Analysis of atropine, its degradation products and related substances of natural origin by means of reversed-phase high-performance liquid chromatography

Chromatographic separation and quantification methods of tropa alkaloids were often described. In order to separate atropine from its degradation products ion-pair chromatography (IPC) has been frequently applied. Beside long equilibration times IPC often suffers from poor robustness. The aim of this study was to develop robust and simple HPLC methods for both stability testing of atropine solutions and limitation of related substances in atropine from plant material. Using a hydrophilic embedded RP18 column and a gradient elution gave baseline separation of all components.     

High-performance purification of quaternary alkaloids from Corydalis yanhusuo W. T. Wang using a new polar-copolymerized stationary phase

An efficient method for purification of alkaloids from Corydalis yanhusuo W. T. Wang using HPLC was developed, featuring a polar-copolymerized stationary phase named C18HCE. As ionizable solutes, the crude alkaloid sample often suffered from serious peak tailing problem on conventional RP-LC columns, and the separation would rapidly became destroyed with the increasing of load amount. However, on the new stationary phase, good peak shapes (asymmetry factor <1.5) as well as good loadability were easily obtained in a commonly used acidic mobile phase condition. The loading amount could reach 10 mg per injection on an analytical C18HCE column for laboratory-scale purification. About 6.8 mg of palmatine (HPLC purity >98%) and 44.4 mg of dehydrocorydaline (HPLC purity >98%) were rapidly derived from 200 mg of the crude alkaloid sample, and the recoveries of these two compounds were 76.5 and 81.7%, respectively. The purified alkaloids were characterized by comparing retention times with standard compounds as well as (1)H-NMR data. The new method is simple and high yielding, and it may provide a promising tool for purification of alkaloids as well as other alkaline compounds.     

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF–MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Preparative isolation and purification of alkaloids from the Chinese medicinal herb Evodia rutaecarpa (Juss.) Benth by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water system (5:5:7:5, v/v) was applied to the isolation and purification of alkaloids from the Chinese medicinal plant Evodia rutaecarpa (Juss.) Benth. Five kinds of alkaloids were obtained and yielded 28 mg of evodiamine (I), 19 mg of rutaecarpine (II), 21 mg of evocarpine (III), 16mg of 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone (IV), 12 mg of 1-methyl-2-dodecyl-4-(1H)-quinolone (V) from 180 mg of crude extract in a one-step separation, with the purity of 98.7%, 98.4%, 96.9%, 98.0%, 97.2%, respectively, as determined by high performance liquid chromatography (HPLC). The structures of these compounds were identified by 1H NMR and 13CNMR.     

Orthogonal separations of nicotine and nicotine-related alkaloids by various capillary electrophoretic modes

The migration behaviour of nicotine and related tobacco alkaloids was investigated using three different capillary electrophoretic (CE) modes. Novel separations were achieved both using microemulsion electrokinetic chromatography (MEEKC) and nonaqueous CE (NACE). Improved resolution compared to previous studies was obtained using free-solution CE (FSCE). Each technique resulted in different, orthogonal separation selectivity. The suitability of each method for application to the analysis of nicotine lozenges is discussed. The FSCE method was applied to the analysis of nicotine lozenges due to its compatibility with an established lozenge extraction solvent. The method used gave good injection precision and linearity. Good agreement of CE and high-performance liquid chromatography (HPLC) results was obtained. The CE method is therefore considered suitable for the quantitative determination of nicotine in nicotine lozenges.     

Fragmentation pathways of heroin-related alkaloids revealed by ion trap and quadrupole time-of-flight tandem mass spectrometry

The electrospray ionization (ESI) ion trap and quadrupole time-of-flight (QqToF) mass spectra of heroin and seven related alkaloids, i.e., morphine, codeine, O-6-monoacetylmorphine (6-MAM), thebaine, acetylcodeine, papaverine and narcotine, have been extensively investigated in this work. The ESI mass spectrometric fragmentation pathways of protonated 6-MAM, heroin, acetylcodeine, and thebaine were comprehensively elucidated for the first time with the aid of high-resolution mass spectrometry. It was found that cleavage of the piperidine ring was the featured fragmentation route of six of the compounds, although not of papaverine and narcotine. In addition, a simple high-performance liquid chromatography (HPLC)-based separation method gave baseline resolution of all eight components. This study could play an important role in the screening for these alkaloids in different matrices by HPLC coupled to tandem mass spectrometry (MS/MS).     

Characterization and online detection of aromatic alkaloids in the ascidian Lissoclinum cf. badium by liquid chromatography/UV detection mass spectrometry

A detection method based on high-performance liquid chromatography coupled with ultraviolet diode-array detection and electrospray ionization ion trap tandem mass spectrometry (HPLC-UV-ESI-MS/MS) was developed to investigate the total alkaloids prepared from the ascidian Lissoclinum cf. badium. The aromatic alkaloids possessing polysulfide structures are the major bioactive constituents isolated from ascidians of the genera Lissoclinum, Eudistoma, and Polycitor. These compounds presented various important biological activities. The ESI-MS fragmentation behavior of this kind of alkaloids was studied, and the fragmentation was characterized by elimination of the NH(CH(3))(2) moiety. The use of reversed-phase HPLC/UV-ESI-MS allowed the online separation and detection of 25 aromatic alkaloids. This approach provided data that can be used for detection of biologically active aromatic alkaloids from marine organisms.     

HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.     

A (−)‐norephedrine‐based molecularly imprinted polymer for the solid‐phase extraction of psychoactive phenylpropylamino alkaloids from Khat (Catha edulis Vahl. Endl.) chewing leaves

A molecularly imprinted polymer (MIP) was prepared using (?)‐norephedrine as the template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross‐linker and chloroform as the porogen. The MIP was used as a selective sorbent in the molecularly imprinted solid‐phase extraction (MIP‐SPE) of the psychoactive phenylpropylamino alkaloids, norephedrine and its analogs, cathinone and cathine, from Khat (Catha edulis Vahl. Endl.) leaf extracts prior to HPLC‐DAD analysis. The MIP was able to selectively extract the alkaloids from the aqueous extracts of Khat. Loading, washing and elution of the alkaloids bound to the MIP were evaluated under different conditions. The clean baseline of the Khat extract obtained after MIP‐SPE confirmed that a selective and efficient sample clean‐up was achieved. Good recoveries (90.0–107%) and precision (RSDs 2.3–3.2%) were obtained in the validation of the MIP‐SPE‐HPLC procedure. The content of the three alkaloids in Khat samples determined after treatment with MIP‐SPE and a commercial Isolute C₁₈ (EC) SPE cartridge were in good agreement. These findings indicate that MIP‐SPE is a reliable method that can be used for sample pre‐treatment for the determination of Khat alkaloids in plant extracts or similar matrices and could be applicable in pharmaceutical, forensic and biomedical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography

An HPLC method is reported for the separation and quantification of five major polyphenolic groups found in fruits and related products: single ring phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid derivatives), flavan-3-ols, flavonols, anthocyanins, and dihydrochalcones. A binary mobile phase consisting of 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55) (solvent A) and acetonitrile (solvent B) was used. The use of sodium acetate was new and key to the near baseline separation of 25 phenolics commonly found in fruits. A photodiode array detector was used and data were collected at four wavelengths (280, 320, 360, and 520 nm). This method was sensitive and gave good separation of polyphenolics in apple, cherry, strawberry, blackberry, grape, apple juice, and a processing by-product. The improved separation has led to better understanding of the polyphenolic profiles of these fruits. Individual as well as total phenolic content was obtained, and the latter was close to and correlated well with that obtained by the Folin-Ciocalteu method (FC). The HPLC data can be used as a total phenolic index (TPI) for quantification of fruit phenolics, which is advantageous over the FC because it has more information on individual compounds.     

Simultaneous determination of seven alkaloids in Phellodendron chinense Schneid by high-performance liquid chromatography

By optimizing the extraction, separation, and analytical conditions, a reliable and accurate HPLC method coupled with a photodiode array detector was developed for simultaneous detection and quantification of seven alkaloids, i.e., (-)-(R)-platydesmin, noroxyhydrastinine, berberine, skimmianine, canthin-6-one, chilenine, and pteleine in "huangbo" (the bark of Phellodendron chinense), a commonly used herb in Traditional Chinese Medicine. The optimal condition for separation was achieved on a reversed-phase Cis column with a stepwise mobile phase gradient prepared from 0.1% phosphoric acid and acetonitrile. For all the alkaloids, a good linear regression relationship (r > 0.9997) was obtained between the peak area and concentration at the range of 0.5-700 microg/mL. The LODs and LOQs for the analytes ranged from 0.07 to 0.28 microg/mL and from 0.28 to 1.12 microg/mL, respectively. The optimized method was applied to the determination of alkaloids in several P. chinense samples, and found to be feasible and reliable. This method and quantitative results can provide scientific and technical bases for setting up QC standards to assure the safety and quality of P. chinense bark raw material, as well as for proprietary Chinese medicine products containing P. chinense bark.     

Chromatographic separation of benzo(a)pyrene isomers on nematic liquid crystal GC and polymeric reversed-phase HPLC phases

Summary The use of two nematic liquid crystals (BABT and BPhBT) as GC stationary phases for the separation of monohydroxybenzo(a)pyrenes. as their TMS ethers, and monomethylbenzo(a)pyrenes was developed and compared with the separation of these isomers by HPLC using a polymeric ODS reversed-phase column. It was found that while HPLC and GC gave comparable separation of the hydroxy isomers, 10 out of 12 separated, better separation of the methyl isomers was obtained using HPLC. A simultaneous use of both HPLC and GC would resolve the twelve hydroxy isomers in about 70min. The results indicated that HPLC, using polymeric reversed-phase columns, is as powerful a tool as GC using nematic liquid crystal phases, for the separation of benzo(a)pyrene isomers. A discussion of the effect of solute length-to-breadth ratio on elution order is presented.Presented in part at the 1981 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ; paper No. 51.     

Separation of drug substances by modern liquid chromatography on silver impregnated silica gels.

The use of silver halide impregnated supports for high-pressure liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analysis of drug substances has been studied. Successful separations of xanthines and mixtures containing barbiturates, xanthines, ergot alkaloids, and tropane alkaloids have been achieved with isocratic conditions or by using simple gradients. The Ag-supports show a similar behavior as many chemically bonded stationary phases, such as modification fo adsorption sites on silica gel to give lower retention but better specificity; rapid reconditioning in connection with gradient elution (3-5 minutes). Reasonable cost, simplicity of preparation and usage, and good chemical and physical stability render the silver halide phases applicable in routine analysis and quite competitive with many commercially available chemically bonded stationary phases.     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silicabased C₁₈ material proved superior to the polymer materials tested.     

HPLC测定植物性农药——0.25%乌头总碱乳油中的乌头类生物碱

 选择十八烷基键合相柱 ,以甲醇 水 氯仿 三乙胺 (体积比为 6 8∶32∶2∶0 1)混合溶液为流动相 ,用高效液相法测定了一种植物性农药 0 2 5 %乌头总碱乳油中的乌头生物碱。实验结果表明中乌头碱、乌头碱及次乌头碱与其他杂质能够得到很好的分离。以安宫黄体酮作内标物 ,用峰面积比测定各生物碱含量 ,在其线性范围内分析结果准确 ,回收率高 (>92 % ) ,重现性好 (RSD <3 2 % ) 。     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silica-based C₁₈ material proved superior to the polymer materials tested.     

Preparative isolation of guaipyridine sesquiterpene alkaloid from Artemisia rupestris L. flowers using high-speed counter-current chromatography

Although the medicinal plant Artemisia rupestris L. has been widely researched for several decades, its alkaloids have never been isolated before. To our surprise, the alkaloids in the plant were not detected in the stems but detected in the flowers. Herein, a novel and strange guaipyridine sesquiterpene alkaloid with a carboxyl group named rupestine was purified successfully from the total alkaloids extracted from the flowers by high-speed counter-current chromatography (HSCCC). The two-phase solvent system used was composed of ethyl acetate-methanol-water (8:1:7, v/v/v). Fifty six milligrams of rupestine was obtained at over 97% purity and 95% recovery from 200 mg of the total alkaloids in one-step separation. Its structure was elucidated by spectroscopic methods including high resolution ESI-MS, (1)H NMR, (13)C NMR, Heteronuclear Multiple Bond Correlation (HMBC), Heteronuclear Single Quantum Coherence (HSQC), and Nuclear Overhauser Enhancement Spectroscopy (NOESY).     

Characterization ofC-glycosylflavones fromDissotis rotundifolia by liquid chromatography — UV diode array detection — tandem mass spectrometry

Summary By high-performance liquid chromatography (HPLC) coupled with UV diode-array detection (DAD) and thermospray mass spectrometry (TSP-MS), four main constituents of a polar, whole plant extract fromDissotis rotundifolia T. were characterized. The fourC-glycosylflavones, isoorientin, orientin, vitexin and isovitexin were detected in the methanolic and hydroalcoholic extract of the plant as well as in the commercial drug preparation ‘Sirop de Dissotis’. Although the UV data and TSP mass spectra allowed rapid characterisation of all fourC-glycosylflavones, exact attribution of the peaks to their structures could not be achieved as neither the UV spectra nor the TSP mass spectra enabled differentiation of one position isomer from the other. Therefore a successful attempt was made to distinguish the 6-C from the 8-C-glycosylflavones by thermospray tandem mass spectrometry (TSP-MS-MS). The collision induced dissociation (CID) spectra of the particular ion [M+H-120]⁺ gave fragments which permitted differentiation of position isomers. To confirm the accuracy of on-line results, reference compounds were included in the HPLC study.