Innovative analysis of diazepam, zolpidem and their main metabolites in human urine by micelle-to-solvent stacking in capillary electrophoresis.

Diazepam and zolpidem are the most widely used medications for managing insomnia. However, significant concerns regarding the potential risks of misuse and abuse problems arose in many literatures. While urine analysis is a valuable diagnostic tool, a challenge arises from the fact that some parent drugs may remain undetectable in urine. This necessitates concurrent monitoring of their metabolites. Here, we described an innovative on-line sample preconcentration technique known as micelle to solvent stacking (MSS) for the analysis of diazepam, zolpidem, and their main metabolites in urine. Several key parameters warrant further discussion to optimize the MSS model, enhancing its performance in terms of sensitivity and resolution. After optimizing the conditions, we conducted a validation test, achieving high correlation coefficients (greater than 0.9977) for intra-day and inter-day regression lines. Additionally, both the relative standard deviation (RSD) and relative error (RE) remained below 6.10% and 12.55%, respectively. The limits of detection (LODs, S/N = 3) for all five analytes ranged from 2.0 to 56 ng/mL. Compared to the conventional capillary zone electrophoresis method, this new approach exhibited remarkable sensitivity enhancements, ranging from 123 to 235-fold. Upon applying this method to actual urine samples from patients, we successfully detected nordiazepam, zolpidem, and its metabolites. This simple and sensitive approach has promising applications in supporting patient medication safety and bolstering forensic investigations.

Two-step micelle-to-solvent stacking of arsenic species from foods in permanently coated tubing for capillary electrophoresis.

An effective and sensitive two-step stacking of arsenic species by electrokinetic injection and micelle-to-solvent stacking (MSS) using zwitterionic surfactant 3-(N,N-dimethylpalmitylammonio) propane sulfonate was successfully developed in a co-electro-osmotic flow (co-EOF) capillary zone electrophoresis (CZE). The fused silica capillaries were coated with 1% hexadimethrine bromide solution to satisfy the condition of the same EOF direction between the test anions and capillary. The background solution was 100 mM borax buffer (pH 9.2). The proposed method was performed by a CZE system equipped with a diode array detector, and the detection wavelength was monitored at 192 nm. The separation voltage was -20 kV. The group of micelles and target analytes was stacked by field-enhanced sample injection at -10 kV for 180 s. The second stacking step used 60% MeOH to serve as an organic solvent where the analytes were brought by the micelles to the MSS boundary. The enrichment factors of As(Ⅴ), As(Ⅲ), disodium methyl arsonate hexahydrate (MMA), and dimethylarsinic acid (DMA) were 1230, 840, 3820, and 1450, respectively, compared to typical injections in CZE. The limits of detection (S/N = 3) ranged from 0.382 to 0.911 ng mL-1. The intracapillary repeatabilities (%RSD, n = 3) were 0.5-1.0% for migration time and 0.3-0.7% for peak areas. The technology of two-step stacking by MSS in co-EOF CZE together with a simple extraction procedure for As(Ⅴ), As(Ⅲ), MMA and DMA were demonstrated in kelp and rice samples.

Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis.

A direct on-line complexion combined with micelle to solvent stacking method was proposed for simultaneous determination of metal ions by capillary electrophoresis coupled diode array detector. During the experiment, a plug of complexing agent was first injected to the inlet of capillary, followed by introducing the micelle-bound metal ions. Then the metal ions produced a micelle-to-solvent stacking effect and interacted with the complexing agent under a positive voltage. Continued application of voltage, the analytes were effectively focused and separated in the capillary zone electrophoresis. Repeatability was ranged from 1.89% to 1.94% for the migration time. The detection limits were 2.66-27.9 ng mL-1 for Ni2+, Co2+, Cu2+, Hg2+ and Cd2+. Furthermore, the developed method showed a great potential for the determination of metal ions in the crayfish, beebread and Dendrobium officinale samples.

On-line double focusing of atenolol and metoprolol in human urine using capillary electrophoresis with the aid of ß-cyclodextrin.

A maneuverable and sensitive on-line double focusing technique combined field amplified sample stacking (FASS) with micelle to solvent stacking (MSS) with the aid of ß-cyelodextrin (ß-CD) is developed to detect the contents of AT and ME in human urine by capillary electrophoresis (CE) with UV detector. Small amount of ß-CD not only increase the critical micelle concentration (CMC) of SDS, but also strengthen the interaction between SDS and the aimed compound by forming inclusion complexes. The result indicates that the addition of ß-CD affords 5.5- and 3.5-fold improvements for atenolol (AT) and metoprolol (ME) in sensitivity than that of in the absence of ß-CD in the double focusing system, respectively. Under the optimal conditions, about 200-fold improvement in sensitivity for analytes is achieved compared with conventional CE method. The limits of detection (LODs) at a signal-to-noise of 3 (S/N = 3) of the two ß-blockers can be reached 3.3 and 3.7 ng mL-1 respectively, which are lower than minimum required performance levels (MRPLs) set by the World Anti Doping Agency. The relative standard deviations (RSDs) of peak areas of intra-day and inter-day are 3.51-3.38% and 2.34-4.28% (n = 6), respectively. AT and ME in urine without special pretreatment and additional instrument are analyzed. The recoveries are 82-98% with RSDs less than 2.0%.

Monitoring of vancomycin in human plasma via portable microchip electrophoresis with contactless conductivity detector and multi-stacking strategy.

A portable microchip electrophoresis (MCE) coupled with on-chip contactless conductivity detection (C4D) system was evaluated for the determination of vancomycin in human plasma. In order to enhance the detection sensitivity, a new online multi-stacking preconcentration technique based on field-enhanced sample injection (FESI) and micelle-to-solvent stacking (MSS) was developed and implemented in MCE-C4D system equipped with a commercially available double T-junction glass chip. The cationic analytes from the two sample reservoirs were injected under FESI conditions and subsequently focused by MSS within the sample-loading channel. The proposed multi-stacking strategy was verified under a fluorescence microscope using Rhodamine 6G as the model analyte and a sensitivity enhancement factor (SEF) of up to 217 was achieved. The developed approach was subsequently implemented in the aqueous-based MCE, coupled to C4D in order to monitor the targeted antibiotic (in this case, vancomycin) present in human plasma samples. The multi-stacking and analysis time for vancomycin were 50s and 250s respectively, with SEF of approximately 83 when compared to typical gated injection. The detection limit of the method for vancomycin was 1.2µg/mL, with intraday and interday repeatability RSDs of 2.6% and 4.3%, respectively. Recoveries in spiked human plasma were 99.0%-99.2%.

Combination of micelle collapse and field-amplified sample stacking in capillary electrophoresis for determination of trimethoprim and sulfamethoxazole in animal-originated foodstuffs.

An on-line preconcentration method combining micelle to solvent stacking (MSS) with field-amplified sample stacking (FASS) was employed for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by capillary zone electrophoresis (CZE). The optimized experimental conditions were as followings: (1) sample matrix, 10.0mM SDS-5% (v/v) methanol; (2) trapping solution (TS), 35mM H3PO4-60% acetonitrile (CH3CN); (3) running buffer, 30mM Na2HPO4 (pH=7.3); (4) sample solution volume, 168nL; TS volume, 168nL; and (5) 9kV voltage, 214nm UV detection. Under the optimized conditions, the limits of detection (LODs) for SMZ and TMP were 7.7 and 8.5ng/mL, and they were 301 and 329 times better compared to a typical injection, respectively. The contents of TMP and SMZ in animal foodstuffs such as dairy products, eggs and honey were analyzed, too. Recoveries of 80-104% were acquired with relative standard deviations of 0.5-5.4%.

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis.

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross-channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.

Field-enhanced sample injection-micelle to solvent stacking in nonaqueous capillary electrophoresis.

The low conductivity of separation electrolytes employed in nonaqueous capillary electrophoresis (NACE) limits the use of on-line sample concentration or stacking by field enhancement. Herein, micelle-to-solvent stacking (MSS) was performed by the simple injection of a micellar solution plug prior to electrokinetic injection of sample prepared under field-enhanced stacking conditions (known as field-enhanced sample injection, FESI). The proposed approach allowed a 214-625-fold improvement in peak signals for targeted anticancer drugs (e.g., tamoxifen) and its major metabolites in NACE using 100% methanol-based separation electrolyte that comprised of 7.5mM deoxycholic acid sodium salt, 15mM acetic acid and 1mM 18-crown-6. These improvements yielded tamoxifen and its metabolites with 2-5 times better stacking efficiency as compared to those obtained without micellar solution injection or FESI only. This is comparable to the results typically achieved when FESI is combined with isotachophoresis (electrokinetic supercharging). The FESI-MSS-NACE was tested for the measuring levels of target drugs in plasma. The analytical figures of merit are also reported.

Online sample concentration and analysis of drugs of abuse in human urine by micelle to solvent stacking in capillary zone electrophoresis.

A sensitive and rapid CZE-UV method was developed to determine drugs and their metabolites' presence in human urine. Ten drugs of abuse were analyzed including four amphetamines, cocaine, cocaethylene, heroin, morphine, 6-monoacetylmorphine, and 4-methylmethcathinone. An MSS (micelle to solvent stacking) approach was evaluated to enhance method sensitivity. This method considers composition of the micellar sample solution matrix and the injection time. Several analytical conditions influencing the resolution of the drugs mixture as pH and buffer concentration, organic solvent content, were also investigated. The base-line separation of all studied analytes in the same run was achieved within 18 min in an uncoated fused silica capillary (50 µm id × 60 cm) using a background solution containing 50 mM phosphate buffer pH 2.5 and 30% ACN v/v. Other experimental parameters such as applied voltage and capillary temperature were set up at 20 kV and 20°C, respectively. LOD values ranging between 15 and 75 ng/mL for all studied compounds were obtained. From a comparison with conventional CZE, the proposed method provides an increase of sensitivity (39- to 55-fold enhancement factor). Under optimal MSS-CZE conditions, good linearity was achieved (R2 ≤ 0.9998). The method was finally applied to the analysis of urine samples spiked with a standard mixture after a sample pretreatment, reaching satisfactory recovery values.

Unusual stacking with electrokinetic injection of cationic analytes from micellar solutions in capillary zone electrophoresis.

Electrokinetic injection (EKI) in capillary zone electrophoresis (CZE) of charged analytes is by the electroosmotic flow (EOF) and electrophoretic mobility of analytes. In most forms of stacking with EKI, the sample ions were introduced via electrophoretic mobility and concentrated in a stacking boundary inside the capillary. In this work, we describe the unusual stacking of cationic analytes via EKI of sodium dodecyl sulfate (SDS) micelles into a fused silica capillary filled with acidic background solution (BGS) with 40-50 % acetonitrile. The analytes prepared with SDS micelles were injected because of their interaction with micelles or effective electrophoretic mobility. We observed two peaks from an analyte, and this suggested the concentration of analytes into two stacking zones. These two adjacent stacking zones were surprisingly maintained inside the capillary during EKI although the EOF was moving towards the inlet. The zones were identified as the SDS micelles (micelles zone) and organic solvent-rich stacking zone (solvent-rich zone) where the micelles zone was closer to the inlet end of capillary. The analytes concentrated in the solvent-rich zone through the mechanism of micelle to solvent stacking (MSS). The concentrated analytes in the micelles zone were from the concentrated analytes that electrophoretically migrated into the micelles zone from the solvent-rich zone during EKI. The analytes in the micelles zone were then re-stacked by MSS and formed the second sharp peak in CZE. This was prevented by reduction of acetonitrile concentration in the inlet BGS. A sensitivity enhancement factor of more than 100 was obtained for model cationic drugs (diphenhydramine and imipramine).

Field-enhanced sample injection micelle-to-solvent stacking capillary zone electrophoresis-electrospray ionization mass spectrometry of antibiotics in seawater after solid-phase extraction.

The synergistic stacking approach of field-enhanced sample injection-micelle-to-solvent stacking was used for high sensitivity CZE-ESI-MS of eight penicillins and sulfonamides. Sensitivity enhancement factors (peak height) were 1629-3328 compared to typical injection, with LODs from 0.11 to 0.55 ng/mL. The analytical figures of merit were acceptable. SPE on a fortified seawater sample resulted in 50-fold enrichment with recoveries of 85-110%. The overall method LODs were 0.002-0.011 ng/mL.

Three-step stacking by field-enhanced sample injection, sweeping, and micelle to solvent stacking in capillary electrophoresis: Anionic analytes.

Three-step stacking by field-enhanced sample injection (FESI), sweeping, and micelle to solvent stacking (MSS) in co-EOF capillary zone electrophoresis (CZE) is presented for anionic analytes. Long FESI produced an overloaded stacked zone of analytes (four model penicillins). Sweeping of the FESI zone was by electrokinetic injection of cetyltrimethylammonium bromide (CTAB) micelles. MSS was by short injection of 60% methanol that released the swept analytes from CTAB micelles. The sensitivity enhancement factors were 146-279 and 519-954 for conductivity ratio of 10 and 100, respectively. The SEF enhancement factors (factor=SEF from three-step stacking/SEF from FESI) were 16-32 and 6-10, correspondingly. The LODs were between 6.6-13.2 ng/mL, repeatability (intraday and interday) was %RSD≤5.4%, and linearity was R(2)≥0.998. Application to real sample was investigated using fortified plasma after liquid-liquid extraction.

Simultaneous electrophoretic concentration and separation of herbicides in beer prior to stacking capillary electrophoresis UV and liquid chromatography-mass spectrometry.

Simultaneous electrophoretic concentration and separation (SECS) was used as a simple and environmental friendly sample preparation strategy for herbicides in beer samples. An electric field was used to facilitate the separation and concentration of the analytes based on their charge from a 20 mL sample of diluted beer into two separate 20 µL aliquots of an acceptor electrolyte housed inside a micropipette. The anionic organophosphonate and cationic quaternary ammonium herbicides were concentrated in the anodic and cathodic pipette, respectively. Under optimized conditions, SECS was completed in 30 min at an applied voltage of 150 V, which provided analyte concentration factors of up to 90. After sample preparation, the SECS concentrate of cationic and anionic herbicides was analyzed by stacking CE with UV detection and also by LC-MS, respectively. The method detection limit for the diluted and undiluted sample was as low as 3 and 15 ng/mL, respectively. The method was linear over two orders of concentration with repeatability and intermediate precision of better than 5.8 and 7.0%RSD, respectively. Accuracy values were between 91.0-115.1%.

Determination of tamoxifen and its metabolites using micelle to solvent stacking in nonaqueous capillary electrophoresis.

Micelle to solvent stacking was implemented for the recently established NACE-C(4) D method to determine tamoxifen and its metabolites in standard samples and human plasma of breast cancer patients. For stacking, the standard samples and extract after liquid-liquid extraction (LLE) were prepared in methanol and the resulting sample solution was pressure injected after a micellar plug of SDS. Factors that affected the stacking such as SDS concentration, micelle, and sample plug length were examined. The sensitivity enhancement factor (peak height from stacking/peak height from typical injection of sample in BGE) was 15-22. The method detection limits with LLE were in the range of 5-10 ng/mL, which was lower than the established method (where the LLE extract was also prepared in methanol) with reported method detection limits of 25-40 ng/mL. The intraday and interday repeatability were in the range of 1.0-3.4% and 3.8-6.5%, respectively.

Three-step stacking of cationic analytes by field-enhanced sample injection, sweeping, and micelle to solvent stacking in capillary electrophoresis.

The sensitivity enhancement factor (SEF) in field enhanced sample injection (FESI) in capillary electrophoresis is dictated by the conductivity ratio. The higher the conductivity ratio (using very low conductivity sample diluents such as water), the higher the SEF. Here, we improved the performance of FESI by combination with sweeping and micelle to solvent stacking (MSS) in a well-defined three-step stacking procedure using model cationic drugs. The separation was by capillary zone electrophoresis (CZE) using 100mM phosphoric acid as background solution (BGS). Under the experimental conditions studied, the SEF (vs. typical injection in CZE) range of FESI using a conductivity ratio of 10, 100, and 1000 (sample diluent with conductivity 10, 100, and 1000× lower than the BGS, respectively) was 5-6, 33-50, and 272-393, respectively. The SEF range of three-step stacking was 308-891, 2188-6463, and 3088-6499, correspondingly. The SEF enhancement factor due to three-step stacking (SEF of three-step stacking divided by SEF of FESI) was from 11 to 161. We evaluated the performance of proposed procedure using a conductivity ratio of 10 (10mM phosphoric acid as diluent) which is the minimum requirement for field-enhancement. The strategy was as follows: long FESI (e.g., 420s at 10kV) to form an overloaded stacked zone; sweeping (e.g., 315s at -10kV) with 10mM sodium dodecyl sulfate micelles; and MSS by injection (6s at 50mbar) of 30% acetonitrile. The strategy was studied in terms of sweeping and MSS conditions, FESI/sweeping time ratio, and FESI time at constant FESI/sweeping ratio. Analytical figures of merit including linearity, LOD (S/N=3), and repeatability (intraday and interday) were determined. Moreover, sample matrix effect was studied using acetone treated plasma sample.

High-sensitivity analysis of anionic sulfonamides by capillary electrophoresis using a synergistic stacking approach.

A synergistic stacking approach whereby field-enhanced sample injection and micelle-to-solvent stacking in capillary zone electrophoresis are combined has been developed and has been applied to the separation and quantification of anionic sulfonamides. Electrokinetic injection of the sample in a low conductivity alkaline diluent was performed for 90s at -15kV. Micelle-to-solvent stacking was then undertaken by hydrodynamic injection of micellar cetyltrimethylammonium bromide solution prior to the electrokinetic injection of sample that also contained 50% methanol. This combined stacking approach, when compared to a typical hydrostatic injection, provided improvements in peak height and corrected peak area in the range of 397-1024 and 758-1246, respectively. Limits of quantification in the range of 0.01-0.03µg/mL were obtained for sulfamerazine, sulfamethazine and sulfamethizole and were sufficient for the determination of these analytes in river water. The percentage recovery and accuracy values obtained for a fortified river water sample that had been subjected to sample preparation by evaporation and reconstitution with diluent were 74-135%. Intra-day and inter-day repeatabilities for migration time, peak height, and corrected peak area were in the range 0.5-5.0% (percentage relative standard deviation, n=8) and these relatively low values were attributed to the use of a stable capillary coating established by the successive multiple ionic-polymer layer technique.

On-line two-step stacking in capillary zone electrophoresis for the preconcentration of strychnine and brucine.

An on-line sample preconcentration method by two-step stacking i.e., sweeping and micelle to solvent stacking, in capillary zone electrophoresis (CZE) has been developed for the determination of strychnine and brucine in traditional Chinese herbal medicines. After experimental optimizations, the best separation was achieved by using 75 mM phosphate buffer (pH 2.5) with 30% methanol (v/v). Compared with normal CZE injection, 51- and 38-fold improvement in concentration sensitivity was achieved for strychnine and brucine, respectively. The calibration curve was linear in the range of 0.1-5.0 µg mL(-1) for both strychnine and brucine, with the correlation coefficients of 0.9998 and 0.9997, respectively. The limits of detection (S/N=3) for both alkaloids were 0.01 µg mL(-1). The inter-day (n=8) and intra-day (n=5) reproducibilities expressed as the relative standard deviations for corrected peak area were less than 9.5%. The method was applied to determine strychnine and brucine in two Chinese herbal medicines, with recoveries ranging from 94.2% to 105.4%. The results indicated that the method is simple, rapid, reliable, and can be applied to determine strychnos alkaloids in traditional Chinese herbal medicines.

Determination of three nitroimidazoles in rabbit plasma by two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking.

This study reports a sweeping and micelle to solvent stacking (MSS) method for fast stacking of nitroimidazoles in capillary zone electrophoresis (CZE). The optimal experimental conditions are run buffer of 25mM sodium phosphate (pH 1.5) and 0.50% (v/v) methanol, micellar solution of 30mM sodium dodecyl sulfate, applied voltage of 28kV, and sample and micellar solution injection of 100s. By applying this new on-line preconcentration technique, the nitroimidazoles content can be determined within 9min with the limit of detection (S/N=3) ranging 2.3-3.0ng/mL, which is lower than that of conventional CZE analysis. The proposed MSS technique affords 20-, 12- and 38-fold improvements in sensitivity for the detection of dimetridazole, metronidazole and secnidazole, respectively. The relative standard deviations (RSDs) of intra-day and inter-day are 2.1-3.6% and 2.7-4.6% (n=6), respectively. The recoveries in pretreated rabbit plasma at spiked levels of 5.0-10.0µg/mL are 92.0-101.1% with RSDs lower than 3.4%. The proposed sweeping and MSS-CZE is a highly sensitive method for the detection of nitroimidazoles in biological and clinical samples and has been successfully applied to analyze nitroimidazoles in pretreated rabbit plasma.