Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Determination of brompheniramine enantiomers in rat plasma by cation‐selective exhaustive injection and sweeping cyclodextrin modified electrokinetic chromatography method

A method consisting of cation‐selective exhaustive injection and sweeping (CSEI‐sweeping) as online preconcentration followed by a cyclodextrin modified electrokinetic chromatography (CDEKC) enantioseparation has been developed for the simultaneous determination of two brompheniramine enantiomers in rat plasma. In this method, analytes were electrokinetically injected at a voltage of 8 kV for 80 s in a fused‐silica capillary. Prior to the injection, the capillary was rinsed with 50 mM phosphate buffer of pH 3.5, followed by a plug of a higher conductivity buffer (150 mM phosphate pH 3.5, 20 psi, 6 min) and a plug of water (0.5 psi, 5 s). Separation was carried out applying –20 kV in 50 mM phosphate buffer, pH 3.5, containing 10% v/v ACN and 30 mg/mL sulfated‐β‐cyclodextrin (S‐β‐CD). Analytical signals were monitored at 210 nm. The detection sensitivity of brompheniramine enantiomers was enhanced by about 2400‐fold compared to the normal injection mode (hydrodynamic injection for 3 s at 0.5 psi, with a BGE of 50 mM phosphate buffer containing 20 mg/mL S‐β‐CD at pH 3.5), and LLOQ of two enantiomers were both 0.0100 μg/mL. In addition, this method had fairly good repeatability and showed promising capabilities in the application of stereoselective pharmacokinetic investigations for brompheniramine enantiomers in rat.     

Impurity profiling of dexamphetamine sulfate by cyclodextrin‐modified microemulsion electrokinetic chromatography

A CD‐modified microemulsion electrokinetic chromatography method has been developed and validated for dexamphetamine sulfate which allows the simultaneous determination of charged and uncharged impurities of the drug including the levorotary (R)‐enantiomer. The optimized background electrolyte consisted of 1.5% w/w SDS, 0.5% w/w ethyl acetate, 3.5% w/w 1‐butanol, 2.5% w/w 2‐propanol and 92% w/w 50 mM sodium phosphate buffer, pH 3.0, containing 5.5% w/w sulfated β‐CD. Separations were performed in a 50.2/40 cm, 50 μm id fused silica capillary at a temperature of 20°C and an applied voltage of ?14 kV. Carbamazepine was used as internal standard. The assay was validated in the range of 0.1–1.0% for the related substances and 0.1–5.0% for levoamphetamine based on a concentration of 3 mg/mL of dexamphetamine sulfate. The LOD of all analytes ranged between 0.05 and 0.2%. In commercial samples of dexamphetamine sulfate, levoamphetamine was found at concentrations between 3.2 and 3.8%, whereas none of the other impurities could be detected.     

Development of a novel dual CD‐MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.—Loranthaceae—extracts

The present work deals with the development and validation of a novel dual CD‐MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.—Loranthaceae—extracts. The BGE consisted of 20 mM pH 8.3 borate buffer, 50 mM SDS, a dual CD system based on the combination of 5 mM β‐CD and 2% w/v S‐β‐CD, and 10% v/v methanol. The proposed method has been successfully applied to the comparative analysis of extracts from aerial parts and different hosts, geographical areas, and extraction procedures in order to establish the flavonoid fingerprint of L. cuneifolia . The method was validated according to international guidelines. LOD and LOQ, intra and interday precision, and linearity were determined for catechin, epicatechin, procyanidin B2, rutin, quercetin‐3‐O‐glucoside, quercetin‐3‐O‐xyloside, quercetin‐3‐O‐rhamnoside, quercetin‐3‐O‐arabinofuranoside, quercetin‐3‐O‐arabinopyranoside, and quercetin. The CD‐MEKC methodology emerges as a suitable alternative to the traditional HPLC for quality control, fingerprinting, and standardization of L. cuneifolia extracts from different sources.     

Enantioseparation of chiral benzimidazole derivatives by electrokinetic chromatography using sulfated cyclodextrins

Baseline separation of 18 new substituted benzimidazole derivatives, potent AMP‐activated protein kinase (AMPK) activators, with one chiral center, was achieved by CD‐EKC using sulfated and highly sulfated CDs (SCDs and HS‐CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The SCDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times around 6 min using 25 mM phosphate buffer at pH 2.5 containing either β‐S‐CD, HS‐β‐CD, HS‐γ‐CD (3 or 4% w/v) at 25°C, with a voltage of 20 kV. The apparent association constants of the inclusion complexes were calculated. The study of the solute structure‐enantioseparation relationships seems to show the high contribution of the interactions between the solutes phenyl ring and the CDs to the enantiorecognition process. The optimized method was briefly validated (LOD less than 1%) and the purity of enantiomers of compound 3 was determined. The enantiomer migration shows reversal order depending on the kind of CD.     

Simultaneous enantioseparation of cyproconazole,bromuconazole, and diniconazole enantiomers by CD‐modified MEKC

An efficient method for the simultaneous enantioseparation of cyproconazole, bromuconazole, and diniconazole enantiomers was developed by CD‐modified MEKC using a dual mixture of neutral CDs as chiral selector. Three neutral CDs namely hydroxypropyl‐β‐CD, hydroxypropyl‐γ‐CD, and γ‐CD were tested as chiral selectors at different concentrations ranging from 10, 20, 30 and 40 mM, but enantiomers of the studied fungicides were not completely separated. The best dual chiral recognition mode for the simultaneous separation of cyproconazole, bromuconazole, and diniconazole enantiomers was achieved with a mixture of 27 mM hydroxypropyl‐β‐CD and 3 mM hydroxypropyl‐γ‐CD in 25 mM phosphate buffer (pH 3.0) containing 40 mM SDS to which methanol‐acetonitrile (10%:5% v/v) was added as organic modifiers. The best separation was based on the appearance of 10 peaks simultaneously, with good resolution (R_s 1.1–15.9), and peak efficiency (N>200 000). Good repeatabilities in the migration time, peak area, and peak height were obtained in terms of RSD ranging from (0.72 to 1.06)%, (0.39 to 3.49)%, and (1.90 to 4.84)%, respectively.     

Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as labeling reagent

A sensitive and effective micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection approach was described for the determination of low molecular‐mass thiols using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as the labeling reagent. After precolumn derivatization, baseline separation of six thiol compounds including cysteine, glutathione, N‐acetylcysteine, homocysteine, 6‐mercaptopurine, and penicillamine were achieved within 18 min. The optimal running buffer was composed of mixtures involving 25 mM sodium dodecyl sulfate, 25% (v/v) acetonitrile and 15 mM sodium phosphate buffer, pH 7.5. The detection limits (S/N = 3) were found as low as 40 pM under argon ion laser‐induced fluorescence detector (λ_ex/λ_em = 488/520 nm), which were much better than the reported approaches. The accuracy and specificity of this assay for real samples were assured by a standard addition method. The proposed method has been applied to the analysis of thiols both in human plasma and plum flower samples with recoveries of 92.0–109.4%.     

A sensitive non‐aqueous capillary electrophoresis‐mass spectrometric method for multiresidue analyses of β‐agonists in pork

Non‐aqueous capillary electrophoresis–mass spectrometry (NACE‐MS) was developed for trace analyses of β‐agonists (i.e. clenbuterol, salbutamol and terbutaline) in pork. The NACE was in 18 mM ammonium acetate in methanol–acetonitrile–glacial acetic acid (66 : 33 : 1, v/v/v) using a voltage of 28 kV. The hyphenation of CE with a time‐of‐flight MS was performed by electrospray ionization interface employing 5 mM ammonium acetate in methanol–water (80 : 20, v/v) as the sheath liquid at a flow rate of 2 μL/min. Method sensitivity was enhanced by a co‐injection technique (combination of hydrodynamic and electrokinetic injection) using a pressure of 50 mbar and a voltage of 10 kV for 12 s. The method was validated in comparison with HPLC–MS‐MS. The NACE‐MS procedure provided excellent detection limits of 0.3 ppb for all analytes. Method linearity was good (r² > 0.999, in a range of 0.8–1000 ppb for all analytes). Precision showed %RSDs of <17.7%. Sample pre‐treatment was carried out by solid‐phase extraction using mixed mode reversed phase/cation exchange cartridges yielding recoveries between 69 and 80%. The NACE‐MS could be successfully used for the analysis of β‐agonists in pork samples and results showed no statistical differences from the values reported by the Ministry of Public Health, Thailand using HPLC‐MS‐MS method. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl‐γ‐cyclodextrin‐modified micellar electrokinetic chromatography

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD)‐modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate–15 mM borate buffer (pH 9.0) containing 30 mM HP‐γ‐CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)‐ and (R)‐enantiomers of regioisomeric 8‐, 11‐, 12‐, and 15‐HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8‐ and 12‐HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8‐HETE was found to be a mixture of 98% (R)‐enantiomer and 2% (S)‐enantiomer, while the 12‐HETE was a mixture of 98% (S)‐enantiomer and 2% (R)‐enantiomer. The present study demonstrates that the HP‐γ‐CD‐modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.     

Enantioseparation of esbiothrin by cyclodextrin‐modified microemulsion and micellar electrokinetic chromatography

A comparison between chiral cyclodextrin‐modified microemulsion electrokinetic chromatography (CD‐MEEKC) and cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) for the enantiomeric separation of esbiothrin was carried out. For both methods, the separation conditions were optimized by varying CD types and concentration, running buffer pH and compositions, organic modifiers, and temperature. The optimal CD‐MEEKC conditions were 0.8% n‐heptane, 2.3% SDS, 6.6% n‐butanol, 90.3% 10 mM sodium tetraborate containing 3% (w/v, the ratio of CD mass to microemulsion volume) methyl‐β‐cyclodextrin, pH 10, 25°C. The optimized CD‐MEKC conditions were 3.3% SDS, 96.7% 10 mM sodium tetraborate containing 5% (w/v) β‐CD, pH 10, 25°C. The difference in physicochemical properties of the buffer and CDs resulted in different optimal CD type. The competitive distribution between the microemulsion (or micelle) and chiral CD contributed to the chiral separation. Both methods provided excellent separation (R_s ～? 3) with similar migration time (ca. 15 min). CD‐MEEKC provided higher separation efficiencies (>300000) than CD‐MEKC (>200000). The LODs for CD‐MEEKC and CD‐MEKC were 4.7 μg/mL and 3.2 μg/mL, respectively. The RSDs of migration time and peak area for CD‐MEEKC were slightly higher than for CD‐MEKC. Both the demonstrated CD‐MEEKC and CD‐MEKC methods provided high efficiencies, low LODs, and reproducible enantioseparations of esbiothrin.     

Simultaneous determination of yohimbine,sildenafil, vardenafil and tadalafil in dietary supplements using high‐performance liquid chromatography‐tandem mass spectrometry

A simple and sensitive method was developed for determination of illegal adulterants (yohimbine, sildenafil, vardenafil and tadalafil) in dietary supplements by HPLC‐MS/MS. The separation was achieved on a C₁₈ column with the mobile phase consisting of acetonitrile and 0.1% acetic acid aqueous solution with a gradient elution at a flow rate of 0.5 mL/min. The analytes were quantified and identified by two characteristic transitions using the multiple‐reaction monitoring mode. The recoveries of the analytes ranged from 77.5 to 109.3% with the RSD less than 8.1% (n=6). The method has been successfully applied to screen illegal adulterations of natural dietary supplements.     

Capillary electrochromatography‐mass spectrometry for the determination of 5‐nitroimidazole antibiotics in urine samples

The separation of eight antibiotics belonging to 5‐nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; Cogent^TM Bidentate C18, 4.2 μm; Pinnacle II? Phenyl, 3 μm; Pinnacle II? Cyano, 3 μm), Cogent? Bidentate C18 (4.2 μm) gave the best performance. For CEC‐MS coupling, a laboratory assembled liquid‐junction‐nano‐spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC‐ESI‐MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica‐60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5‐nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67–103% range were obtained. Furthermore, for the selected antibiotics, CEC‐MS² spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Enrichment and separation of antitumor triterpene acids from the epidermis of Poria cocos by pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography

Triterpene acids were extracted from the epidermis of Poria cocos (Schw.) Wolf. These acids were found to inhibit the growth of lung cancer cells in vitro and in vivo. An efficient method for the preparative separation of antitumor triterpene acids was established that involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography. We used pH‐zone‐refining counter‐current chromatography to concentrate the triterpene acids using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (3:7:5:5, v/v/v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer, and ammonia (10 mM) was added to the lower phase as an eluter. As a result, 200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high‐speed counter‐current chromatography using a solvent system composed of petroleum ether/ethyl acetate/methanol/water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively. The inhibitory activity of the major compound on lung A549 cells was examined and poricoic acid A was found to significantly inhibit the growth of A 549 cells.     

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‐, β‐CD and phosphated‐γ‐CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α‐ and β‐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‐β‐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.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Sensitive LC‐MS‐MS method for the determination of tiopronin in human plasma

A highly selective and sensitive LC‐MS‐MS method was developed and validated to quantify tiopronin in human plasma, using fudosteine as the internal standard (IS). L ‐Cysteine and 1,4‐dithiothreitol (DTT) were used as the reducer and the stabilizer to release and stabilify tiopronin from a dimmer and mix forms with endogenous thiols in the treatment of plasma samples. After a simple liquid–liquid extraction with ethyl acetate in acidic condition, the post‐treatment samples were analyzed on a C₁₈ column interfaced with a triple‐quadruple tandem mass spectrometer using negative electrospray ionization. Methanol and water (40:60, v/v) were used as the isocratic mobile phase, with 0.2% formic acid and 1.0 mM tris (hydroxymethyl) aminomethane (Tris) in water. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy and precision of measurements. The assay was linear over the concentration range 0.078–10 μg/mL. The correlation coefficients for the calibration curves ranged from 0.9980 to 0.9990. The intra‐ and inter‐day precisions, calculated from quality control samples, were not more than 10.49%. The method was employed in a pharmacokinetic study after oral administration of 200 mg tiopronin tablets to 24 healthy volunteers. Copyright © 2009 John Wiley & Sons, Ltd     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole,nicotine and DMXAA

A selective and low‐cost CD‐MEKC method under acidic conditions was developed for investigating the N‐oxygenation of tamoxifen (TAM) by flavin‐containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N‐oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β‐CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis–Menten constant (K_m) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V_max) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K_i) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti‐tumour agent, were investigated.