A statistical experimental design to study factors affecting enantioseparation of propranolol by capillary electrophoresis with cellobiohydrolase (Cel7A) as chiral selector

The capillary electrophoretic enantioseparation of rac-propranolol using cellobiohydrolase Tr Cel7A as selector was optimized by an unbiased statistical experimental design. A set of pre-experiments was performed in order to identify critical experimental factors. In the definitive chemometric design pH, ranging from 5 to 7, ionic strength ranging between 0.01 and 0.02 and organic solvent additive in concentration from 1 to 19% v/v were studied. The response surface plot revealed a separation optimum in the pH interval studied. When all parameters were taken into account, a background electrolyte consisting of 0.016 M bistris-acetate buffer with pH 6.5 and 17% v/v acetonitrile gave the optimum separation. The significance of the statistical design was confirmed by the generally good agreement obtained between predicted response and actual experimental data.     

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.     

Baseline separation of α and β‐acids homologues and isomers in hop (Humulus lupulus L.) by CD‐MEKC‐UV

An alternative method for simultaneous baseline separation of α and β‐acids homologues and isomers in hop by CD‐MEKC with UV detection was proposed. The optimized background electrolyte was composed of 30 mmol/L sodium tetraborate solution, 45 mmol/L sodium dodecyl sulfate, 20 mmol/L β‐cyclodextrin and 10% v/v acetonitrile. The instrumental conditions were evaluated by using a 3³ Box‐Benhken experimental design. In order to demonstrate the applicability of the method, 21 hop samples from different varieties were analyzed. The repeatability intra‐ and interday tests were performed and relative standard deviations lower than 7% for area and migration times were observed. The present method comprehended 8 min analysis time and revealed to be faster and more efficient when compared to previous reports from literature.     

Preparative and scaled‐up separation of high‐purity α‐linolenic acid from perilla seed oil by conventional and pH‐zone refining counter current chromatography

α‐Linolenic acid is an essential omega‐3 fatty acid needed for human health. However, the isolation of high‐purity α‐linolenic acid from plant resources is challenging. The preparative separation methods of α‐linolenic acid by both conventional and pH‐zone refining counter current chromatography were firstly established in this work. The successful separation of α‐linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n‐heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α‐linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by‐products. The scaled‐up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α‐linolenic acid by pH‐zone refining counter current chromatography was also satisfactory by the solvent system n‐hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH₄OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α‐linolenic acid from 18 000 mg free fatty acid sample. pH‐zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.     

Development of a stability-indicating capillary electrophoresis method for norfloxacin and its inactive decarboxylated degradant

A simple, accurate, precise, rapid and sensitive stability-indicating capillary electrophoresis (CE) method was optimized and validated for the simultaneous determination of norfloxacin and its inactive decarboxylated degradant in pharmaceuticals. The univariant method was used to optimize electrophoretic factors including injection time, separation voltage and column temperature. Electrolyte concentration and pH were optimized using the factorial design and response surface methods. The optimum conditions obtained were: 10 mmol l^− 1 phosphate at pH 2.5, hydrodynamic injection time of 8 s at pressure 0.5 p.s.i., separation voltage 25 kV and column temperature 25 °C. The separation was carried out into a fused-silica capillary column (31.2 cm length × 50 μm i.d.) with detection at 301 and 285 nm for the intact drug and the degradant, respectively using a diode array detector. For both analytes, the method enjoys wide dynamic range (1-50 μg ml^− 1) with good detectability (limits of detection 0.11 μg ml^− 1). In addition, acceptable accuracy (recovery > 95%); and good repeatability and intermediate precision (RSD < 3.5%) were obtained.     

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics

A method of capillary electrophoresis with contactless conductivity detection has been developed for non‐enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 μm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 μmol/L) for ketamine and 75 ng/mL (0.3 μmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 μg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 μL of plasma with 30 μL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.     

Examination of colour inkjet printing inks by capillary electrophoresis

The possibility of comparing inkjet printing inks by micellar electrokinetic capillary electrophoresis (MECC) with diode array detection was studied. An analytical procedure was designed and successfully applied to discriminate between the electrophoretic profiles of inks (extracted from paper) produced by five well-known manufacturers. The separation process was conducted in a polyimide-coated fused silica capillary (ID 50 μm, 60 cm total/50 cm effective length) with +30 kV high voltage applied. Background electrolyte was used of the following optimum composition: 40 mM sodium borate buffer, 20 mM sodium dodecyl sulphate(IV) (SDS) and 10% (v/v) acetonitrile (pH 9.56). The experimental conditions were adjusted in terms of resolution and analysis time. The best results were obtained at 10 and 25 °C storage and capillary temperature, respectively, using 25 dots (ø 0.8 mm) cut from printouts as the sample and BGE diluted with water (1:99, v/v) as the injecting solution. The MECC separation of main printing ink components by the proposed method showed excellent precision - the RSD value of the migration time calculated for each of the investigated peaks did not exceed 3.3%. The optimized method was applied to group identification and differentiation of: (a) three colours of printing inks, (b) inks from different manufacturers (Hewlett-Packard, Epson, Brother, Lexmark and Canon) and (c) inks from different printer models. In all these cases, inks were successfully differentiated on the basis of position (migration time) and shape of their characteristic peaks.     

Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

Capillary electrophoresis fingerprints combined with chemometric methods to evaluate the quality consistency and predict the antioxidant activity of Yinqiaojiedu tablet

The quality consistency of Yinqiaojiedu tablets (YQJDTs) was monitored by extracting their electrophoretic fingerprint and marker compound data that were obtained using capillary electrophoresis. To select the suitable background electrolyte, wing‐shape method was proposed. A background electrolyte composed of 103.1 mM boric acid, 51.6 mM sodium borate, 9.8 mM disodium hydrogen phosphate, and 15.6 mM sodium dihydrogen phosphate was used to separate compounds. Under the optimized conditions, the content of three marker compounds was determined in 25 YQJDT samples. The capillary electrophoresis fingerprints were developed simultaneously, then 25 samples from two manufacturers were clearly divided into two clusters based on the principal component analysis. In fingerprint assessments, systematic quantified fingerprint method was adopted for integrative quality discrimination of YQJDTs from both qualitative and quantitative perspectives, by which the qualities of 25 samples were well differentiated. In addition, partial least squares model was established to explore fingerprint–efficacy relationship between the fingerprints and the antioxidant activities in vitro, providing clinically useful information for quality control. The proposed method was reliable and comprehensive, which could be used for a valuable reference to monitor the quality consistency of traditional Chinese medicines.     

Investigation of sport supplements quality by Raman spectroscopy and principal component analysis

In this work, sport supplements were investigated by Raman spectroscopy. Samples were obtained from health foods shops, gyms and sports centers covering a wide range of available supplement powders. A systematic comparison of Raman spectra of the analyzed supplements allowed identifying the supplement type through the characteristic vibrational modes of carbohydrates and proteins. The protein supplements were identified by Raman bands at 1650, 1250 and 1004 cm⁻¹, while the spectral range between 1200 and 800 cm⁻¹ was useful to identify the carbohydrate supplements. Due to the diversity in composition of sport supplements, a chemometric tool such as principal component analysis (PCA) was employed to assist in the interpretation of Raman spectra, allowing also the identification of compounds present in sport supplements. Especially, the Raman scattering of aromatic and aliphatic amino acids residues contributes to the existence of bands characteristic for the different types of proteins. This kind of information is very important for the quality control of these products, for detecting the presence of fraud or a sample composition in disagreement with the label, thus ensuring the provenance of the supplements.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

Monitoring of circulating amino acids in patients with pancreatic cancer and cancer cachexia using capillary electrophoresis and contactless conductivity detection

Branched chain amino acids (BCAAs), alanine and glutamine are determined in human plasma by capillary electrophoresis with contactless conductivity detection (CE/C⁴D). The baseline separation of five amino acids from other plasma components is achieved on the short capillary effective length of 18 cm in 3.2 mol/L acetic acid with addition of 13% v/v methanol as background electrolyte. Migration times range from 2.01 min for valine to 2.84 min for glutamine, and LODs for untreated plasma are in the interval 0.7–0.9 μmol/L. Sample treatment is based on the addition of acetonitrile to only 15 μL of plasma and supernatant is directly subjected to CE/C⁴D. Circulating amino acids are measured in patients with pancreatic cancer and cancer cachexia during oral glucose tolerance test. It is shown that patients with pancreatic cancer and cancer cachexia syndrome exhibit low basal circulating BCAAs and glutamine levels and loss of their insulin-dependent suppression.     

Ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography for screening and isolating potential α‐glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α‐glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α‐glucosidase‐ and xanthine oxidase binding activities and one lactone with α‐glucosidase‐binding activity. Subsequently, with the help of high‐speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low‐toxicity two‐phase solvent system composed of ethyl acetate/n‐butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography is not only a powerful tool for screening and isolating α‐glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.     

New perspective of diabetes response to an antioxidant treatment through metabolic fingerprinting of urine by capillary electrophoresis

Benefits of capillary electrophoresis to provide a comprehensive snapshot of multiple metabolites in biological samples have been exploited. Afterwards, multivariate statistical methods can be employed in order to mine additional information from the data. Urine fingerprints of control and diabetic rats have shown the clear effects of an antioxidant treatment on diabetic animals, which were not seen in controls, in a rapid, simple and cost-effective way without identifying a single marker. The procedure involves the measurement of samples with a relatively inexpensive tool such as CE-UV, without any previous treatment other than filtration and the application of chemometric tools [PCA (principal components analysis) and PLS-DA (partial least squares discriminant analysis)]. Data pre-treatment of electrophoretic profiles (alignment, normalization and baseline correction) has shown to be key for further chemometric treatment. Once developed, the methodology can easily be applied for a rapid in vivo screening of extracts with potential in vitro activity. Classification was supported by that produced after PCA and PLS-DA of target variables obtained with selectively designed, time and reagent consuming methods.     

Fast GC analysis of major volatile compounds in distilled alcoholic beverages: Optimisation of injection and chromatographic conditions

The principal secondary flavour compounds in distilled spirits can be successfully quantified by split injection to a 0.15 mm internal diameter (I.D.) capillary column. Initial conditions for split ratio, gas velocity, initial oven temperature and oven ramp rate are given by method translation from a similar method on a standard 0.25 mm internal diameter column with the same phase. These parameters were then investigated in an experimental design comprising a series of experiments in which the responses were the resolution of two critical peak pairs, the analysis time and the limit of quantification (LOQ) of the eight major compounds. The LOQ is the concentration corresponding to a signal 10 times greater than the noise. The experiments were replicated at two different concentration levels, which encompassed the natural levels of the compounds of interest found in distilled spirits. From the chemometric evaluation of these data, a validated model was constructed, which allowed the prediction of conditions for optimum chromatographic analysis. Three additional concentration levels were then added to the model to establish linearity, repeatability and sensitivity. Modern gas chromatographic hardware allows the use of these narrow-bore capillary columns for routine use without operational difficulties. Major advantages are a substantial decrease in analysis time allowing high throughput processing of samples.     

Determination of xanthohumol in hops (Humulus lupulus L.) by nonaqueous CE

Xanthohumol (XN) is a prenylated chalcone with antimutagenic and anticancer activity from hops. A nonaqueous reverse polarity capillary electrophoretic method for the determination of XN in hop extract was developed and validated. The optimal parameters were a 64.5 cm long fused-silica capillary with 50 microm id at 25 degrees C; 30 kV negative voltage (anode at detector side of the capillary); nonaqueous buffer with 75 mM NaOH and 50 mM boric acid in methanol; hydrodynamical injection with 10 mbar for 40 s; and detection at 440 nm. XN, isoxanthohumol (IX), colupulone, adlupulone, and n-lupulone were well resolved on the electropherogram. The LOD for XN was 0.05 mg/L and RSD for peak area was below 3%. The amount of XN in different samples of hop pellets varied from 0.14 to 0.42%.     

Development and validation of a capillary electrophoretic method for the determination of amiodarone and desethylamiodarone

Summary A simple, sensitive and rapid capillary electrophoretic method has been developed for the separation and quantification of amiodarone and its metabolite, desethylamiodarone. The compounds were separated in a capillary of 45 cm effective length and 75 μm i.d., by use of an applied voltage of 25 kV and an electrolyte containing 15mm ADA buffer (pH 7.5), 10mm SDS, and 70% (v/v) acetonitrile. The selectivity, precision, linearity, range, sensitivity, and robustness of the method were good. The applicability of the assay was demonstrated by analyzing these drugs in serum. Electrokinetic injection with field-amplified sample-stacking was used to increase sensitivity. The limit of detection of the serum assay was 6.46 ng mL⁻¹ and the precision 3.7%.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.