Computer simulation of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers with a free or immobilized neutral chiral selector.

A detailed computer simulation study of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers for cases with the neutral selector added to the leader, immobilized to the capillary wall or support, or partially present in the separation column is presented. The electrophoretic transport of the analytes from the sampling compartment into the separation medium with the selector, the formation of a transient mixed zone, the separation dynamics of the stereoisomers with a free or immobilized selector, the dependence of the leader pH, the ionic mobility of norpseudoephedrine, the complexation constant and selector immobilization on steady-state plateau zone properties, and zone changes occurring during the transition from the chiral environment into a selector free leader are thereby visualized in a hitherto unexplored way. For the case with the selector dissolved in the leading electrolyte, simulation data are compared to those observed in experimental setups with coated fused-silica capillaries that feature minimized electroosmosis and zone detection with conductivity and absorbance detectors.

Enantiomeric separation of R,S-tolterodine and R,S-methoxytolterodine with negatively charged cyclodextrins by capillary electrophoresis.

The methods for separation of R,S-tolterodine and R,S-methoxytolterodine enantiomers using sulfated α-, ß-CD and phosphated-γ-CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α- and ß-CD allow anodic detection while phosphated-γ-CD allows only cathodic detection of the separated enantiomers. The influence of chiral selector (CS)'s concentration as well as the influence of composition and concentration of BGE on resolutions were studied. Reversal migration order of tolterodine and methoxytolterodine enantiomers was observed, when sulfated-α- and sulfated-ß-CD were used. The developed methods with all three studied CSs, were validated and compared. All proposed methods enable determination of 0.2% of S-tolterodine as an optical impurity in pills, however the method with phosphated-γ-CD provided lower detection limit, better repeatability of peak areas and migration times, and also lower consumption of CS. Developed method employing phosphated-γ-CD that was applied for the determination of optical purity of R-tolterodine in commercial pills.

Separation of enantiomers of norephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE and NMR studies.

In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α-CD, ß-CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD), and heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD). EMO was reversed in the presence of α-CD and ß-CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS-ß-CD, and HDAS-ß-CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS-ß-CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.

Simultaneous identification of the enantiomers and diastereomers of N,O-di-trifluoroacetylated ephedrine and norephedrine in blood plasma using chiral capillary gas chromatography-mass spectrometry with selected ion monitoring.

A method for identifying the enantiomers of N,O-di-trifluoroacetylated ephedrine (EP) and norephedrine (NE) and the enantiomers of pseudoephedrine (PEP) and pseudonorephedrine (PNE) in plasma was developed using chiral capillary gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). N,O-Di-trifluoroacethyl (TFA) derivatization was accomplished in a dried hydrochloride extract of plasma (minimum quantity of 0.2 mL). An SIM GC-MS method with a ß-cyclodextrin chiral capillary column allowed the successful and simultaneous detection of each TFA-derivatized stereoisomer of EP, NE, PEP, PNE, and an internal standard (IS; S-(+)-ethylamphetamine). Each TFA-drivatized stereoisomer was identified using four mass fragment ions (m/z 140, 154, 168, and 230). The TFA-derivatized stereoisomers of EP, NE, PEP, PNE, and IS were separated completely and were detected with sufficient sensitivity. The assay allowed the stereoisomers to be determined in a linear range of 12.5-1250 ng/mL for the EP stereoisomers and a linear range of 5-1250 ng/mL for the PEP, NE, and PNE stereoisomers. The detection limits were 7.5 ng/mL for the EP stereoisomers and 2.5 ng/mL for the PEP, NE, and PNE stereoisomers. The intra- and interday precisions were less than 5.9% and 8.2%, respectively. This chiral capillary SIM GC-MS method was sufficiently effective in the analysis of plasma from users of over-the-counter cold medicines and was also fully applicable to the plasma analysis of guinea pigs following their treatment with racemic EP.

[Storage proteins from seeds of Pinus pinea L]. / Les protéines de réserve du pin pignon (Pinus pinea L.).

The Mediterranean stone pine Pinus pinea L. (gymnosperm, Pinaceae) is much appreciated for its seed production, widely used in food preparation in the Mediterranean Basin. Seeds contain 25% proteins on a dry-weight basis. Pinus pinea accumulate globulins as major storage proteins in seeds (75% of total storage proteins), composed of several subunits of 10 to 150 kDa, revealed by SDS-PAGE. The albumin fraction (15%) represents three subunits of 14, 24 and 46 kDa. Glutelins, the least soluble fraction, represents a small proportion (10%). Their constitutive units have frequent PM of 43 kDa. Prolamins also represent a very small percentage (1 to 2%).

Enantiomeric purity methods for three pharmaceutical compounds by electrokinetic capillary chromatography utilizing highly sulfated-gamma-cyclodextrin as the chiral selector.

Methods for enantiomeric purity by electrokinetic chromatography were developed and validated for three pharmaceutical compounds, each utilizing highly sulfated-gamma-cyclodextrin (HS-gamma-CD) as the chiral recognition agent. Two of the compounds are weak bases, hence charged at low pH, and the third is a quaternary nitrogen compound, charged at all pH. In each instance quantification was via an authentic reference standard with addition of an internal standard. Separation was on a 61 cm x 50 microm untreated capillary under reverse polarity with a background electrolyte of 5% HS-gamma-CD in pH 2.50 lithium phosphate buffer. Each method was validated with respect to the usual validation parameters, notably recovery and precision, yielding results, including limits of detection and quantitation, that allow reporting the minor enantiomer to 0.1% and less. In applying the methods, all batches of bulk drug tested were shown to be of enantiomeric purity > or =99.9%.

Rational method development strategies on a fluorinated liquid chromatography stationary phase: mobile phase ion concentration and temperature effects on the separation of ephedrine alkaloids.

Fluorinated, silica-based stationary phases are becoming increasingly popular alternatives to traditional alkyl phases owing to their differential selectivity and retention for a variety of analyte classes. In this report, the ion-exchange mechanisms characteristic of a fluorinated phase are exploited to rapidly develop separation conditions for ephedrine alkaloids and synephrine using a mobile phase compatible with mass spectrometry. A linear relationship of basic analyte retention with the reciprocal of ammonium acetate concentration is first established. This linear relationship can then be used to optimize retention and selectivity in just two experiments. The relationship of retention with temperature is also explored. Greater retention with increasing temperature is demonstrated on the fluorinated phase at high percentages of organic modifier, which is in contrast to behavior observed in typical reversed-phase separations. The unexpected observation is explicated based on the reduction in solvent solvating power with increasing temperature. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger interactions between analyte and stationary phase. Both mobile phase ion concentration and temperature are shown to be powerful tools for the manipulation of analyte retention and selectivity.

Application of dimethyl-beta-cyclodextrin as a chiral selector in capillary electrophoresis for enantiomer separation of ephedrine and related compounds in some drugs.

Dimethyl-beta-cyclodextrin (DM-beta-CD) modified capillary electrophoresis has been developed for chiral separation of ephedrine and related compounds, such as (+/-)-norephedrine, (+/-)-N-methylephedrine, (+/-)-ephedrine and (+)-pseudoephedrine. The influence of some crucial parameters such as buffer concentration, pH value, DM-beta-CD concentration, applied voltage and separation temperature on the separation was investigated. Under the optimum conditions, i.e. 40 mM DM-beta-CD in 75 mM Tris (pH 2.5) as the running electrolyte, separation voltage +25 kV and temperature 25 degrees C, a satisfactory separation of the enantiomers was accomplished. The detection limits (S/N = 3) ranged from 65 to 161 ng/mL and the linear range was 0.15 to 101.0 microg/mL for pressure injection. The present method was successfully applied for the analysis of a series of drugs such as anti-tussive, the drug for rheum, the drug for rhinitis and a Chinese traditional herbal medicine, Ephedrae herba (Ma-Huang in Chinese). The recoveries of ephedrine and related compounds in real samples ranged from 97.6 to 103.5%. This method is useful in the simple and rapid analysis of ephedrine derivatives in marketed products.

Liquid-phase microextraction based on carrier mediated transport combined with liquid chromatography-mass spectrometry. New concept for the determination of polar drugs in a single drop of human plasma.

Recently, we demonstrated for the first time liquid-phase microextraction (LPME) of polar drugs based on carrier mediated transport. In this new extraction technique, selected analytes were extracted as ion-pairs from small volumes of biological samples, through a thin layer of a water immiscible organic solvent immobilised in the pores of a porous hollow fibre (liquid membrane), and into a microl volume of an acidic aqueous acceptor solution placed inside the lumen of the hollow fibre. In the current paper, this new extraction technique was combined with liquid chromatography-mass spectrometry (LC-MS) for the first time. Carrier mediated LPME was evaluated for several new model drugs (0.01

Determination of ephedrine and related compounds in pharmaceutical preparations by ion chromatography with direct conductivity detection.

An ion chromatographic method with conductivity detection for the simultaneous determination of ephedrine, pseudoephedrine and norephedrine was developed. A mixture of 2.0 mmol/L HNO3 and 2% (v/v) acetonitrile was used as eluent. The three ephedrine-like compounds were separated and determined within 20 min. The linear ranges were 0.08-50 microg/mL for ephedrine, 0.08-40 microg/mL for pseudoephedrine and 0.06-40 microg/mL for norephedrine. The detection limits were 0.03 microg/mL for ephedrine and pseudoephedrine, and 0.02 microg/mL for norephedrine. The method has been applied successfully to the determination of these sympathomimetics in pharmaceutical preparations and in Ephedra herbs.

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.

Separation and determination of ephedrine alkaloids and tetramethylpyrazine in Ephedra sinica Stapf by gas chromatography-mass spectrometry.

A simple, sensitive, and reliable method using gas chromatography (GC)-mass spectrometry (MS) is developed for the simultaneous determination of ephedrine alkaloids and 2,3,5,6-tetramethylpyrazine (TMP) in Ephedra sinica Stapf. The sample is extracted with ethyl ether and submitted to GC-MS for identification and quantitation without derivatization. The column used for GC is an HP-5 (30.0 m x 250 microm x 0.25 microm, 5% phenyl methyl siloxane), and the carrier gas is helium. The detection limits for ephedrine, pseudoephedrine, and TMP are 0.4 ng 0.7 ng, and 0.02 ng (signal-to-noise ratio of 3), respectively. The reproducibility of the total procedure is proved to be acceptable (RSD < 2%), and the recoveries are above 93%.

Separation of cold medicine ingredients by capillary electrophoresis.

This study demonstrates the separation of cold medicine ingredients (e.g., phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol) by capillary zone electrophoresis and micellar electrokinetic chromatography. Factors affecting their separations were the buffer pH and the concentrations of buffer, surfactant and organic modifiers. Optimum results were obtained with a 10 mM sodium dihydrogen-phosphate-sodium tetraborate buffer containing 50 mM sodium dodecyl sulfate (SDS) and 5% methanol (MeOH), pH 9.0. The carrier electrolyte gave a baseline separation of phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol with a resolution of 1.2, and the total migration time was 11.38 min.

[Separation and determination of ephedrine alkaloids and 2,3,5,6-tetramethyl pyrazine in Ephedra herba by HPLC].

A sensitive and reliable high performance liquid chromatographic method(HPLC) has been developed for the first time for the simultaneous determination of the active ingredients of ephedrine alkaloids and 2,3,5,6-tetramethyl pyrazine (TMP) in Ephedra herba crude drug and two Chinese traditional medicines (Xiao-er qingfeiwan and Lu-si kewan). The HPLC assay was performed on a reversed phase C18 column (Nova-Pak C18, 3.9 mm i.d. x 150 mm) by using methanol-0.02 mol/L KH2PO4-acetic acid-triethyl amine (4:96:0.2:0.01, V/V) as mobile phase for the ephedrine alkaloids analysis and methanol-H2O-acetic acid (35:65:0.5, V/V) as mobile phase for TMP analysis. Regression equations revealed the linear relationships (correlation coefficients: 0.991-0.998) between the peak area of each constituent (E, PE, NE, NPE, TMP) and its concentration. The detection limits for E, PE, NE, NPE and TMP were 0.4 mg/L, 0.1 mg/L, 0.03 mg/L, 0.02 mg/L and 0.03 mg/L, respectively, and the recoveries ranged between 92%-103%. The contents of E, PE, NE, NPE, TMP in Ephedra herba, traditional medicine Xiao-er qingfeiwan and Lu-si kewan were determined respectively. The relative standard deviations (RSD) of the contents ranged between 1.1%-3%.

On-chip chiral and achiral separation of amphetamine and related compounds labeled with 4-fluoro-7-nitrobenzofurazane.

Amphetamine and analogous compounds have been labeled with 4-fluoro-7-nitrobenzofurazane and analyzed on a microfabricated chip. Separation of norephedrine, ephedrine, cathinone, pseudoephedrine, methcathinone, amphetamine and methamphetamine is demonstrated using micellar electrokinetic capillary chromatography (MEKC) and laser-induced fluorescence (LIF) detection. Chiral separations of individual drugs were studied using neutral and negatively charged cyclodextrins (CDs) with and without the addition of an organic modifier and/or sodium dodecyl sulfate (SDS). The best results were obtained using a highly sulfated gamma-CD (HS-gamm-CD) in combination with a low concentration of SDS. To obtain complete separation of a mixture of (+/-)-norephedrine, (+/-)ephedrine, (+/-)-pseudoephedrine, (+/-)-methcathinone, (+/-)-amphetamine and (+/-)-methamphetamine it was necessary to add a small amount (1.5 mM) of SDS to the separation buffer. Optimized chiral separation was achieved within 7 min using an S-folded separation channel, a separation voltage of 8 kV and a buffer consisting of 50 mM phosphate (pH 7.35), 10 mM HS-gamma-CD and 1.5 mM SDS.

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein.

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.

Direct enantioseparation of adrenergic drugs via thin-layer chromatography using molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) of (-)-pseudoephedrine and (-)-norephedrine were prepared to use as chiral stationary phases (CSPs) in thin layer chromatography (TLC). The resolution of the enantiomers of adrenergic drugs, including pseudoephedrine, ephedrine, norephedrine, and epinephrine were investigated on these CSPs. In preparation of MIPs, two monomers: (1) methacrylic acid and (2) itaconic acid were employed as functional monomers. Mobile phase system of either methanol or acetonitrile was used and the effects of acetic acid content of the mobile phases were also investigated. The best resolution was achieved for enantioseparation of norephedrine on plates based on MIP of (-)-norephedrine using itaconic acid as functional monomer (alpha = 5.1) in mobile phase 1% acetic acid in methanol. Moreover, these MIPs were able to resolve the racemates of compounds whose structures corresponded to print molecule. The results obtained showed that TLC based on MIPs could succeed the direct separation of enantiomers of adrenergic drugs as a method of separation. The method offers a rapid, sensitive and reliable method for quality control of optically active compounds.

Coupled achiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds.

A multicolumn approach was developed to address the limited achiral selectivity of chiral stationary phases. Groups of structurally related compounds, including beta-blockers and 1,4-benzodiazepines, were separated using coupled achiral/chiral stationary phases under subcritical fluid conditions. The achiral selectivity of amino and cyano stationary phases was used to modify the resolution of compounds on a Chiralcel OD chiral stationary phase by combining the achiral and chiral columns in series. In the case of the benzodiazepines, separation of achiral compounds was performed concurrently with the enantioseparation of chiral molecules. The separation of components of a multidrug cough and cold medication was also demonstrated on a cyano column coupled with a Chiralpak AD chiral stationary phase. The use of modified carbon dioxide eluents eliminated the mobile phase incompatibility problems associated with column coupling in liquid chromatography and incorporated the high efficiency of sub- and supercritical fluid chromatography.

[Separation of norephedrine enantiomers by high performance liquid chromatography (HPLC) with urea derivative as chiral stationary phase].

By using normal-phase HPLC with urea derivative as chiral stationary phase(CSP), the direct separation of racemic norephedrine has been investigated. The resolution was optimized by varying the concentration of iso-propanol in a ternary mobile phase which consists of two other components: hexane and 1,2-dichloroethane. The lower the percent of iso-propanol, the better the resolution is. But the retention time is longer as the percent of iso-propanol decreases. In an attempt to reduce the retention time without reducing the stereoselectivity, we chose the mixture of hexane, 1,2-dichloroethane and iso-propanol as the mobile phase with a proportion of 73.5:25:1.5 in vol. We also altered the organic modifier by substituting ethanol, acetonitrile and tetrahydrofuran (THF) for iso-propanol. The addition of ethanol appears to increase the hydrogen bond interactions between the solute and solvent, which results in lower k' values and an increase in alpha and Ra. Acetonitrile is only a kind of proton-accepter and the interactions of hydrogen bonding between the solute and solvent are weaker than iso-propanol, which results in higher k' values and a reduction in alpha and Ra. These separations are in agreement with the three-point rule. When THF is used as organic modifier, a reversed elution order and a more favorable separation can be obtained. The specific behavior of THF in HPLC has not been explained satisfactorily yet.

[Rapid determination of active constituents in compound ibuprofen tablets by capillary zone electrophoresis].

A rapid capillary zone electrophoresis (CZE) method for the determination of ibuprofen and pseudoephedrine hydrochloride in compound ibuprofen tablets has been developed. CZE was performed in 0.025 mol/L phosphate buffer solution (pH 8.1). The two compounds were completely separated within 3 minutes, and quantified with a UV-detector at 210 nm by means of external standard method. Relative standard deviations of 2.9% for pseudoephedrine hydrochloride and 1.9% for ibuprofen were achieved for 11 determinations of the sample solution containing 9.5 mg/L pseudoephedrine hydrochloride and 66.7 mg/L ibuprofen, and recoveries of 103.1% and 97.6% were obtained respectively for the spiked pseudoephedrine hydrochloride and ibuprofen. The proposed method has been applied for the analysis of compound ibuprofen tablets, and the analytical results are in good agreement with those obtained by HPLC method.