Ion Fast Atom Bombardment Mass Research Articles

Study of Host‐Guest Complexation of Alkaline Earth Metal Ion, Aluminum Ion and Transition Metal Ions with Crown Ethers by Fast Atom Bombardment Mass Spectrometry

AbstractComplexations of crown ethers with alkali metal ions have been investigated extensively by FAB mass spectrometry over the past decade, but very little attention has been paid to reactions of crown ethers with other classes of metal ions such as alkaline earth metal ions, transition metal ions and aluminum ions. Although fast atom bombardment ionization mass spectrometry has proven to be a rapid and convenient method to determine the binding interactions of crown ethers with metal ions, problems in reliabilities for quantitative measurements of” binding strength for the host‐guest complexes have been described in the literature. Thus, in this paper, applications of FAB/MS for investigating the complexation of crown ethers with various classes of metal ions is discussed. Extensive fragmentations for neutral losses such as C2H4O or C2H4 molecules from the host‐guest complexes could be observed. The reason is attributed to the energetic bombardment processes of FAB occuring in the formation of these complexes. Complexes of cyclen with metal ions also show neutral losses of C2H4NH molecules leading to fragment ions. Transition metal ions usually form (Crown + MCl)+ type of ions, alkaline earth metal ions can form both (Crown + MCl)+ and (Crown + MOH)+ type of ions. But for aluminum ions, only (Crown + Al(OH)2)+ type of ions could he observed.

Epitope dissection of receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus.

Receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus were chemically modified and analyzed by negative ion fast atom bombardment mass spectrometry (FAB MS) or electron ionization mass spectrometry (EI MS) after permethylation. Derivatizations included mild periodate oxidation of the sialic acid glycerol tail or conversion of the carboxyl group to primary alcohol or amides. The modified gangliosides were then tested for binding affinity using thin-layer plates overlaid with labeled microbes or microbe-derived proteins. Mild periodate oxidation, which shortens sialic acid tail without destruction of sugar cores, abolished or drastically reduced binding of H. pylori and avian influenza virus to sialyl-3-paragloboside (S-3-PG). The same effect was observed in the case of binding of the human influenza virus to receptor-active gangliosides of human leukocytes. Conversion of S-3-PG or leukocyte gangliosides to primary alcohols or amides also abolished the binding. However, mild periodate oxidation had no effect on binding of NAP (neutrophil-activating protein of H. pylori) to the active ganglioside.

BILE ACID SYNTHETIC DISORDER IN A SYMPTOMATIC PATIENT WITH NORMAL LIVER ENZYMES

173 The bile acid synthetic disorders (BASD) are due to inherited defects in cholate and chenodeoxycholate biosynthesis. Abnormal hepatotoxic and cholestatic bile acids result in severe liver injury, which commonly presents in the neonatal period. Patients with diagnosis in childhood typically have biochemical evidence of significant hepatic injury. We report a patient with a BASD presenting with profound malabsortion of fat soluble vitamins, but with normal hepatic aminotransferases. Case report: A 3 year old female born to non-consanguineous Liberian parents presented following a one week history of epistaxis. She was previously healthy except for rickets at age one year. She had frequent nosebleeds and easy bruisability, but no fever, diarrhea, bleeding gums or jaundice. There was no known toxic ingestion. On examination, she was a slightly pale child with height and weight both at the 75th percentile. She had hepatomegaly with a liver span of 10 cm at the right costal margin. The remainder of the physical exam was normal. The hemoglobin was 7.4 (11.5-13.5 gm/dl), platelets 553 (150-400 K/ul), PT 33.1(11.0-13.0 sec), PTT 70.0 (27.0-38.0 sec). Levels of factors VII, IX and X were low at 9%, 2% and 2% respectively. (normal - 50-150%) Factor VIII and factor XII levels were at or above normal at 231% and 87%. Warfarin level was zero. At the time of admission, total bilirubin was 0.7 (0.2-1.4 mg/dl), alkaline phosphatase 247 (145-320 U/I), ALT 39 (5-45 U/I), AST 41 (20-60 U/I) and GGT 23 (6-19 U/I) After 5 mg IV Vitamin K, PT and PTT corrected to 12.4 and 26.4 seconds. Levels of fat soluble vitamins were as follows: Vitamin E< 0.5 (3.0-12.0), 25-OH Vitamin D < 5.0 (10-55 ng/ml), Vitamin A 74(30-95 mcg/dl). Commercial choleglycine was 97 (< 68 mcg/dl). Urine was screened for abnormal bile acids by fast atom bombardment ionization mass spectrometry (FAB-MS). Results showed elevated levels of sulfate and glycosulfate conjugates of unsaturated dihydroxy- and trihydroxy-cholanoic acids, consistent with a diagnosis of 3β-hydroxy-C27-steroid dehydrogenase/isomerase deficiency. Percutaneous liver biopsy showed a moderate inflammatory infiltrate in the portal tracts and lobules, portal tracts expanded by bands of fibrosis and occasional bile plugs. Cholic acid therapy was instituted. Conclusions: Inherited defects in bile acid synthesis should be considered in patients with fat soluble vitamin deficiency. In patients with these disorders, significant hepatotoxicity from abnormal bile acids may exist even in the presence of normal liver enzymes. Maintaining a high index of suspicion for bile acid synthetic defects may result in earlier diagnosis and a better prognosis for these children.

Iron Salts in the Post-Metallization of Mordant Dyes

Iron (II) and iron (III) salts are used as mordanting agents in lieu of chromium ions, as a source reduction approach to eliminating chromium in dye effluents following the post-metallization of acid mordant dyes. Specifically, wool fabric is dyed with Mordant Yellow 8, Yellow 30, Orange 3, Orange 6, Red 7. Red 19, Blue 1, Blue 9, Blue 13, Brown 40, Black 1, and Black 11, then treated with iron salts. The complexes are characterized by negative ion fast-atom bombardment mass spectrometry and absorp tion spectrophotometry, and the after-treated fabrics are evaluated for lightfastness and washfastness. Mordant dyes possessing either ortho, ortho'-bis(hydroxy)- phenylazopyrazole or ortho-hydroxy-phenylazonaphthol substituted chromophoric sys tems form the corresponding 1:2 metal : dye complex and undergo significant improve ments in lightfastness and washfastness following metallization.

Characterization of a novel [Sm(HMPA) 4I 2]I complex by positive ion fast atom bombardment and high resolution mass spectrometry

A positive-ion Fast Atom Bombardment (FAB) study was performed to determine the structure of samarium(III) triiodine complex ( I). Collision Induced Dissociation (CID) and High Resolution Mass Spectrometry were used to establish the fragmentation pattern of the C 24 H 72 O 4 N 12 P 4 I 3Sm compound. The structure of ( I) was confirmed by X-ray analysis.

Fast Atom Bombardment Mass Spectra ofN-Phosphorylated Peptide Analogs

A series of N-(N-alkyl)- and N-(O-alkyl)phosphopeptide analogs were determined by positive and negative ion fast atom bombardment mass spectrometry and their fragmentation pathways were investigated. Their mass spectra gave abundant [MH - NH z R'] + , [MH - HOR] + , [MH - H 2 O] or [M - H - NH 2 R'] - , [M - H - HOR] - ions, which contained pentavalent tricoordinate phosphorus ions through the loss of amine or water. In the positive ion mass spectra, + NH 3 R' and + NH 3 CHRCONHR' were found, while [MH - NH 2 CHRCONHR'] + was suppressed. In the negative ion mass spectra, the pseudomolecular ion [M - H] - was the base peak, and almost all the fragment ions contained a phosphoryl group.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo.

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.

Fast-atom bombardment mass spectrometry of brassinosteroid analogs

Positive ion fast-atom bombardment mass spectra of brassinosteroid analogs have been systematically obtained for the first time. The spectra of six brassinosteroid analogs and their corresponding 22S,23S isomers included the protonated molecule in medium to high relative intensity. The fragmentation pattern is dominated by side chain cleavage. There is a marked preferential loss of acetic acid from the [M + H]+ ion of 5α-hydrogen-3β-acetylated derivatives compared to the 5α-hydroxy-3β-acetylated analogs.

Disulfated oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis

Twenty novel disulfated oligosaccharides were purified in nanomolar quantities from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, followed by anion-exchange chromatography, size-exclusion chromatography, and high-performance liquid chromatography (HPLC). In addition to positive ion fast-atom-bombardment mass spectrometry (FABMS), proposed structures for the resulting purified disulfated oligosaccharides were also based on carbohydrate permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, and parallel analysis of desulfated products. Sulfate esters were found to reside on C-3 or C-6 of terminal d-galactose and on C-6 of internal d-galactose or 2-acetamido-2-deoxy- d-glucose residues. For this group of oligosaccharides, ranging in size from tri- to undeca-saccharides and possessing linear, di- and tri-antennary forms, it was also observed that sulfate esters could be located on the same or on different branches and that branched oligosaccharides can possess sulfate esters on C-3 and C-6 of different terminal galactose residues within the same structure.

CE/ES-MS ANALYSIS ON MACROCYCLIC SCHIFF BASES DERIVED FROM A SODIUM TEMPLATE CYCLOCONDENSATION OF 2,6-DIFORMYL-4-METHYLPHENOL WITH ALKYLDIAMINES

Abstract The template reaction of sodium 2,6-diformyl-4-methylphenolate with alkyl diamines H2NRNH2 [R = (CH2)n, n = 4, 5, 6] led to the production of macrocyclic Schiff bases which were characterized by elemental analyses, IR and proton NMR spectra. Positive ion fast atom bombardment (FAB) and electron spray ionization (ES) mass spectroscopic investigations on the above products showed the presence of a [3 ± 3] (H3L3) cyclocondensation mode in addition to the common [2 ± 2] (H2L2) mode. The product in a typical reaction was analysed by capillary electrophoresis (CE) coupled to a mass spectrometer (MS) via an electrospray (ES) ionization interface, confirming the independent formation of both tetraimine and hexaimine macrocycles as a mixture of Na2L2 and Na3L3.

Le(b), a novel glycosphingolipid antigen of human distal colorectal cancer

The expression of the glycosphingolipid antigens was investigated in human colorectal carcinomas and adjacent normal tissues obtained by surgical operation from six patients. The glycosphingolipids, including both non-acidic glycolipid and ganglioside, were extracted from the tissue specimens and separated by two-dimensional thin-layer chromatography of silica gel. A novel neutral glycolipid antigen migrating lower than Le(a) blood group antigen was detected in the cancerous tissues of all six patients, but not in normal regions in the same patients. The structure of the glycosphingolipid was partially characterized by gas chromatography and gas chromatography/mass spectrometry of alditol acetate and negative ion fast atom bombardment mass spectrometry (FAB/MS) of the intact glycolipid, and TLC-immunostaining with monoclonal anti-Le(a), -Le(b), -sialyl Le(a), and -Le(Y) antibody, respectively. The novel neutral glycosphingolipid in colorectal cancer was identified to be the III(4)IV(2)Fuc(2)Lc(4)Cer, Le(b) antigen of the human Lewis blood system. Direct FAB/MS showed the ceramide was composed mainly of 2-hydroxy tetradecanon and C18 phytosphingosine. In such individuals, the expression of Le(b) antigen in normal tissues is absent or minimal. Based on this study, we suggest that this Le(b) antigen may be a human distal colorectal cancer-associated antigen, which will be helpful for diagnosis and treatment of distal colorectal cancer.

BISDISELENOLATE METAL COMPLEXES. SYNTHESIS, STRUCTURE AND ELECTROCHEMICAL PROPERTIES

Abstract The multi-selenium donor ligand 2,2-dicyanoethylene-1,1-diselenolate (DCNSe) has been prepared. The dianionic complex of DCNSe with Ni(II) has been isolated as the tetrabutylammonium (TBA) salt. The compound is characterized by negative ion Fast Atom Bombardment (FAB) mass spectral analysis, Fourier Transform Infrared (FT-IR) spectral analysis, UV-Vis spectral analysis and cyclic voltammetry. The electrochemical properties and crystal structure are studied as well. In addition, the corresponding Cu(II) complex has been isolated as the TBA salt and characterized by negative ion FAB mass spectral analysis and FT-IR spectral analysis.

Liquid secondary ion mass spectra and fast atom bombardment mass spectra of diquaternary pyridinium oxime salts

AbstractPositive‐ion mass spectra of diquaternary pyridinium oxime salts were measured using fast atom bombardment and liquid secondary ion mass spectrometry in different laboratories to establish both the characteristic behavior and the spectral variability of this class of compounds. Molecular ions observed in these mass spectra include the one‐electron reduced doubly charged cation, the deprotonated double charged cation and the anion‐reduced doubly charged cation. Common fragment ions include the products of simple cleavages along the aliphatic bridge between the heterocyclic rings. Concomitant loss of hydrogen and cyclization of the bridge can occur, along with losses of OH, NOH and CHNOH from various fragment ions. A nitrobenzyl alcohol matrix generally gave superior results to glycerol and thioglycerol in all laboratories, although the use of additive surfactant acids such as p‐toluenesulfonic acid or camphorsulfonic acid in glycerol could provide useful results.

Negative ion fast atom bombardment and collision-induced dissociation mass spectrometry of the 2-, 3-, 4- and 6-deoxy derivatives from methyl β-D-galactopyranoside and related compounds

The fast atom bombardment collision-induced dissociation mass spectra of the [M-H]- ions of the 2-, 3-, 4- and 6-deoxy derivatives from methyl beta-D-galactopyranoside and some related compounds have been recorded. The fragmentation reactions of these quasimolecular ions and of OD-labelled analogues have been examined and related to the molecular structure. In some cases distinct and common mechanisms can be derived, but it is also clear from these experiments that not only the site of deprotonation of the molecules, but also the anticipated charge localization in the fragment ions strongly direct the fragmentation reactions.

N-Alkylnicotinium halides: a class of cationic matrix additives for enhancing the sensitivity in negative ion fast-atom bombardment mass spectrometry of polyanionic analytes

The addition of some surfactants to the fast-atom bombardment (FAB) matrix previously has been demonstrated to enhance analyte signals in fast-atom bombardment mass spectrometry. In particular, cationic surfactants appear to enhance the negative ion FAB detectability of analytes that exist as anionic species in the matrix solution. It has been proposed that the charged surfactant concentrates the oppositely charged analyte near the surface, which results in larger signals for the analyte. Cationic surfactants that contain a fixed positive charge and an additional basic site were prepared with different hydrophobic moieties and were evaluated for their effectiveness as FAB matrix additives. The compound N-octylnicotinium bromide (ONBr) is shown to improve greatly the analyte-related signals in negative ion fast-atom bombardment mass spectrometry for a variety of polyanionic analytes, relative to other surfactants (e.g., cetylpyridinium salts). This surfactant not only enhances detectability, but also simplifies the pseudomolecular ion region of the resulting spectra by reducing or eliminating metal cation adduct peaks. The simple mechanism of enhancement via surface activity is evaluated, and alternative mechanisms are considered. It is clearly shown that ONBr, as a FAB matrix additive, will allow mass spectrometry to be used for the analysis of anionic compounds that normally exhibit very low responses.

High Performance Liquid Chromatographic Isolation, Spectroscopic Characterization, and Immunosuppressive Activities of Two Rapamycin Degradation Products

Abstract A high performance liquid chromatographic method has been developed for the isolation of two degradation products of rapamycin which is currently under development as an immunosuppressive agent. Prior to isolation, the drug was incubated at 37°C in rat bile or ammonium acetate (pH 8.0). The isolation was achieved by a Supelco, PLC-18 21.2 × 250 mm, 18 μm column using methanol/ammonium acetate gradient mobile phase. After evaporation of methanol, the remaining eluates were lyophilized. The isolated degradation products were characterized by negative ion fast atom bombardment mass spectrometry (FAB MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). Degradation product A was found to be a macrolide ring-opened hydrolysis product of rapamycin where the C25 ester bond had been hydrolyzed. Degradation product B was determined to be a ring-opened isomer of rapamycin. B had less than 4% of the potency of rapamycin in a thymocyte proliferation assay, while A had minimal activity at concentra...

Metabolism and effect of 7-oxo-lithocholic acid 3-sulfate on bile flow and biliary lipid secretion in rats

Significant amounts of oxo and sulfated bile acids are excreted in the urine of patients with cholestatic liver diseases, but these bile acid species are found only in traces in bile. Several possible explanations for this finding include poor canalicular secretion, rapid biotransformation by oxidoreduction and extrahepatic sulfation of oxo bile acids. To assess the physiological actions and hepatic metabolism of oxo and sulfated bile acids, we synthesized 7-oxo-lithocholic acid 3-sulfate, examined the effect of intravenous infusion of this bile acid (5.0 μmol/min/kg body wt) on bile flow and biliary lipid secretion in Sprague-Dawley rats with a bile fistula and compared this effect with that of the unconjugated analog, 7-oxo-lithocholic acid, and the primary bile acid conjugate, chenodeoxycholic acid 3-sulfate. Bile flow and bile acid secretion increased immediately after infusion of all three bile acids. The highly polar 7-oxo-lithocholic acid 3-sulfate significantly reduced the biliary secretion of phospholipids (p < 0.05) and cholesterol (p < 0.05), but by contrast the more hydrophobic analog, 7-oxo-lithocholic acid, caused a significant increase in the biliary phospholipids (p < 0.01) and cholesterol (p < 0.01) secretion. Infusion of chenodeoxycholic acid 3-sulfate also resulted in an increase in secretion of biliary cholesterol and phospholipids, but the magnitude of the change was less than that induced by 7-oxo-lithocholic acid infusion. Biliary lipid secretion was inversely related to the polarity of the infused bile acid. Negative ion fast atom bombardment—mass spectrometry and gas chromatography—mass spectrometry analyses indicated that the major bile acids excreted in the bile after infusion of 7-oxo-lithocholic acid were taurine and glycine conjugates of the parent compound and the corresponding conjugates of its metabolite, ursodeoxycholate, whereas chenodeoxycholate and its conjugates were minor metabolites. By contrast, 7-oxo-lithocholic acid 3-sulfate and chenodeoxycholic acid 3-sulfate were only partially amidated with taurine and glycine and did not undergo further metabolic transformation of the steroid nucleus. These data demonstrate that sulfation of 7-oxo-lithocholic acid significantly alters its physiological properties and hinders biotransformation, including oxidoreduction, hydroxylation and amidation. The findings that the sulfate conjugates of 7-oxo-lithocholic acid and chenodeoxycholic acid are efficiently and preferentially secreted into bile, and not into urine, would strongly indicate that the bile acid sulfates, found in relatively high proportions in the urine of patients with cholestasis, are of extrahepatic and most probably of renal origin.

Structural Elucidation by Fast Atom Bombardment Mass Spectrometry of Multisulfated Oligosaccharides Isolated from Human Respiratory Mucous Glycoproteins

Abstract Underivatized mono- and multisulfated oligosaccharides obtained by the alkalineborohydride treatment of human respiratory mucous glycoproteins were analyzed by positive ion fast atom bombardment mass spectrometry (FAB MS). Employing three unique and structurally homologous groups, the FAB mass spectra of a mono- and a disulfated tri- and tetrasaccharide and a mono-, di- and trisulfated branched hexasaccharide were compared. Each produced mass spectra displaying molecular weight-related and structurally significant fragment ions including fragments differing in mass by multiples of 102 amu reflecting the loss of one or more sulfate esters. From these data, combined with monosaccharide composition, the carbohydrate sequence and number and location of sulfate esters can readily be determined. These findings, with other chemical and enzymatic analyses, make FAB MS a valuable tool applicable to the unambiguous structural elucidation of underivatized reduced, linear and branched, mucous glycoprotein ol...

A Revised Structure for the Disialosyl Globo-Series Gangliosides of Human Erythrocytes and Chicken Skeletal Muscle

Disialosyl globo-series gangliosides have previously been isolated from chicken skeletal muscle (E. L. Hogan, R. D. Happel, and J.-L. Chien (1982) Adv. Exp. Med. Biol. 152, 273-278; S. Dasgupta, J.-L. Chien, E. L. Hogan, and H. van Halbeek (1991) J. Lipid Res. 32, 499-506) and human erythrocytes (S. K. Kundu, B. E. Samuelsson, I. Pascher, and D. Marcus (1983) J. Biol. Chem. 258, 13857-13866). In both cases, the structure of this ganglioside was proposed to be NeuAcα2 → 3(NeuAcα2 → 6)Galβ1 → 3GalNAcβ1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer (V 3NeuAcV 6NeuAcGb 5Cer). We have reinvestigated the human erythrocyte antigen and now propose an alternative structure differing in the location of the NeuAcα2 → 6 residue: NeuAcα2 → 3Galβ1 → 3(NeuAcα2 → 6)GalNAcβ1 → 3Gal α1 → 4Galβ1 → 4Glcβ1 → 1Cer (V 3NeuAcIV 6NeuAcGb 5Cer). This novel structure is supported by results of 1H-NMR spectroscopy, negative ion fast atom bombardment mass spectrometry, and methylation linkage analysis with capillary gas chromatography-mass spectrometry in both electron impact and chemical ionization modes. Furthermore, based on new results from negative ion fast atom bombardment mass spectrometry and linkage analysis, we propose that the chicken skeletal muscle antigen also has this revised structure, differing only in ceramide composition. The terminal tetrasaccharide of these gangliosides is identical to that of G D1α NeuAcα2 → 3Galβ1 → 3(NeuAca2 → 6)GalNAcβ1 → 4Galβ1 → 4Glcβ1 → 1Cer(IV 3NeuAcIII 6NeuAcGg 4Cer), previously identified in a rat ascites hepatoma cell line (T. Taki, Y. Hirabayashi, H. Ishikawa, S. Ando, K. Ken, Y. Tanaka, and M. Matsumoto (1986) J. Biol. Chem. 261, 3075-3078) and a murine lymphoma cell line with low metastatic potential (K. Murayama, S. B. Levery, V. Schirrmacher, and S. Hakomori (1986) Cancer Res. 46, 1395-1402), although they appear to be immunologically distinct.

Characterization and differentiation of diglycosyl flavonoids by positive ion fast atom bombardment and tandem mass spectrometry

This study shows that fast atom bombardment (FAB) in combination with collisional activation and tandem mass spectrometric techniques is useful for the characterization of, and the differentiation between, O-diglycosyl, O-C-diglycosyl and di-C-glycosyl flavonoids. A quick differentiation between the three types of glycosyl flavonoids is possible by simply examining FAB mass spectra. Low-energy product ion spectra of [M+H]+ ions have been used for determining the carbohydrate sequence in O-diglycosyl flavonoids and characterizing the terminal monosaccharide in O-C-diglycosyl flavonoids. The data obtained for per-O-deuterated precursor ion species of O-diglycosyl flavonoids indicate that O-linked hydrogen atoms are involved in the formation of Yn+ ions. High-energy product ion spectra of [M+H]+ ions enable further characterization and differentiation of isomeric di-6,8-C-glycosides.