Analysis of ten abused drugs in urine by large volume sample stacking-sweeping capillary electrophoresis with an experimental design strategy.

A statistical tool equipped with Plackett-Burman design (PBD) and central composite design (CCD) was used for fast stacking analysis of ten frequently consumed drugs, namely codeine, morphine, methamphetamine, ketamine, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam and oxazepam, by capillary electrophoresis (CE). This statistical design is expected to help quick analysis with few procedures, avoiding tedious work required because of the large number of variables or parameters. A large volume sample stacking (LVSS)-sweeping CE is developed for concentrating and analyzing the 10 abused drugs. First, phosphate buffer (50 mM, pH 2.3) containing methanol was filled into a capillary and then the extracted urine sample was loaded (1 psi, 200 s) to enhance sensitivity. The sweeping and separating steps were completed simultaneously by phosphate buffer (50 mM, pH 2.3) containing methanol and sodium dodecyl sulfate, within 15 min. Better resolution was obtained by the experimental design than the "one factor at a time" (OFAT) approach. During method validation, calibration plots were linear (r>0.998), over a range of 25-1500 ng/mL for the six benzodiazepines, methamphetamine and ketamine, and 50-3000 ng/mL for codeine and morphine. The RSD of precision and absolute RE of accuracy in intra-day and inter-day assays were below 14.54% and 16.61%, respectively. The minimum limits for detection (S/N=3) of analytes were in the range of 7.5-30 ng/mL. This stacking method increased sensitivity more than 200-fold and can be applied for detection of the presence of methamphetamine in an abuser's urine (3600 ng/mL), which was confirmed by GC-MS. The method is considered feasible for fast screening of abused drugs in urine.

Analysis of multiple abused drugs and hypnotics in urine by sweeping CE.

Multiple drugs usage is very common in addicts (AD). However, some parent drugs were undetectable in urine, it was necessary to monitor their metabolites simultaneously. A sweeping CE was established for the determination of several kinds of abused drug and their metabolites in AD urine. This method was developed using chemometric experimental design to simplify the CE optimization. The capillary was filled by separation buffer (phosphate buffer (75 mM, pH 2.5) and methanol (70:30 v/v)) and then hydrodynamically injected large volume of samples into capillary (1 psi, 200 s). Following was using sweeping buffer (phosphate buffer (75 mM, pH 2.5) and methanol (90:10 v/v) containing 65 mM SDS) to sweep and stack analytes. The separation voltage was set at -15 kV (anode at detector end). During method validation, calibration plots were linear (r > or = 0.992) over a range of 0.1-3 microg/mL for codeine, ketamine, and methamphetamine, 0.15-3 microg/mL for morphine, 0.1-1 microg/mL for alprazolam and oxazepam, and 0.1-1.2 microg/mL for other other benzodiazepines and its metabolites. During intra- and inter-day analysis, relative standards and relative errors were less than 14%. The analytes could be simultaneously analyzed and have a detection limit down to 20-50 ng/mL (S/N=3). The results showed good coincidence with GC-MS or LC-ESI-MS. This method was feasible for application to detect trace levels of abused drugs in AD' urine.

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 microm 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 > or = 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.

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.

On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designs.

A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4 min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9 s at a voltage of -5 kV were stacked in a water plug (0.5 psi, 9 s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate-triethanolamine at low pH, and a separation voltage of -10 kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies.

Hair analysis for methamphetamine, ketamine, morphine and codeine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography.

We established a capillary electrophoretic method with high sensitivity and specificity for testing hair taken from addicts. After pretreatment of hair sample, the cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was used to test for the presence of abused drugs in human hair. These drugs include morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, an uncoated fused-silica capillary (40 cm, 50 microm I.D.) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, followed by high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s). Electrokinetic injection (10 kV, 600 s) was used to load samples and to enhance sensitivity. Stacking steps and separations were performed at -20 kV with detection at 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 the level of picogram per milligram hair. During method validation, calibration plots were linear (r > or = 0.999) over a range of 0.15-80 ng/mg hair for MA and K, 0.3-30 ng/mg hair for C and 0.5-50 ng/mg hair for M. The limits of detection were 50 pg/mg hair for MA and K, 100 pg/mg hair for C and 200 pg/mg hair for M (S/N=3, sampling 600 s at 10 kV). Our method was applied for analysis of real hair samples taken from addicts. The addicts' specimens were also analyzed by LC-MS, and showed good coincidence of results. This method has proven feasible for application in detecting trace levels of abused drugs in forensic analysis.

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.

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.

Field-amplified sample stacking in capillary electrophoresis for the determination of clozapine, clozapine N-oxide, and desmethylclozapine in schizophrenics' plasma.

A method of field-amplified sample stacking in capillary electrophoresis is described for the simultaneous determination of clozapine (CZP) and its metabolites, clozapine N-oxide (CNO), and desmethylclozapine (DMC), in human plasma. Plasma (0.2 mL) was extracted with organic solvents (ethyl acetate/n-hexane/isopropyl alcohol, 8/1/1 by volume) and centrifuged. An aliquot of supernatant was evaporated and suitably reconstituted with water for CE analysis. An untreated fused-silica capillary was used (31.2 cm; effective length, 20 cm; 50 microm i.d.) for the analysis. The background buffer was phosphate buffer (400 mM, pH 3.0) containing 50% ethylene glycol. The separation voltage was 25 kV with a detection wavelength of 214 nm. In the method validation, the calibration curves were linear (r > or = 0.98) over a range of 50-800 ng/mL for CZP, 30-180 ng/mL for CNO, and 25-600 ng/mL for DMC. The relative standard deviation (R.S.D.) and relative error (R.E.) were all less than 11% for the intra- and inter-day assays. The limits of detection (S/N = 3, electric-driven injection, 99.9s) of CZP, DMC, and CNO were 5, 5, and 10 ng/mL, respectively. After continuing treatment with the CZP tablets, a blood sample from one male schizophrenic patient (41-year-old, 62 kg) who had been receiving ongoing treatment with the CZP tablets was prepared and analyzed. The levels of CZP, DMC, and CNO were determined and the feasibility of the method's application in clinical treatment was proven.