Determination of Homoisoflavonoids in Ophiopogon japonicus by RP-HPLC

The homoisoflavonoid content of 14 samples of Ophiopogon japonicus collected from different parts of China have been investigated. Three principal homoisoflavonoids, methylophiopogonanone A, methylophiopogonanone B, and 6-aldehydo-isoophiopogonanone A, were analyzed simultaneously by reversed-phase high-performance liquid chromatography with a binary mixture of acetonitrile and 0.3% aqueous acetic acid as mobile phase. The recovery of the method was 98.5–102.6% and good linearity (r > 0.9990) was obtained for all the homoisoflavonoids over a relatively wide concentration range. The results indicated that the homoisoflavonoid content of Ophiopogon japonicus varied significantly from species to species, and from locality to locality. The quality and identity of Ophiopogon japonicus from the herb market can be determined from the homoisoflavonoid content.     

Analysis of terpenes fromGinkgo biloba L. extracts by reversed phase high-performance liquid chromatography

Summary The main terpenes ofGinkgo biloba L. extracts (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) have been separated by isocratic elution on a 3 μm C₁₈ Spherical column using 2-propanol:water (10∶90) as eluent.     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

Quantitative analysis of the compounds in synthesis mixtures of 2,2-dimethyl-3-hydroxypropionaldehyde by RP-HPLC and GC

Summary RP-HPLC and GC methods are described for the determination of the compounds in synthesis mixtures of 2,2-dimethyl-3-hydroxypropionaldehyde (DHPAL). Special attention is paid to the analysis of DHPAL. Conditions for analysis are developed so that no derivatization is needed despite the equilibrium reactions typical of hydroxy aldehydes. Different solvent matrices, pH and sample concentrations are tested in sampling and analysis. Linearity of the calibration lines and precision of the methods are evaluated for all compounds. Detection limits of different compounds are calculated for both methods.     

Identification of the major compounds in extracts ofVerbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization

Summary A qualitative reversed-phase HPLC method has been developed for the analysis of 50% MeOH extracts ofVerbena officinalis L. (Verbenaceae) leaves. The method enables separation of the main constituents: iridoids, flavonoids and phenolic acid derivatives. Simultaneous detection at different wavelengths, measurement of the UV spectrum of each separated compound during elution and co-injection of reference substances facilitated easy and rapid identification of verbenalin, hastatoside and verbascoside. As some of these compounds, mainly flavonoids, have closely related structures, however, characterization by derivatization with reagents inducing a shift of UV absorption maxima was required. This furnished additional structural information. The reagents were adapted for compatibility with the solvent system used for the chromatographic separation.     

A study of the behaviour of some new column materials in the chromatographic analysis ofCatharanthus alkaloids

Summary New column-packing materials specially designed for the HPLC analysis of basic compounds have been tested for the analysis ofCatharanthus alkaloids. Mobile phase optimization was performed for each column tested. The influence of mobile phase pH, nature and content of the organic modifier and salt concentration on retention, selectivity and resolution was studied. An important factor in the separation proved to be the pH of the eluent, because of the widely different pK _a values of the analytes. Complete separation was easily achieved on ODS columns, but polymeric materials also gave acceptable results. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Stability of anthocyanins extracted from grape skins

Summary The extractability and stability of anthocyanins from the skins ofVitis vinifera were determined at different pH values. Anthocyanins were extracted using acetone, partitioned with chloroform and pre-purified by solid-phase extraction (SPE). They were analysed by RP-HPLC, and the kinetic parameters of decomposition were calculated. The total monomeric anthocyanin content was determined by spectrophotometry. Anthocianins were well separated by RP-HPLC. The efficiency of extraction depended strongly on the pH of the extracting agent and on the character of the pigment to be extracted. The amount of anthocyanins decreased with increasing duration of storage, more so at elevated temperatures. Presented at Balaton Symposium '01 on High-Performance Separation Methods Siófok, Hungary, September 2–4, 2001.     

High-performance liquid chromatographic analysis of ginsenosides inPanax ginseng extracts using glass-ODS column

Summary Octadecyl-porous glass was prepared and used as the packing for reversed-phase high-performance liquid chromatography. A mixture of ginsenosides, saponins of ginseng was analyzed with detection at 203 nm. Ginsenosides Rf, Rg₂, Rb₁, Rc, Rb₂, and Rd were separated with acetonitrile-water (27.5:72.5) as the mobile phase. A well-resoluted chromatogram of ginsenosides Ro, Rg₁ and Re was also obtained with acetonitrile-water (16.5:83.5). The whole separation was achieved in 12 min with a flowrate of 1 ml/min. Calibration curves of ginsenosides Rb₁, Rc, Rb₂, Rd, Rg₁ and Re were linear up to 5 μg. It can be concluded that the rapid and accurate analysis of ginsenosides is possible by the described method.     

Reverse-phase liquid chromatography ofp-tert-butylcalixarenes

Summary Thep-tert-butylcalix[n]arenes (n=4,6,7,8) andp-tert-butyldihomooxacalix[4]arene have been separated by reverse phase liquid chromatography. Optimum conditions have been obtained on a Spherisorb ODS1, 5 μm C18 column by isocratic ambient elution with acetonitrile-methyltert-butyl ether. Calibration plots have been obtained from purified calixarenes and the reliability of the method is confirmed from test mixtures of calixarenes of known composition.     

Determination of hypericin and pseudohypericin in extracts fromHypericum Perforatum L. and pharmaceutical preparations by liquid chromatography-fluorescence detection

Summary An isocratic reversed-phase liquid chromatographic method for the simultaneous determination of hypericin and pseudohypericin, two of the main constituents ofHypericum Perforatum L., has been developed. The compounds were eluted from an Inertsil ODS-3, column by triethylammonium acetate-methanol-acetonirile (5:15:80) eluent and detected fluorimetrically, excitation 478, emission 598 nm. Hypericin and pseudohypericin were extracted from flowring tops by Soxhlet and pseudohypericin was isolated from the extract by collecting its chromatographic peak from the eluent flow. Identification of peaks was by HPLC coupled to a diode array detector and electrospray MS. The method was applied to the determination of hypericin and pseudohypericin in plant extract and in pharmaceutical tablets. For the latter a solid-phase extraction procedure was adopted. Riboflavin (0.1 ng.μL⁻¹) was used as internal standard. The linear working range of the method is 0.025–4 ng.μL⁻¹ and limit of detection 0.2 ng injected on-column. A comparative SPE study for hypericin is presented.     

A stability-indicating HPLC method for the determination of 17β-estradiol-3-phosphate in an ophthalmic solution

Summary Thep-tert-butylcalix[n]arenes (n=4,6,7,8) andp-tert-butyldihomooxacalix[4]arene have been separated by reverse phase liquid chromatography. Optimum conditions have been obtained on a Spherisorb ODS1, 5 m C18 column by isocratic ambient elution with acetonitrile-methyltert-butyl ether. Calibration plots have been obtained from purified calixarenes and the reliability of the method is confirmed from test mixtures of calixarenes of known composition.     

Relationship between the lipophilicity and specific hydrophobic surface area of some pesticides by RP-HPLC and HPTLC

Summary Using methanol-water mixtures as the mobile phases, the retention behaviors of thirty-seven pesticides were determined in RP-HPLC and RP-HPTLC. Regular retention behavior was observed for all the investigated pesticides: theirR _m and logk values decreasing linearly with increasing concentration of methanol in the mobile phase. The lipophilicity and specific hydrophobic surface area values for each compound were obtained and they have a good linear relationship. Although the chemical structures of these pesticides were different, factor analysis proved that the lipophilicity and specific hydrophobic surface area of these compounds have much in common, and the insecticides, fungicides and herbicides could not be distinguished from each other according to their lipophilicity parameters obtained from chromatography method.     

RP-HPLC as an evaluation method for the behaviour of urinary trypsin inhibitors

Summary Human urine contains a family of trypsin inhibitors. Procedures for their purification and characterization involve laborious techniques and the conclusions are different in the identification of the separated compounds. We report results obtained by applying our RP-HPLC method to some procedures adopted by different authors.     

HPLC separation of opium alkaloids on porous and non-porous stationary phases

Summary A new reversed-phase (RP) HPLC method has been developed and validated for the separation of the main opium alkaloids morphine, codeine, thebaine, papaverine and noscapine on a non-porous (micropellicular) stationary phase. On this phase quantification of the compounds by internal standardization with brucine was achieved extremely rapidly, in ca 1.5 min, only. Thus, the analysis time for the opium alkaloids was approximately one tenth of that on porous stationary phases. Different opium samples were investigated using non-porous and porous packings. The correlation between the results was excellent. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Simultaneous Determination of Six Active Compounds in Rhodiola L. by RP-LC

Rhodiola L. has a long history as traditional Chinese medicine (TCM) with a medicinal efficacy similar to Ginseng and Manyprickle Acathopanax roots. There exist three classes of important active constituents, i.e., phenylethanoids (salidroside, p-tyrosol), phenylpropanoides (rosarin, rosavin, rosin), and monoterpene (rosiridin) in this TCM. In this study, by optimizing the extraction, separation and analytical conditions, a sensitive and accurate high performance liquid chromatographic method has been developed for the simultaneous determination of the six active compounds in the different species of Rhodioa L. for the first time. The analysis was performed on a Purospher STAR C18 column at 30 °C using 20 mmol L⁻¹ aqueous ammonia acetate/methanol gradient system at a flow rate of 1.0 mL min⁻¹ and photodiode array detection (DAD ) at wavelengths 276, 250 and 205 nm, respectively. The optimized method provided good linear relation (r ² > 0.9993 for all the target compounds), satisfactory precision (RSD values less than 1.53%) and good recovery (from 96.3–104.8%). The limits of detection ranged between 0.012 and 0.047 μg mL⁻¹ for the different analytes. The developed method has been successfully applied to analysis and quality control of Rhodiola L.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Isolation of some quaternary alkaloids from the extract of roots ofChelidonium Majus L. by column and thin-layer chromatography

Summary A chromatographic system comprising untreated silica gel and an aqueous buffer—methanol eluent was investigated for the micropreparative separation of the alkaloids ofChelidonium majus L. The relationship between the logarithm of the retention factorsk of the alkaloids and volume fraction of methanol at pH^* 6 was linear for volume fractions in the range of 0.1 to 0.4. From this relationship it is possible to estimate the value of the retention factor for a given alkaloid in pure buffer and then the maximum volume of alkaloid extract that can be sampled on micropreparative column filled with silica gel.     

Simultaneous Determination of Nine Flavonoids in Dalbergia odorifera by LC

A rapid and accurate HPLC method has been developed for the simultaneous determination of nine major flavonoids, namely 3-hydroxymelanettin, melanettin, stevenin, butein, isoliquiritigenin, dalbergin, 2,4-dihydroxy-5-methoxybenzophenone, pinocembrin, 4-methoxydalbergione in Dalbergia odorifera. The samples were separated on an Aglient Zorbax SB-C₁₈ column (250 × 4.6 mm, 5 m) with a gradient of acetonitrile and 1% aqueous acetic acid (/) at a flow rate of 1.0 mL min^–1 and detected at 350 nm. The complete separation was obtained within 30 min for the nine target flavonoids. All calibration curves showed good linearity (r² > 0.999) within test ranges. The repeatability was evaluated by intra- and inter-day assays and RSD values were less than 4.0%. The recoveries were between 92.0% and 104.5%. The developed method was successfully applied to the analysis of 32 commercial samples of D. odorifera.     

Simultaneous HPLC Determination of Five Flavonoids in Flos Inulae

A simple and accurate reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous determination of five flavonoids, spinacetin, quercetin, luteolin, 6-methoxyluteolin, and isorhamnetin, in an extract of the flowers of Inula britannica L., an important Traditional Chinese Medicine (TCM). Samples were extracted with 80% ethanol. Optimum separation and detection were achieved on an ODS-3 column with a methanol–acetonitrile gradient containing 0.49% (v/v) citric acid as mobile phase. The flow rate was 1.0 mL min⁻¹ and detection was at 360 nm. All calibration plots revealed linearity was good (r ² = 0.999) within the concentration ranges tested. Repeatability was evaluated by performing intra-day and inter-day assays; relative standards deviations (RSD) were less than 2.8%. Recovery of the five flavonoids was between 91.5 and 103.6%, with RSD less than 6.5%. The method was successfully used for analysis of seven samples of Flos Inulae from different parts of China and was found to be simple and efficient.     

Urtica dioica Agglutinin (UDA) – Separation and Quantification of Individual Isolectins by Reversed Phase High Performance Liquid Chromatography

Summary Stinging nettle (Urtica dioica and Urtica urens) roots are traditionally used as a remedy against BPH in Europe, and one of the plant's active principle discussed is UDA (a mixture of different isolectins). This paper reports on the first HPLC-method permitting the qualitative and quantitative analysis of individual isolectins in Urtica plant material. Optimum results were obtained by using a cyano column, and a mobile phase comprising of 10 mM phosphate buffer (pH 6.75) and acetonitrile. Temperature and detection wavelength were adjusted to 25 °C and 228 nm, respectively. The method was successfully validated for linearity, accuracy and precision. Several U. dioica samples were analysed, and except for one, all contained lectins (isolectins II, I, V and VI in varying composition). Individual compounds were assigned by comparison with reference material or based on data obtained from LC-MS experiments. The quantitative results showed variations in the lectin content from 0.016 to 0.401%; in three commercial products, which were additionally analyzed, no lectins were found.