Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.     

Separation of perfluorocarboxylic acids using capillary electrophoresis with UV detection

A capillary electrophoretic method with UV detection for separation and quantitation of perfluorocarboxylic acids (PFCAs) from C6-PFCA to C12-PFCA has been developed. The optimization of measurement conditions included the choice of the most appropriate type and concentration of buffer in the background electrolyte (BGE), as well as the type and the content of an organic modifier. The optimal separation of investigated PFCAs was achieved with 50 mM phosphate buffer and 40% isopropanol in the BGE using direct UV detection. The optimum wavelength for direct UV detection was optimized at 190 nm. For indirect detection, several chromophores were studied. Five mM 3,5-Dinitrobenzoic acid (3,5-DNBA) in 20 mM phosphate buffer BGE and indirect UV detection at 280 nm gave the optimal detection and separation performance for the investigated PFCAs. The possibility of on-line preconcentration of solutes by stacking has been examined for indirect detection. The detection limits (LODs) determined for direct UV detection ranged from 2 microg/mL for C6-PFCA to 33 microg/mL for C12-PFCA. The LODs obtained for indirect UV detection were comparable to those obtained for direct UV detection.     

Determination of chlorophenols in human urine based on the integration of on-line automated clean-up and preconcentration unit with micellar electrokinetic chromatography.

A new method was developed and validated for the determination of chlorophenols in human urine by using micellar electrokinetic chromatography (MEKC) coupled via a mechanic arm to an on-line automatic clean-up and preconcentration unit for urine samples. Separation is accomplished by using a selective buffer consisting of 15 mM borate, 25 mM phosphate and 100 mM sodium dodecyl sulfate (SDS) at pH 9.1 in addition to a positive power supply of 25 kV at 18 degrees C. The proposed capillary electrophoresis (CE) method allows the separation of 11 chlorophenols within 7 min with a reproducibility as relative standard deviation (RSD) between 2.6% and 7.2%, and limits of detection (LODs) between 0.08 and 0.46 microg/mL for all chlorophenols. Urine samples were previously hydrolyzed with 37% HCl at 80 degrees C for 60 min and then cleaned on a C-18 mini-column. Recoveries ranged from 58% to 103%. The preconcentration treatment affords limits of determination between 4 and 12 ng/mL for all chlorophenols except pentachlorophenol and 4-chlorophenol, which could not be determined. The overall analysis time, including on-line clean-up, preconcentration and electrophoretic separation is 20 min per sample.     

Determination of synthesized alpha-vitamin E by micellar electrokinetic chromatography

We developed a micellar electrokinetic chromatography method (MEKC) for the direct determination of the content of synthesized alpha-vitamin E. It was found that under the optimum separation conditions 7 mM borate + 14 mM phosphate + 15 mM sodium dodecyl sulfate (SDS) + 10 mM sodium cholate (NaCh) + 8% acetonitrile (pH 9.2) with UV detection wavelength at 214 nm, 16 kV constant voltage, and 26 degrees C constant temperature, alpha-vitamin E and its isomers can be baseline separated and alpha-vitamin E was quantitatively analyzed. In addition, the sample recovery, the limit of detection and the repeatability of the method were investigated. The influence of various parameters on the separation such as SDS concentration, NaCh concentration, buffer pH and acetonitrile percentage were also discussed.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Quantitation of transdermal tadalafil in human skin by reversed-phase high-performance liquid chromatography

A reliable and sensitive HPLC method was developed for the quantitation of tadalafil transdermal permeation through human skin. An RP column with UV detection at 290 nm was used for chromatographic separation at ambient temperature. The mobile phase was acetonitrile-water containing 20 mM pH 7 phosphate buffer (35/65, v/v) with a flow rate of 1.0 mL/min. The LOQ achieved was 1 ng/mL, and the calibration curve showed good linearity over the concentration range of 5-2000 ng/mL for tadalafil, with a determination coefficient (R2) of 0.998. The RSD values of intraday and interday analyses were all within 7%. Parameters of validation proved the precision of the method; this validated method was applied for the determination of tadalafil in transdermal permeation and drug deposition in human skin studies.     

Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking–sweeping capillary electrophoresis

This study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)–sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60 cm) was filled with a high concentration of background electrolyte (150 mM phosphate, pH?3.5, containing 15 % methanol), followed by a large volume of samples (10 psi, 20 s) with low-concentration buffers (5 mM phosphate, pH?3.5, with 5 % methanol). When high voltage was applied (?20 kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150 mM phosphate, pH?3.5) with 15 % methanol and 50 mM SDS added, and the detection was performed by UV at 214 nm. The method was validated for linearity (r?≧?0.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03–25 and 0.03–3 μg/mL. The limits of detection (S/N?=?3) were 0.01 μg/mL for each analyte. Compared with simple MEKC (0.5 psi, 5 s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200 mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82 μg/mL.

Detection of caffeine and its main metabolites for early diagnosis of Parkinson's disease using micellar electrokinetic capillary chromatography

Caffeine (CA) is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of CA and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease (PD) patients. In this study, we demonstrated a new MEKC method for determining CA and its three main downstream metabolites, paraxanthine (PX), theobromine (TB), and theophylline (TP), in human plasma. Plasma samples were collected, and analyzed using MEKC, after SPE. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for CA, 5.0 ng/mL for TB, and 4.0 ng/mL for both PX and TP. The recoveries were between 88.0% and 105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early PD than in control subjects (p < 0.05). The area under curve was improved to 0.839 when CA and its three main metabolites were included, suggesting that MEKC testing of CA, TP, TB, and PX may serve as a potential method for early diagnosis of PD.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

Analysis of green tea extract dietary supplements by micellar electrokinetic chromatography

Dietary supplements are growing in popularity as a source of catechins such as epigallocatechin gallate (EGCG). The first determination of five catechins in green tea extract dietary supplements using an extraction followed by micellar electrokinetic chromatography (MEKC) with UV detection is presented here. The optimum run buffer is 5 mM borate-60 mM phosphate with 50 mM SDS at pH 7.00 with detection at 210 nm. The limit of detection is 2-3 microg/mL (S/N=3) and the limit of quantitation is 6-8 microg/mL (S/N = 10). Results indicate that the amount of catechins varies greatly among manufacturers, between capsules of the same manufacturers, and between batches.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

Analysis of repaglinide enantiomers in pharmaceutical formulations by capillary electrophoresis using 2,6-di-o-methyl-β-cyclodextrin as a chiral selector

This study used the general applicability of 2,6-didi-o-methyl-β-cyclodextrin (DM-β-CD) as the chiral selector in capillary electrophoresis for fast and efficient chiral separation of repaglinide enantiomers. A systematic study of the parameters affecting separation was performed with UV detection at 243 nm. The optimum conditions were determined to be 1.25% (w/v) DM-β-CD in 20 mM sodium phosphate (pH 2.5) as the running buffer and separation voltage at 20 kV. DM-β-CD had the best enantiomer resolution properties under the tested conditions, whereas other β-cyclodextrins showed inferior performances or no performance. The proposed method had a linear calibration curve in the concentration range of 12.5-400 μg/mL. The limit of detection was 100 ng/mL. The intra-day and inter-day precisions were 2.8 and 3.2%, respectively. Recoveries of 97.9-100.9% were obtained. The proposed method was fast and convenient, and was determined to be efficient for separating enantiomers and applicable for analyzing repaglinide enantiomers in quality control of pharmaceutical production.     

Using sweeping micellar electrokinetic chromatography to analyze Delta9-tetrahydrocannabinol and its major metabolites

We have applied sweeping micellar electrokinetic chromatography (sweeping-MEKC) to the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). We monitored the effects of several of the sweeping-MEKC parameters, including the proportion of organic modifier, the concentration of sodium dodecyl sulfate (SDS), the pH, and the sample injection volume, to optimize the separation process. The optimal buffer for the analysis of the three analytes was 25 mM citric acid/disodium hydrogenphosphate (pH 2.6) containing 40% methanol and 75 mM SDS. Under the optimized separation parameters, the enrichment factors for THC, THC-COOH, and THC-OH when using sweeping-MEKC (relative to MEKC) were 77, 139, and 200, respectively. The limits of detection (LODs) for the three compounds in standard solutions ranged from 3.87 to 15.2 ng/mL. We combined the sweeping-MEKC method with solid-phase extraction to successfully detect THC, THC-COOH, and THC-OH in human urine with acceptable repeatability. The LODs of these analytes in urine samples ranged from 17.2 to 23.3 ng/mL. Therefore, this sweeping-MEKC method is useful for determining, with high sensitivity, the amounts of THC and its metabolites in the urine of suspected THC users.     

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(?) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(?) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 μg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.     

Determination of valproic acid (2-propylpentanoic acid) in human plasma by capillary electrophoresis with indirect UV detection

A rapid capillary zone electrophoresis method with indirect UV detection was developed and validated for the determination of valproic acid (VPA) in human plasma. The analyses were carried out under optimized conditions, using a buffer system composed of 15 mM benzoate and 0.5 mM cetyltrimethylammonium bromide at pH 6.0, and 25% v/v methanol; 2-hydroxybutyric acid was selected as the internal standard (IS). The capillary electrophoresis (CE) separation was carried out at a negative potential of 30 kV and the indirect UV detection was operated at 210 +/- 20 nm for all assays. The influence of buffer pH, ionic strength, concentration of electroosmotic flow (EOF) modifier and organic modifier on indirect signal response and migration behavior of the organic acid was investigated. Isolation of VPA from plasma was accomplished by a carefully implemented procedure using methanol as the precipitant agent. Using a high ratio of methanol to plasma for deproteinization (4:1), good absolute recovery of the analyte and satisfactory selectivity was obtained. The calibration line for VPA was linear over the 1-100 microg/mL concentration range. Sensitivity was high; in fact, the limit of detection (LOD) of VPA was 150 ng/mL and 450 ng/mL the limit of quantitation (LOQ). The results obtained analyzing real plasma samples from schizophrenic patients under polytherapy with VPA as well as antipsychotic drugs were satisfactory in terms of precision, accuracy and sensitivity.     

On-line preconcentration techniques in determination of melatonin and its precursors/metabolites using micellar electrokinetic chromatography

Determination of melatonin (MT) (N-acetyl-5-methoxytryptamine) and related indole compounds using standard capillary electrophoresis (CE) system with UV detection was investigated. Satisfactory separations of six analytes i.e. l-tryptophan (l-TRP), 5-methoxyindoleacetic acid (5-MIAA), 6-hydroxymelatonin (6-HMT), MT, serotonin (SER) and 5-methoxytryptamine (5-MTRA) were performed employing micellar electrokinetic chromatography (MEKC). The optimal background electrolytes (BGE) used for separations were 20mM tetraborate buffer (pH 9.2) and 20mM phosphate buffer (pH 3.3) when employing techniques with normal and reverse migration of micelles, respectively. Fifty millimolar sodium dodecyl sulfate (SDS) was employed as the pseudostationary phase and voltage of +/-20kV was used throughout the investigation. On-line preconcentration techniques, stacking and sweeping, were applied in order to overcome high detection limits that are a serious drawback of CE with UV detection. A comparison of used techniques, concerning enhancement factors and limits of detection (LOD), is presented. Obtained results show that the use of stacking with reverse migrating micelles (SRMM) as one of preconcentration techniques allows obtaining the lowest estimated LODs for MT at the level of 30ng/mL with injection time of 99s at 0.5psi. Estimated LODs for other analytes in these conditions were, 21, 26 and 100ng/mL for l-TRP, 5-MIAA and 6-HMT, respectively. Signals of 5-MTRA and SER obtainable only with 10s injection allowed reaching estimated LODs of 62.5 and 130ng/mL, respectively. Analysis of spiked, diluted human serum was carried out as a preliminary application illustration of developed procedure.     

Surfactant‐coated graphitized multiwalled carbon nanotubes as the pseudostationary phase in electrokinetic chromatography for the analysis of phytochemical compounds in biological fluids

This report describes the use of surfactant‐coated graphitized multiwalled carbon nanotubes (SC‐GMWNTs) as a novel pseudostationary phase in CE with diode array detection for the determination of phenolic acids and tanshinones in herbal and urine samples. Several parameters influencing the separation were studied, such as the concentrations of SDS, GMWNTs, and isopropanol; choice of carbon nanotubes; sodium borate content; and buffer pH. The results revealed that the presence of SC‐GMWNTs in buffer enhanced the separation efficiency for the target analytes relative to conventional micelles due to the strong interaction between the surface of the GMWNTs and the target compounds. Under the optimum conditions, the method showed good linearity, with correlation coefficients higher than 0.9950. LODs were in the range of 0.71–3.10 μg/mL. Furthermore, satisfactory separations were achieved with good recovery values in the range of 89.97 and 103.30% when 10 mM borate, 30 mM SDS, 10% isopropanol, and 6 μg/mL SC‐GMWNTs were introduced into the buffer solution.