Investigation of the Enantioselectivity of Tetramethylammonium L‐hydroxyproline Ionic Liquid as a Novel Chiral Ligand in Ligand‐Exchange CE and Ligand‐Exchange MEKC

Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE‐CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L‐hydroxyproline ([TMA][L‐OH‐Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand‐exchange capillary electrophoresis (LE‐CE) and ligand‐exchange micellar electrokinetic capillary chromatography (LE‐MEKC). In the LE‐CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4‐dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE‐MEKC system was established to further study the enantioselectivity of [TMA][L‐OH‐Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE‐CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. Chirality 27:58–63, 2015. © 2014 Wiley Periodicals, Inc.     

Investigation of maltodextrin‐based synergistic system with amino acid chiral ionic liquid as additive for enantioseparation in capillary electrophoresis

The combined use of chiral ionic liquids (ILs) and chiral selectors in capillary electrophoresis (CE) to establish a synergistic system has proven to be an effective approach for enantioseparation. In this article, tetramethylammonium‐L‐arginine, a kind of amino acid chiral IL, was applied to investigate its potential synergistic effect with maltodextrin in CE enantioseparation. The established maltodextrin‐based synergistic system showed markedly improved enantioseparations compared with the single maltodextrin system. Parameters such as the chiral IL concentration, maltodextrin concentration, buffer pH, applied voltage, and capillary temperature were optimized. Satisfactory enantioseparation of the five studied drugs, including nefopam, duloxetine, ketoconazole, cetirizine, and citalopram was achieved in 50 mM Tris‐H₃PO₄ buffer solution (pH 3.0) containing 7.0% (m/v) maltodextrin and 60 mM tetramethylammonium‐L‐arginine. In addition, the chiral configuration of tetramethylammonium‐L‐arginine was also investigated to demonstrate the existence of a synergistic effect between chiral ILs and maltodextrin.     

Enantiomeric Separation of 13 New Amphetamine‐Like Designer Drugs by Capillary Electrophoresis,Using Modified‐‐Cyclodextrins

An easy‐to‐prepare chiral CE method for the enantiomeric separation of 13 new amphetamine‐like designer drugs, using CDs as chiral selectors, was developed. Sulfated‐β‐CD was found to be the best chiral selector among the three used (sulfated‐β‐CD, caroboxymethyl‐β‐CD, dimethyl‐β‐CD). The separation of the analytes was achieved in a fused‐silica gel capillary at 20 °C using an applied voltage of +25 kV. The optimized background electrolyte consisted of 63.5 mM H₃PO₄ and 46.9 mM NaOH in water. Several electrophoretic parameters such as CD type, CD concentration (1 ? 40 mg/mL), buffer pH (2.6, 3.6, 5.0, 6.0), length of the capillary (70 ? 40 cm total length), amount of the organic solvent (methanol and acetonitrile) were investigated and optimized. Chirality 25:617–621, 2013. © 2013 Wiley Periodicals, Inc.     

Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5‐s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 27:223–227, 2015. © 2014 Wiley Periodicals, Inc.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Chiral Separation of Uncharged Pomalidomide Enantiomers Using Carboxymethyl‐β‐Cyclodextrin: A Validated Capillary Electrophoretic Method

The racemic mixture of pomalidomide (POM), a second‐generation immunomodulatory uncharged drug, was separated into enantiomers by capillary zone electrophoresis for the first time. Seven different chargeable cyclodextrin (CD) derivatives were screened as complexing agents and chiral selectors, investigating the stability of the POM‐CD inclusion complexes and their enantiodiscriminating capacities. Based on preliminary experiments, carboxymethyl‐β‐CD (CM‐β‐CD) was found to be the most effective chiral selector. Factors influencing enantioseparation were systematically optimized, using an orthogonal experimental design. Optimal parameters (background electrolyte [BGE]: 50 mM Tris‐acetate buffer, pH 6.5, containing 15 mM CM‐β‐CD; capillary temperature: 20°C; voltage applied +15 kV) allowed baseline separation of POM enantiomers with a resolution as high as 4.87. The developed method was validated, in terms of sensitivity (limit of detection and limit of quantification), linearity, accuracy, repeatability, and intermediate precision. Chirality 28:199–203, 2016. © 2015 Wiley Periodicals, Inc.     

Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis

In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-β-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HP-β-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE.     

Electrically Assisted Liquid‐Phase Microextraction Combined With Capillary Electrophoresis for Quantification of Propranolol Enantiomers in Human Body Fluids

In this study, electromembrane extraction (EME) combined with cyclodextrin (CD)‐modified capillary electrophoresis (CE) was applied for the extraction, separation, and quantification of propranolol (PRO) enantiomers from biological samples. The PRO enantiomers were extracted from aqueous donor solutions, through a supported liquid membrane (SLM) consisting of 2‐nitrophenyl octyl ether (NPOE) impregnated on the wall of the hollow fiber, and into a 20‐μL acidic aqueous acceptor solution into the lumen of hollow fiber. Important parameters affecting EME efficiency such as extraction voltage, extraction time, pH of the donor and acceptor solutions were optimized using a Box‐Behnken design (BBD). Then, under these optimized conditions, the acceptor solution was analyzed using an optimized CD‐modified CE. Several types of CD were evaluated and best results were obtained using a fused‐silica capillary with ammonium acetate (80 mM, pH 2.5) containing 8 mM hydroxypropyl‐β‐CD as a chiral selector, applied voltage of 18 kV, and temperature of 20°C. The relative recoveries were obtained in the range of 78–95%. Finally, the performance of the present method was evaluated for the extraction and determination of PRO enantiomers in real biological samples. Chirality 26:260–267, 2014. © 2014 Wiley Periodicals, Inc.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Separation of Tryptophan Enantiomers by Ligand‐Exchange Chromatography With Novel Chiral Ionic Liquids Ligand

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu²⁺ to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L‐Pro][CF₃COO], [L‐Pro][NO₃], [L‐Pro]₂[SO₄], and [L‐Phe][CF₃COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L‐Pro][CF₃COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H₂O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)₂, 4.0 mmol/L [L‐Pro][CF₃COO] ,and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. Chirality 26:160–165, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral separation of catechin by capillary electrophoresis using mono‐, di‐, tri‐succinyl‐β‐cyclodextrin as chiral selectors

The chiral separation of (±)‐catechin was investigated by capillary electrophoresis using characterized succinyl‐β‐cyclodextrins (Suc‐β‐CDs) with one to three degree of substitution values. The effects of nature and concentration of Suc‐β‐CDs and running buffer pH on the migration time and resolution of (±)‐catechin are discussed. All three kinds of Suc‐β‐CDs show a clear baseline separation of (±)‐catechin in capillary electrophoresis. Mono‐Suc‐β‐CD effectively separated (±)‐catechin, and additional substituted CDs (di‐ and tri‐Suc‐β‐CD) were capable of chiral separation at a broad pH range. The optimum running conditions were found to be 100 mM borate buffer (pH 9.8) containing 5 mM mono‐Suc‐β‐CD with no methanol organic modifier. Chirality, 2009. © 2009 Wiley‐Liss, Inc.     

A chiral enantioseparation generic strategy for anti‐Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach

The present study describes a generic strategy using capillary electrophoretic (CE) method for chiral enantioseparation of anti‐Alzheimer drugs, namely, donepezil (DON), rivastigmine (RIV), and antifungal drugs, namely, ketoconazole (KET), Itraconazole (ITR), fluconazole (FLU), and sertaconazole (SRT) in which these drugs have different basic and acidic properties. Several modified cyclodextrins (CDs) were applied for enantioseparation of racemates such as highly sulfated α, γ CDs, hydroxyl propyl‐β‐CD, and Sulfobutyl ether‐β‐CD. The starting screening conditions consist of 50‐mM phosphate‐triethanolamine buffer at pH 2.5, an applied voltage of 15 kV, and a temperature of 25°C. The CE strategy implemented in the separation starts by screening prior to the optimization stage in which an experimental design is applied. The design of experiment (DOE) was based on a full factorial design of the crucial two factors (pH and %CD) at three levels, to make a total of nine (3²) experiments with high, intermediate, and low values for both factors. Evaluation of the proposed strategy pointed out that best resolution was obtained at pH 2.5 for five racemates using low percentages of HS‐γ‐CD, while SBE‐β‐CD was the most successful chiral selector offering acceptable resolution for all the six racemates, with the best separation at low pH values and at higher %CD within 10‐min runtime. Regression study showed that the linear model shows a significant lack of fit for all chiral selectors, anticipating that higher orders of the factors are most likely to be present in the equation with possible interactions.     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Determination of arecoline in areca nut based on field amplification in capillary electrophoresis coupled with electrochemiluminescence detection

A sensitive capillary electrophoresis–electrochemiluminescence (CE–ECL) assay with an ionic liquid (IL) was developed for the determination of arecoline in areca nut. The IL, 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMImBF₄), was an effective additive improved not only the separation selectivity but also the detection sensitivity of the analyte. BMImBF₄ in the separation electrolyte made the resistance of the separation buffer much lower than that of the sample solution, which resulted in an enhanced field amplified electrokinetic injection CE. ECL intensity of arecoline is about two times higher than that of the analyte with phosphate–IL buffer system. Resolution between arecoline and other unknown compounds in real samples was improved. Under the optimized conditions (ECL detection at 1.2 V, 16 kV separation voltage, 20 mmol/L phosphate with 10 mmol/L BMImBF₄ buffer at pH 7.50, 5 mmol/L Ru(bpy)₃²⁺ and 50 mmol/L phosphate buffer in the detection reservoir), a detection limit of 5 × 10^–9 mol/L for arecoline was obtained. Relative standard deviations of the ECL intensity and the migration time were 4.51% and 0.72% for arecoline. This method was successfully applied to determination of the amount of arecoline in areca nut within 450 s. Copyright © 2012 John Wiley & Sons, Ltd.     

Enantioseparation of ofloxacin and its four related substances with ligand exchange–micellar electrokinetic chromatography using copper(II)‐L‐isoleucine complex as chiral selector

A ligand‐exchange micellar electrokinetic capillary electrophoresis system with copper(II)‐L‐isoleucine complexes as the chiral selector incorporated in micelles of sodium dodecyl sulfate (SDS) was developed for the enantioseparation of ofloxacin and its four related substances (impurities A, C, E, and F). The effects of important parameters affecting separation such as buffer pH, SDS concentration, chiral selector concentration, and organic additive were investigated in detail. Under optimum experimental conditions, enantioseparation of ofloxacin, impurities A, C, E, and F enantiomers was accomplished with resolutions of 4.28, 2.83, 3.40, 3.58, and 2.46, respectively. Further, simultaneous separation of impurities A, C, E, and F enantiomers was achieved using 10 mmol/L NH₄OAc as the running buffer containing 4 mmol/L copper sulfate,20 mmol/L L‐isoleucine, 20 mmol/L SDS, and 5% methanol at pH 8.5. To the best of our knowledge, the simultaneous enantioseparation of four impurities of ofloxacin has not been reported previously.     

Analysis of optical purity and impurity of synthetic D-phenylalanine products using sulfated beta-cyclodextrin as chiral selector by reversed-polarity capillary electrophoresis

A new capillary electrophoresis (CE) method has been achieved for simultaneous separation and quantification of phenylalanine, N-acetylphenylalanine enantiomers, and prochiral N-acetylaminocinnamic acid, possibly co-existent in reaction systems or synthesized products of D-phenylalanine. The separation was carried out in an uncoated capillary under reversed-electrophoretic mode. Among the diverse charged cyclodextrins (CDs) examined, highly sulfated (HS)-beta-CD as the chiral selector exhibited the best enantioselectivity. The complete separation of the analytes was obtained under the optimum conditions of pH 2.5, 35 mM Tris buffer containing 4% HS-beta-CD, applied voltage -15 kV, and capillary temperature 25 degrees C. Furthermore, the proposed method was applied to the determination of optical purity and trace impurities in three batches of the asymmetric synthetic samples of D-phenylalanine, and satisfactory results were obtained. The determination recoveries of the samples were in the range of 97.8-103.8%, and precisions fell within 2.3-5.0% (RSD). The results demonstrate that this CE method is a useful, simple technique and is applicable to purity assays of D-phenylalanine.     

Enantiomeric separation of bupropion enantiomers by electrokinetic chromatography: quantitative analysis in pharmaceutical formulations

The first CE method enabling the quantitation of the two enantiomers of bupropion was developed in this work. Electrokinetic chromatography (EKC) mode using cyclodextrins as chiral selectors was employed. A study on the enantiomeric separation ability of different neutral and anionic CDs was carried out. Sulfated-beta-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, temperature, and separation voltage on the enantiomeric separation of bupropion was also studied. The use of 10 mM sulfated-beta-CD in 50 mM borate buffer (pH 9.0) with an applied voltage of 30 kV and a temperature of 30 degrees C enabled the separation of the enantiomers of bupropion with high resolution (Rs > 7) and short analysis time (approximately 3.5 min). Finally, the method was successfully applied to the quantitation of bupropion in two pharmaceutical formulations.     

Maltodextrins as Chiral Selectors in CE: Molecular Structure Effect of Basic Chiral Compounds on the Enantioseparation

Prediction of chiral separation for a compound using a chiral selector is an interesting and debatable work. For this purpose, in this study 23 chiral basic drugs with different chemical structures were selected as model solutes and the influence of their chemical structures on the enantioseparation in the presence of maltodextrin (MD) as chiral selector was investigated. For chiral separation, a 100‐mM phosphate buffer solution (pH 3.0) containing 10% (w/v) MD with dextrose equivalent (DE) of 4‐7 as chiral selector at the temperature of 25°C and voltage of 20 kV was used. Under this condition, baseline separation was achieved for nine chiral compounds and partial separation was obtained for another six chiral compounds while no enantioseparation was obtained for the remaining eight compounds. The results showed that the existence of at least two aromatic rings or cycloalkanes and an oxygen or nitrogen atom or –CN group directly bonded to the chiral center are necessary for baseline separation. With the obtained results in this study, chiral separation of a chiral compound can be estimated with MD‐modified capillary electrophoresis before analysis. This prediction will minimize the number of preliminary experiments required to resolve enantiomers and will save time and cost. Chirality 26:620–628, 2014. © 2014 Wiley Periodicals, Inc.     

Liquid chromatographic determination of acetylcholine based on pre‐column alkaline cleavage reaction and post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre‐column alkaline cleavage of ACh to form trimethylamine (TMA) and the post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180°C in the presence of lithium hydroxide, and the produced TMA was separated on a cation‐exchange/reversed‐phase dual‐functional column using a mixture of 0.2 m potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2′‐bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal‐to‐noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 µL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations. Copyright © 2009 John Wiley & Sons, Ltd.