Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced fluorescence detection

A capillary electrophoresis method for the simultaneous separation and enantioseparation of the antibacterial drug ofloxacin and its metabolites desmethyl ofloxacin and ofloxacin N-oxide in human urine has been developed and validated. Enantioseparation was achieved by adding sulfobutyl β-cyclodextrin to the running buffer. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd-laser with an excitation wavelength of 325 nm. In comparison with conventional UV detection, LIF detection provides higher sensitivity and selectivity. The separation can be performed after direct injection of urine into the capillary without any sample preparation, because no matrix compounds interfere with the assay. Additionally, the high sensitivity of this method allows the quantification of the very low concentrations of enantiomers of both metabolites. The limit of quantification was 250 ng/ml for ofloxacin enantiomers and 100 ng/ml for each metabolites’ enantiomers. This method was applied to the analysis of human urine samples collected from a volunteer after oral administration of 200 mg of (±)-ofloxacin to elucidate stereoselective differences in the formation and excretion of the metabolites. It could be demonstrated that the renal excretion of the S-configured metabolites, especially S-desmethyl ofloxacin, within the first 20 h after dosage, is significantly lower than that of the R-enantiomers.     

Determination of gabapentin in human plasma by capillary electrophoresis with laser-induced fluorescence detection and acetonitrile stacking technique

A sensitive analytical method for gabapentin [1-(aminomethyl) cyclohexaneacetic acid] (GBP) in human plasma based on capillary electrophoretic separation and laser-induced fluorescence (LIF) detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent drug in plasma. Optimal separation and detection were obtained with an electrophoretic buffer of 50mM sodium borate (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). A calibration curve ranging from 0.3 to 150 microM was shown to be linear. The concentration limit of detection (LOD) in plasma was 60 nM. We also demonstrate how the detection limit can be enhanced by using acetonitrile stacking technique. With stacking, the limit of detection for gabapentin in plasma was 4.8 nM. A calibration curve ranging from 0.03 to 15 microM was shown to be linear. Both the within-day and day-to-day reproducibility and accuracy were 相似文献   

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT_8–13), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise = 3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT_8–13, NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT_8–13 in human cerebrospinal fluid sample.     

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.     

Separation of the glucuronides of entacapone and its (Z)-isomer in urine by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method was developed for the separation of the 3-O-glucuronides of entacapone and its (Z)-isomer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intended for use as an adjunct in the treatment of Parkinson’s disease. Urine samples spiked with synthetic 3-O-glucuronides were used to study the effects of running buffer pH, composition and applied voltage on separation of the closely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved by increasing the running buffer ionic concentration. Changes in pH had little effect on the separation, whereas increase in sodium dodecyl sulfate (SDS) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphate (25 mM)–borate (50 mM)–SDS (20 mM) running buffer, pH 7.0, in a 75 μm×60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in the UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 μg/ml (5.2·10 ⁻⁷M) (injection 0.5 p.s.i./8 s). The linear detection range was 2–100 μg/ml (r²>0.999). Good repeatability of injection and relative migration times were obtained.     

Ultrasensitive capillary electrophoresis of sulfated disaccharides in chondroitin/dermatan sulfates by laser-induced fluorescence after derivatization with 2-aminoacridone

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived Δ-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated Δ-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized Δ-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.     

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4×10⁻⁸ M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Trace analysis of D-tyrosine in biological samples by microchip electrophoresis with laser induced fluorescence detection

A rapid and sensitive microchip electrophoresis (MCE) method with laser induced fluorescence (LIF) detection has been developed for the quantification of D-tyrosine (Tyr) in biological samples. The assay was performed using a MCE-LIF system with glass/poly(dimethylsiloxane) (PDMS) hybrid microchip after pre-column derivatization of amino acids with fluorescein isothiocyanate (FITC). Chiral separation of the derivatives was achieved by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using γ-CD as chiral selector in the running buffer. D/L-Tyr enantiomer was well separated in less than 140s. The limit of detection (S/N=3) was 3.3 × 10(-8) M. Using the present method, D-Tyr level in human plasma was found to vary significantly from normal humans to patients suffering from renal failure.     

Quantitation of the diastereoisomers of l-buthionine-(R,S)-sulfoximine in human plasma: a validated assay by capillary electrophoresis

An assay for the diastereoisomers of the biochemical modifier $\text{l}\text{-buthionine}\text{-(R,S)-}\text{sulfoximine}$ (BSO) in human plasma has been developed using capillary electrophoresis (CE). Separation of the diastereoisomers is achieved by the micellar electrokinetic chromatography (MEKC) mode of CE. Plasma is injected directly onto the separation capillary without any extraction step, and BSO is detected directly by ultraviolet absorbance measurements at 190 nm without prior derivatization. The whole assay, including capillary conditioning, takes approximately 30 min. Intra- and inter-day R.S.D. values are approximately 7% at sample concentrations around 25 μg ml⁻¹, and approximately 3% at sample concentrations around 500 μg ml⁻¹. The limit of detection in plasma is 3.9 μg ml⁻¹ (S/N = 2). The assay has been used to quantitate the diastereoisomers of BSO in patient samples in a pharmacokinetic study.     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

Analysis of amino acids in individual wheat embryonic protoplast

Summary. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl₂) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68×10⁻⁵ mol/L to 18.18×10⁻⁵ mol/L in single protoplast.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

Determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine by capillary electrophoresis using ultraviolet absorbance and laser-induced fluorescence detection

Two capillary electrophoresis methods have been developed for the direct determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine. Analytes were detected using conventional UV detection as well as laser-induced fluorescence (LIF) detection with an HeCd-laser operating at a wavelength of 325 nm. The results of both detection techniques were compared. Indeed, the limit of quantification was eightfold lower using LIF detection (50 ng/ml) in comparison to UV detection (400 ng/ml). As no interference due to endogenous urine compounds was observed, direct urine analysis was feasible. Analysis was very simple and fast-one run could be performed within less than 10 min (CE-UV method) and 2.5 min (CE-LIF method), respectively. Both assays were fully validated and applied to urine samples from a human volunteer. The results of the application of the CE-LIF method to human urine samples are presented in this publication.     

Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Immunological analysis of methamphetamine antibody and its use for the detection of methamphetamine by capillary electrophoresis with laser-induced fluorescence

An accurate, simple and rapid immunoassay is demonstrated for the detection of methamphetamine in urine by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). An aminobutyl derivative of methamphetamine was conjugated with proteins, and used as an immunogen to produce antibodies for the assay. The methamphetamine derivative was also labeled with fluorescein isothiocyanate (FITC) to compete with free methamphetamine in the sample for the antibody binding site. Levels of free and antibody-bound FITC-labeled methamphetamine were monitored by performing CE–LIF using an untreated fused-silica column. This competitive immunoassay used antiserum instead of purified antibody or antibody fragment, yet was found to have good precision with a sensitivity of lower than 20 ng/ml. Various antibodies were also screened, and cross-reactivity of anti-MA antibody with methamphetamine analogues were also investigated. The results indicate that CE–LIF-based immunoassay is a powerful tool for the screening and characterization of antibody and may have possible applications in the detection of abused drugs in urine.     

High-performance liquid chromatographic determination of tazobactam by precolumn derivatization

A new reversed-phase high-performance liquid chromatography (RP-HPLC) method for the detection and quantification of tazobactam in serum and haemofiltration fluid is described. The assay for these biological fluids involves an extraction with diethyl ether followed by derivatization using 1,2,4-triazole. The mobile phase consisted of phosphate buffer-methanol and the detection wavelength was 325 nm. The limit of detection was 0.05 μg/ml in the two fluids and the calibration curves were linear over the range 0.1–50 μg/ml. For a tazobactam concentration equal to 1, 5 or 20 μg ml⁻¹, the coefficients of variation were less than 5%. The assay was successfully applied to the analysis of samples from drug monitoring in a patient with renal insufficiency undergoing continuous venovenous haemofiltration (CVVH).     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Determination of kanamycin in serum by solid-phase extraction,pre-capillary derivatization and capillary electrophoresis

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.