Cyclodextrin-Modified Micellar UPLC for Direct, Sensitive and Selective Determination of Water Soluble Vitamins in Milk.

Cyclodextrin-modified micellar ultra pressure liquid chromatography (CD-MUPLC) was firstly developed and directly applied to the simultaneous determination of water-soluble vitamins thiamine hydrochloride (VB1), pyridoxine hydrochloride (VB6) and ascorbic acid (VC) in milk samples. A hybrid isocratic mobile phase consisting of ß-cyclodextrin (ß-CD, 5.0 mmol L-1) and cetyltrimethylammonium bromide (CTAB, 0.1 mol L-1) in the presence of acetic acid (0.1 mol L-1) at pH 2.9 on a RP-C18 column at 25.0°C was successfully used. The separation of vitamins was achieved in less than 10 min at a 0.2 mL min-1 flow rate showing adequate linearity at 245 nm in the ranges of 5.0-500.0 µg L-1 for VB1, 5.0-1000.0 µg L-1 for VB6 and 5.0-10000.0 µg L-1 for VC with coefficients of variation (r2) of 0.9999, 0.9987 and 0.9971, respectively. In addition, limits of detection obtained were 0.885, 1.352 and 1.358 µg L-1 and limits of quantification were 2.681, 4.096 and 4.115 µg L-1 for VB1, VB6 and VC, respectively. The high sensitivity of the proposed CD-MUPLC-UV method permitted its applications to the determination of water-soluble vitamins VB1 (32-488 µg L-1), VB6 (82-95 µg L-1) and VC (790-45000 µg L-1) in breast and bovine milk samples. The relative standard deviations and recoveries ranged between 0.07 and 2.14% and between 85.27 and 114.8%, respectively, indicating the accurate and precise measurements without any negative impact of matrix. The current analytical method illustrated several advantages including direct, sensitive, selective and non-consuming organic solvents over the hitherto published methods. These features could be attributed to the four-point competitive interactions among analytes, pseudostationary phases and modified C18 stationary phases.

Ultrasound-assisted temperature-controlled ionic liquid dispersive liquid-phase microextraction combined with reversed-phase liquid chromatography for determination of organophosphorus pesticides in water samples.

A rapid and sensitive ultrasound-assisted temperature-controlled ionic liquid (IL) dispersive liquid-phase microextraction (UTILDLPME) combined with reversed-phase liquid chromatography-ultraviolet (RPLC-UV) was developed for the determination of five organophosphorus pesticides (OPPs; azinphos-methyl, chloropyriphos, parathion-methyl, diazinon, and phosalone) in water samples. Parameters including IL type, IL volume, ionic strength, sonication time, heating/cooling temperature, centrifugal time, and speed were investigated. The extraction procedure was induced by the formation of cloudy solution, which was composed of 75 µL of 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM]PF6 ) dispersed entirely into 5 mL sample solution with the assistance of ultrasound for 3 min and temperature at 40°C. Under optimal conditions, linearity of the five OPPs was obtained in the range of 0.09-200 ng/mL with correlation coefficients of 0.998 or more. Limits of detection and limits of quantitation ranged from 0.01 to 0.1 ng/mL and from 0.05 to 0.4 ng/mL, respectively. Compared with conventional microextraction techniques, the proposed UTILDLPME exhibited the highest extraction efficiency ranging between 90 and 98% for targeted OPPs. Furthermore, the proposed UTILDLPME/RPLC was successfully applied to different water samples (tap, well, and lake water) showing relative recoveries ranging from 96.9 to 103.2%. Therefore, UTILDLPME/RPLC-UV could be a simple, rapid, sensitive, and efficient routine technique for determination of OPPs in water.

Ionic liquids in enhancing the sensitivity of capillary electrophoresis: Off-line and on-line sample preconcentration techniques.

The popularity of ionic liquids (ILs) has grown during the last decade in enhancing the sensitivity of CE through different off-line or on-line sample preconcentration techniques. Water-insoluble ILs were commonly used in IL-based liquid phase microextraction, in all its variants, as off-line sample preconcentration techniques combined with CE. Water-soluble ILs were rarely used in IL-based aqueous two phase system (IL-ATPS) as an off-line sample preconcentration approach combined with CE in spite of IL-ATPS predicted features such as more compatibility with CE sample injection due to its relatively low viscosity and more compatibility with CE running buffers avoid, in some cases, anion exchange precipitation. Therefore, the attentions for the key parameters affecting the performance of IL-ATPSs were generally presented and discussed. On-line CE preconcentration techniques containing IL-based surfactants at nonmicellar or micellar concentrations have become another interesting area to improve CE sensitivity and it is likely to remain a focus of the field in the endeavor because of their numerous to create rapid, simple and sensitive systems. In this article, significant contributions of ILs in enhancing the sensitivity of CE are described, and a specific overview of the relevant examples of their applications is also given.

Stabilizing proteins for affinity capillary electrophoresis using ionic liquid aqueous two phase systems: Pharmaceuticals and human serum albumin.

Recently, ionic liquids (ILs) are finding ever broader scope within pharmaceutical and bioanalytical applications. In the current work, ACE binding measurements of tryptophan (Try)-HSA, chlorambucil (CHL)-HSA, and dacarbazine (DTIC)-HSA complexes were estimated in the absence or presence of several short chain imidazolium ILs within the range of concentrations of 10.0-1000.0 µmol/L that are far below the critical micelle concentrations of ILs. Results indicated that the value of binding constant of Trp-HSA was dramatically deviated in the presence of 1000.0 µmol/L 1-decyl-3-methylimidazolium bromide (DMIMBr) IL. However, interestingly, there is no any deviation for the Trp-HSA binding constant with 100.0 µmol/L 1-butyl-3-methylimidazolium bromide (BMIMBr) IL as an adjuvant additive in 67.0 mmol/L phosphate buffer at pH 7.4. This finding was further used to estimate the binding constants of important but weakly binding substances of CHL and DTIC antitumors with HSA; their binding constants were also estimated by HPAC giving data in good agreement with that revealed by ACE. These achievements were attributed to the significant improvement of HSA stability by combination with BMIMBr IL through hydrogen bond, electrostatic, and π-π forces. In addition, the use of 100.0 µmol/L BMIMBr extended the stability of native HSA solution stored under the ambient lab conditions up to 25 days with significant improvements in the precision of ACE binding data.

Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).

Data quality in drug discovery: the role of analytical performance in ligand binding assays.

Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

Recent advances in affinity capillary electrophoresis for binding studies.

The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry.

Synthesis-microstructure-performance relationship of layered transition metal oxides as cathode for rechargeable sodium batteries prepared by high-temperature calcination.

Research on sodium batteries has made a comeback because of concern regarding the limited resources and cost of lithium for Li-ion batteries. From the standpoint of electrochemistry and economics, Mn- or Fe-based layered transition metal oxides should be the most suitable cathode candidates for affordable sodium batteries. Herein, this paper reports a novel cathode material, layered Na1+x(Fey/2Niy/2Mn1-y)1-xO2 (x = 0.1-0.5), synthesized through a facile coprecipitation process combined with subsequent calcination. For such cathode material calcined at 800 °C for 20 h, the Na/Na1+x(Fey/2Niy/2Mn1-y)1-xO2 (x = 0.4) electrode exhibited a good capacity of 99.1 mAh g(-1) (cycled at 1.5-4.0 V) and capacity retention over 87% after 50 cycles. Optimization of this material would make layered transition metal oxides a strong candidate for the Na-ion battery cathode.

Alkyl imidazolium ionic liquid based sweeping-micellar electrokinetic chromatography for simultaneous determination of seven tea catechins in human plasma.

Determination of tea catechins in human plasma might provide a means of better evaluation of their benefits. The main difficulty in their analysis is the low catechins concentrations in plasma and their susceptible to oxidation during sample pretreatment. In the current work, a sweeping-micellar electrokinetic chromatography (sweeping-MEKC) by long alkyl chain ionic liquid was investigated for the simultaneous determination of seven principal naturally-occurring tea catechins in human plasma under acidic conditions after the intake of green tea beverage. The effects of type and concentration of three 1-tetradecyl-3-methylimidazolium ionic liquids, namely bromide, acetate and hydrogen sulfate salts were studied. The seven catechins were successfully separated within 5min by micellar running buffer of 5mmolL(-1) 1-tetradecyl-3-methylimidazolium bromide and 15mmolL(-1) phosphate buffer at pH 4.5 under optimal parameters of 50mbar injection for 150s, 10kV, 25°C and 200nm. To prevent the possibility of IL adsorption, an appropriate rinsing protocol was established. The method has analytical ranges from 0.5, 1, 0.5, 1, 2, 1 and 1 to 500ngmL(-1) for GC, C, EC, EGCG, GCG, ECG and EGC, respectively (r ranged from 0.995 to 0.999). The intraday precision and accuracy were 0.1-0.9% RSD (n=10) and 97-106% recovery, respectively. Limits of detections of analytes were ranged from 0.2 to 1.2ngmL(-1). The current sweeping-MEKC achieved sensitivity enhancement factor (SEF) up to 183-fold of analytes concentrations compared to other hitherto published CE reports that is suitable to find out the trace amounts of catechins in plasma.

Free-standing hierarchically sandwich-type tungsten disulfide nanotubes/graphene anode for lithium-ion batteries.

Transition metal dichalcogenides (TMD), analogue of graphene, could form various dimensionalities. Similar to carbon, one-dimensional (1D) nanotube of TMD materials has wide application in hydrogen storage, Li-ion batteries, and supercapacitors due to their unique structure and properties. Here we demonstrate the feasibility of tungsten disulfide nanotubes (WS2-NTs)/graphene (GS) sandwich-type architecture as anode for lithium-ion batteries for the first time. The graphene-based hierarchical architecture plays vital roles in achieving fast electron/ion transfer, thus leading to good electrochemical performance. When evaluated as anode, WS2-NTs/GS hybrid could maintain a capacity of 318.6 mA/g over 500 cycles at a current density of 1A/g. Besides, the hybrid anode does not require any additional polymetric binder, conductive additives, or a separate metal current-collector. The relatively high density of this hybrid is beneficial for high capacity per unit volume. Those characteristics make it a potential anode material for light and high-performance lithium-ion batteries.

Investigating effects of sample pretreatment on protein stability using size-exclusion chromatography and high-resolution continuum source atomic absorption spectrometry.

In this study, size-exclusion chromatography and high-resolution atomic absorption spectrometry methods have been developed and evaluated to test the stability of proteins during sample pretreatment. This especially includes different storage conditions but also adsorption before or even during the chromatographic process. For the development of the size exclusion method, a Biosep S3000 5 µm column was used for investigating a series of representative model proteins, namely bovine serum albumin, ovalbumin, monoclonal immunoglobulin G antibody, and myoglobin. Ambient temperature storage was found to be harmful to all model proteins, whereas short-term storage up to 14 days could be done in an ordinary refrigerator. Freezing the protein solutions was always complicated and had to be evaluated for each protein in the corresponding solvent. To keep the proteins in their native state a gentle freezing temperature should be chosen, hence liquid nitrogen should be avoided. Furthermore, a high-resolution continuum source atomic absorption spectrometry method was developed to observe the adsorption of proteins on container material and chromatographic columns. Adsorption to any container led to a sample loss and lowered the recovery rates. During the pretreatment and high-performance size-exclusion chromatography, adsorption caused sample losses of up to 33%.

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis.

Since the introduction about 30 years ago, CE techniques have gained a significant impact in pharmaceutical analysis. The present review covers recent advances and applications of CE for the analysis of pharmaceuticals. Both small molecules and biomolecules such as proteins are considered. The applications range from the determination of drug-related substances to the analysis of counterions and the determination of physicochemical parameters. Furthermore, general considerations of CE methods in pharmaceutical analysis are described.

Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 3: anions.

The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, ß-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well.

Eco-friendly ionic liquid based ultrasonic assisted selective extraction coupled with a simple liquid chromatography for the reliable determination of acrylamide in food samples.

Acrylamide in food has drawn worldwide attention since 2002 due to its neurotoxic and carcinogenic effects. These influences brought out the dual polar and non-polar characters of acrylamide as they enabled it to dissolve in aqueous blood medium or penetrate the non-polar plasma membrane. In the current work, a simple HPLC/UV system was used to reveal that the penetration of acrylamide in non-polar phase was stronger than its dissolution in polar phase. The presence of phosphate salts in the polar phase reduced the acrylamide interaction with the non-polar phase. Furthermore, an eco-friendly and costless coupling of the HPLC/UV with ionic liquid based ultrasonic assisted extraction (ILUAE) was developed to determine the acrylamide content in food samples. ILUAE was proposed for the efficient extraction of acrylamide from bread and potato chips samples. The extracts were obtained by soaking of potato chips and bread samples in 1.5 mol L(-1) 1-butyl-3-methylimmidazolium bromide (BMIMBr) for 30.0 and 60.0 min, respectively and subsequent chromatographic separation within 12.0 min using Luna C18 column and 100% water mobile phase with 0.5 mL min(-1) under 25 °C column temperature at 250 nm. The extraction and analysis of acrylamide could be achieved within 2h. The mean extraction efficiency of acrylamide showed adequate repeatability with relative standard deviation (RSD) of 4.5%. The limit of detection and limit of quantitation were 25.0 and 80.0 ng mL(-1), respectively. The accuracy of the proposed method was tested by recovery in seven food samples giving values ranged between 90.6% and 109.8%. Therefore, the methodology was successfully validated by official guidelines, indicating its reliability to be applied to analysis of real samples, proven to be useful for its intended purpose. Moreover, it served as a simple, eco-friendly and costless alternative method over hitherto reported ones.

Determination of catechin isomers in human plasma subsequent to green tea ingestion using chiral capillary electrophoresis with a high-sensitivity cell.

A simple and reliable analytical electrophoretic method using chiral capillary electrophoresis (CCE) with a high-sensitivity cell of special design has been established for simultaneous determination of (+)-catechin (C) and (-)-epicatechin (EC) in aqueous and human plasma media. The application of a capillary with high-sensitivity cell has led to an improvement of 10-fold and 5-fold time-corrected peak area over a standard cell and a capillary with bubble cell, respectively. Analysis has involved the electrophoretic separation of C and EC in less than 4.0 min at 210 nm. The running buffer consist of 50.0 mmol L(-1) borate buffer with 1.0 mmol L(-1) beta-cyclodextrin at pH 8.5. CCE system has been proved for its intended use by applying procedure starting from calibration of CE instrument into validation of all experimental parameters. The resolution between catechin isomers under optimal conditions has been found to be more than 3.0. The detection limits of C and EC have been calculated to be 3.2 and 1.0 ng mL(-1), respectively. Good linearity has been obtained with correlation coefficient (r(2)) ranging between 0.995 and 0.996 at 99% confidence level (CL). Application of the proposed method to human plasma after ingestion of green tea has successfully been achieved and has statistically been proved. The unchanged amounts of C and EC in plasma were about 17.4 and 1.8% of the administered dose after 2 h of starting tea ingestion. The detection limits of C and EC in human plasma at 210 nm were 4.1 and 1.5 ng mL(-1), respectively.

Selective determination of catechin, epicatechin and ascorbic acid in human urine using chiral capillary electrophoresis.

Chiral CE was successfully applied to the separation and quantification of catechin, epicatechin and ascorbic acid in some commercial drinks and human urine. Analysis involved the separation of analytes in less than 5.0 min at 240 nm with an untreated fused-silica capillary under hydrodynamic injection mode. The running buffer consisted of 50 mM borate buffer with 3 mM beta-CD at pH 8.35. Detection limits for catechin, epicatechin and ascorbic acid were 0.028, 0.011 and 0.004 microg/mL, respectively. Linearity was investigated by selecting the ranges of calibration according to the amount of analytes in urine giving correlation coefficient percent (% r(2)) ranging between 99.4 and 99.6 at 99% confidence level. The maximum urinary excretion of catechin and epicatechin were noted at 2.0 and 4.0 h of the administrated dose. Unchanged catechin, epicatechin and ascorbic acid amounted to about 1.500, 8.696 and 0.003% of the administered dose in the 48.0 h urine collection. The proposed method achieved 99.2% completeness (n = 20) in urine media.

Selective and sensitive hydroxypropyl-beta-cyclodextrin based sensor for simple monitoring of (+)-catechin in some commercial drinks and biological fluids.

(+)-Catechin (CAT) was considered as a polyphenolic compound abundantly contained in plants. It exerts protective effect against cancer, inflammatory and cardiovascular diseases. These protective effects are mainly attributed to its antioxidative activity by scavenging free radicals. Therefore, the need of simple, selective and sensitive monitoring of (+)-catechin in commercial drinks and biological fluids is crucial. A new selective and sensitive voltammetric quantification of (+)-catechin was investigated at low cost hydroxypropyl-beta-cyclodextrin modified carbon paste sensor in acidic solutions. The constructed sensor was treated in simple and fast manner to increase its stability for catechin determination. The effect of solution and instrumental parameters was investigated by using osteryoung square-wave anodic voltammetry (OSWAV) at pH 2.20 and differential pulse cathodic voltammetry (DPCV) at pH 4.40 in 0.10 M Britton-Robinson buffer. Acidic solutions were chosen to increase the stability of (+)-catechin, reduce its adsorption on the sensor surface and increase the selectivity of proposed method. Cyclic voltammetry (CV) was used to elucidate the electrochemical mechanism of catechin at the modified electrochemical sensor. A linear range up to 7.20 and 4.20 microg mL(-1) of catechin was achieved in anodic and cathodic voltammetry, respectively. The method gave reproducible and reliable results with 1.50 g mL(-1) catechin (S.D. 0.062). Limit of detection of 0.12 and 0.30 ng mL(-1) and limit of quantification (LOQ) of 1.10 and 2.80 ng mL(-1) were easily achieved using anodic and cathodic voltammetry, respectively. Selectivity of the proposed procedure was estimated by testing recovery and adding the most interfering metal ions and/or organic compounds. The proposed method was applied successfully to selective determination of catechin in some commercial drinks like tea, cocoa and coffee with acceptable recovery range (98-102%). The extraction of catechin was rather simple, making it suitable for studies with a large number of commercial samples. Furthermore, the application to urine samples without pretreatment was achieved and statistically confirmed at 95% confidence level. It was easy to analyze catechin in urine down to 0.55 ng mL(-1).

Simultaneous micellar electrokinetic chromatography and liquid chromatography of adriblastina and tarabine PFS Their application to some biological fluids.

The current work presents analytical procedures for simultaneous determination of tarabine PFS and adriblastina by micellar electrokinetic chromatography (MEKC) and liquid chromatography (LC). For MEKC analysis, separations and identifications were accomplished using uncoated fused-silica capillary with hydrodynamic injections in the presence of 50mM borate/phophate pH 8.7 and 100mM SDS. The migration times of tarabine PFS and adriblastina were found to be 2.70 and 6.40min, respectively. Calibration curves were established for 10-300ng/mL (r=0.998) tarabine PFS and for 8-120microg/mL (r=0.999) adriblastina. For LC analysis, separations were performed on teicoplanin stationary phase with reversed mobile phase containing methanol:buffer pH 4.05 (20:80%, v/v) at 285nm. The retention times of tarabine PFS and adriblastina were 5.18 and 7.20min, respectively. Calibration curves were established for 3-90microg/mL (r=0.998) tarabine PFS and for 10-120microg/mL (r=0.999) adriblastina. Both MEKC and LC methods were applied for the simultaneous determination of analytes in urine samples.

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography.

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.