Modified method for the determination of capillary electrophoresis nitric oxide-correlated nitrate in tissue homogenates

A modified capillary electrophoretic method for the determination of nitric oxide correlated nitrate in several tissue homogenates is described in this study. The method was developed using a running buffer consisting of 200 mM lithium chloride and 10 mM borate buffer at pH 8.5, in a fused-silica column total 82 cm, effective 43 cm length and 75 μm I.D. The signal was measured at 214 nm and controlled current of 200 μA (equivalent to 12.7 kV) was applied in the reversed polarity direction. The sample was injected by vacuum pressure 50 ms (25 nl). In these conditions, bromide as internal standard and nitrate appeared at 7.2 and 8.9 min, respectively. Whole validation procedures were applied and satisfactory results were obtained. The nitrate levels of the tissue homogenates of control and -NAME applied (heart, brain, kidney, stomach, lung, testis and liver) were monitored by the present method and it was decided that the method is precise and accurate.     

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm × 75 μm I.D.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25°C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10⁵-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm×75 μm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine

A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl⁻ and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl⁻ were not separated at the optimum detection pH of 5.2. The signal from Cl⁻ was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl⁻ from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl⁻ in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 μl injected).     

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.     

Enhanced separation and detection of serum bilirubin species by capillary electrophoresis using a mixed anionic surfactant—protein buffer system with laser-induced fluorescence detection

The four major bilirubin species in serum are separated by capillary electrophoresis and detected using laser-induced fluorescence detection. The optimum buffer system consists of 40 mM sodium dodecyl sulfate (SDS)—0.012 mM bovine serum albumin (BSA). The use of the SDS—BSA mixture in the mobile phase allows for the separation of four major bilirubin species at physiological pH with untreated capillaries. The results show that the use of BSA as a run buffer modifier in SDS solution improves separation efficiency and increases sample solubility via pH changes of the run buffer. The limits of detection for the bilirubin species using laser-induced fluorescence are between 30 and 150 nM, depending on the bilirubin species; not only is this approximately two orders of magnitude lower than with visible-light absorption methods, it allows the bilirubin species in normal sera to be quantitatively measured without sample pretreatment.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

Calcium-dependent proteolysis in neural tissue is activated at physiologic intracellular Ca2+ levels and inhibited by some anticonvulsants

The level of calcium-activated neutral protease (CANP) activity in the brain and nerve cord of the crayfish Procambarus clarkii was assayed by measuring the degradation of casein yellow by tissue homogenates. When care was taken to maintain the ionic strength of all incubation media at 0.15M and to buffer the Ca²⁺activity of the media with 5mM EGTA, CANP was found to be very sensitive to Ca²⁺; maximal activity was achieved at 1 × 10^?3M Ca²⁺, with 50% of this maximum present at the physiologic intracellular Ca²⁺activity of 1 × 10^?7M. We found that the anticonvulsant agent phenytoin was without effect on CANP activity while pentobarbital and a relatively new anticonvulsant agent, valproic acid (an eight-carbon branched fatty acid), significantly inhibited CANP activity in a dose-dependent manner. The inhibitory effect of valproic acid was shared by a straight-chain eight-carbon fatty acid, caprylic acid. These findings demonstrate that inhibition of CANP activity is not limited to agents with a specific molecular structure. They also suggest that CANP plays a role in the normal turnover of proteins that control various cellular functions.     

Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis–mass spectrometry

This paper describes the development of an analytical method for the separation and identification of neuropeptide Y (NPY) and two important NPY fragments by capillary electrophoresis (CE) and mass spectrometry (MS). A satisfactory separation and the highest sensitivity were obtained with formic acid at high concentrations (250 mM, pH 2.75). The addition of 25 or 50 mM triethylamine (TEA) improved the separation. When applying full scan CE–MS, the separated peptides could be detected and identified using the spectra of each peak. The use of TEA as an additive to the formic acid slightly decreased the sensitivity but was compensated by the improved efficiency. The best compromise for optimal separation and MS detection was found to be 50 mM formic acid to which 50 mM TEA was added. CE–MS could be used for identification of the decomposition products of NPY. Decomposition products with one amino acid difference, which could not be distinguished with CE–UV, could be distinguished with CE–MS.     

Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-β-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen–phosphorus detection (GC–NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179°C at 4°C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10 000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within ±15%.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10⁻¹⁰ M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

Nitrite and Nitrate Levels in Individual Molluscan Neurons: Single-Cell Capillary Electrophoresis Analysis

Abstract: Cell and tissue concentrations of NO₂^? and NO₃^? are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO₂^? and NO₃^? levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-µm separation capillary and the NO₂^? and NO₃^? were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO₂^? and NO₃^? were <200 fmol (<4 µM in the neurons under study). The NO₂^? and NO₃^? levels in individual neurons varied from 2 mM (NO₂^?) and 12 mM (NO₃^?) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO₂^? was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO₃^? averaged 1.8 ± 0.2 × 10^?3M. Hemolymph NO₃^? in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 ± 0.2 × 10^?5M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 × 10^?4M). Capillary electrophoresis methods provide utility and convenience for monitoring NO₂^?/NO₃^? levels in single cells and small amounts of tissue.     

Measurement of hydroxyl radical in rat blood vessel by microbore liquid chromatography and electrochemical detection: an on-line microdialysis study

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C₁₈, 150×1 mm I.D., 5 μm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).     

In vitro cleavage of paracetamol glucuronide by human liver and kidney β-glucuronidase: determination of paracetamol by capillary electrophoresis

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human β-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 μM (38 ng/ml) and a limit of quantitation of 1 μM (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human β-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human β-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.     

Kinetic Analysis of Leucine-Enkephalin Cellular Uptake at the Luminal Side of the Blood-Brain Barrier of an In Situ Perfused Guinea-Pig Brain

Abstract: The uptake of enkephalin-(5-L-leucine) (Leu-en-kephalin) at the luminal side of the blood-brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, K_in, for [tyrosyl-3,5-³H]Leu-enkephalin was estimated graphically from the multiple-time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose-dependent self-inhibition was demonstrated. Analysis of unidirectional influx of blood-borne Leu-en kephalin into the brain revealed Michaelis-Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with V_max between 0.14 and 0.16 nmol min^?1 g^?1 and K_m ranging from 34 to 41 μM, for the saturable component, whereas the estimated diffusion constant, K_d, was not significantly different from zero. Entry of [³H]Leu-enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L-tyrosine, tyro-sylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood-brain barrier does not involve uptake of the peptide's N-terminal amino acid and/or its tyrosine-containing fragments. The specific δ-opioid antagonist, allyl²-Tyr-AIB-Phe-OH, and μ-opioid receptor agonist, Tyr-D-Ala-Gly-Me-Phe-NH(CH₂)20H, at concentrations in the perfusate above the K_m value for the saturable transport of Leu-enkephalin, did not affect significantly uptake of [³H]Leu-enkephalin. The present study provides, for the first time, a characterization of the kinetic parameters of the unidirectional uptake of a peptide from the luminal side of the blood-brain barrier