Determination of free aspartic acid enantiomers in rat brain by capillary electrophoresis with laser-induced fluorescence detection

Quantification of aspartic acid enantiomers in rat brain by using a chiral capillary electrophoresis procedure is described. Amino acids were pre-column derivatized with naphthalene-2,3-dialdehyde. Enantiomeric separation was achieved by micellar electrokinetic chromatography in the presence of methanol and beta-cyclodextrin as chiral selector. The chiral separation was coupled with laser-induced fluorescence detection. Contents of D- and L-aspartic acids in rats at different stages of growth (from 1 day before birth to 90 days after birth) were determined. D-Aspartic acid was detected in all the brain tissue samples tested, but at different levels. In the cerebrum of rats 1 day before birth, D-aspartic acid was found to be at the highest concentration of 81 nmol/g wet tissue. The level of D-aspartic acid in rat brain falls rapidly after birth, while the L-aspartic acid level increases with age.     

Direct quantification of AD-36 adenovirus DNA by capillary electrophoresis with laser-induced fluorescence

An adenovirus, AD-36, has been linked to human adiposity and a sensitive and reliable quantitative method is required to assess AD-36 viral loads. This report describes direct detection of AD-36 viral DNA, which is the first method to quantitate DNA without amplification. Total genomic DNA is hybridized with an AD-36 specific fluorescently labeled probe and analyzed by capillary electrophoresis with laser-induced fluorescence. The minimum detectable quantity is 10.3 ng/ml, corresponding to 282 copies of AD-36 with a precision of 1-6%. These results indicate that direct detection with capillary electrophoresis with laser-induced fluorescence (CE-LIF) is a reliable and sensitive method for quantifying AD-36 viral DNA.     

Determination of morphine by capillary zone electrophoresis immunoassay combined with laser-induced fluorescence detection

A competitive immunoassay for detecting morphine in bio-samples was established by capillary zone electrophoresis combined with laser-induced fluorescence detection (CZE-LIF). The antigen of morphine was labeled with isothiocyano-fluorescein (FITC) and then incubated with morphine monoclonal antibody and samples. The linear range was 50-1000 ng/mL, which was suitable for clinical and forensic applications. The detection limit can reach 40 ng/mL, based on S/N = 2. The recoveries of morphine from serum were satisfactory.     

Determination of salicylate, gentisic acid and salicyluric acid in human urine by capillary electrophoresis with laser-induced fluorescence detection

Acetylsalicylic acid (Aspirin) is rapidly metabolized to salicylic acid (salicylate) and other compounds, including gentisic acid and salicyluric acid. Monitoring of salicylate and its metabolites is of toxicological, pharmacological and biomedical interest. Three capillary electrophoresis (CE) methods featuring alkaline aqueous buffers, laser-induced fluorescence (LIF) detection and no solute extraction or derivatization have been explored. A competitive binding, electrokinetic capillary-based immunoassay is developed that recognizes the presence of salicylate and gentisic acid in urine. Differentiation of the two compounds, however, is problematic. With appropriate ultraviolet excitation, many salicylate-related compounds are fluorescent so that CE with direct urine injection and LIF detection permits the determination of salicylate, gentisic acid and salicyluric acid. Using a HeCd laser with 325 nm produces interference-free monitoring of all three compounds. Using 257 nm excitation from a frequency doubled Ar ion laser, native fluorescence of an endogenous urinary compound that co-migrates with gentisic acid is observed. With wavelength-resolved fluorescence detection, however, the two substances are distinguished. Furthermore, this technique, with comparison to literature data, permits the putative assignment of several peaks to other salicylate metabolites, namely glucuronide conjugates of salicylate and salicyluric acid. All three CE-LIF techniques have been applied to toxicological patient urines and urines collected after ingestion of 500 mg acetylsalicylic acid. CE results compare favorably with those obtained by a commercial fluorescence polarization immunoassay and by a conventional photometric assay.     

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 beta-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.     

Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection.

Indocyanine green (ICG) is a negatively charged, water-soluble, tricarbocyanine dye used primarily for medical imaging. ICG is only weakly fluorescent in the near-infrared region in its free (unbound) state in dilute aqueous solution. However, when non-covalently bound to protein, its fluorescence is greatly enhanced, making it a candidate for diode laser-induced fluorescence (diode-LIF) detection of proteins in capillary electrophoresis (CE). This paper investigates the suitability of ICG as a fluorescent label for the separation and detection of human serum albumin (HSA) by CE with diode-LIF detection. Specifically, we have considered the separation conditions necessary to resolve free ICG from ICG-HSA complexes; the limits of detection for free and HSA-bound ICG; the stability of aqueous ICG and ICG-HSA solutions over time; and the stoichiometry of the ICG-HSA complex.     

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 x 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.     

On-column derivatization for the analysis of homocysteine and other thiols by capillary electrophoresis with laser-induced fluorescence detection

On-column derivatization and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection has been developed for the fully automated assay of homocysteine and other thiols. The unique feature of this CE technique comes from the direct injection of a sample including homocysteine, enabling the derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadizole (ABD-F) to be accomplished in the capillary. After the derivatization for 10 min at 50 degrees C, the homocysteine was analyzed within 7 min under an applied electric field of 333 V cm(-1). The detection limit obtained for homocysteine with on-column LIF detection was 5.0 nM, as compared to 2.5 nM with pre-column LIF detection. The method is a very simple, fast, and practical approach for the fully automated assay of homocysteine and other thiols contained in low-volume and low-concentration samples.     

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 delta-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated delta-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 delta-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.     

Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa

Human oocytes can be fertilized with high rates of success underin-vitro conditions even if only low numbers of spermatozoaare used. A culture system has been developed in which fertilizationis performed in haematocrit capillary tubes (length 75 mm; i.d.0.8–0.9 mm). Oocytes were fertilized in 5–10µlof different sperm suspensions containing a total of 500, 1000,2000 and 4000 spermatozoa per oocyte (0.1–0.4 x 10⁶ spermatozoa/ml).Oocytes were obtained from 10 patients participating in an in-vitrofertilization programme; of these, 32 oocytes were fertilizedin capillary tubes and 32 oocytes were cultured using standardmethods (1 ml culture medium in tissue culture tubes; 0.1–0.2x 10⁶ spermatozoa/ml). The overall fertilization rate of oocytescultured in tissue culture tubes was 78% (25/32) and the fertilizationrates in capillary tubes using 4000, 2000, 1000 or 500 spermatozoaper oocyte were 71% (5/7), 86% (6/7), 60% (6/10)and 50% (4/8),respectively. The fertilization rate of mature oocytes was highercompared with immature oocytes when fertilization was performedin culture tubes (83 and 63%) or in capillary tubes (74 and44%). Fertilization in capillary tubes using a 10µl ofoocyte and spermatozoa suspension compared to 5µl seemedto provide better culture conditions, resulting in higher fertilizationand cleavage rates. These preliminary results indicate thatfertilization of human oocytes under in-vitrio conditions canbe achieved even with very low numbers of spermatozoa     

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection.

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.     

Direct chiral assay of tramadol and detection of the phase II metabolite O-demethyl tramadol glucuronide in human urine using capillary electrophoresis with laser-induced native fluorescence detection

A chiral separation using carboxymethyl-beta-cyclodextrin and methyl-beta-cyclodextrin for the direct assay of tramadol in human urine by capillary electrophoresis (CE) with laser-induced native fluorescence detection was developed. Furthermore, the phase II metabolite O-demethyl tramadol glucuronide was determined from the urine samples and the ratio of the diasteromers was determined. The chiral method was validated. Correlation coefficients were higher than 0.999. Within day variation showed accuracy in the range 96.1-105.8% with a RSD less than 6.00%. Day to day variation present an accuracy ranging from 100.2 to 103.5% with a RSD less than 5.4%. After oral administration of 150 mg tramadol hydrochloride to a healthy volunteer, the urinary excretion was monitored during 24 h. About 11.4% of the dose was excreted as 1S,2S-tramadol, 16.4% as 1R,2R-tramadol and 23.7% as O-demethyl tramadol glucuronide. The amount of 1S,2S O-demethyl tramadol glucuronide was more than three fold higher as IR,2R-O-demethyl tramadol glucuronide. The enantiomeric ratio of tramadol and the diastereomeric ratio of O-demethyl tramadol glucuronide was deviated from 1.0 showing that a stereoselective metabolism of tramadol occurs.     

Determination of amoxicillin in plasma samples by capillary electrophoresis.

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C18 cartridges followed by elution with water-methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 micrograms l-1 by the FASI technique.     

Quantification of daunorubicin and daunorubicinol in plasma by capillary electrophoresis

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane-2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile-0.12 M NH4H2PO4-tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5-150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze-thaw cycles and after 7 months frozen storage below -20 degrees C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

Subcutaneous microdialysis: a simple technique for monitoring the extracellular biochemical environment. Combination with capillary electrophoresis and laser-induced fluorescence detection

Microdialysis is a simple technique that allows monitoring endogenous or exogenous substances in any extracellular compartment. It has many useful experimental and clinical applications. The sampling of the extracellular fluid of the subcutaneous compartment is especially useful for metabolic evaluation in critically ill patients, pharmacokinetic studies and blood glucose monitoring. We built a subcutaneous microdialysis probe, with a cellulose hollow fiber (13,000 molecular weight cut off, 200 microns outside diameter) glued to stainless steel tubing (26 ga. outside diameter). It was implanted in the subcutaneous tissue of a critically ill child or anesthetized mice to obtain amino acids patterns by means of capillary electrophoresis with laser induced fluorescence detection (CE-LIFD). The probe was also implanted in ambulatory volunteers to monitor glucose. The results confirmed that subcutaneous microdialysis is a very simple, inexpensive and not aggressive method with advantages over repeated venipuncture sampling and endovenous microdialysis sampling. The present report shows that subcutaneous microdialysis with the proper analytical technique can be used to monitor the chemical composition of the interstitial compartment in very different preclinical or clinical conditions.     

Separation of eleven central nervous system drugs by capillary zone electrophoresis.

Several strategies to improve the separation of 11 central nervous system drugs (antipsychotics and antidepressants) with capillary zone electrophoresis were applied: the variation of the pH of the buffering background electrolyte, its ionic strength, addition of inclusion-complex forming beta-cyclodextrin or polyvinylpyrrolidone (PVP), respectively, as a replaceable, soluble, polymeric pseudo-stationary phase. Best separation was achieved at pH 2.5 and 35 mmol/l ionic strength (phosphate buffer), with 0.5% (w/v) PVP.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C(18) cartridge using a mixture of methanol-water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0+/-4.2% and 123.3+/-4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5-20 mg l(-1) and detection limits were between 1.1 and 2.4 mg l(-1).     

Separation of olanzapine, carbamazepine and their main metabolites by capillary electrophoresis with pseudo-stationary phases

Conditions were worked out for the separation of carbamazepine, olanzapine, and their main metabolites carbamazepine 10,11-epoxide, 10-hydroxycarbamazepine, and desmethylolanzapine. The separation was based on electrokinetically driven methods in the capillary format. The main difficulty in separating these compounds is related to their different chemical classes. Whereas the carbamazepine members are amides, and are electrically neutral, the olanzapine members have aliphatic amino groups and are thus cationic under most experimental conditions. Different additives were applied as pseudo-stationary phases to implement selectivity. Poly(diallyldimethylammonium), PDADMA, is a polycationic replaceable and soluble polymer, that interacts mainly according to the polarisability of the analyte molecules. The MEKC principle was applied with the common SDS as micelle former. In both systems, only partial resolution of the analytes was obtained. The most favorable system consisted of a charged, oligomeric additive: full separation of all analytes within 4 min was achieved with heptakis-6-sulfato-beta-cyclodextrin (7 mM) in 30 mM borate buffer, pH 8.5.     

Separation of promethazine and thioridazine using capillary electrophoresis with end-column amperometric detection

Promethazine and thioridazine were separated and detected by capillary electrophoresis with end-column amperometric detection. The influence of pH value on oxidation potential, the peak current and the resolution were studied and the following conditions was selected: 0.03 M Na2HPO4 and 0.015 M citric acid at pH 3.0, detection potential at 1.10 V. The detection limits of these two substances were in the range of 10(-8) mol/l. The linear range spanned two to three orders of magnitude. This method was applied to the detection of promethazine and thioridazine spiked in urine.     

Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis

Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cm x 75 microm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 microM (or 6 x 10(-15) mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 microM. The analysis time was 20 min per injection and the day-to-day precision was +/-2-3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40.