Analysis of polyamines in higher plants by high performance liquid chromatography

A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) through a 5-μm C₁₈ column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations.     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。     

Cytochalasin B: preparation, analysis in tissue extracts, and pharmacokinetics after intraperitoneal bolus administration in mice

Cytochalasin B (CB) was prepared by methanol extraction of dehydrated mold (Drechslera dematioidea) matte, reverse-phase C18 silica gel batch adsorption, selective elution with 1:1 (v/v) hexane:tetrahydrofuran (THF), crystallization, preparative TLC, and recrystallization. Unit gravity silica gel normal phase chromatography afforded additional CB. Yield per liter of medium was 300 mg of CB greater than 95% pure by NMR, HPLC (60:40 hexane:THF, Lichrosorb Si60 silica gel, 230 nm), and TLC. CB added exogenously to mouse organs at 1 and 5 micrograms/organ was recovered 70 to 100% by methanol extraction, adsorption to C18 silica gel Sep-Pak cartridges, elution with ethyl acetate, and analysis by TLC and/or HPLC. Limiting sensitivity (micrograms/extract) was 0.5 TLC; 1.0 HPLC. Quantitative extraction was confirmed with 3H-labeled CB. CB ip in mice at 50 mg/kg (LD10) distributed rapidly into liver, renal fat, kidney, intestines, mesentery, pancreas, spleen, and blood cells and was cleared from all but liver within 24 h. CB was below detectable levels in thymus, lymph nodes, heart, brain, bone marrow, and lungs. Cytochalasin A is fixed to tissues and not extractable. This work affords a source of CB in quantities permitting in vivo study, provides methods for extraction and analysis, and reveals the pharmacokinetics of ip bolus CB.     

Meetings & Symposia

Optimization of the extraction process of phenolics from Bryophyllum pinnatum was carried out using response-surface methodology (RSM). The effect of different variables such as ratio of solvents, plant material/solvent ratio, extraction time, and temperature were investigated. An optimal phenolics yield of 7.952 mg/g gallic acid equivalence (GAE) was achieved at reduced levels of methanol/water ratio (1:1, v/v). During optimization, the product yield was enhanced by ～2-fold at reduced extraction solvent (methanol/water) up to 37%. Validation of the RSM model for extraction of total phenolic content (TPC) was confirmed by high-performance liquid chromatography (HPLC) analysis. The obtained experimental values were in good agreement with the predicted values, thereby indicating the appropriateness of the model generated. Phenolic extracts from B. pinnatum were further examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods for determining the radical scavenging activities. EC₅₀ values of B. pinnatum extracts (BPEs) obtained by these methods were in accordance with the amount of phenolics present in the extract. Significant correlation was found between total phenolic content and antioxidant activities (p < 0.05).     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of colchicine in human plasma

A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050-10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.     

Enantioseparation of nine indanone and tetralone derivatives by HPLC using carboxymethyl‐β‐cyclodextrin as the mobile phase additive

High‐performance liquid chromatography (HPLC) is a powerful method in the area of chiral separation. In this study, a method of HPLC using carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) as chiral selector was developed for enantioseparation of nine indanone and tetralone derivatives. The separation was performed on a conventional C₁₈ column. The optimal mobile phase was a mixture of methanol and 0.05 mol/L phosphate buffer at pH 1.8 (55:45, v /v) containing 22.9 mmol/L CM‐β‐CD. Under such conditions, the resolutions of all analytes were over 1.8 except for Compound F. The results of the study indicate the presence of a complex with 1:1 stoichiometry of the inclusion complex. In addition, it can be inferred from thermodynamic analysis that the behavior of formation of the inclusion complex and enantioseparation occurred simultaneously, while they were driven by different forces. The effect of analyte structure is also discussed.     

2-Methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent for determination of phenols in high-performance liquid chromatography and application for determination of urinary phenol and p-cresol

Highly sensitive fluorescent labeling reagents, 2-(alkyloxy)-4-(2-phthalimidinyl)phenylsulfonyl chlorides (alkyloxy = methoxy, ethoxy, and propoxy; MPS-Cl, EPS-Cl, and PPS-Cl, respectively), for determination of phenols by high-performance liquid chromatography (HPLC) have been developed. These reagents react with phenols in basic medium to produce the corresponding fluorescent sulfonyl esters. The maximum fluorescence wavelengths of the derivatives were 308 nm for excitation and 410 nm for emission. The peaks due to phenol labeled with MPS-Cl, EPS-Cl, and PPS-Cl eluted at 6.3, 8.8, and 12.8 min, respectively, on a reversed-phase column with isocratic elution of methanol/water (2:1, v/v), and the detection limits (signal-to-noise = 3) of the derivatives were 10, 12, and 17 fmol per injection, respectively. Among these reagents, MPS-Cl was advantageous because its derivatives had shorter retention times and higher sensitivities in HPLC. The efficiency of converting phenol to the fluorescent derivative by MPS-Cl was about 100%. When MPS-Cl was applied to the determination of urinary phenol and p-cresol by HPLC using p-ethylphenol as an internal standard, the derivatives were separated at retention times of 6.3, 8.7, and 12.3 min, respectively, under the HPLC conditions described above. The concentrations of phenol and p-cresol in normal human urine were 11.9-293.5 and 8.2-346.1 nmol/mg creatinine, respectively.     

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS) after one single liquid—liquid extraction with chloroform—methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml–5 μg/ml with good accuracy and precision and is used in pharmacokinetic studies.     

Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection

A fast, cost-efficient, sensitive and accurate assay method for zearalenone in edible oils is described, as an alternative to gel permeation chromatography (GPC). Oil samples were extracted with an alkaline mixture of methanol and water (methanol +10 g/l aqueous ammonium carbaminate solution, pH 9; 9 + 1, v+v). The pH of the extract was neutralized with hydrochloric acid and then concentrated to dryness. The residue was dissolved with HPLC solvent, and zearalenone was determined by high-performance liquid chromatography with fluorometric detection (HPLC-FLD). The method was successfully validated for two matrices, maize oil and rapeseed oil. The recovery rate was 87%, and the coefficient of variation was 2.8% in a rapeseed oil sample contaminated with 27 μg zearalenone/kg. At a signal-to-noise ratio of 3:1, the method detection limit was 10 μg/kg, which was considered to be adequate in view of the present European Union maximum level of 400 μg/kg.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of Secondary Metabolites of Microscopic Fungi of the Genus Penicillium by Chromatographic Techniques

Combinations of various systems of thin-layer chromatography and high-performance liquid chromatography (HPLC) were efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems: (a) chloroform, methanol, and 25% NH₄OH (90 : 10 : 1, 90 : 10 : 0.1, or 80 : 20 : 0.2); (b) chloroform and acetone (9 : 1); and (c) ethyl acetate, methanol, and 25% NH₄OH (85 : 15 : 10); staining was performed using Ehrlich's reagent. Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS-2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C₁₈ column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H₂O–H]^?, m/z 331.1 for andrographolide and [M?H]^?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.     

High-pressure liquid chromatographic separation of a mixture of corticosteroids, androgens, and progestins

Fifteen steroids including corticosteroids, androgens, progestins, and their derivatives were completely separated by reverse-phase high-pressure liquid chromatography on a ChemcoPak 7 ODS-H column in 50 min. The elution procedures were first with water:methanol:acetonitrile:isopropanol 55:32:6.5:7.5 (v/v) for 15 min and followed with a linear gradient elution for 35 min from 0 to 80% of water:methanol:n-butanol 40:40:20 (v/v). The applicability of this method was successfully demonstrated in the analyses of the biological samples of carp plasma, testis, and head kidney.     

Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C₁₈ column and a Vydac C₁₈ column in series are used with an isocratic solvent system of acetonitrile–methanol containing 50 mM acetate ammonium–dichloromethane–water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5–8.3%) and inter-day (1.3–12.7%) coefficients of variation are suitable for routine clinical determinations.     

Simultaneous quantification of components of neoglycolipid-coated liposomes using high-performance liquid chromatography with evaporative light scattering detection

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and a neoglycolipid, mannopentaose-conjugated dipalmitoylphosphatidylethanolamine (Man5-DPPE), have been shown to have a strong adjuvant effect in inducing the antigen-specific cellular immunity. In this study, a rapid and simple analytical method using a HPLC system with an evaporative light scattering detector was developed for simultaneous quantification of the liposome components Man5-DPPE, cholesterol and DPPC. The chromatographic separation of these components was performed using a trimethylsilane column with an isocratic mobile phase of chloroform–methanol–water (1:33:6, v/v) after disrupting the liposomes with chloroform–methanol–water (10:10:3, v/v). This HPLC method provided sufficient reproducibility and linearity of calibration curves for the quantification of the liposome constituents. In addition, this method can be used for the quantification of various neoglycolipids with different carbohydrate structures.     

Biosynthesis,isolation, purification and seperation of Zearalenone,deoxynivalenol and 15-Acetyldeoxynivalenol

Fusarium graminearum KF-376 isolate was found to be able to form simultaneously three toxic metabolites: zearalenone (FF-2), deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-AcDON). Toxins were extracted with methanol — water 3:1 (v/v) and purified by liquid chromatography on charcoal — Kieselgel 60 column (preliminary) and Aluminiumoxid 90 column. Final separation of the metabolites was achived on Kieselgel 60 — Aluminiumoxid 90 column.     

Simultaneous measurement of pazufloxacin,ciprofloxacin, and levofloxacin in human serum by high-performance liquid chromatography with fluorescence detection

In this study, three fluoroquinolones, pazufloxacin, ciprofloxacin and levofloxacin, were simultaneously determined in spiked human serum by high-performance liquid chromatography (HPLC) method with fluorescence detection. Chromatography was performed using a C₈ column with an isocratic mobile phase consisting of 1% triethylamine (pH 3.0)/acetonitrile (86/14, v/v). Protein precipitation was conducted using perchloric acid and methanol. The calibration curves for the three fluoroquinolones were linear over concentrations ranging from 0.1 to 20.0 μg/mL. The within-day and between-day coefficients of variation obtained from three fluoroquinolones were less than 7%, and relative errors ranged from −1.6% to 9.3%. Mean recoveries of pazufloxacin, ciprofloxacin, and levofloxacin from spiked human serum were 97%, 88%, and 90%, respectively. The proposed method proved to be simple and reliable for the determination of three fluoroquinolones.     

A low molecular weight substance purified from human placenta inhibits cAMP-dependent protein kinase and activates protein kinase C

We have purified from human placenta a low molecular mass substance that inhibits cAMP-dependent protein kinase and activates protein kinase C. This protein kinase regulator was purified in three steps: (1) homogenizing placentas in chloroform/methanol and extracting the regulator into water; (2) eluting a strong anion exchange high performance liquid chromatography (HPLC) column with a quaternary gradient; and (3) eluting a reversed-phase HPLC column with a binary gradient. The regulator was found to be highly purified by HPLC, thin-layer chromatography (TLC) and laser desorption ionization mass spectrometry with a molecular mass of 703 Daltons by the latter procedure. The physical and biochemical properties of this protein kinase regulator suggest that it is a phospholipid but it did not co-elute by HPLC or by TLC with any of the known phospholipid activators of protein kinase C.     

Production of indole-3-acetic acid by several wild-type strains of Ustilago maydis

Indole-3-acetic acid (IAA) was identified and quantitated in spent media from cultures of ten Ustilago maydis strains. IAA was identified by thin-layer chromatography, high performance liquid chromatography (HPLC) and u.v. spectroscopy, and was quantitated by HPLC. All strains produced IAA in a tryptophan (Trp)-supplemented minimal medium at levels of 0.1 to 4.0 g IAA/ml of spent medium as assessed by HPLC. The highest levels of IAA were found in strains I2 and P2. The latter was also capable of producing IAA without addition of Trp to the medium.