A karyological study of some Dianthus L. species (Caryophyllaceae) in Northeast of Iran

This study focuses on karyological investigation of four taxa of Dianthus (Caryophyllaceae) including D. crossopetalus, D. orientalis subsp. stenocalyx, D. orientalis subsp. gorganicus, and Dianthus sp. distributed in Northeast of Iran. The karyotype asymmetry/symmetry was evaluated using two methods: 1) CVCL (coefficient of variation of chromosome length) and CVCI (heterogeneity of the centromeric index); and 2) MCA (mean centromeric asymmetry). The karyotype asymmetry was also used to investigate the relationships among the taxa. Results obtained from the current study revealed that there are two different ploidy levels (2n = 2x = 30 and 2n = 4x = 60) among the investigated taxa. The indices CVCL and MCA described karyotype asymmetry correctly based on variation in chromosome length. Diagram of CVCL vs. MCA seems to be appropriate for karyological delimitation and taxonomic relationships among the Dianthus taxa under study. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantification of nepetalactones in catnip (Nepeta cataria L.) by HPLC coupled with ultraviolet and mass spectrometric detection

Nepetalactones, the major chemical components of catnip (Nepeta cataria L.), were analysed by reversed-phase HPLC coupled with UV and MS detection. Two major nepetalactones, Z,E-nepetalactone and E,Z-nepetalactone, were successfully identified and quantified. The linearity range for Z,E-nepetalactone was determined as 0.00655-0.655 mg/mL with a correlation coefficient of 0.9999, and the linearity range of E,Z-nepetalactone was found to be 0.00228-0.456 mg/mL with a correlation coefficient of 0.9999, under UV detection at 228 nm. The linearity ranges were from 0.00164 to 0.0328 mg/mL, with a correlation coefficient of 0.9999, for ZE-nepetalactone and 0.00114-0.0228 mg/mL, with a correlation coefficient of 0.9999, for E,Z-nepetalactone by MS detection with selected ion monitoring of ion peak m/z 167. The MS detection was found to be more sensitive than UV detection and this method was validated as simple, reliable and sensitive for catnip nepetalactone analysis. This method can be used for identification and fingerprinting of catnip products.     

Simultaneous quantification of five triterpenoid saponins in Clematis L. spp. by high-performance liquid chromatography with evaporative light scattering detection

A simple and accurate method involving high-performance liquid chromatography with evaporative light scattering detection was developed for the simultaneous determination of five triterpenoid saponin components in Clematis L. spp. for the first time. The analysis was performed on a Zorbax SB-C(18) column and gradient elution with acetonitrile and water with 0.1% formic acid was utilised. All the calibration curves exhibited good linear characteristics with correlation coefficients in the range from 0.9979 to 0.9997. The limits of detection and limits of quantification were less than 0.152 and 0.506 microg, respectively. The overall recoveries for the five analytes were between 91.3 and 99.5%. A total of 10 samples from Clematis L. spp. were analysed under optimised conditions and the chemical profiles provided information for the identification of botanical origin, the development of new medicinal resources and chemotaxonomic investigation.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of colchicine in Colchicum steveni and C. hierosolymitanum (Colchicaceae): comparison between two analytical methods

The amounts of colchicine present in two Jordanian species of Colchicum, namely, C. steveni Kunth and C. hierosolymitanum Feibrun (Colchicaceae), have been determined. An HPLC-UV (photodiode array) method employing gradient elution was developed and the results compared with those obtained using a simple TLC-spectrophotometric method. The levels of colchicine as measured by these methods were not significantly different (p < 0.05) indicating that the spectrophotometric method is an acceptable alternative to HPLC. With respect to C. steveni, the leaves contained the largest amount of colchicine (0.204/100 g), whilst in C. hierosolymitanum corms showed the highest colchicine content (0.126/100 g). As a source of colchicine, the two investigated species showed levels comparable with those found in C. autumnale, the traditional source of colchicine.     

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection

An analytical procedure was developed for the simultaneous determination of total hypericin (protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.     

Practical enantioresolution of alcohols with 2-methoxy-2-(1-naphthyl)propionic acid and determination of their absolute configurations by the (1)H NMR anisotropy method.

The enantioresolution of racemic alcohols as esters of 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1) and the determination of their absolute configurations on the basis of (1)H NMR anisotropy effect are described. The enantiopure MalphaNP acid (S)-(+)-1 was allowed to react with racemic 2-alkanols and 1-octyn-3-ol, yielding diastereomeric mixtures of esters, which were easily separated by HPLC on silica gel. To determine the absolute configurations of the first-eluted diastereomeric esters by the (1)H NMR anisotropy method, the general scheme was proposed. Separated esters were reduced with LiAlH(4) or hydrolyzed with KOH/EtOH to recover enantiopure alcohols.     

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MS(n). In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol. Furthermore, the experimental results demonstrate that the stability of DDA depends on pH values of the buffer. Aconine as hydrolysate has been only found in pH 10.0 buffer, and the other hydrolysates and the pyrolyzates of DDA, such as benzoylaconine and deacetoxy aconitine, have been observed in all pH aqueous solutions. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine.     

Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe‐emodine,emodine, rheine,chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae)

Introduction – Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the flora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of different methods of extraction. Objective – After a partial validation of an HPLC method for the simultaneous determination of five anthraquinones, aloe‐emodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three different methods of extraction. Methodology – Anthraquinones were extracted from the bark of R. alpinus using different techniques (methanol maceration, ultrasonic and supercritical CO₂ extraction). Separation and quantification of anthraquinones were accomplished using a reversed‐phase C₁₈ column with the mobile phase of H₂O–methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm. Results – All calibration curves were linear over the concentration range tested (10–200 mM) with the determination coefficients ≥0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All five anthraquinones were found in the samples tested at concentrations reported in experimental data. Conclusion – The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantification of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays. Copyright © 2009 John Wiley & Sons, Ltd.     

Contribution of chiral HPLC in tandem with polarimetric detection in the determination of absolute configuration by chemical interconversion method: Example in 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomer series

We report the determination of the absolute configuration of eight stereoisomers in the series of chiral 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomers 1-3, from the known absolute configuration of one stereoisomer, determined by X-ray crystallography. The method uses the affiliation between signs of rotation of polarised light during chemical transformations which preserve the absolute configuration and also during rotation around a single pivot bond producing a compound of known configuration. The use of chiral HPLC in tandem with a chirality detector gives a decisive advantage since such correlation can be performed on a mixture of a very limited quantity of compounds, without tedious purification steps. The method shown as an example in this article, which uses chiral HPLC with chirality detection, may prove useful in many other cases where the determination of the absolute configuration is necessary and where a chemical interconversion method can be used on a microscale.