Mass assay for inositol 1-phosphate in rat brain by high-performance liquid chromatography and pulsed amperometric detection.

A high-performance liquid chromatographic method for direct mass measurement of inositol 1-phosphate (I(1)P) in rat brain is described. Separation of I(1)P from its isomers and from endogenous components is achieved by polymeric anion-exchange chromatography with a sodium hydroxide/sodium acetate mobile phase. Detection is performed at high pH by pulsed amperometric detection at a gold electrode. Sample preparation involves liquid-liquid extraction and ion-exchange solid-phase extraction, prior to HPLC. The method is sufficiently sensitive and selective to enable facile determination of basal levels of I(1)P in small amounts of brain tissue. The applicability of the method is demonstrated by the in vivo monitoring of I(1)P levels in rat brain after administration of the inositol monophosphatase inhibitor lithium and the cholinergic agonist pilocarpine. The method is a significant improvement over existing published mass assays for I(1)P by virtue of its simplicity, speed, sensitivity, and ruggedness.     

Determination of indoles and catechols in rat brain and pineal using liquid chromatography with fluorometric and amperometric detection

Tryptophan, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid were determined in rat brain by the direct injection of a centrifuged tissue homogenate into a liquid chromatographic—fluorometric/amperometric system. The above indoles, along with melatonin, were also determined in single rat pineal glands. The utility of the system in determining several additional catechols and indoles in brain was examined.     

Determination of erythromycin concentrations in rat plasma and liver by high-performance liquid chromatography with amperometric detection

A simple method for the quantitative determination of erythromycin (EM) concentrations in rat plasma and liver by high-performance liquid chromatography with amperometric detection was developed. EM was extracted from 200 μl of plasma or liver homogenate sample under sodium hydroxide alkaline conditions with tert.-butyl methyl ether. Oleandomycin was used as an internal standard. The recovery rate of EM was up to 100%. The detector cell potential for the oxidation of EM was +1100 mV. The calibration curves were linear over the concentration ranges 0.1–20.0 μg/ml for plasma and 0.5–100.0 μg/g for liver. The method was applied to the determination of the plasma and liver concentrations of EM in rats after intravenous administration (50 mg/kg dose). The method presented here has proved to be of great use for the investigation of the pharmacokinetic characteristics of EM in small animals such as rats.     

Simple determination of riluzole in rat brain by high-performance liquid chromatography and spectrophotometric detection

A simple method was developed for separation and quantification of riluzole in rat brain. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 264 nm. The mobile phase consisted of methanol-water containing 1% triethylamine adjusted with orthophosphoric acid to pH 3.2. The retention time was 8.6 min. A simple liquid-liquid extraction with ethyl acetate was used to obtain riluzole from brain samples. The limit of quantification was 10 ng/g. The recovery was about 80%. The relationship between peak areas and concentrations was linear over the range between 0.01 and 0.8 microg/g, with r2 value over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of riluzole.     

Pineal and plasma melatonin as determined by high-performance liquid chromatography with electrochemical detection.

A rapid and sensitive method for the routine quantitative determination of melatonin in pineal and plasma is described. The assay used reversed-phase high-performance liquid chromatography (RP-HPLC) separation combined with either amperometric (system A) or coulometric (system B) detection. The method gave satisfactory reproducibility and accuracy, and detection limits for melatonin were as low as 8.5 pg (system A) and 1 pg (system B). This high sensitivity, together with the short analysis time (less than 10 min), and the simplicity of sample procedure make the present RP-HPLC method suitable for a wide range of studies concerning melatonin measurements. Melatonin values obtained in this study from both rat pineal and human plasma agree with those reported previously, and clearly determined a circadian pattern.     

Determination of retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection

A method for separating and detecting retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection is described. Packed columns with an inner diameter of 180 μm were employed for the separation using a C₁₈ stationary phase and a mobile phase containing acetonitrile-water-methanol (65:32.5:2.5, v/v/v) with 1% tetrabutylammonium perchlorate and 0.174 M acetate buffered at pH 5. The detection cell consisted of a carbon fiber barrel electrode held at 0.9 V versus an Ag/AgCl reference. Injection volumes of 2 μl produced detection limits of 2.73, 0.472, 0.428, and 0.267 fmol (or 410, 64.1, 60.9, and 38.2 pg ml⁻¹) for 13-cis-retinoic acid, all-trans-retinoic acid, retinaldehyde, and retinol, respectively. This represents an improvement in detection limits of at least three orders of magnitude for similar analyses using liquid chromatography and UV absorbance detection. The detector signal was linear over two orders of magnitude of analyte concentration. Retinoid concentrations in bovine serum were determined and found to be in good agreement with previously reported values.     

Determination of gamma-aminobutyric and glutamic acids in rat brain by liquid chromatography with electrochemical detection

The determination of glutamic and gamma-aminobutyric acids in rat brain regions by derivatization with o-phthaldialdehyde-thiol, isocratic separation by liquid chromatography, and quantification by electrochemical detection provides a simple and precise method for assessing changes in glutamatergic and GABAergic neuronal systems. Gamma-aminobutyric acid was eluted in 30-35 minutes followed by a washout step with 90% methanol to remove all amino acid derivatives with longer retention times. Homoserine was used as an internal standard. Significant increases in gamma-aminobutyric acid content in nucleus accumbens and substantia nigra could be detected 20 minutes after injection of 400 mg/kg valproic acid.     

Analysis of acetaminophen metabolites in urine by high-performance liquid chromatography with UV and amperometric detection

Acetaminophen and several of its metabolites are separated isocratically on a reversed-phase (C₁) column using a mobile phase of 7% methanol and 0.75% glacial acetic acid in 0.1 M KH₂PO₄. Metabolites that can be separated include the sulfate, glucuronide, cysteine, and mercapturic acid conjugates of acetaminophen, as well as 3-hydroxyacetaminophen, 3-methoxyacetaminophen, and 3-methylthioacetaminophen. Although all of the metabolites can be detected by UV spectrophotometry, the sensitivity limits of detection are improved significantly for acetaminophen and all of the metabolites except the sulfate and glucuronide, by amperometric detection (electrochemical) of the same sample as it elutes from the UV detector. Minimal detectable limits (signal-to-noise ratio 2) for acetaminophen and its metabolites, other than the glucuronide and sulfate conjugates, were in the order of 1–2 ng injected on column using UV detection at 248 nm, and 0.1–0.5 ng using electrochemical detection at + 0.60 V with reference to an Ag/AgCl standard electrode.     

Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection

A sensitive assay was developed for the measurement of olanzapine in rat brain tissue using HPLC with electrochemical detection. The assay has a lower limit of quantitation of 0.5 ng/ml in tissue homogenate and utilizes a liquid–liquid extraction followed by reversed-phase HPLC for the quantitative analysis of olanzapine. The method provided a linear response for olanzapine over a concentration range of 0.5–100 ng/ml with a coefficient of determination (r²) greater than 0.9995. The extraction efficiencies of olanzapine and internal standard (LY170158) were greater than 82% in brain tissue. The intra-assay and inter-assay relative errors ranged from −5.38 to 17.60% and −3.25 to 10.53%, respectively. The intra-assay and inter-assay RSD values were in the range of 1.12 to 6.96% and 3.78 to 6.68%. Long-term stability studies showed that brain tissue homogenate samples spiked with olanzapine and internal standard are stable at −70°C for at least 110 days. However, a room temperature stability study showed that olanazapine was not stable in brain homogenate if the sample was exposed at 25°C longer than 2 h. This method has been used for the study of the disposition and pharmacokinetics of olanzapine in male Sprague–Dawley rats.     

Simultaneous determination of guanine nucleotides in rat brain by high-performance liquid chromatography with dual-electrochemical detection

A new, sensitive, and specific assay method for guanine nucleotides using high-performance liquid chromatography with dual-electrochemical detection was developed. GTP, GDP, GMP, and cyclic GMP were separated with reversed-phase "ion-pair" chromatography and detected by a dual-electrochemical detector. Only guanine nucleotides among all purine and pyrimidine nucleotides responded to the electrochemical detector at 0.95 V. The peak heights for these guanine nucleotides were linear at concentrations between 0.5 pmol and 1 nmol. The regional distribution of these guanine nucleotides in the rat brain was studied by this new assay method.     

Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection

The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch-Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60-6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 microM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.     

High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection.

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Quantification of l-stepholidine in rat brain and plasma by high performance liquid chromatography combined with fluorescence detection

A sensitive and reliable assay for the quantification of l-stepholidine (SPD) in rat plasma and brain was developed using high performance liquid chromatography (HPLC) combined with fluorescence detection. Brain regions (prefrontal cortex, striatum, and cerebellum) and plasma from rats treated with SPD (10 mg/kg s.c.) 20, 40, 60, or 90 min prior to euthanasia were analyzed for SPD levels. Brain samples were homogenized in ice-cold 0.1M perchloric acid and centrifuged to remove proteins. The supernatants and diluted plasma samples, to which O-desmethylvenlafaxine was added as a process standard, were basified and extracted with ethyl acetate. The organic phase was taken to dryness and the residue taken up in mobile phase. The samples were then injected into an HPLC equipped with a fluorescence detector (excitation and emission wavelengths set at 280 and 320 nm, respectively). The mean recovery of SPD was 74.6%, and reliability studies confirmed the reproducibility of the assay (intra- and inter-assay coefficients of variation of 4.8% and 5.3%, respectively). The assay was readily applicable to the brain and plasma samples obtained from rats injected with SPD as described above; the levels and patterns of disappearance of SPD in brain regions and plasma are shown.     

III. simultaneous determination of catecholamines in rat brain by reversed-phase liquid chromatography with electrochemical detection

Recently, an assay method for the determination of norepinephrine and dopamine in biological specimens by application of high performance liquid chromatography with electrochemical detection has been developed. Application of a microparticle reversed-phase column has made clean separation and detection of trace amounts of each amine possible. This paper presents an assay method for the simultaneous measurement of norepinephrine, dopamine and epinephrine with N-methyldopamine as the internal standard. Incorporation of an effective sensitivity-shift technique resulted in a remarkable increase in sensitivity with this method. The assay limit for quantitation was approximately 50 pg for all amines. Applicability was also studied following administration of reserpine or fusaric acid.     

Determination of piretanide and furosemide in pharmaceuticals and human urine by high-performance liquid chromatography with amperometric detection

A high-performance liquid chromatographic method with electrochemical detection (ED) has been developed for the determination of two diuretics: 4-phenoxy-3-(1-pyrrolidinyl)-5-sulfamoylbenzoic acid (piretanide) and 4-chloro-2-furfurylamino-5-sulfamoylbenzoic acid (furosemide). The chromatographic separation was performed on a μBondapak C₁₈ column with a mobile phase of acetonitrile-water (40:60) containing 5 mM KH₂PO₄/K₂HPO₄ and with a flow-rate of 1 ml/min (69 bar). The temperature was optimized at 30 ± 0.2°C. The amperometric detector equipped with a glassy carbon electrode was operated at + 1200 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds in two concentration ranges (ppm and ppb), obtaining a reproducibility in terms of relative standard deviations lower than 1% for within-day and 4% for day-to-day and determination limits of 15 ppb for both compounds. Recoveries greater than 90% were obtained for spiked urine samples, using a liquid-liquid extraction method in the sample clean-up procedure. The LC-ED method was applied to commercially available pharmaceuticals (Seguril, furosemide 40 mg, and Perbilén, piretanide 6 mg) and urine samples obtained from healthy volunteers and hypertensive patients.     

Microanalysis of beta-cyclodextrin and glucosyl-beta-cyclodextrin in human plasma by high-performance liquid chromatography with pulsed amperometric detection.

High-performance liquid chromatography with pulsed amperometric detection was applied to the determination of beta-cyclodextrin (beta-CD) and glucosyl (G)-beta-CD in human plasma. They were well resolved from each other and from background components of plasma on a polymer-based reversed-phase column with 0.6% acetonitrile aqueous solution containing 1 mM sodium hydroxide as an eluent. The samples in the effluent were detected with a pulsed amperometric detector after postcolumn alkalization. The detection limits of beta-CD and G-beta-CD in plasma at a signal-to-noise ratio of 3 were 11 and 5 pmol, respectively.     

Effect of ageing on human plasma glutathione concentrations as determined by high-performance liquid chromatography with fluorimetric detection

A convenient method for the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves direct addition of human plasma to methanolic monobrombimane. for simultaneous protein precipitation and thiol derivatization. The assay was validated by addition of authentic GSH and GSSG to plasma samples. Plasma glutathione levels in Chinese male and female volunteers were found to decrease with increasing age (age groups, 20–30, 30–40, 40–50, 50–60, and >60; mean ± S.E.M. 0.97 ± 0.03, 0.77 ± 0.02, 0.67 ± 0.03, 0.51 ± 0.02, 0.48 ± 0.02 μM for male volunteers and 1.11 ± 0.06, 0.76 ± 0.03, 0.61 ± 0.03, 0.53 ± 0.04 and 0.43 ± 0.04 μM for female volunteers). GSSG levels, in both males and females, did not show a correlation with age. There were no significant differences in GSH or GSSG levels among male and female volunteers of the same age group. These results suggest that elderly persons might be more susceptible to oxidative injury due to decreased plasma glutathione levels.     

Sugar analysis of glycoproteins and glycolipids after methanolysis by high-performance liquid chromatography with pulsed amperometric detection

A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uronic acid in a single chromatographic step at the subnanomolar level.