Liquid Chromatography for Separation and Quantitative Determination of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Stages of Growth

The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. They have also been used to treat gastric and duodenal ulcers. Chemical analysis of extracts of Poncirus trifoliatus Raf. fruit at different stages of maturation revealed variations in the concentrations of flavonoids present. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, and tangeretin) and five amines (synephrine, octopamine, N-methyltyramine, hordenine, and tyramine) in extracts from the fruit of Poncirus trifoliatus Raf. were analyzed by HPLC with a C₁₈ reversed-phase column. The concentrations of the four flavonoids naringin, poncirin, narirutin, and neoponcirin was maximum during the first stage of growth and gradually decreased until the fruits were fully developed. The paper also discusses the anatomical variations observed at different stages of development of the fruit.     

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.     

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.     

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.     

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.     

Separation of urea and other polar compounds by HPLC

Summary Urea, glycolic and aminoxyacetic acid amides are the polar metabolites of 2-acetyl-3-phenyl-tetrahydro-1,2,4-oxadiazin-5-one (RGH 4615). They cannot be separated on octadecyl-, cyanosilyl- or amino-phase columns, but silica, used with C₃-C₄ alcohol and water mixtures as the eluent permits their separation. Besides refractive index detection and on-line radioactivity measurement the metabolic formation of¹⁴C-urea was proved by enzymatic cleavage into¹⁴CO₂.     

Determination of Diterpenoids and Flavonoids in Isodon rubescens by LC-ESI-MS-MS

The diterpenoids and flavonoids in Isodon rubescens were analyzed simultaneously by high-performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) for the first time. The fragmentation pathway of rubescensin A (oridonin) and rubescensin B (ponicidin) in an electrospray ion trap mass spectrometer was also investigated. A total of 10 compounds, including five diterpenoids and five flavonoids, were identified or preliminarily characterized based on their mass spectra. Six of them were reported from Isodon species for the first time.     

Qualitative and Quantitative LC Profile of Embelin and Rapanone in Selected Lysimachia Species

Qualitative and quantitative determination of the pharmacologically active benzoquinones, embelin and rapanone, in different organs of eight Lysimachia species has been conducted by reversed-phase high-performance liquid chromatography. An analytical Hypersil BDS C-18 column and a mobile phase of water containing 0.1% v/v H₃PO₄ and acetonitrile (10:90) at a flow rate of 1.0 mL min⁻¹ were used. UV detection was at 286 nm. The recovery of the method was 81.5% for embelin and 80.5% for rapanone. Good linearity (r > 0.999) was obtained for both compounds. The leaves of L. ephemerum had the highest amount of rapanone (1.69%) while the roots of L. punctata had the highest amount of embelin (1.28%).     

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.     

Preparative Separation and Analysis of Complex Mixtures of Leucomycins and Desmycarosyl Leucomycins Using HPLC and Mass Spectrometry

Composition and hydrolysis products of a biotechnical available complex of macrolides were analyzed by HPLC and ESI-CID-MSⁿ. Major components are leucomycin-type antibiotics (leucomycins A₁ (5), A₇ (3), A₉ (2), 9-desoxy-9-oxo-turimycin H₃ (4) and niddamycin B (6). Hydrolysis of the complex mixture yielded 9-, 13-isoforocidins (7, 8, 9, 11) and 9-oxo-forocidin (10). A preparative separation procedure was elaborated furnishing compounds 3 - 11 for semisynthetic experiments.Prof. Dr. Udo Gräfe deceased on 14.2.2003. For obituarity see: Schlegel B (2003) J Peptide Sci 9: 661–661.     

HPLC analysis of flavonoids and phenolic acids and aldehydes inEucalyptus spp.

Summary Standards of the polyphenols occurring in wood, bark and leaf extracts ofEucalyptus spp. (i.e. flavonoids and phenolic acids and aldehydes) have been analyzed by HPLC using reversed phase columns, gradient elution and diode-array detection. The conditions used are reported.     

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.     

Large-Scale Isolation and Purification of Scoparone from Herba artemisiae scopariae by High-Speed Counter-Current Chromatography

A preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol- water (1:1:0.45:1.55, v/v/v/v) was successfully performed to isolate scoparone (6,7-dimethoxycoumarin, 6,7-dimethylesculetin, 6,7-DME) from the plant of Herba artemisiae scopariae, a traditional Chinese medicine. 233.5 mg Scoparone with the purity of 96.8% (determined by HPLC) was obtained in one-step elution from 800 mg crude extract. The recovery of scoparone was 91.8%, and the chemical structure of this compound was identified by IR, MS, ¹H NMR and ¹³C NMR spectrum.     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Separation ofAstragalus saponins by reversed phase high performance liquid chromatography and evaporative light scattering detection

Summary A new HPLC method permitted the separation of 13 triterpene lycosides isolated from differentAstragalus species within 40 min. A water/acetonitrile gradient was used as eluent and 5 μm RP-18 material as stationary phase. By using an evaporative light scattering (ELS) detector, the main saponins ofA. membranaceus could be detected at levels as low as 20.0 μg·mL⁻¹. This method facilitated distinction of differentAstragalus species as well as the analyses of market products containingA. membranaceus. The results showed variations from 0.019 to 0.184% in the total saponin content of the market products.     

Quantitative HPLC Determination and Stability Studies of Pyridostemin in Extracts and Water Dispersible Granule Formulations of Stemona curtisii

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the determination of pyridostemin, the major pesticidal alkaloid found in Stemona curtisii. This methodology was applied to the investigation of plant extracts and water dispersible granule formulations. Stability indicating procedures have also been carried out. The chromatographic separation was on a C₁₈ column with a mixture of acetonitrile–water–triethylamine (30:70:0.12, v/v/v), using UV detection at 300 nm. Validation procedures showed that the method was specific, accurate and precise. The response was linear over a range of 5–25 μg mL⁻¹ with recoveries in the range of 98.28–102.85%. The RSD for intra- and inter-day precision were <0.72 and <1.29%, respectively. Extraction of plant material with dichloromethane gave a significantly higher pyridostemin content in the crude extracts when compared with extractions in methanol. Partial purification of the crude extracts by silica gel column chromatography was used to concentrate the mixture about fourfold. Degradation behavior of pyridostemin in the partially purified extracts followed first-order kinetics. The main pathways for its decomposition were base hydrolysis and oxidation.     

Enantioselective HPLC method for quantitative determination of tramadol andO-desmethyltramadol in plasma and urine: Application to clinical studies

Summary A sensitive, enantioselective high-performance liquid chromatographic method has been developed for the separation and individual quantitative determination of (+)-and (-)-tramadol and (+)- and (-)-O-desmethyltramadol (M1) in plasma and urine. Extraction from plasma and urine was performed by solid-phase extraction (SPE) on disposable butyl silica (100 mg) extraction cartridges. Separation of the enantiomers of tramadol and M1 was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine, 97:2.8:0.1 (v/v). Quinidine was used as internal standard. The analytes were detected by use of fluorescence detection. The limit of quantification for tramadol and M1 as 5 nM in plasma and 25 nM in urine. Recoveries were approximately 90% for tramadol and M1 in both plasma and urine. Linearity was observed for both enantiomers of tramadol and M1 in both plasma (r ²>0.999) and urine (r ²>0.997). The intra and inter-day precision (CV) did not exceed 6.0%. The applicability of the method was demonstrated by means of two clinical studies—a pharmacokinetic study in which a healthy volunteer received 150 mg tramadol hydrochloride as a single oral dose and a study in which poor and extensive CYP2D6 metabolizers received 50 mg tramadol hydrochloride as a single oral dose.     

Column purification and HPLC determination of teicoplanin and A40926

Summary A simple HPLC method for quali- and quantitative determination of teicoplanin and A40926 in fermentation broths and in biological fluids is described. The sample is purified on affinity chromatography and then analyzed on a reverse phase column using a gradient phosphate buffer-acetonitrile as mobile phase and UV detection at 254 nm. No interference from endogenous sources has been observed. The analytical method, with partial modifications, was also used to isolate on semipreparative scale the single components of these antibiotics, in order to investigate their structure and their biological activity.     

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     

Rapid Determination of Aristolochic Acids I and II in Some Medicinal Plants by High-Performance Liquid Chromatography

Aristolochic acids (AA) are toxic components of Aristolochia plants which result in diseases of the kidney such as urothelial cancer. It is, therefore, essential to monitor the amount of aristolochic acid in herbal medicines. In this study a reversed-phase high-performance liquid-chromatographic (HPLC) method has been developed for rapid determination of aristolochic acids I and II. Baseline separation was achieved within five minutes by use of an ODS C₁₈ column with methanol–water, 60:40, as mobile phase. Two kinds of aristolochic acid were successfully determined in 31 herbal samples of Aristolochia fangchi Wu and Caulis Aristolochiae Manshuriensis. The results indicated that in most samples the aristolochic acid I content is much higher than that of aristolochic acid II. The two kinds of aristolochic acid were not detected in Aristolochia fangchi from the Guangdong region, so Aristolochia fangchi from this region is recommended for use in herbal remedies.