Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method

The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.     

Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS

The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as ‘uña de gato’, have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC‐ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on ?ow injection ES/MS, and a more complete analytical technique using HPLC‐MS. Copyright © 2004 John Wiley & Sons, Ltd.     

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.Abbreviations ACC 1-Aminocyclopropane-1-carbonic acid - EDTA Ethylenediaminetetraacetic acid - LS-medium Linsmaier and Skoog medium - Pmg Phytophthora megasperma     

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content.     

Simultaneous determination of four sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. extracts by high-performance liquid chromatography

Wilfortrine, wilfordine, wilforgine and wilforine are four major bioactive sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. The first analytical determination of the four major bioactive alkaloids is described. The four alkaloids are well-resolved within 15 min using the developed HPLC method. The identity of the analytes was confirmed by an HPLC-MS experiment, with all compounds being clearly assignable by atmospheric pressure chemical ionization (APCI) positive mode analysis. The method was validated for limit of qualification, linearity and inter-day variation of precision and accuracy. Seven T. wilfordii samples (extracts and commercial product) were successfully analysed.     

Supercritical carbon dioxide extraction of colchicine and related alkaloids from seeds of Colchicum autumnale L

A method for the extraction of the alkaloids colchicine, 3-demethylcolchicine and colchicoside from seeds of Colchicum autumnale by supercritical carbon dioxide has been established. Several parameters such as pressure, temperature, percentage of modifier and extraction time have been examined. Two extraction steps with constant carbon dioxide density (0.90 g/mL) and flux (1.5 mL/min) were required to extract the alkaloids in 110 min using 3% methanol as modifier. The quantitative determination of the alkaloids was performed by HPLC; the percentages of recovery were higher than 98% for the three alkaloids. This extraction procedure was compared with a conventional method involving maceration and sonication, and the same levels of alkaloids were obtained in each case. The supercritical carbon dioxide method is, however, very efficient, more rapid and more environmentally friendly than conventional methods.     

Determination of deracoxib in feline plasma samples using high performance liquid chromatography

A new HPLC procedure for the determination of deracoxib, a selective cyclooxygenase-2 inhibitor, has been developed and validated. Following a liquid-liquid extraction using isopropyl alcohol and chloroform, samples were separated by isocratic reversed-phase HPLC on an Atlantis C18 column and quantified using UV detection at 252 nm. The mobile phase was a mixture of 10 mM potassium phosphate (pH 4.5) and acetonitrile, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 10-1500 ng/ml. The development of the assay allowed the determination of pharmacokinetic parameters after oral administration of deracoxib in cats and would be suitable for other pharmacokinetic studies.     

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.     

白毛茛中生物碱的提取和HPLC分析

白毛茛根粉末经室温萃取后用HPLC分析其中的生物碱成分,色谱柱为Zorbax Eclipse XDBC18快速分离,该方法可准确测定白毛茛中主要的生物碱,包括小檗碱（berberine）和白毛莨碱（hydrastine）。萃取和HPLC分析还可用于其他几种生物碱的测定,包括氢化小蘖碱（canadine）、白毛莨分碱（hydrastinine）和巴马亭（palmatine）,也可以应用于其它含小檗碱的植物根。Eclipse XDBC18快速分离柱采用等梯度分离,所有组分在15min内获得了高的分离度。     

Quantitative analysis by HPLC-MS2 of the pyrrolizidine alkaloid adonifoline in Senecio scandens

A quantitative method using HPLC-MS(2) has been developed for the determination of adonifoline, one of the retronecine-type hepatotoxic pyrrolizidine alkaloids in Senecio scandens Buch.-Ham. ex D. Don., a traditional Chinese herb. Using an orthogonal design test, a simple and rapid sample extraction method was developed. HPLC analysis was conducted using a C(18) column as stationary phase and a mixture of acetonitrile and aqueous formic acid as mobile phase. Good linearity for adonifoline was found in the concentration range 0.12-4.18 microg/mL, and the HPLC-MS/MS method was shown to be appropriate, in terms of sensitivity, precision and reproducibility. The quantities of adonifoline in extracts of 18 plant samples from different collection sources and from different parts (flowers, leaves, thick stems, slim stems and roots) of S. scandens were determined using the newly developed HPLC/MS(2) analysis.     

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

Analysis of Catharanthus roseus alkaloids by HPLC

Catharanthus roseus is a medicinal plant from which secondary metabolites used in chemotherapy to treat diverse cancers are extracted. The well known high value metabolites vincristine and vinblastine are just 2 of 130 alkaloids that can be found in C. roseus. However, only few (∼11) of this high number of chemical entities are frequently analyzed and even fewer (∼8) are available commercially. For more than 30 years, different analytical techniques have been developed to isolate and identify C. roseus metabolites, and then allowing revealing the therapeutic potential of C. roseus metabolites. Among few approaches, high performance liquid chromatography (HPLC) technique is still widely used for the separation and analysis of secondary metabolites such as those from C. roseus. This article thus reviews the most recent developments in HPLC analysis of alkaloids from C. roseus. Diverse considerations that are crucial to the efficiency of secondary metabolites separation and identification steps, such as biomass manipulation, extraction phase and protocols, HPLC separation and analysis protocols are reviewed in details. Examples of spectra obtained using the most common detectors are also shown and suggestions are made on how to proceed in developing efficient separation and identification methods at the analytical and semi-preparative scales.     

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambda(ex)=330 nm, lambda(em)=460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were 相似文献   

Analysis of goldenseal, Hydrastis canadensis L., and related alkaloids in urine using HPLC with UV detection

A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl(3):2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C(18) (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r(2)=0.9990) and hydrastine (r(2)=0.9983) over a range of 11.80 ng/mL to 17.64 μg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.     

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.     

Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

Summary Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g^–1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g^–1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l^–1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O₂ l^–1 h^–1 and 0.95 mmol CO₂ l^–1 h^–1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g^–1 dw) by day 15.     

Determination of methotrexate, several pteridines, and creatinine in human urine, previous oxidation with potassium permanganate, using HPLC with photometric and fluorimetric serial detection

A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10(-3) M KMnO4 (pH 5.0) and 35min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris-HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at lambda(em) = 444 nm and lambda(ex) = 280 nm and creatinine was monitored by absorption measurements at lambda(abs) = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml(-1) for MTX, less than 1 ng ml(-1) for all of the pteridines, and 4 microg ml(-1) for CREA.     

An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

Simultaneous determination of OSI-774 and its major metabolite OSI-420 in human plasma by using HPLC with UV detection

A new method was developed and validated for quantitating OSI-774 and its metabolite OSI-420 in human plasma. Sample preparation involved initial extraction with methyl t-butyl ether followed by back extraction with HCl and re-extraction with methyl t-butyl ether. This extraction process resulted in significant improvement in the specificity, reproducibility and sensitivity. The analytes were separated on a Water Symmetry C18 analytical column and the mobile phase consisted of acetonitrile-0.05 M potassium phosphate buffer (42:58, v/v) (pH 4.8), and monitored at a wavelength 345 nm. Values of between- and within-day precision and accuracy for both OSI-774 and OSI-420 were <20%. This method was successfully applied to study steady-state pharmacokinetics of OSI-774 and OSI-420 in a phase II clinical trial.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.