Preparation of chondroitin sulfate-g-poly(ε-caprolactone) copolymers as a CD44-targeted vehicle for enhanced intracellular uptake.

Chondroitin sulfate-g-poly(ε-caprolactone) (CP) copolymers were synthesized via atom transfer radical addition (ATRA). The CP copolymers self-assembled into micelles in water, and the micelles could be used to encapsulate a hydrophobic anticancer drug, camptothecin (CPT), in the core for tumor targeting delivery. The physicochemical properties of the micelles and CPT-loaded micelles were thoroughly characterized. For the in vitro test, the CPT release, the protection of the lactone ring of CPT from hydrolysis and the cellular uptake of CPT were studied. The cell-killing and apoptosis-inducing effects using the CPT-loaded micelles were significantly better than using free CPT against CRL-5802 cells. The micellar internalization into CRL-5802 cells was primarily via CD44 and clathrin dual-mediated endocytosis. For the in vivo test, the therapeutic efficacy of the CPT-loaded micelles was studied in a non-small-cell lung cancer xenograft animal model. The CPT-loaded micelles showed good inhibition in tumor growth as compared with a commercial product, CPT-11, in CRL-5802 tumor-bearing mice. The in vitro and in vivo data suggested the CP-based micelles are promising anticancer drug vehicles for lung cancer targeting.

Concentration of donepezil to the cognitive response in Alzheimer disease.

BACKGROUND: Donepezil has been approved, and higher dosages are recommended for the treatment of Alzheimer disease (AD). However, a few studies have reported different cognitive responses in patients with AD treated with donepezil without measuring the concentration. METHODS: We evaluated the relationships between the therapeutic responses and plasma concentrations of donepezil in various cognitive domains using the Cognitive Ability Screening Instrument among 37 patients with newly diagnosed mild stage AD taking donepezil 5 mg/d. RESULTS: Among the 9 cognitive domains in the Cognitive Ability Screening Instrument, the long-term memory domain had the highest improvement ratio (81.1%) compared with the other domains. An increased donepezil plasma concentration [mean (SD), 75.14 (32.16) ng/mL] was significantly associated with the improvement of long-term memory (P = 0.045; odds ratio, 0.959; 95% confidence interval, 0.920-0.999) after adjusting for age, sex, education, and apolipoprotein E genotype. CONCLUSIONS: Although there are some limitations in our study, these findings indicate that a higher concentration of donepezil improves long-term memory in patients with mild stage AD and imply the possible benefits in the advanced stage of AD for relatively preserved long-term memory.

Trace analysis of acetylcholinesterase inhibitors with antipsychotic drugs for Alzheimer's disease by capillary electrophoresis with on column field-amplified sample injection.

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid-liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in <20 min at 25 °C with a fused-silica capillary column of 60.2 cm × 50 µm I.D. (effective length 50 cm) and a run buffer containing 120 mM phosphate (pH 4.0) with 0.1 % γ-cyclodextrin, 40 % methanol (MeOH), and 0.02 % polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with the capillary wall. Using phenformin as an internal standard (40.0 ng/mL), the linear ranges of the proposed method for the simultaneous determination of donepezil, rivastigmine, aripiprazole, quetiapine, risperidone, clozapine, ziprasidone, and trazodone were over the range 4.0-80.0 ng/mL, and olanzapine was over the range 1.0-20.0 ng/mL. The method was applied for concentrations monitoring of AChEIs and antipsychotic drugs in ten Alzheimer's disease patients with behavioral and psychological symptoms of dementia after oral administration of the commercial products.

Sample stacking by field-amplified sample injection and sweeping for simultaneous analysis of acidic and basic components in clinic application.

In this study, online sample concentration method, which coupled field-amplified sample injection (FASI) and sweeping technology with micellar electrokinetic chromatography (MEKC), was used to detect and analyze acidic and basic components in a single run. In order to concentrate the acidic and basic components simultaneously in a single run sweeping step, a combination of successive anion- and cation-selective injections were used. Before sample loading, a rinse buffer containing 50 mM Tris buffer (pH 3) with 41% MeOH and 0.1% polyethylene oxide (PEO) was injected in order to suppress the electroosmotic flow (EOF). Sample loading of anionic components was achieved by electrokinetic injection at a negative voltage of -2.5 kV for 80 s, and then the cationic components were injected at a positive voltage of +5 kV for 120 s. Finally, sweeping with SDS micelles from the separation buffer (25 mM Tris buffer with 60 mM SDS, pH 3) was performed at a negative voltage of -20 kV. This capillary electrophoretic methodology was applied to the quantification of acidic and basic drugs in commercial tablets and in plasma samples. The precision and accuracy of the proposed method at different concentrations ranging from high, medium, to low were evaluated on spiked plasma samples. The intra and interday precision and accuracy values at three concentrations were all below 6.1%. The method was also successfully applied to monitor the tested drugs in the plasma of nine elderly cardiovascular and/or Alzheimer's disease patients after oral administration of the commercial products.

Simultaneous determination of memantine and amantadine in human plasma as fluorescein derivatives by micellar electrokinetic chromatography with laser-induced fluorescence detection and its clinical application.

A nonionic surfactant MEKC method with LIF detection was developed for the simultaneous determination of memantine, an anti-Alzheimer's disease agent, and amantadine, an anti-Parkinson's disease drug, in human plasma. Before analysis, the plasma samples were pretreated by liquid-liquid extraction with ethyl acetate, and derivatized with 6-carboxyfluorescein N-hydroxysuccinimide ester. The chemical derivatization is performed with 6-carboxyfluorescein N-hydroxysuccinimide ester in ACN - 5 mM pH 9.0 borate buffer (40:60, v/v) at 35 degrees C for 3 h. After the derivatization reaction, hydrodynamic injection (0.5 psi, 8 s) was used to introduce the derivatized solution, and the separation was performed in borate buffer (30 mM, pH 9.5) with the nonionic surfactant Brij-35 (0.07%, w/v); the separation voltage was 6 kV. The linear ranges of the method for the determination of memantine and amantadine in human plasma were over a range of 2.0-60.0 ng/mL. The detection limit was 0.5 ng/mL (S/N=3). This method was applied successfully to monitor the concentration of memantine or amantadine in patients with Alzheimer's disease or Parkinson's disease.

Determination of free and total levels of phenytoin in human plasma from patients with epilepsy by MEKC: an adequate alternative to HPLC.

Phenytoin is a widely used anti-seizure agent, and a good correlation is observed between its concentration in plasma and the clinical effect. We developed a selective CE with UV detection at 200 nm for analysis of free and total levels of phenytoin in human plasma based on MEKC. A sample pretreatment by liquid-liquid extraction with ethyl acetate for determination of total level of phenytoin and serum ultrafiltrate was prepared by ultrafiltration technique (ultrafiltration membrane 30 kDa; 2000 g, 10 min) for determination of free level of phenytoin and subsequent quantification by MEKC was used. MEKC was performed in Tris buffer (25 mM; pH 10.5) containing SDS (180 mM) and EG (13%) as BGE. Hydrodynamic injection for phenytoin determination (0.5 psi 5 s for total level, 2 psi 5 s for free level) was used to introduce samples. The separation voltage was set at 20 kV. Data obtained by MEKC were compared with the results by a validated HPLC method. The MEKC assay of phenytoin exhibited a very good correlation with respect to HPLC by Bland-Altman method. The equations for the Passing-Bablok regression line were as follows: for total level: MEKC=1.0143 x HPLC+0.0976, r(2)=1; for free level: MEKC=1.0013 x HPLC-0.0016, r(2)=1. The proposed method was applied successfully to monitor free and total levels of phentoin in 20 patients with seizures.

Simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in plasma by capillary electrophoresis combining on-column field-amplified sample injection and application in schizophrenia.

A sensitive high-performance CZE combining on-column field-amplified sample injection (FASI) has been developed for simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in human plasma. A sample pretreatment by means of liquid-liquid extraction (LLE) (diethyl ether) with subsequent quantitation by FASI-CZE was used. The separation of aripiprazole and dehydroaripiprazole was performed using a BGE containing 150 mM phosphate buffer (pH 3.5) with 40% methanol and 0.02% PVA as a dynamic coating to reduce interaction of analytes with the capillary wall. Before sample loading, a methanol plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (10 kV, 30 s) to introduce sample cations and the applied voltage was 20 kV with on-column detection at 214 nm. Several parameters affecting the separation and sensitivity of the drug and its active metabolite were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer. The linear ranges of the method for test drug and its active metabolite, in plasma using amlodipine as an internal standard, were over the range 5.0-100.0 ng/mL. One female volunteer (25 years old) was orally administered a single dose of 10 mg aripiprazole (Abilify, Otsuka) and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied to monitor the concentration of aripiprazole and dehydroaripiprazole in plasma collected after oral administration of 20 or 30 mg aripiprazole (Abilify, Otsuka) daily at steady state in one schizophrenic patient.

Analysis of quinapril by two solvent-saving methods: application of capillary column high-performance liquid chromatography with ultraviolet absorbance detection and LDI-TOF-MS.

A capillary column high-performance liquid chromatography (CapLC) method and a laser desorption ionization-time of flight (LDI-TOF)-MS method are described for the determination of quinapril, an angiotensin-converting enzyme inhibitor. Effective separation was achieved by using a C18 capillary column at a flow rate of 10 microL/min. For CapLC, quinapril and 7-hydroxycoumarin (internal standard) were detected at 210 and 320 nm, respectively. Phenformin replaced 7-hydroxycoumarin as the internal standard for the LDI-TOF-MS method successfully developed to detect quinapril. The calibration curves showed good linearity in the range of 1-100 micro/mL in these two methods. For high throughput purposes, the LDI-TOF-MS method was simpler and faster than the CapLC method. Both green methods were suitably validated and successfully applied to determine quinapril in commercial tablets.

Simultaneous determination of galantamine, rivastigmine and NAP 226-90 in plasma by MEKC and its application in Alzheimer's disease.

A simple and sensitive MEKC with UV detection was developed and validated for the simultaneous determination of acetylcholinesterase inhibitors including galantamine, rivastigmine and major metabolite NAP 226-90 in plasma. A sample pretreatment by liquid-liquid extraction with diethylether and subsequent quantification by MEKC was used. The optimum separation for these analytes was achieved in <10 min at 25 degrees C with a fused-silica capillary column of 30.2 cm x 50 microm id (effective length 20 cm) and a run buffer containing 25 mM Tris buffer (pH 5.0) with 160 mM sodium octanesulfonate, 20% ACN and 0.01% PVP as a dynamic coating to reduce analytes' interaction with the capillary wall. For sensitivity consideration regarding the determination of linearity, LOD, quantitation and monitoring drugs concentration in patients, the detection wavelengths for galantamine or rivastigmine and NAP 226-90 were set at 214 or 200 nm, respectively. One male volunteer (26-year-old) was orally administered a single dose of 4.5 mg rivastigmine (Exelon, Novartis) in capsule, and blood samples were drawn over a 12 h period for concentration-time profile study. The method was also successfully applied for monitoring galantamine or rivastigmine and its metabolite NAP 226-90 in 11 Alzheimer's disease patients' plasma after oral administration of the commercial products Reminyl (8 mg galantamine/capsule) or Exelon (3 mg rivastigmine/capsule), respectively.

CE with direct sample injection for the determination of metformin in plasma for type 2 diabetic mellitus: An adequate alternative to HPLC.

We developed a simple and selective CE with UV detection at 233 nm for the analysis of metformin in plasma based on direct sample injection without any pretreatment. The sample was employed with an electrokinetic injection of 10 kV for 100 s. CE separation of metformin from biological matrix was performed at 25 degrees C using a BGE consisting of 25 mM Tris buffer at pH 4.0. The linear range of the CE method for the determination of metformin in plasma was over the range of 0.1-2.0 mug/mL; the LOD of the drug in plasma (S/N = 3; injection 10 kV, 100 s) was 30 ng/mL. Data by CE were compared with the results obtained by a validated HPLC method. CE assay of metformin exhibited a very good correlation (r(2 )= 1) with respect to HPLC. CE determination of metformin is a robust, sensitive, and reproducible method with the advantage over HPLC of being fast, without prior extraction, or precipitation of proteins, also enabling quick assessment of metformin for pharmacokinetic and clinical studies.

Development of a new HPLC method for the simultaneous determination of ticarcillin and clavulanic acid in pharmaceutical formulations.

A new HPLC method has been developed and validated for the simultaneous determination of ticarcillin (TIC) and clavulanic acid (CA) in pharmaceutical formulations. The HPLC separation was achieved on a beta-cyclodextrin column (Cyclobond I, 250 x 4.6 mm, 5 microm) with methanol-16 mM pH 6.0 ammonium acetate buffer (50 + 50, v/v) mobile phase at a flow rate of 0.8 mL/min. Detection was at 220 nm. Validation of the method was performed by evaluating specificity, robustness, accuracy, and precision. The calibration curves were linear in the range of 1-100 microg/mL for CA and 2-200 microg/mL for TIC. The LOQs based on the standard regression lines were 0.42 and 1.42 microg/mL for CA and TIC, respectively, and the LOD were 0.14 and 0.47 microg/mL, respectively. Total recoveries of synthetic mixtures (CA:TIC = 1:10, 1:15, and 1:30) were 99.25-100.99% for CA and 99.54-100.82% for TIC. Compared with the U.S. Pharmacopeia method, the proposed method has the advantage of a relatively low flow rate and short analysis time. The proposed method was successfully applied for the simultaneous determination of these two drugs in sterilized H20 and 5% dextrose injection solutions.

Enantioseparation of cetirizine by sulfated-beta-cyclodextrin-mediated capillary electrophoresis.

A sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated beta-CD (w/v) as chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and the concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear range of the method for the determination of levocetirizine was over 1.0 to 50.0 microg/mL; the detection limit (signal-to-noise ratio = 3) of levocetirizine was 0.5 microg/mL. The method allowed the enantioseparation of cetirizine in bulk samples and enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal), and it was also found to be suitable for enantioseparation in human plasma.

Enantioselective determination of aspartate and glutamate in biological samples by ultrasonic-assisted derivatization coupled with capillary electrophoresis and linked to Alzheimer's disease progression.

A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0-20.0 µg mL-1l-Glu and 0-2.0 µg mL-1d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85-0.96 µg mL-1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer's disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = -0.158) between plasma l-Asp concentration and AD severity.

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients.

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection.

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.

Determination of indomethacin in plasma by micellar electrokinetic chromatography with UV detection for premature infants with patent ducts arteriosus.

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3-10.0 microg/mL; the detection limit (signal-to-noise ratio=3; injection 0.5 psi 5s) was 0.1 microg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.

Effect of atorvastatin withdrawal on circulating coenzyme Q10 concentration in patients with hypercholesterolemia.

Statin therapy can reduce the biosynthesis of both cholesterol and coenzyme Q10 by blocking the common upstream mevalonate pathway. Coenzyme Q10 depletion has been speculated to play a potential role in statin-related adverse events, and withdrawal of statin is the choice in patients developing myotoxicity or liver toxicity. However, the effect of statin withdrawal on circulating levels of coenzyme Q10 remains unknown. Twenty-six patients with hypercholesterolemia received atorvastatin at 10 mg/day for 3 months. Serum lipid profiles and coenzyme Q10 were assessed before and immediately after 3 months and were also measured 2 and 3 days after the last day on the statin. After 3 months' atorvastatin therapy, serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and coenzyme Q10 (0.43 +/- 0.23 to 0.16 +/- 0.10 microg/mL) were all significantly reduced (all p<0.001). On day 2 after the last atorvastatin, the coenzyme Q10 level was significantly elevated (0.37 +/- 0.16 microg/mL) and maintained the same levels on day 3 (0.39 +/- 0.18 microg/mL) compared with those on month 3 (both p< 0.001), while TC and LDL-C did not significantly change within the same 3 days. These results suggest that statin inhibition of coenzyme Q10 synthesis is less strict than inhibition of cholesterol biosynthesis.

Distinct CPT-induced deaths in lung cancer cells caused by clathrin-mediated internalization of CP micelles.

We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells.

Enantiomeric analysis of (+)-menthol and (-)-menthol by fluorogenic derivatization and liquid chromatography.

A simple and sensitive liquid chromatography is described for the quantitative analysis of enantiomeric (+)-menthol and (-)-menthol that are lack of chromophore. The method is based on the derivatization of (+)-menthol and (-)-menthol with a fluorescent reagent, naproxen acyl chloride, in toluene. The resulting diastereomic derivatives were separated on a C8 column with methanol-water-tetrahydrofuran (80:18:2, v/v) as a mobile phase; they were sensitively monitored with a fluorimetric detector (excitation 235 nm and emission 350 nm). The linear range for the quantitation of the enantiomers was 5.0-50 microM with a detection limit (signal to noise ratio = 3, injected volume 10 microl) of about 1 microM. Application of the method to the enantiomeric analysis of menthol in mint plants proved simple and feasible. Toluene was used for the extraction of menthol from the leaves of mint, and the resulting toluene extract was directly used for subsequent derivatization without solvent replacement.

Trace analysis of zotepine and its active metabolite in plasma by capillary electrophoresis with solid phase extraction and head-column field-amplified sample stacking.

A sensitive high-performance capillary zone electrophoresis (CZE) with head-column field-amplified sample stacking (FASS) in binary system has been developed for the simultaneous determination of zotepine and its active metabolite, norzotepine, in human plasma. The separation of zotepine and norzotepine was performed using a background electrolyte consisting of 50% ethylene glycol-borate buffer (20mM, pH 8.0) solution with 20% methanol as the running buffer and on-column detection at 200 nm. Under the optimal FASS-CZE condition, good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation and sensitivity of the drug were studied, including sample matrix, pH and concentrations of the borate buffer, ethylene glycol and methanol. Using clozapine as an internal standard, the linear ranges of the method for the determination of zotepine and norzotepine in human plasma were over 3-100 ng/mL; the detection limits of zotepine and norzotepine in plasma were 2 and 1 ng/mL, respectively. A sample pretreatment by means of solid-phase extraction (SPE) with subsequent quantitation by FASS-CZE was used. The application of the proposed method for determination of zotepine and norzotepine in plasma collected after oral administration of 125 mg zotepine in one schizophrenic patient was demonstrated.