Optimized Method for the Determination of Flavonoid Glycosides and Aglycones

The relationship R_M=f (log % S; S- modifier) of several flavonoids and their sugar derivatives was examined by using chromatographic systems chosen by preliminary experiments. The dependence for flavonoid aglycones was flat in the whole mobile phase concentration. Two types of the R_M=f (log % S) plots, related to the size of sugar molecule, were found. For glycosides with smaller sugar moiety (arabinose or glucose) the plot was almost parallel, while plots of derivatives with larger sugar moiety (rutose) were convergent. After plotting the relationship R_M=f (log % S), even if standards are not available, it is possible to qualify the examined compound as containing single or complex sugar moiety. For all mentioned above compounds the R_M=f (log %S) values were given. Silica gel Si60 was used as polar adsorbent. Mobile phase composition was determined in preliminary experiments; it was composed of two solvents: methanol in ethyl acetate in increasing concentration of methanol: 5 – 20%.     

Use of a Graphical Method to Predict the Retention Times of Selected Flavonoids in HPLC from Thin-Layer Chromatographic Data

Similarities and differences between the retention characteristics of octadecylsilica wettable with water used in TLC and RP-18 used in HPLC have been elucidated by use of the linear relationships between log k and R_M. The stationary phases compared were investigated with the same mobile phases—binary mixtures of methanol and water, acetonitrile and water, and tetrahydrofuran and water. For these adsorbents of the same type but differing in specific surface area the correlation line was shifted by log (_systemI/_systemII). High values of the correlation coefficients obtained over the whole range of mobile phase organic modifier concentration examined indicated that the TLC systems could be used to predict HPLC conditions for flavonoid separation.     

Synthesis of amino-silane modified magnetic silica adsorbents and application for adsorption of flavonoids from <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> Fisch

Magnetic separation technology was applied in the separation of flavonoids from the licorice root in this work. Licorice flavonoids (LF) displayed a remarkable array of biological and pharmacological activities. The magnetic adsorbents with functional —NH₂ groups were synthesized by immobilization of amino-silane on the surface of the magnetic silica supports, which were prepared by co-precipitation method. The adsorption and desorption characteristics of the magnetic adsorbents for the separation of LF have been evaluated. The purity of an enriched extract with this method was 16.7% while the crude extract only had about 6.8% purity. Therefore, it can be concluded that these kinds of magnetic adsorbents have selectivity to the flavonoids to some extent. The affinity selectivity of the adsorbents is based on the formation of hydrogen bonding between the —NH₂ on the magnetic adsorbents and —OH, —CO on the flavonoids. Supported by the National High Technology Research and Development Program of China (Grant No. 2002AA302211) and the National Science Fund for Creative Research Groups of China (Grant No. 20221603)     

Quasi-column chromatography of barbituric acid derivatives II. Separation on Non-polar adsorbents using binary water-organic solvent mixtures as the mobile phase

Summary Twenty barbituric acid derivatives having four different types of substitution were separated in TLC-S chambers. Using non-polar adsorbents (silanized silica gel or silica gel coated with paraffin oil) and binary water-organic solvent mixtures as the mobile phase. Linear relationships between R_M and the concentration of the organic solvent were observed for the majority of the investigated compounds. The non-polar absorbents ensure a better separation than untreated silica gel especially for the therapeutically useful C₅ disubstituted barbiturates. The results can be used for the optimization of the systems for the chromatography of barbiturates. The R_f values were correlated with the number of carbon atoms of the substitutions, molecular connectivity and a parameter associated with the molecular volume. The best correlations were obtained for this last parameter.     

Liquid chromatographic enantioseparation of β-blocking agents with (1R,2R)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propyl isothiocyanate as chiral derivatizing agent

The applicability of (1R,2R)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propyl isothiocyanate [(R,R)-DANI] as a recently developed chiral derivatizing agent for the enantioseparation of a series of β-blockers is described. The thiourea diastereomers formed were analyzed by reversed-phase high-performance liquid chromatography, mixtures of water and methanol or acetonitrile being used for elution. Conditions of derivatizations (temperature, reagent excess and reaction time) were optimized, and the effects of organic modifiers on the retention and separation were investigated; the diastereomers could readily be baseline separated with methanol-containing mobile phases with resolutions between 1.58 and 2.72.     

Evaluation of phenyldimethylethoxysilane treated high-performance thin-layer chromatographic plates. Application to analysis of flavonoids inScutellariae radix

Summary The retention and selectivity of flavonoids (baicalin, baicalein, wogonin, oroxylin A) inScutellariae radix have been studies by high-performance thin-layer chromatography on phenyldimethylethoxysilane-treated silica plates. The silica plates treated with phenyl groups were used for physical and chemical analysis. From elemental carbon analysis, the maximum number of bonded phenyl surface groups per gram was calculated to be 0.467×10²¹ (Oginal silica plate: Merck Art. 15109, Silica gel 100 F₂₅₄). With methanol-1/15 M phosphate buffer (pH 6.2) mixtures as mobile phase, baicalin, baicalein, wogonin, and oroxylin A inScutellariae radix were separated. It has been shown that phenyl-treated plates are more suitable for selective separation of baicalin, baicalein, wogonin, and oroxylin A than octadecyl-treated plates.     

Computer-aided optimization of stepwise gradient profiles in thin-layer chromatography

Summary Two mixtures, one containing model coloured solutes and the other glycosides from the Digitalis species were separated by stepwise gradient thin-layer chromatography, the first in the system silica-toluene + ethyl acetate, the second in the system silica-ethyl acetate + methanol. The optimal gradient programs (i.e., for which maximal spread of spots along the plate is observed) were determined using a computer program reported in an earlier paper. Good separation and satisfactory agreement of predicted and experimental R_F values were obtained; the non-aqueous system for the separation of lanatosides permits considerable shortening of elution time owing to the lower viscosity of ethyl acetate-methanol mixtures.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

SPE–TLC Profiling of Impurities in 1-(3,4-Methylenedioxyphenyl)-2-nitropropene, an Intermediate in 3,4-Methylenedioxy- methamphetamine (MDMA) Synthesis

Impurity profiling of 1–(3,4-methylenedioxyphenyl)-2-nitropropene, an intermediate in the synthesis of 3,4-methylenedioxymethamphetamine (MDMA), has been studied by SPE combined with TLC. Extraction of impurities was performed on C₁₈ columns. TLC separation was performed on 0.2 mm silica gel 60 plates, with fluorescent indicator F₂₅₄, in a horizontal developing chamber. To improve the quality of the profile (increase the TLC sensitivity) SPE extracts were concentrated by evaporation in a stream of nitrogen. The usefulness of methanol, ethanol, ethyl acetate, chloroform, acetonitrile, and their mixtures as mobile phases was tested by use of Gibbss triangle. Spots of the separated impurities were observed under UV light (_exc=254 and 366 nm). The proposed characteristics of profile quality (optimization criteria) are based on matrix presentation of the TLC pattern. They take into account, simultaneously, the number of spots revealed, differences between R_F values, and the intensity of fluorescence.     

HPLC Phospholipid Separation Mechanisms on Silica,Amino, and Diol Columns

Abstract

The HPLC separation of phospholipid mixtures was investigated on silica, amino, and diol columns, using mobile phases consisting of acetonitrile, methanol, and phosphoric or trifluoroacetic acids in varying proportions. An explanation of the mechanism of these separations is given with regard to the individual column types.     

Use of a database of plots of pesticide retention (R F) against mobile-phase composition.Part I. Correlation of pesticide retention data in normal- and reversed-phase systems and their use to separate a mixture of ten pesticides by 2D TLC

Summary Ten pesticides have been completely separated by two-dimensional (2D) development on TLC plates coated with coupled layers of octadecyl silica (reversed-phase, RP) and plain silica (normal-phase, NP). The binary mobile phases, aqueous-organic for RP chromatography and nonaqueous for NP chromatography, were chosen from plots ofR _F against mobile-phase composition and graphicalR _F(RP)-R _F(NP) correlations. The different selectivity of the RP and NP systems enabled dispersion of spots over the plate area and good separation.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Correlation between excess adsorption data measured for solvent mixture/adsorbent systems and RM values obtained for different solutes chromatographed in binary mobile phases

This paper concerns the application of excess adsorption isotherms, measured for solvent mixture/adsorbent systems, to the characterization of TLC data. For this purpose the excess adsorption isotherms for three liquid mixtures: cyclohexane/ benzene, benzene/acetone, and carbon tetrachloride/ethyl acetate on silica gel at 20°C have been measured. These mixtures have been used as binary mobile phases in TLC measurements. It has been shown for a given solute in binary mobile phase that the quantity R_M is a simple function of the excess adsorption. Parameters of this function have been used to characterize chromatographic systems with binary mobile phases.     

Enantioselective separation of thalidomide on an immobilized α1-acid glycoprotein chiral stationary phaseglycoprotein chiral stationary phase

Summary An easy and rapid enantioselective separation for assay of racemic thalidomide on an immobilized α₁-acid glycoprotein chiral stationary phase (GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modifier, buffer concentration to control the ionic strenth, and mobile phase pH were studied. These variations have consequences in terms of chromatographic retention (k), resolution (R _s), selectivity (α), and peak asymmetry (USP tailing factor). The main condition affecting chromatographic retention was mobile phase pH. At pH 4.5, no separation of thalidomide enantiomers was achieved whereas at pH 7.9 chiral separation was optimum. Peak tailing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF concentration-dependent, and that the separation factor (α) is the best parameter for evaluating enantioselectivity. The best mobile phase was pH 7.0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions validation including linearity, recovery, and precision was performed. The suitability of this method has been successfully proved in a limited in-vivo study after intravenous administration of thalidomide to a New Zealand male rabbit.     

Analysis and retention behavior of isoflavone glycosides and aglycones in Radix Astragali by HPLC with hydroxypropyl‐β‐cyclodextrin as a mobile phase additive

In order to determine isoflavone glycosides (calycosin‐7‐O‐β‐d ‐glucoside and formononetin‐7‐O‐β‐d ‐glucoside) and aglycones (calycosin and formononetin), a simple HPLC method with isocratic elution employing hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as a mobile phase additive was developed. Various factors affecting the retention of isoflavone glycosides and aglycones in the C₁₈ reversed‐phase column, such as the nature of cyclodextrins, HP‐β‐CD concentration, and methanol concentration, were systematically studied. The results show that HP‐β‐CD, as a very effective mobile phase additive, can markedly reduce the retention of isoflavone glycosides and aglycones, and the decrease magnitudes of isoflavone aglycones are more than those of their glycosides. The role of HP‐β‐CD in the developed HPLC method is attributed to the formation of the inclusion complexes between isoflavone glycosides (or aglycones) and HP‐β‐CD. So, the apparent formation constants of the isoflavone glycosides (or aglycones)/HP‐β‐CD inclusion complexes also were investigated. Isoflavone glycosides (and aglycones) form the 1:1 inclusion complexes with HP‐β‐CD, and the isoflavone aglycones/HP‐β‐CD complexes are more stable than the isoflavone glycosides/HP‐β‐CD complexes. Finally, the optimized method was successfully applied for the determination of isoflavone glycosides and aglycones in Radix Astragali samples.     

Mixture designs for exploring class diversity and metabolite fingerprinting: an efficient column chromatographic strategy

The effects of four solvents, hexane, dichloromethane, ethyl acetate, methanol, and their mixtures on the separation of metabolites in crude extracts of Erythrina speciosa Andrews leaves were investigated using two strategies for open column chromatography. The classical extraction procedure was compared with mobile phases prepared according to a mixture design in order to explore the effects of solvent interactions on metabolite separations. Principal component analysis was used to compare the UV spectra obtained from RP-HPLC-DAD and to estimate the number of independent factors contained in the chromatographic data of the extracts. The results showed that, in addition to solvent polarity, solvent mixtures play an important role in metabolite separation. When pure solvents are used, larger groups of similar spectra are observed in the factor analysis score graphs indicating the same or a limited number of metabolite classes. In contrast solvent mixtures produced score graphs with a larger number of clusters indicating greater metabolic diversity. Besides resulting in more peaks than the pure solvents the chromatographic data of the design mixtures resulted in larger numbers of significant principal components confirming the greater chemical diversity of their extracts. Thus, if the objective of an analysis is to obtain metabolites of the same class, one should use pure solvents. On the other hand, binary and ternary solvent mixtures are recommended for more efficient investigations of class diversity and richer metabolite fingerprints.     

Identification of Free and Glycosidically Bound Volatiles and Glycosides by Capillary GC and Capillary GC‐MS in “Lulo del Chocó” (Solanum topiro)

The volatile constituents of lulo del Chocó (Solanum topiro) fruit pulp obtained by liquid‐liquid extraction were analyzed by capillary GC and capillary GC‐MS. In total, 30 components were identified with methyl salicylate, hexadecanoic acid, hexanal, guaiacol, ethyl butanoate, and ethyl acetate being the major components. Chirospecific MDGC analysis revealed the predominance of (R)‐ethyl‐3‐hydroxybutanoate (ee 40%) and the presence of racemic mixtures both of δ‐octalactone and of δ‐decalactone. For γ‐hexalactone, γ‐octalactone, and γ‐decalactone enantiomeric distributions of 22.4 : 77.6, 22.9 : 77.1, and 20.0 : 80.0, (R) : (S), respectively, were determined. Glycosidically bound aroma compounds were identified by capillary GC and capillary GC‐MS after isolation of the glycosidic fraction obtained by Amberlite XAD‐2 adsorption and methanol elution followed by hydrolysis with a commercial pectinase enzyme. In total 13 bound aroma compounds (aglycones) were identified. These aglycones mainly consisted of compounds exhibiting aromatic structures. Additionally, with the aid of capillary GC and capillary GC‐MS (EI and NCI) of trifluoroacetylated derivatives we identified eight glucosides: the novel 3,6‐epoxy‐7‐megastigmen‐5,9‐diol β‐D‐glucopyranoside and the hexyl, benzyl, linalyl oxide (furanic), 2‐phenylethyl, vomifolyl (isomer 1), (6S,9R)‐vomifolyl, and scopoletin β‐D‐glucopyranosides.     

Gradient thin-layer chromatography of extracts fromDigitalis lanata andDigitalis purpurea

Summary Glycosides isolated fromDigitalis lanata andDigitalis purpurea (1985–1991 crops) were chromatographed on High-Performance precoated silica layers using stepwise gradient elution, the eluents containing increasing concentrations of methanol in ethyl acetate. The extracts were separated into about 20 components, presumably glycosides, some of which were identified using a mixture of standards. Densitograms at 365 nm were also obtained. The adsorbent-eluent system was very efficient and the separation was rapid owing to the use of a non-aqueous mobile phase. The method is suitable for the comparative analysis of lanatosides in plant extracts.     

Synthesis and characterization of novel polar-embedded silica stationary phases for use in reversed-phase high-performance liquid chromatography

We have developed a series of new C₁₀ dipeptide stationary phases via a simple and effective synthetic method. The preparation of the new phases involves the synthesis of silanes and the surface modification of silica. Chromatographic evaluations of these columns were performed using the Engelhardt, Tanaka, and Neue test mixtures. The applicability of these new stationary phases was also evaluated using a series of diagnostic probes including acids, bases or neutral compounds and several generic applications. These new C₁₀ dipeptide stationary phases showed excellent hydrolytic stability over a wide pH range. Like other existing amide-embedded columns, these new stationary phases exhibit higher retention for polar and hydrophilic compounds and different selectivity as compared to conventional C₁₈ columns. These new phases are compatible with 100% aqueous mobile phases, and also provide high column efficiency and good peak shapes for both acidic and basic compounds.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.