Enhancement of chiral drugs separation by a novel adjustable gravity mediated capillary electrophoresis combined with sulfonic propyl ether ß-CD polymer.

A water-soluble negative sulfonic propyl ether ß-CD polymer (SPE-ß-CDP) to be used as chiral selector in capillary electrophoresis (CE) was polymerized. The sulfonic substitution degree of each ß-CD in SPE-ß-CDP was statistically homogenized. The only one negative peak in electrophoretogram with indirect ultraviolate method proved its uniformity of electrophoretic behavior. There were 7.12 sulfonic substitution in ß-CD unit and 164 µmole ß-CD units in each gram of SPE-ß-CDP, which corresponded a molecular weight of 7000 or more. Compared with monomer, SPE-ß-CDP was lower effect on electrical current of CE, indicating a high concentration of SPE-ß-CDP could be added. Its separation ability was verified by 12 chiral drugs. SPE-ß-CDP also showed advantages of good water solubility, easy preparation and recovery to reduce the overall cost. However, five of 12 chiral drugs were hardly to be fully separated which was normal for any kind of chiral selector. A newly adjustable gravity mediated capillary electrophoresis (AGM-CE) technology was proposed and combined with SPE-ß-CDP to enhance the chiral separation efficiencies of propranolol, salbutamol, omeprazole, ofloxacin and phenoxybenzamine which were markedly improved to 3.02, 1.17, 7.63, 4.14, and 2.81, respectively. Furthermore, its gradient mode (AGMg-CE) was also used to improve resolution through utilizing the zero mobility point, at which the effective apparent mobility of one racemate was zero. Resolutions of five chiral drugs were significantly improved, especially resolution of carvedilol changed from 0.43 to 1.0. These indicated SPE-ß-CDP as chiral selector, AGM-CE and AGMg-CE as new CE technologies had a great potential in chiral separation.

Preparation of a phenylboronic acid and aldehyde bi-functional group modified silica absorbent and applications in removing Cr(vi) and reducing to Cr(iii).

Cr(vi) is a great threat to the ecological environment and human health, so it is urgent to remove Cr(vi) from the environment. In this study, a novel silica gel adsorbent SiO2-CHO-APBA containing phenylboronic acids and aldehyde groups was prepared, evaluated and applied for removing Cr(vi) from water and soil samples. The adsorption conditions including pH, adsorbent dosage, initial concentration of Cr(vi), temperature and time were optimized. Its ability in removing Cr(vi) was investigated and compared with three other common adsorbents, SiO2-NH2, SiO2-SH and SiO2-EDTA. Data showed SiO2-CHO-APBA had the highest adsorption capacity of 58.14 mg g-1 at pH 2 and could reach adsorption equilibrium in about 3 h. When 50 mg SiO2-CHO-APBA was added in 20 mL of 50 mg L-1 Cr(vi) solution, more than 97% of Cr(vi) was removed. A mechanism study revealed that a cooperative interaction of both the aldehyde and boronic acid groups is attributed to Cr(vi) removal. The reducing function was gradually weakened with the consumption of the aldehyde group, which was oxidized to a carboxyl group by Cr(vi). This SiO2-CHO-APBA adsorbent was successfully used for the removal of Cr(vi) from soil samples with satisfactory results which indicates a good potential in agriculture and other fields.

A magnetically recyclable superparamagnetic silica supported Pt nanocatalyst through a multi-carboxyl linker: synthesis, characterization, and applications in alkene hydrosilylation.

To simplify separation procedures, improve the reusability and decrease the loss of Pt, two Pt catalysts anchored on superparamagnetic silica (Fe3O4@SiO2-EDTA@Pt and Fe3O4@SiO2-DTPA@Pt) were prepared for the first time. The stable magnetic properties made them easily recyclable using a magnet rather than filtration, decantation or centrifugation. After 12 catalytic runs for both 30-50 nm Pt catalysts, the yield of 1-heptylmethyldichlorosilane was still up to 90%. The average loss of Pt in each reaction was only 0.87% for Fe3O4@SiO2-EDTA@Pt and 0.66% for Fe3O4@SiO2-DTPA@Pt owing to the strong interaction between Pt and carboxyl. The unprecedented activity and selectivity of the two Pt nanoparticle catalysts were observed in the hydrosilylation of alkenes. The turnover number in the reaction between 1-hexene and methyldichlorosilane using 5 × 10-8 mol of the Pt approached 662 733 for Fe3O4@SiO2-EDTA@Pt and 579 947 for Fe3O4@SiO2-DTPA@Pt over 12 h. The corresponding hydrosilylation products in excellent yields were obtained when we employed a broad range of alkenes as substrates, including 5 isomerous hexenes and 14 important industry raw materials. Fe3O4@SiO2-DTPA@Pt showed a better activity. They have potential for catalyzing more reactions and replacing the current homogeneous Pt catalysts in industry.

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.

Synthesis and applications of sulfopropyl ether γ-cyclodextrin polymer as chiral selector in capillary electrophoresis.

A novel sulfopropyl ether γ-cyclodextrin polymer (SPE-γ-CDP) through polycondensating sulfated cyclodextrins (SCDs) was synthesized. This synthesis approach also has the potential of preparing other derived cyclodextrins (CDs) polymers. The polymerized SCDs took on both the properties of SCDs and certain characteristics of polymers, such as chiral selectivity and high viscosity. Synthesis parameters, including reactions sequence, sulfation, and polycondensation conditions were investigated systematically. The product was characterized by elemental analysis, infrared spectroscopy (IR), and indirect UV detections prior to use as background electrolytes additive. The separation conditions, including the concentration of SPE-γ-CDP, the concentration and pH of running buffer, separation voltage, as well as the additional organic solution were optimized during chiral separation of neutral, acidic, and basic enantiomers in capillary electrophoresis (CE). SPE-γ-CDP was proven to be an effective chiral resolving agent in CE with the advantages of simple synthesis process, low cost, similar ratio of charge-to-mass, low current, great reproducibility, and reusability. Graphical Abstract Synthesis and applications of sulfopropyl ether γ-cyclodextrin polymer.

Development and application of a high-throughput sample cleanup process based on 96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography-triple quadrupole mass spectrometry.

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography-triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20% sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6-300.0 pg(.)mL(-1) in plasma, 3.0-270.0 pg·mL(-1) in milk, and 2.2-210.0 pg(.)mL(-1) in urine. The recoveries of the 16 steroids were 81.9-97.9% in plasma (relative standard deviation 1.0-8.0%), 80.6-97.7% in milk (relative standard deviation 0.8-5.4%), and 87.3-98.7% in urine (relative standard deviation 1.0-4.9%). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises.

A method developed to fractionate intact proteins based on capillary electrophoresis.

Reduction in the sample complexity enables more thorough intact protein analysis using MS-based proteomics. A capillary electrophoresis method, namely the velocity gap mode of capillary electrophoresis (VGCE), is proposed to separate protein mixtures with high resolution. Although the separation mechanism of VGCE is also based on the difference of the mass-to-charge ratios of the proteins, it fractionates the sample zone into small pieces of subunits. In this way, the resolution can be dramatically improved due to less longitudinal dispersion of the sample. The effect of the new approach is evaluated by separation of three groups of reference protein mixtures, i.e. a mixture of lysozyme and BSA; a mixture of lysozyme, ß-lactoglobulin, and ribonuclease A; and a mixture of cytochrome C, lysozyme, BSA, ß-lactoglobulin, ribonuclease A, conalbumin, carbonic anhydrase, and hemoglobin. The results indicate that the new approach shows great potential to couple with MS for top-down analysis of complex mixtures.

An approach to the determination of the enantiomeric excess at the extreme case by capillary electrophoresis.

Capillary electrophoresis (CE) has been applied to determine the percentage of enantiomeric excess (ee%) of chiral compounds. In such assays, the quality of chiral selectors (CSs) plays vital roles in resolving the enantiomers for accurate determination of the ee%. Selecting an efficient CS is usually by trial and error, and is, if ever possible, time-consuming and costly. Here we propose a new approach by using the velocity gap mode of CE (VGCE) method, to simplify the method development process for ee% determination. With VGCE, it is still possible to measure ee% even when the CS has a weak resolving power. This is especially important at the extreme cases where one of the enantiomers is significantly higher than the other one. The key point of VGCE in this case is to fractionate the small part of the mixture containing both enantiomers from the major component of the enantiomer, which is already enantiopure. Baseline separations can be achieved between the two enantiomers for the small mixture due to less longitudinal dispersion, making it possible to determine the ee%. The feasibility of this VGCE approach was confirmed by the ee% measurements of amlodipine and ofloxacin, respectively. And the practical application of VGCE was tested by analyzing levamlodipine besylate tablet.

The development of a high-throughput measurement method of octanol/water distribution coefficient based on hollow fiber membrane solvent microextraction technique.

This paper describes the development of a novel high-throughput hollow fiber membrane solvent microextraction technique for the simultaneous measurement of the octanol/water distribution coefficient (logD) for organic compounds such as drugs. The method is based on a designed system, which consists of a 96-well plate modified with 96 hollow fiber membrane tubes and a matching lid with 96 center holes and 96 side holes distributing in 96 grids. Each center hole was glued with a sealed on one end hollow fiber membrane tube, which is used to separate the aqueous phase from the octanol phase. A needle, such as microsyringe or automatic sampler, can be directly inserted into the membrane tube to deposit octanol as the accepted phase or take out the mixture of the octanol and the drug. Each side hole is filled with aqueous phase and could freely take in/out solvent as the donor phase from the outside of the hollow fiber membranes. The logD can be calculated by measuring the drug concentration in each phase after extraction equilibrium. After a comprehensive comparison, the polytetrafluoroethylene hollow fiber with the thickness of 210 µm, an extraction time of 300 min, a temperature of 25 °C and atmospheric pressure without stirring are selected for the high throughput measurement. The correlation coefficient of the linear fit of the logD values of five drugs determined by our system to reference values is 0.9954, showed a nice accurate. The -8.9% intra-day and -4.4% inter-day precision of logD for metronidazole indicates a good precision. In addition, the logD values of eight drugs were simultaneously and successfully measured, which indicated that the 96 throughput measure method of logD value was accurate, precise, reliable and useful for high throughput screening.

Velocity gap mode of capillary electrophoresis developed for high-resolution chiral separations.

A new CE method based on velocity gap (VG) theory has been developed for high-resolution chiral separations. In VG, two consecutive electric fields are adopted to drive analytes passing through two capillaries, which are linked together through a joint. The joint is immersed inside another buffer vial which has conductivity communication with the buffer inside the capillary. By adjusting the field strengths onto the two capillaries, it is possible to observe different velocities of an analyte when it passes through those two capillaries and there would be a net velocity change (NVC) for the same analyte. Different analytes may have different NVC which may be specifically meaningful for enantioseparations because enantiomers are usually hard to resolve. By taking advantage of this NVC, it is possible to enhance the resolution of a chiral separation if a proper voltage program is applied. The feasibility of using NVC to enhance chiral separation was demonstrated in the separations of three pairs of enantiomers: terbutaline, chlorpheniramine, and promethazine. All separations started with partial separation in a conventional CE and were significantly improved under the same experimental conditions. The results indicated that VG has the potential to be used to improve the resolving power of CE in chiral separations.

Development of a novel 96-well format for liquid-liquid microextraction and its application in the HPLC analysis of biological samples.

A novel 96-well liquid-liquid microextraction system combined with modern HPLC was developed and used for the simultaneous analysis of 96 biological samples. The system made use of hollow fibers, a 96-well plate, and a plastic base with a center hole and a side hole. One end of the hollow fiber was sealed, while the other end was attached to one of the holes positioned at the center for the plastic base. The needle was inserted into the liquid from inside or outside of the hollow fiber through the center or the side holes, respectively. The system was tested with plasma samples containing three compounds, acidic indomethacin, neutral dexamethasone, and basic propafenone. Some parameters, such as the kind and dimension of hollow fiber, pH and salt concentration of the donor phase, the selection of organic solvent for the acceptor phase, and the extraction time were investigated. Under the optimization conditions, the Log D and drug concentration of indomethacin, dexamethasone, and propafenone in plasma and urine samples were analyzed. Then, the methodology was validated. The results demonstrated that ng/mL levels could be exactly and rapidly analyzed by our system, which was equipped with an auto-injection sampler, making sample analysis more convenient.

Rapid determination of protein binding constant by a pressure-mediated affinity capillary electrophoresis method.

A new pressure-mediated affinity capillary electrophoresis method for the rapid and accurate determination of drug-protein binding constants is described. A special combination of pressure and electrophoresis is used to shorten the electrophoresis and the overall analysis time to only a few minutes. At the same time, the suitability of this method is checked against a traditional fluorescence spectroscopy method. The binding constants of bovine serum albumin and a total of eight drugs with different pK(a) have been evaluated and compared with those determined by the fluorescence spectroscopy method and other methods in literature. The results indicate that the P-ACE method is well suited for the determination of binding constants with weak interaction (K(b) <10(5) M(-1)).

Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector.

An effective, rapid and reliable capillary electrophoresis-laser induced fluorescence (CE-LIF) procedure was built to study the characterization of tyrosine kinase (TK), which was a target for drug screening. In this procedure, CE separated the sample of the TK reaction and LIF selectively detected the fluorescence-labeled polypeptide substrate and product. The precise TK activity was quantitated by introducing the transformation ratio of the substrate (T%) to avoid the deviation resulted from the detection sensitivity and the injection amounts in different runs and different capillaries. By measuring the T%, the effects of various reaction conditions were optimized. Meanwhile, the progression of the enzyme reaction was monitored. The K(m) and V(max) were calculated for TK under the optimized experimental conditions. In addition, the inhibition effectiveness of two model inhibitors, Staurosporine and SU6656 were evaluated. The results indicated that the screening platform based on electrophoresis was suitable for TK analysis and laid a foundation for the HTS of TK inhibitors.

Preparation and evaluation of amphiphilic silica-based monolithic column having surface-bound octanoyl-aminopropyl moieties for capillary electrochromatography.

A novel amphiphilic silica-based monolithic column having surface-bound octanoyl-aminopropyl moieties was successfully prepared by a one-step in situ derivatization process. As expected, the amphiphilic monolithic column exhibited RP chromatographic behavior toward non-polar solutes (e.g., alkyl benzenes) with high column performance. As the pH of the buffer inside the column increases, the EOF changed from -2.65 x 10(-8) m(2) V(-1) s(-1) at pH 3.0 to 1.20 x 10(-8) m(2) V(-1) s(-1) at pH 8.0 with the reversion of EOF at about pH 6.4. Using acidic mobile phase, five aromatic acids can be efficiently separated in less than 6 min under co-EOF conditions. For basic compounds, symmetrical peaks were obtained due to the existence of hydrophilic acyl amide group, which can effectively minimize the adsorption of the positively charged basic analyte to the silica-based surface of the capillary column.

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.

Separation of aromatic acids by wide-bore electrophoresis with nanoparticles prepared by electrospray as pseudostationary phase.

A model mixture of six aromatic acids has been separated using a laboratory-made wide-bore electrophoretic device with aminopropyl-modified nanoparticles used as pseudostationary phase. Optimization of preparation of nanoparticles by an electrospray (ES) method is described. With the optimized electrophoretic method, 30 mmol/L acetate running buffer, pH 4.5, containing 1.0 mg/mL of nanoparticles as an additive was used, and 3.0 kV applied potential, improved resolution was achieved. The average theoretical plate number obtained was above 5.0 x 10(4) theoretical plates per meter with the highest value achieved in certain runs exceeding 1.0 x 10(5) theoretical plates per meter, which was better than previously reported results (approximately 6.7 x 10(4) theoretical plates per meter). Furthermore, repeatabilities of 2, 6.5, and 6% were obtained for the migration time, peak areas, and peak height, respectively. Additionally, sample capacity and sensitivity were improved by hundredfold using the novel wide-bore electrophoresis system compared to traditional CE.

Wide-bore monolithic column for electrochromatography.

A new wide-bore electrophoresis (WE) system adopting an inner cooling device was set up to perform electrochromatography. In this system, a quartz tube of 1.2 mm inner diameter was used as the separation channel. The Joule heat generated during electrophoresis was removed timely through the outer surface of the quartz tube and a cooling capillary inserted into the quartz tube. A proper coolant passed through the cooling capillary to further improve the cooling efficiency. In the primary research, a polyacrylamide monolithic column was successfully prepared in this quartz tube. Then it was evaluated in the electrochromatographic mode. An electric field strength as high as 625 V/cm can be applied to this system without obvious deviation of the current from the linear curve of the Ohm plot. Sample volume as high as 1 microL was injected into the WE system and reasonable efficiency was obtained for separation of the test compounds.

WITHDRAWN: An improved direct pumping approach to eliminate sample bias in capillary electrokinetic injection.

The EOF pump was successfully used as a means of introducing samples into a capillary system. An improved sample injection device has been developed using a Teflontrade mark union (TU) to link the two capillaries together. The capillary applied high voltage served as the EOF pump to pull the liquid inside while the other one served the purposes of isolating the electric field. Using the bias degree (BD) and SD of bias (SDB), it was possible to quantitatively determine the sampling bias and evaluate the effectiveness of injection approaches in bias elimination. Several related factors were evaluated, and it was found that TU approach could fully eliminate the bias under the optimal conditions. The fracture did not affect the efficiency, leak, or dilute the sample significantly. This approach was effective under both normal and reverse EOF situations and adapted to real samples. Finally, a TU method using grounded injection electrode was proposed and shown to be suitable for samples with low conductivity and high injection voltage.

An electrical pumping approach to eliminate sample bias in capillary electrokinetic injection.

A general pumping injection (PI), which involves the use of two capillaries with different diameters, was taken to evaluate systematically the effects on eliminating sample bias associated with the electrokinetic injection process in CE. One end of the separation capillary of the smaller diameter was inserted into another pumping capillary of larger diameter. When a high voltage was applied to the pumping capillary, the EOF generated inside will act as a pump to drive the solution stream in the separation capillary. The results have demonstrated that PI is suitable for both normal and reverse EOF situations. Second, the bias degree (BD) and SD of bias we presented were used to evaluate the degree of the bias under different conditions, and the factors of bias elimination have been investigated. Under optimal conditions, the bias was satisfactorily eliminated by PI. This EOF pumping system was successfully applied to the analysis of samples in CEC for a bias-free injection. Moreover, this two-capillary pumping system did not significantly affect the EOF, current, and the column efficiency of the separation process. Finally, a PI with grounded electrode was proposed and shown to be suitable for samples with low conductivity and ions with different mobility.

A hollow fiber solvent microextraction approach to measure drug-protein binding.

A new direct method has been developed to determine protein-drug binding based on hollow fiber membrane solvent microextraction. Hollow-fiber membrane solvent microextraction coupled with high-performance chromatography with UV detection was employed to evaluate the binding characteristics of drugs to bovine serum albumin (BSA) and blood serum. It was found that the BSA and matrix in the blood serum did not interfere with the measurement. The method is simple and fast. It lacks the drawbacks of some conventional analytical techniques, such as taking much long time and requiring large volume sample consumption.