chromatography. The working ranges, LOD and LOQ, recovery, precision and measurement uncertainty were calculated. The method is suitable for samples from buckwheat. The highest content of rutin was found in flowers of both kinds of buckwheat (99 400 mg/kg in F. esculentum, 108 000 mg/kg in F. tataricum). The free quercetin occurs in flowers and achenes of F. esculentum, whereas flowers and achenes of F. tataricum contained quercitrin.     

Some properties of 4-nitrobenzoates of saccharides and glycosides; application to high-pressure liquid chromatography

Summary A correlation between the extinction coefficient and the number of hydroxy groups reacting with 4-nitrobenzoylchloride (4-NBCl) has been established for 11 digitalis glycosides and 12 saccharides. The structures of the derivatives have been confirmed by U.V. and NMR-studies. The -values are independent of the structure of the compound studied and strictly additive as a function of the degree of substitution. For a mono-substituted molecule is 14800 and for the 11-fold substituted raffinose was found to be 162000. This additivity offers interesting possibilities for a quantitative determination by high-pressure liquid chromatography (HPLC). A demonstration of this principle has been given with a mixture of five digitalis glycosides.

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Einige Eigenschaften der 4-Nitrobenzoate von Sacchariden und Glykosiden. Anwendung auf die Hochdruck-Flüssigkeits-Chromatographie

Zusammenfassung Der Zusammenhang zwischen Extinktionskoeffizienten und der Anzahl der mit 4-Nitrobenzoylchlorid (4-NBCl) veresterten Hydroxylgruppen wurde an 11 Digitalisglykosiden und 12 Sacchariden studiert. Die Struktur der Derivate konnte durch UV- und NMR-Spektroskopie bestätigt werden. Die Extinktionskoeffizienten im Absorptionsmaximum sind unabhängig von der Struktur des Substrates und streng additiv eine Funktion des Substitutionsgrades von =14800 für ein einfach verestertes Molekül bis =162000 für die elffach substituierte Raffinose. Diese Additivität ergibt interessante Möglichkeiten für eine quantitative Bestimmung dieser Substanzen mittels HPLC. Dies wird am Beispiel der Bestimmung von 5 Digitalisglykosiden aus einem synthetischen Gemisch gezeigt.

     

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata using high-speed counter-current chromatography

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata was conducted using high-speed counter-current chromatograph (HSCCC) with a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:6:1.5:7.5, v/v). In a single operation, 28 mg of 5,7-dihydroxy-3',4'-trihydroxyflavone-3-O-6'-rhamnose and 18 mg of 5,7-dihydroxy-3',4'-dihydroxyflavone-3-O-6'-rhamnose was obtained from 150 mg of the extract. The chemical structure of the two compounds was elucidated by electrospray ionization (EIS) MS and NMR.     

Preparative isolation and purification of three flavonoid glycosides from Taraxacum mongolicum by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) was successively applied to purify three flavonoid glycosides from the aerial part of Taraxacum mongolicum, a traditional Chinese medicine. Subsequent UV, MS, and NMR analyses have led to the characterization of three flavonoid glycosides including two new compounds isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside and isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and a known compound, isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside, which were first isolated from T. mongolicum. The two-phase solvent system composed of ethyl acetate/n-butanol/water (2:1:3, v/v/v) was performed in HSCCC. Consequently, a total of 25.7 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside, 19.1 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and 10.6 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside were obtained with purity of 98.7, 98.3, and 99.1%, respectively, as determined by HPLC from 500 mg enriched extract after cleaning-up by polyamide resin.     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.