Chiral characterization of deprenyl-N-oxide and other deprenyl metabolites by capillary electrophoresis using a dual cyclodextrin system in rat urine

A chiral capillary electrophoresis method has been developed for the simultaneous separation of the enantiomers of deprenyl and eight of its metabolites, among them the recently described metabolite deprenyl-N-oxide. Although heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) was suitable for the enantioresolution of deprenyl and its dealkylated derivatives, the enantiomers of deprenyl-N-oxide were just partly resolved. Carboxymethyl-beta-cyclodextrin (CMBCD) in as low as 2 mM concentration was capable of the enantiomer separation of all the nine examined compounds, however co-migration of 1R,2S-(-)-norephedrine and 1R,2R-(-)-pseudoephedrine, as well as 1S,2R-(+)-ephedrine and R-(-)-amphetamine was observed. This problem could be overcome by the use of a dual cyclodextrin system containing 4 mM DIMEB in addition to 2 mM CMBCD; simultaneous separation of all the compounds could be achieved. The optimized method was used for the analysis of rat urine samples after 10 days of treatment of animals with either R-(-)- or S-(+)-deprenyl. The stereospecific biotransformation of both deprenyl enantiomers was confirmed, and the stereoselectivity of N-oxide formation was demonstrated.     

Simultaneous determination of two active ingredients in Flos daturae by capillary electrophoresis with electrochemiluminescence detection

The coupling of Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the simultaneous determination of the two major active ingredients (atropine and scopolamine) in Flos daturae. Parameters related to the separation and detection were discussed and optimized. It was proved that 20 mM phosphate buffer at pH 8.48 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.2 V. Under the optimized conditions: ECL detection at 1.2 V, 20 mM phosphate buffer at pH 8.48, 5 mM Ru(bpy)₃²⁺ and 50 mM phosphate buffer at pH 7.48 in the detection reservoir, detection limits of 5 × 10⁻⁸ mol/l for atropine and 1 × 10⁻⁶ mol/l for scopolamine were obtained. Relative standard derivations of the ECL intensity and the migration time were 5.16 and 0.71% for atropine and 5.07 and 1.22% for scopolamine, respectively. Developed method was successfully applied to determine the amounts of both alkaloids in Flos daturae. A baseline separation for atropine and scopolamine was achieved within 11 min.     

Separation of homologues and isomers of linear alkylbenzenesulfonates by capillary electrophoresis with sodium dodecyl sulfate,carboxylic acids and bile salts

The ability of several anionic compounds, including carboxylic and dicarboxylic acids, sodium dodecyl sulfate (SDS), and sodium deoxycholate (SDC) and other bile salts, to separate the C(10)-C(13) homologues and the corresponding 20 positional isomers of linear alkylbenzenesulfonates (LAS) by capillary electrophoresis was studied. Up to 19 peaks and a shoulder were observed with a background electrolyte (BGE) containing 10 mM phosphate (pH 6.8), 30% acetonitrile and 40 mM SDS, and 18 peaks were obtained with a BGE containing 10 mM borate (pH 9), 40% ethanol and 40 mM palmitic acid (PA). Resolution increased with the alkyl chain length of the carboxylic acid. Dicarboxylic acids with a short alkyl chain, as azelaic acid, were useful to separate the homologues without distinguishing between the isomers. Up to 16 peaks and a shoulder were distinguished with SDC. Resolution decreased with the other bile salts. The 6-C(11)/5-C(11) isomer pair was better resolved with SDC than with SDS, and the 2-C(12) isomer was isolated using both PA and SDC, but not with SDS. Only the 7-C(13)/6-C(13) pair could not be resolved with any of the discriminating agents used.     

Determination of atropine in biological specimens by high-performance liquid chromatography

A sensitive and selective method for the determination of atropine in biological specimens has been developed. Samples alkalinized with sodium hydroxide were extracted with dichloromethane, and the organic phase was evaporated in a water-bath at 50 degrees C for ca. 10 min. The residue was dissolved in the mobile phase and injected into a reversed-phase column (TSK gel ODS-120A). The retention time for atropine could be varied by changing either the acetonitrile-water ratio in the mobile phase or the pH of the mobile phase. Acetonitrile-water (2:8, v/v) containing 6 mM phosphoric acid was used as mobile phase. Samples of 200 microliters or less were injected into the chromatography and measured at 215 nm. The recoveries of atropine added to drug-free specimens were satisfactory with coefficients of variation of 4% or less. Ninety-two compounds tested did not interfere with the assay of atropine. The method has been applied for monitoring atropine concentrations in cases of organophosphate and drug poisoning.     

Enantioseparation of atropine by capillary electrophoresis using sulfated beta-cyclodextrin: application to a plant extract

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.     

Analysis of choline and atropine in hairy root cultures of Cannabis sativa L. by capillary electrophoresis-electrospray mass spectrometry

We describe a capillary zone electrophoresis method coupled to electrospray ionization (ion trap) mass spectrometry (CZE-ESI-MS) for the identification and determination of choline and atropine compounds in hairy root extracts from Cannabis sativa L. Fused-silica capillary and an alkaline solution of 20 mM ammonium acetate at pH 8.5 are used being the most suitable for the analysis of choline and atropine in less than 10 min. Under the optimized conditions, including CE and ESI-MS parameters, the method resolved both compounds with very high sensitivity. The system exhibited good linear response in the range of 25-500 mg/L and 500-1000 microg/L for choline and atropine, respectively. The detection limit of choline was 18 mg/L and 320 microg/L for atropine. Finally, the developed method was applied to the analysis of these compounds in transgenic root cultures of Cannabis sativa L.     

用两种相似多糖类手性固定相拆分阿托品外消旋对映体的比较

分别涂敷纤维素-三（3，5-二甲基苯基氨基甲酸酯）（CDMPC）与直链淀粉-三（3，5-二甲基苯基氰基甲酸酯）（ADMPC）于自制的球形氨丙基硅胶上，制备了两种多糖类手性崮定相。用高效液相色谱法（HPLC）在正相条件下，用两种固定相直接拆分了阿托品（atmpine）外消旋体、在正己烷中加入了不同的醇类改性剂对阿托品进行拆分，并优化了流动相中醇类改性剂的比例：结果发现，阿托品在CDMPC固定相上可以得到基线拆分，而在ADMPC固定相上只能得到部分拆分。     

Chiral separation of anticholinergic drug enantiomers in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoretic (NACE) method for the separation of nine structurally similar chiral anticholinergic drugs was developed. The eight drug enantiomers were separated on baseline within 18 min using 20mM phosphoric acid and 10 mM NaOH, containing 10 mM heptakis(2,3-dimethyl-6-sulfato)-4beta-cyclodextrin (HDMS-beta-CD) in methanol. The results were compared with those obtained in the high performance liquid chromatography system.     

Determination of sequestering agents in cosmetics and synthetic detergents by high-performance liquid chromatography with ultraviolet detection

Using a fast reversible reaction of aminopolycarboxylic acids (APCAs) into Fe(III)-APCA complexes in the presence of Fe(III) ions, seven kinds of APCAs [nitrilotriacetate (NTA), N-(2-hydroxyethyl)ethylenediamine-triacetate (HEDTA), ethylenediamine-tetraacetate (EDTA), 1,3-propanediamine-tetraacetate (PDTA), diethylenetriamine-pentaacetate (DTPA), 1,2-diaminopropane-tetraacetate (MeEDTA), and O,O'-bis(2-aminoethyl)ethyleneglycol-tetraacetate (GEDTA)] in cosmetics and synthetic detergents were separated on two reversed-phase C30 columns connected in series and detected with ultraviolet detection. Simple pretreatment, consisted of thousand times dilution of samples and addition of 100 microl of the Fe(III) solution containing 10 mM Fe(III) chloride and 0.5 M sulfuric acid to 10 ml of diluted samples, permitted the determination of APCAs in cosmetics and synthetic detergents at concentration level of 0.1 mM, except 0.3 mM for GEDTA. APCAs except GEDTA could be detected at concentration level of 0.03 mM and GEDTA could be detected at concentration level of 0.09 mM. Good recoveries (95-110%) were obtained for each APCA by the standard addition method on two diluted samples with high accuracy (RSD 0.2-9.1%). Three APCAs (EDTA, HEDTA and NTA) were detected in various concentrations in cosmetics and synthetic detergents and the other APCAs were not detected in any of the samples. This method requires no tedious pretreatment and takes only 15 min for one analysis, so it is useful for determination of APCAs.     

Simultaneous determination of atropine,anisodamine, and scopolamine in plant extract by nonaqueous capillary electrophoresis coupled with electrochemiluminescence and electrochemistry dual detection

A rapid and simple method was demonstrated for the analysis of atropine, anisodamine, and scopolamine by nonaqueous capillary electrophoresis (NACE) coupled with electrochemiluminescence (ECL) and electrochemistry (EC) dual detection. The mixture of acetonitrile (ACN) and 2-propanol containing 1 M acetic acid (HAc), 20 mM sodium acetate (NaAc), and 2.5 mM tetrabutylammonium perchlorate (TBAP) was used as the electrophoretic buffer. Although a short capillary of 18 cm was used, the decoupler was not needed and the separation efficiency was good. The linear ranges of atropine, anisodamine, and scopolamine were 0.5–50, 5–2000, and 50–2000 μM, respectively. For six replicate measurements of 100 μM scopolamine, 15 μM atropine, and 200 μM anisodamine, the RSDs of ECL intensity, EC current, and migration time were less than 3.6%, 4.5%, and 0.3%, respectively. In addition, because the organic buffer was used, the working electrode (Pt) was not easily fouled and did not need reactivation. The method was also applied for the determination of these three alkaloids in Flos daturae extract.     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.     

Synthesis and Resolution of 10, 11-Dihydro- 10-hydroxycyproheptadine

10, 11-Dihydro-10-hydroxycyproheptadine was synthesized in a new and facile synthetic route and resolved by L-(-)-2-(1, 3)-dioxo-1, 3-dihydroisoindol-2-yl)-propionic acid and D-( )-tartaric acid, respectively. The free base of the two enantiomers has the same absolute specific rotation value.     

Determination of RS,E/Z-tocotrienols by HPLC

Synthetic alpha-tocotrienol was separated into four geometrical E/Z side chain isomers by preparative HPLC (permethylated beta-cyclodextrin phase). The isolated isomers were resolved in ethylene glycol dimethyl ether, converted into the corresponding methyl ether using dimethyl sulfate, and the tocotrienol methyl ethers were extracted with n-hexane. A subsequent HPLC separation on a chiral phase (adsorbent cellulose derivated with 3,5-dimethyl phenyl carbamate) discriminates between the enantiomers of each E/Z side chain isomer, achieving the complete resolution of the eight occurring synthetic RS,E/Z-alpha-tocotrienols. The method can be shortened by omitting the preparative separation of the E/Z tocotrienol isomers prior to the chromatography on the chiral dimethyl phenyl carbamate phase. The simplified method achieved the following separation: RS,E/Z-alpha-tocotrienol separated into five peaks, RS,E/Z-beta-tocotrienol into eight, RS,E/Z-gamma-tocotrienol into six and RS,E/Z-delta-tocotrienol into eight peaks. The naturally occurring R,E-E-tocotrienol isomer could be identified within the synthetic RS,E/Z-isomers by co-chromatography with tocotrienol methyl ethers derived from natural sources, respectively.     

Capillary electrophoresis separation of heterocyclic nitrosoamino acid-conformers at sub-ambient temperatures

The separation of a selected group of naturally occurring, heterocyclic nitrosoamino acids was achieved by capillary zone electrophoresis and the resolution of the syn and anti conformers improved as the temperature was lowered to 5 °C. The double peaks observed for each nitrosoamino acid are probably caused by the slow kinetics of conformational isomerism. The experimental setup consisted of a P/ACE 5510 electrophoresis system, a 57 cm polyacrylamidecoated capillary, and a 10 mM phosphate buffer, pH 7.2, containing 2 mM of 3-(N-N-dimethylmyristylammonio)propanesulfonate (DMMAPS) and 0.1% Tween 20. Our study shows that (a) the lower the temperature, the greater the resolution and the longer the migration times; and (b) different nitrosoamino acid conformers were resolved at different temperatures because of differences in the rate of isomerization. For example, the conformers of N-nitrosothiazolidine-4-carboxylic acid were partially resolved at 30 °C while those of N-nitrosoproline were well resolved at the same temperature.     

Direct Measurement of Enantiomeric Excess of Synthetic Tert-Leucine with a Chiral-Ligand-Exchange Chromatography

Racemic tert-leucine can be completely resolved by ligand exchange chromatography (LEC) using a Chiralpak WH column. The retention time of the tert-leucine was decreased by increasing concentration of Cu²⁺, or decreasing pH of the eluent whereas it was unaffected by varying the temperature of eluent. Optimal conditions for complete separation of the D.L-tert-leucine were found to be (Cu²⁺, 5.0 mM, pH 5.5, 25 °C). The enantiomeric excess of synthetic tert-leucine, a chiral auxiliary reagent, was determined.     

Construction and analytical application of ion-selective piezoelectric sensor for atropine sulfate

The method describes the use of a piezoelectric quartz crystal (PQC) as a substitute for ion-selective electrodes. The approach is feasible when the membrane materials are electrically non-conductive and membrane potential measurements are consequently not possible. An ion-selective piezoelectric sensor sensitive to atropine sulfate was constructed by coating a PVC membrane containing activant on one the side of a PQC. On the basis of selective adsorption of atropine ions across the modified film and the sensitive mass response of PQC, the method exhibits a sensitive, rapid response and is easy to operate without pretreatment of the sample. The logarithm of the frequency shift gave a linear relationship with the logarithm of atropine sulfate concentration in the 1.0 x 10(-8)-1.0 x 10(-3) M range with a detection limit of 5.0 x 10(-9) M at pH 7.0. Recoveries were from 98.7-102.2%. Two activants, atropine tetraphenylborate and atropine dipicrylaminate, were synthesized and investigated. Influencing factors were also examined and optimized. The results for real samples obtained by the proposed method agreed with those obtained by conventional methods.     

Ion-interaction chromatography of several metallocyanide complexes stabilized by adding cyanide in a neutral mobile phase.

Standard solutions (at 10(-5) M levels) of Cu(I)- and Fe(II)-cyanide complexes were stabilized for at least 5 h using 0.5 mM cyanide solution (around pH 9) as a medium. Complexes of Cu(I)- and Fe(III)-cyanide also could be stabilized without any dissociation by adding 1 mM cyanide to an acetonitrile-water (18:82, v/v) mobile phase (pH 7.0) containing 10 mM tetra-n-propylammonium salt (TPA). Under the optimal conditions, the six complexes of Cu(I)-, Ag(I)-, Ni(II)-, Fe(II)-, Fe(III)- and Au(I)-cyanides were resolved from their mixtures within about 45 min, with well-shaped chromatographic peaks.     

Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers

A cyclodextrin-modified capillary zone electrophoresis method was developed for the separation and determination of three isomeric compounds (ursolic acid, oleanolic acid and betulinic acid), caffeic acid, p-coumaric acid, rosmarinic acid, rutin and quercetin. Without the addition of beta-cyclodextrin (beta-CD) and methanol, the separation of these analytes was poorly resolved. These eight compounds, however, were well separated from each other within 20 min with a borax running buffer (40 mM of borax, pH 9.4) containing 2mM beta-CD and 4% (v/v) methanol at the voltage of 25 kV, temperature of 25 degrees C and detection wavelength of 210 nm. The relative standard deviations (RSDs) of migration time ranged from 0.16 to 0.74% while those of the peak area ratios ranged from 2.17 to 4.61% for six determinations of the analytes at concentration of 10 and 25 microg mL(-1). The correlation coefficients of the calibration curves of the analytes were all >0.998, and the recoveries were from 96.8 to 103.6%. The method was successfully applied to determine these bioactive components in the samples of Prunella vulgaris L. and its beverage drink products. Our results reveal that only the isomeric compounds and rosmarinic acid could be detected in the spikes of P. vulgaris L.; other components were either too low to be detected or not present while only rosmarinic acid was detected in the beverage products.     

Ionic liquids as additives for separation of benzoic acid and chlorophenoxy acid herbicides by capillary electrophoresis

Ionic liquids (ILs) were tested as additives to phosphate-acetate buffer for the separation of chlorophenoxy and benzoic herbicide acids. The effects of buffer concentration, buffer pH, IL concentration, and concentration of organic solvent were investigated. It was found that in the presence of 40 mM phosphate-acetate containing 10% acetonitrile at pH 4.5, addition of 10 mM 1-butyl-3-methylimidazoium could reverse EOF. The shoulder-merged peaks of two herbicide acids, 2,4-dichlorobenzoic acid and 3,5-dichlorobenzoic acid, were successfully resolved by the addition of IL cation. Apart from these, results showed different IL cations had different influences on the migration behavior of some of the analytes, while IL anions did not lead to obvious difference on the separation.     

Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle-forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV-active chromophore in the metabolites, they were derivatized with 2-nitrobenzaldehyde (2-NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2-NBA) were extracted by using C(18) SPE cartridges. After washing with H(2)O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2-NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (t(mig)) was achieved for all analytes using the developed MECC approach, with absolute t(mig) <1% RSD and t(mig) ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19-2.0 microg/mL.