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.     

Thin-layer chromatography (TLC) as pilot technique for HPLC. Utilization of retention database (R F) vs. eluent composition of pesticides

Summary TLC and HPLC are frequently unjustly regarded as competitive methods. Using the advantages of both methods, a combination of TLC and HPLC leads to a considerable saving of time and expense in analysis. Experimental data are presented aslog k (HPLC) vs.R _M (TLC) correlations; high correlation coefficients indicate that TLC using octadecyl silica, wettable with water (RP-18W) can be applied as a pilot technique for HPLC (RP-18).     

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 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.     

The Use of Encoded Structural Data to Predict the Liquid Chromatographic Retention Behavior of Tetradentate Metal Complexes

Abstract

Retention data for tetradentate metal complexes of copper and nickel are reported for microparticulate silica and reversed phase columns. An attempt is made to relate structure and stability constant data to the retention volume by means of multiple linear regression. The results indicate that if the structure is encoded by means of dummy variables it may be possible to predict retention volumes of metal complexes. There is not a strong correlation between the value of the stability constants for the metal diamine and metal ketone complexes and the retention volume.     

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.     

HPLC Separation of O-Acylated Flavonoids and Biflavones from Some Species of Gymnospermae

Flavonoids present in the extracts from leaves of Pseudotsuga menziesii (Pinaceae), Ginkgo biloba (Ginkgoaceae) and Podocarpus dacrydioides (Podocarpaceae) were separated by use of the reversed phase HPLC method. The analysed compounds belong to different groups of flavonoids – biflavones (amentoflavone, bilobetin, 5–methoxybilobetin, podocarpusflavone A, sequoiaflavone, podocarpusflavone B, ginkgetin, isoginkgetin, sciadopitysin, kayaflavone, hinokiflavone, 2,3–dihydrosciadopitysin, 2,3–dihydroisoginkgetin), O–acylated flavonol glycosides (daglesiosides I, II, III, IV, trans–tiliroside, trans–ditiliroside), flavonol O–glycosides (astragalin, isoquercetin) and flavonol aglycones (kaempferol, quercetin, isorhamnetin). The conditions for flavonoid separation were optimized using various RP–18 columns. The chromatographic resolution was performed with isocratic or gradient elution – optimized by Drylab program or by traditional trial-and-error method, depending on the composition of flavonoid complex.     

Chromatographic investigations of flavonoid compounds in the leaves and flowers of some species of the genusAlthaea

Summary The flavonoids present in the leaves and flowers ofAlthaea armeniaca Ten., A. cannabina L., A. narbonesis Pourr. andA. broussonetiifolia Iljin were investigated and compared to the flavonoids present in the leaves and flowers ofA. officinalis L. The inliquid chromatography and two-dimensional paper chromatography. The same flavonoids were found in the flowers of all the investigated species while differences could be noted in the flavonoid composition of the leaves. Both the qualitative and quantitative difference was the greatest in the flavonoids of the leaves ofA. cannabina L.     

Optimization of chromatographic systems for the high-performance thin-layer chromatography of flavonoids

Summary To characterize the retention and selectivity of separations of 23 flavonoids (aglycones and glycosides) relationships betweenR _F and modifier concentration were determined for silica and diol adsorbents (with mixtures of ethyl acetate and methanol as mobile phases), for cyanopropyl silica (with mixtures of ethyl acetate and dichloromethane as mobile phases), for aminopropyl silica (with mixtures of ethyl acetate, methanol and water as mobile phases) and for octadecyl silica (with mixtures of methanol and water as mobile phases). Owing to large polarity differences between aglycones and glycosides, these groups of compounds cannot be separated other than by use of reversed-phase systems, for which the selectivity is lower. It follows from correlation plots ofR _F1 againstR _F2 that for some pairs of adsorbents (e. g. silica and diol) selectivity differences are small; for others the points in the plot are widely dispersed, indicating selectivity differences. The chemometric database obtained can be used to choose optimum chromatographic systems for the separation of given sets of flavonoids and for planning gradient elution programs for separation of flavonoid aglycones and glycosides in a single TLC experiment.     

Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir

Summary A method for post-column derivatization of the highly carcinogenic aflatoxins with iodine has been developed. It involves splitting of the mobile phase used for the reversed phase HPLC separation. One part flows through the injection valve and the C₁₈ analytical column to achieve the separation. The other part flows through a column packed with solid iodine. The iodine-containing solution is recombined with the flow coming from the analytical column. The derivatization reaction takes place in a knitted open tubular reactor maintained at 60 °C. Detection is done by fluorescence measurement. Due to the low solubility of iodine in the mobile phase, the iodine solid-phase column can be used for very long periods of time before refilling is necessary. The analytical system consists of only one pump and therefore gives the opportunity to carry out low-cost post-column reaction detection. The method yields reproducible results, a linear response over at least two orders of magnitude and detection limits of about 1 ppb, both for standard solutions and for peanut butter samples.     

HPLC-DAD and TLC-Densitometry for quantification of hypericin inHypericum perforatum L. Extracts

Summary Hypericum perforatum L. is a spontaneous perennial herbaceous plant, widely distributed in Europe, Asia, Northern Africa, and North America. The dried flowers or dried aerial parts are used to prepare the drug Hyperici Herba or St. John's Wort. Nowadays this drug is largely used as a natural antidepressant; hypericin and hypericin-like substances are considered the main active ingredients. In this work the hypericin and pseudohypericin content of hydroalcoholic extracts both of Hyperici Herba and ofHypericum perforatum L. dried flowers were measured by two techniques, TLC-densitometry with fluorescence detection and reversed-phase HPLC-DAD (diode-array detection). The quantitative data obtained by applying these techniques were compared and the identification of the main flavonoid constituents was performed by HPLC-DAD for characterization of the extracts. The quantitative data obtained by use of the two techniques were in good agreement and statistical analysis of the findings was indicative of the equivalence of the techniques.     

A Reliable Gas Capillary Chromatographic Determination of Lactulose in Dairy Samples

The rhizome of Polygonum cuspidatum is an important Chinese medicine used against infectious hepatitis, leucorrhagia, pruritus vulvae of the dampness-heat type, burns, snake bite, carbunculosis, amenorrhea, dysmenorrhea, trauma with blood stasis, and rheumatism, etc. Emodin, resveratrol, and polydatin are main active components of the rhizome. We report a simple densitometric HPTLC method for quantification of these compounds. The method was validated for precision, repeatability, and accuracy. The method was found to be precise, with RSD of 0.23, 0.25, and 0.32 (interday) and 0.45, 0.57, and 0.48 (intraday) for different concentrations of emodin, resveratrol, and polydatin, respectively. Instrument precision was 0.25, 0.23, and 0.34 (%CV) for emodin, resveratrol, and polydatin, respectively. The accuracy of the method was checked by measuring the recovery of the three compounds at three different levels; the average recoveries were 102.56%, 100.21%, and 100.27%, respectively. The amounts of emodin, resveratrol and polydatin in Polygonum cuspidatum, as estimated by the proposed method, were 4.96 mg g^–1, 1.81 mg g^–1, and 13.02 mg g^–1. The HPTLC method proposed for estimation of emodin, resveratrol and polydatin was found to be simple, precise, specific, sensitive, and accurate and can be used for quality control of Polygonum cuspidatum.     

高效液相色谱法测定牛奶中黄曲霉毒素M1含量的不确定度评定

根据方法GB 5009.24-2016《食品安全国家标准食品中黄曲霉毒素M族的测定》,建立高效液相色谱法测定牛奶中黄曲霉毒素M₁含量不确定度的数学模型,分析检测过程中的不确定度来源,并对不确定度分量进行量化和合成.当牛奶中黄曲霉毒素M₁的质量分数为0.842 μg/kg时,其扩展不确定度为0.048 4 μg/kg（k=2）.方法相对不确定度主要来源于标准品纯度和样品回收率.     

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.     

Chromatographic Retention of Epigallocatechin Gallate on Oligo-β-Cyclodextrin Coupled Sepharose Media Investigated Using NMR

The retention of epigallocatechin gallate (EGCG) on oligo-β-cyclodextrin (oligo-β-CD) bonded agarose chromatographic media was investigated. NMR spectroscopy in solution showed that the EGCG immerses into the β-CD cavity. The association constant calculated by NMR titration was used to estimate a retention factor which accurately reflected chromatographic behaviour. This correlation suggests that oligo-β-CD forms inclusion complexes with EGCG via the same mechanism as monomeric β-CD. Revised: 14 March and 25 April 2006     

The Use of Bovine Serum Albumin as a Ligand in Affinity Chromatographic Clean-up of Non-Steroidal Anti-Inflammatory Drugs from Bovine Plasma

The European Union regulates the use of non-steroidal anti-inflammatory drugs (NSAID) in animal production and official national analytical monitoring programmes have been set up in each member state to detect residues of the drugs in bovine plasma. In this work, we describe the development and application of affinity chromatography for molecular recognition of NSAID in bovine plasma. Bovine serum albumin (BSA), the plasma protein which binds such drugs, was covalently bound to a polymeric support via its glycosyl moieties. Loading capacities and specificity were tested both on standard solutions and on spiked bovine plasma for nine drugs—ketoprofen, naproxen, phenylbutazone, oxyphenylbutazone, acetylsalicylic acid, salicylic acid, tolfenamic acid, diclofenac, and nimesulide—chosen as being the most representative of the different chemical sub-classes of NSAID. The BSA columns could bind up to 150 × 10^–6 g total of a mixture of these nine NSAID, with mean recoveries ranging from 74.0% (tolfenamic acid) to 96.0% (nimesulide). HPLC separation on an RP C₁₈ column with a linear gradient enabled resolution of the drugs; identification was achieved by coupling with photodiode array detection (DAD) operated at 240 and 280 nm. Results showed that all the compounds except acetylsalicylic acid were bound effectively by the BSA affinity columns. When plasma samples were spiked at 2.5 g mL^–1 with a mixture of the NSAID the chromatographic profile and UV spectra recorded (in the range 220–350 nm) indicated the procedure was sufficiently selective for identification of the drugs at the concentrations expected according to their pharmacokinetics. No memory effects and matrix interferences were observed.Revised: 18 December 2003 and 16 April 2004     

High-throughput analytical strategy with combined planar and column liquid chromatography for improvement of the poppy (Papaver somniferum L.) with a high alkaloid content

Summary Poppy capsules with a high alkaloid content are of great importance to the pharmaceutical industry, because approximately 35 000 tons of straw (capsule with short stem), 350 tons of straw concentrate, and 1000 tons of opium are used annually for extraction of morphinane alkaloids. The combined use of four different liquid chromatographic methods is proposed for determination of alkaloid content. In the first, semi-quantitative, method screening is performed by multilayer overpressured-layer chromatography (MLOPLC) in which 142 samples are separated on silica as stationary phase within 5 s per sample. If the morphine content is >1.3% it is then measured quantitatively by NPHPTLC (normal-phase high-performance thin-layer chromatography) combined with densitometric evaluation. A second, quantitative, determination is always performed by means of a rapid RPHPLC (reversed-phase high-performance liquid chromatography) method in which the separation time is less than 4 min per sample. If the difference between the alkaloid content measured by use of the last two methods is >12% a confirmatory RPHPLC method with increased selectivity and analysis time (15 min) must be used. The high throughput strategy presented has been used for analysis of ca. 15 000 samples per year, per genotype. Systematic selection, cross-breeding, and this analytical strategy with combined planar and column liquid chromatographic methods resulted in the discovery of two new candidate cultivars with high morphine (ca. 2%) and thebaine (ca. 1.5%) content. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

The Use of Proton Nuclear Magnetic Resonance Spectroscopy for the Determin- ation of pK a Values in Aqueous/Organic Solutions for Basic Analytes Employed in Column Characterisation Protocols

Proton nuclear magnetic spectroscopy has been used to measure pK _a values of two basic analytes in a range of aqueous/organic eluents. The results support the hypothesis that the poor correlation in terms of ion exchange capacity with values greater than pH 7 may be attributed to the difference in the ionisation of the silica surface and not differences in ionisation of the basic analytes under the differing chromatographic conditions (i.e. temperature, type and amount of modifier) employed in two commonly used HPLC stationary phase characterisation procedures (Tanaka and Snyder).     

Classification of structurally related compounds fromAstragalus extract by correlation of the logk w andS

Summary The linear relationship between logk _w andS derived from molecular interactions and statistical thermodynamics was investigated by four series of different polar probe solutes. For each series of similar polar solutes, structurally related compounds with similar dipolarity/polarizability and hydrogen bonding energy, a linear relationship between logk _w andS was obtained. The more similar the solutes, the greater were the regression coefficients obtained. For two series of solutes with different strong polar groups resulting in different dipolarity/polarizability and hydrogen bonding energy, two parallel lines were observed in the logk _w-S plots. Each line represented one series of compounds and the distance between them indicated the difference in the dipolarity/polarizability and hydrogen bonding energy. Based on the parallel lines implying information on structurally related compounds in the logk _w-S plot, a method of classification of structurally related compounds was put forward and the linear logk _w-S correlation for unknown components in nonaqueous RP-HPLC analysis ofAstragalus extract with isopropanol-methanol mobile phase was studied. Two nearly parallel lines were obtained in the logk _w-S plot and two series of structurally related compounds were classified in this way.     

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%).