Metabolic fingerprinting of Schistosoma mansoni infection in mice urine with capillary electrophoresis

Schistosoma mansoni infection in mice has been fingerprinted using CE to study the capabilities of this technique as a diagnostic tool for this parasitic disease. Two modes of separation were used in generating the electrophoretic data, with each untreated urine sample the following methods were applied: (i) a fused-silica capillary, operating with an applied potential of 18 kV, in micellar EKC (MEKC) and (ii) a polyacrylamide-coated capillary, operating with an applied potential of -20 kV under zonal CZE conditions. By combining normal and reverse polarities in the data treatment we have extracted more information from the samples, which is a better approach for CE metabolomics. The traditional problems associated with variability in electrophoretic peak migration times for analytes were countered by using a dynamic programming algorithm for the electropherograms alignment. Principal component analyses of these aligned electropherograms and partial least square discriminant analysis (PLS-DA) data are shown to provide a valuable means of rapid and sample classification. This approach may become an important tool for the identification of biomarkers, diagnosis and disease surveillance.     

Improvements in pentosan polysulfate sodium quality assurance using fingerprint electropherograms

Complex samples from polymer production, plant extracts or biotechnology mixtures can be characterized by fingerprints. Currently, the standard approach for sample characterization employs near-infrared (NIR) spectroscopy fingerprinting. Up to now, however, fingerprints obtained by chromatography or electrophoresis could only be visually evaluated. This type of inspection is very labor-intensive and difficult to validate. In order to transfer the use of fingerprints from spectroscopy to electrophoresis, spectra-like properties must be obtained through a complete alignment of the electropherograms. This has been achieved by interpolation and wavelet filtering of the baseline signal in the present work. The resulting data have been classified by several algorithms. The methods under survey include self-organizing maps (SOMs), artificial neural networks (ANNs), soft independent modeling of class analogy (SIMCA) and k-nearest neighbors (KNNs). In order to test the performance of this combined approach in practice, it was applied to the quality assurance of pentosan polysulfate (PPS). A recently developed capillary electrophoresis (CE) method using indirect UV detection was employed in these studies [1]. All algorithms were well capable of classifying the examined PPS test batches. Even minor variations in the PPS composition, not perceptible by visual inspection, could be automatically detected. The whole method has been validated by classifying various (n = 400) unknown PPS quality assurance samples, which have been correctly identified without exception.     

Forensic analysis of condom and personal lubricants by capillary electrophoresis

Condoms may offer sexual assailants a simple and relatively effective means by which they may remove and dispose of the biological evidence of their contact with the victim. Without this valuable probative evidence, the investigator may need to turn to such alternative forms of physical evidence, such as the residue remaining from the condom lubricant, and any other additional lubricating substances, which may have been used by the assailant. In this study, 68 different condom and personal lubricant samples were analysed using micellar electrokinetic capillary chromatography (MEKC) with ultraviolet absorbance detection. The electropherograms were processed by principal component analysis (PCA) and classified with linear discriminant analysis. This classified 233 samples out of 263 into their correct sample group. An alternative method of classification in which the sample electropherograms were converted to vectors and a correlation was determined by the cosine of the angles between these vectors. This correctly classified 172 samples of the 296. Results indicate that the combination of CE with chemometric data treatment offers the potential of being a rapid and efficient means by which condom and personal lubricant samples may be differentiated. Although this analytical method at present lacks the sensitivity required for sexual assault swab analysis, it is hoped that future developments in instrumentation detection capabilities will permit its use one day as a routine casework tool.     

Determination of orotic acid in aqueous solutions by micellar electrokinetic capillary chromatography using sensitized lanthanide-ion luminescence detection

Summary A sensitive and selective laser-induced luminescence detection scheme for orotic acid in urine, separated by micellar electrokinetic capillary chromatography (MEKC) has been developed. The 325 nm line from a helium cadmium laser is used to excite orotic acid, which transfers its energy to terbium. Resultant luminescence of terbium is linear with orotic acid concentration over more than 2.5 orders of magnitude. This novel and practical system enables the detection of 50 nm orotic acid in urine in less than 1.5 minutes while using only nanoliters of sample. The significant decrease in analysis time over traditional methods (spectrophotometric and chromatographic) comes from the high efficiency of MEKC. A dramatic improvement in sensitivity and selectivity over UV detection in capillary electrophoresis is achieved through the use of laser-induced lanthanide ion energy transfer luminescence detection. Finally, no sample pretreatment is needed and the method is free from any known interferences in urine.     

Partial-filling micellar electrokinetic chromatography and non-aqueous capillary electrophoresis for the analysis of selected agrochemicals

Selected agrochemicals (s-triazines and phenoxy acids) have been investigated with partial-filling micellar electrokinetic chromatography (PFMEKC) and non-aqueous capillary electrophoresis (NACE). Because these two techniques are compatible for coupling of capillary electrophoresis with mass spectrometry, different conditions affecting the separation efficiency (reproducibility, method linearity) were systematically tested, and the results were compared with those from classical MEKC. The conditions tested included buffer molarity, pH, the concentrations of the organic modifier and surfactant, the applied voltage, the injection time of the sample, and the length of the partial-filling plug. The respective limits of detection (LOD) using UV-detection were determined. Reduction of the electrophoretic raw data using the mobility scale transformation (micro-scale) improved qualitative comparison of the electropherograms and the reproducibility of quantitative data (integrated peak area) thus extending this data treatment from CZE to other endoosmotic flow-driven CE-techniques such as PFMEKC and NACE.     

Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were applied to the simultaneous separation of cyanobacterial toxins (anatoxin-a, microcystin-LR, cylindrospermopsin). The analytical performance data of both methods, optimized for the three toxins, were similar with a precision of migration times smaller than 0.8 RSD% and a detection limit in the range of 1-4 microg/mL, using spectrophotometric detection at 230 nm. Both methods were applied to an analysis of cyanotoxins in water bloom samples and crude cyanobacterial extracts. The results obtained indicate that, for complex matrices, the sequential application of CZE and MEKC is necessary. It is recommended to use both CE techniques for the analysis of the same sample in order to confirm the results by an orthogonal approach.     

Analysis of substances to be used as internal standards in MEKC

The use of internal standards (ISs) improves the quantitative performance of CE. However, ISs chosen for use in CZE often cannot be used for micellar EKC (MEKC). Therefore 22 substances were investigated as potential ISs in MEKC. These substances were selected based upon a literature search. The substances were investigated using a method similar to the standard operating conditions for MEKC as recommended by S. Terabe. Furthermore, the migration time and the corrected migration time (k(S)) were determined for each substance to establish the migration position relative to other peaks in the electropherograms. A combination of eight substances, selected according to the obtained results (t(m) = 4 up to 12 min), was tested for practical benefit and applicability. The peak area precision was in the range of 0.8 and 1.2% (n = 60), and the peaks were well shaped for all the investigated substances. The selected substances covered a wide migration time window and therefore they can be regarded as suitable for future analysis at any required migration position.     

Pyrene-assisted synthesis of size-controlled gold nanoparticles in sodium dodecyl sulfate micelles

Gold nanoparticles prepared by chemical reduction in sodium dodecyl sulfate (SDS) solution are size-controlled with the addition of pyrene. Micellar electrokinetic capillary chromatography (MEKC) is applied to the system to examine the size and polydispersity of gold nanoparticles and to show that pyrene has the extraordinary effect in decreasing the size and narrowing the dispersity of gold nanoparticles. The MEKC electropherograms further suggest that pyrene could be oxidized by the aqueous Au(III) complexes first. All the reduced Au complexes were then solubilized in the pyrene-SDS micelles. The growth of gold nanoparticles beyond the embryonic stage was subsequently inhibited by the encapsulating SDS and electrophilic pyrene.     

UV light-emitting diode-induced fluorescence detection combined with online sample concentration techniques for capillary electrophoresis.

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.     

Simultaneous resolution of overlapping peaks in high-performance liquid chromatography and micellar electrokinetic chromatography with diode array detection using augmented iterative target transformation factor analysis

In this paper, augmentation has been applied to data matrices, which originate from hyphenated methods that share the same mode of detection, but use different separation methods, HPLC-DAD and MEKC-DAD. A novel method, wavelength shift eigenstructure tracking (WET), has been proposed for the alignment between the wavelength scale of both detectors. WET proves to be suitable for the detection as well as correction of wavelength shift between both detectors. After correction of the wavelength scale, data obtained on both systems have been augmented and submitted to iterative target transformation factor analysis. Augmented curve resolution provides significantly better estimates of the chromatographic and electrophoretic profiles and spectra than the use of non-augmented curve resolution on HPLC and MEKC data separately. It is particularly useful when the pure fraction of a chromatographic peak is less than 0.10. Finally, the relative weight of MEKC versus HPLC in augmentation may be increased using intensity and noise normalisation. However, since noise normalisation and its accompanying decrease in signal-to-noise ratio leads to a loss of information, and, since intensity normalisation may cause a failure of the augmented curve resolution algorithm, benefits and drawbacks of normalisation should be weighed on a case-by-case basis.     

Characterization of polyethylene glycols and polypropylene glycols by capillary zone electrophoresis and micellar electrokinetic chromatography

Methods based on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) have been developed and optimized for the separation of polyethylene glycols (PEGs) and polypropylene glycols (PPGs). To provide for charge and detectability both types of polymeric compounds were derivatized with phthalic anhydride (PhAH) or 1,2,4-benzenetricarboxylic anhydride (BTA) before the separation. Derivatization with BTA yielded more complex electropherograms, due to the occurrence of different isomeric reaction products for every PEG or PPG species. Effective mobilities of the PhAH derivatives were related to the number of monomer units in the polymers in a straightforward way. The CZE method could also be used to determine the monomer-number distribution of random and block PEG-PPG copolymers. For an MEKC analysis the PEGs and PPGs were derivatized with phenyl isocyanate. Oligomers of PEGs could be separated up to molecular masses of 5,000, while for the more hydrophobic PPGs oligomeric separation was only accomplished for molecular masses of up to 1,500. Due to a strongly different separation mechanism for the PEG and PPG derivatives in the MEKC system. a complete group separation of the two types of polymer molecules could be obtained.     

Determination of 5-nitroimidazoles and metabolites in environmental samples by micellar electrokinetic chromatography

A method based on micellar electrokinetic chromatography (MEKC) with UV detection has been developed for the determination of nine 5-nitroimidazoles (5-NDZs), including metabolites in river water samples. Due to the relative insensitivity of UV detection in MEKC, a solid-phase extraction (SPE) method has been proposed that preconcentrates water samples fiftyfold and cleans them up off-line. An on-line preconcentration approach based on sweeping and the use of an extended light path fused-silica capillary (64.5?cm?×?50?μm i.d., 56?cm effective length) was also found to improve the sensitivity of the method. Separation was carried out in <21?min using 20?mM phosphate buffer (pH 6.5) and 150?mM SDS as the background electrolyte (BGE). The temperature of the capillary was kept constant at 20°C, a voltage of 25?kV was applied (normal mode), and a detected wavelength of 320?nm was utilized. Hydrodynamic injection (50?mbar for 15?s) of the samples, which were dissolved in 20?mM phosphate (pH 6.5), was employed. The limits of detection were lower than 1.1?μg?L(-1). Recoveries of >80% from spiked river water samples were obtained for most of the analytes at three different concentration levels with acceptable precision. This method could provide an efficient and economical alternative to the use of chromatographic methods to monitor nitroimidazole residues, thus supplementing the relatively few methods available for the analysis of these compounds in environmental samples.     

Using micellar electrokinetic chromatography for the highly efficient preconcentration and separation of gold nanoparticles

In this paper, we report the use of micellar electrokinetic chromatography (MEKC) for the highly efficient preconcentration and separation of gold nanoparticles (Au NPs). We used the reversed electrode polarity stacking mode (REPSM) of the MEKC system for the on-line enhancement and separation of the Au NPs. Several parameters had dramatic effects on the systems’ performance, including the concentration of sodium dodecylsulfate (SDS) surfactant, the presence of salts in the NP solution, the pH of the running electrolyte, and the temperature of the capillary. Under the optimized conditions [buffer: SDS (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 10.0; applied voltage: 20 kV; operating temperature: 25 °C; additive: sodium dihydrogenphosphate (NaH₂PO₄, 10 mM); REPSM strategy for sample preconcentration], the number of theoretical plates for the 5.3- and 40.1-nm-diameter Au NPs were 3000 and (an ultrahigh) 2.1 × 10⁶, respectively; in addition, the detection sensitivities toward the Au NPs were enhanced ca. 20- and 380-fold, respectively, relative to those obtained using standard MEKC analysis conditions. Furthermore, monitoring the electropherograms using diode-array detection allowed us to identify and characterize the sizes of the separated NPs from their UV–vis spectra. Our findings suggest that MEKC is a highly efficient tool for both the preconcentration and separation of NPs.     

Determination of itraconazole and hydroxyitraconazole in human serum and plasma by micellar electrokinetic chromatography

The electrokinetic separation of the hydrophobic antimycotic drug itraconazole (ITC) and its major metabolite, hydroxyitraconazole (HITC), by a binary aqueous-organic solvent medium containing sodium dodecylsulfate, by microemulsion electrokinetic chromatography (MEEKC) and by micellar electrokinetic chromatography (MEKC) was studied. The results suggest that the first approach is difficult to apply and that there is no substantial difference between separations performed using MEEKC and MEKC modified with n-butanol. The simpler MEKC method is more than adequate and was thus employed for the analysis of ITC and HITC in human serum and plasma. Separation was achieved in plain fused-silica capillaries having a low-pH buffer (pH 2.2) with sodium dodecyl sulfate micelles and reversed polarity. The addition of 2-propanol and n-butanol enhanced analyte solubility and altered the selectivity of the separation by influencing the magnitude of the electrophoretic component in the separation mechanism. Under optimised conditions and using head-column field-amplified sample stacking, an internal standard, ITC and two forms of HITC could be separated in under 9 min, with detection limits less than 0.01 microg/mL. Analysis of samples from patients currently prescribed ITC revealed a different HITC peak area ratio to that of the standards, suggesting a stereoselective component of ITC metabolisation. Comparison of MEKC data with those of a HPLC method employed on a routine basis showed excellent agreement, indicating the potential of this approach for therapeutic drug monitoring of ITC.     

Molecularly imprinted SPE and MEKC with in-capillary sample preconcentration for the determination of digoxin in human urine

Molecularly imprinted solid-phase extraction (MISPE) combined with MEKC was used for clean-up, preconcentration and determination of digoxin in the presence of its aglycon digoxin (digoxigenin) in human urine samples. In addition, the use of an in-capillary sample concentration electrophoretic technique by sweeping was investigated to enhance the concentration sensitivity in MEKC. The highly selective, fast and effective sample pretreatment by MISPE along with the preconcentration by sweeping could overcome the low sensitivity of the highly efficient capillary electrophoresis separation with UV detection. The optimization of the variables affecting the separation as well as MISPE conditions procedure was carried out to select the best conditions of selectivity and sensitivity to determine digoxin at low concentration levels in urine. To demonstrate the suitability of the developed method several analytical characteristics (selectivity, linearity, accuracy, precision, and LOD) were evaluated. Satisfactory results were obtained in terms of linearity (r > 0.99), recovery (95.4-96.5% with RSD from 1.3% to 2.6%), precision (RSD from 0.3% to 1.7% for migration times and from 2.1% to 7.3% for corrected peak areas), and sensitivity (LODs of 6 μg/L with 5 mL of sample or 1.2 μg/L with 25 mL). The proposed MISPE-MEKC method was satisfactorily applied to the analysis of spiked human urine samples achieving a concentration factor up to 7500-fold.     

Low-parachor solvents extraction and thermostated micro-thin-layer chromatography separation for fast screening and classification of spirulina from pharmaceutical formulations and food samples

The goal of this paper is to demonstrate the separation and detection capability of eco-friendly micro-TLC technique for the classification of spirulina and selected herbs from pharmaceutical and food products. Target compounds were extracted using relatively low-parachor liquids. A number of the spirulina samples which originated from pharmaceutical formulations and food products, were isolated using a simple one step extraction with small volume of methanol, acetone or tetrahydrofuran. Herb samples rich in chlorophyll dyes were analyzed as reference materials. Quantitative data derived from micro-plates under visible light conditions and after iodine staining were explored using chemometrics tools including cluster analysis and principal components analysis. Using this method we could easily distinguish genuine spirulina and non-spirulina samples as well as fresh from expired commercial products and furthermore, we could identify some biodegradation peaks appearing on micro-TLC profiles. This methodology can be applied as a fast screening or fingerprinting tool for the classification of genuine spirulina and herb samples and in particular may be used commercially for the rapid quality control screening of products. Furthermore, this approach allows low-cost fractionation of target substances including cyanobacteria pigments in raw biological or environmental samples for preliminary chemotaxonomic investigations. Due to the low consumption of the mobile phase (usually less than 1 mL per run), this method can be considered as environmentally friendly analytical tool, which may be an alternative for fingerprinting protocols based on HPLC machines and simple separation systems involving planar micro-fluidic or micro-chip devices.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

On-line preconcentration and determination of ketamine and norketamine by micellar electrokinetic chromatography. Complementary method to gas chromatography/mass spectrometry

We have investigated a rapid, simple, and highly efficient on-line preconcentration method using in micellar electrokinetic chromatography (MEKC) for the analysis of abused drugs. Ketamine is an anesthetic that has been abused as a hallucinogen. We applied the sample sweeping technique first to ketamine and its major metabolite, norketamine, and separated the analytes with MEKC. Several of the sweeping MEKC parameters to effect successful separations, such as the concentration of sodium dodecyl sulfate (SDS), the injection time, and the applied voltage were optimized. The improvements in the number of theoretical plates under the different separation conditions are presented clearly in a three-dimensional representation. The limits of detection were 2.8, 3.4, and 3.3 ng/mL for ketamine, norketamine, and ketamine-D(4), respectively. The enrichment factor for each compound was within the range of 540-800. Experimental results are in agreement with those of analysis conducted by gas chromatography/mass spectroscopy (GC/MS). Therefore, we believe that sweeping, combined with MEKC, represents a suitable complementary method to GC/MS for use in clinical and forensic analyses of ketamine and norketamine.     

Determination of nikethamide by micellar electrokinetic chromatography

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)–UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000 g ), separation by MEKC and ultraviolet‐absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 μA) using a standard fused‐silica capillary (effective length 51 cm, 75 μm i.d.). The determined limit of detection for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4–280 μmol/L (0.71–49.90 μg/mL), with R² 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.     

Concentration and determination of cotinine in serum by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), combined with on-line concentration techniques, cation-selective exhaustive injection (CSEI) and sweeping, was developed for the analysis of cotinine, the primary biomarker for exposure to secondhand smoke. Experimental parameters including sample matrix, surfactant concentration, injection length and concentration of high-conductivity buffer, and sample electrokinetic injection time were optimized for electrophoretic enrichment and separation processes. Under the optimal conditions, the detection sensitivity of cotinine was enhanced by about 5000-fold using CSEI-sweeping MEKC compared to normal MEKC. The limit of detection for cotinine was found to be 0.2 ng/mL using ultraviolet absorbance detection. Furthermore, the developed method was successfully applied to the detection of cotinine in mouse serum samples.