Determination of monohydroxyethylrutoside in heart tissue by high-performance liquid chromatography with electrochemical detection

7-Monohydroxyethylrutoside (monoHER) is one of the components of the registered drug Venoruton. It showed a good protection against the cardiotoxic effects of doxorubicin. The analysis of monoHER was developed to study the pharmacokinetic profile of the drug in heart tissue. MonoHER was extracted from heart tissue homogenate with methanol. The supernatant was diluted 1:1 (v/v) with 25 mM phosphate buffer and injected onto a reversed-phase ODS column. The mobile phase consisted of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogenphosphate (pH 3.4), 10 mM acetic acid and 36 μM EDTA. The retention time of monoHER was about 5.2 min and no endogenous peaks were interfering. The lower limit of quantification was 0.072 nmol g⁻¹ wet heart tissue. The calibration line was linear up to 24 nmol g⁻¹. The within-day accuracy and precision of the quality controls (0.12, 1.2 and 12.0 nmol g⁻¹) were smaller than 17 and 19%, respectively. The between-day accuracy and precision were better than 6 and 11%, respectively. The recovery of monoHER from heart tissue ranged from 104.1 to 114.3% and was concentration independent. MonoHER was stable in heart tissue when stored at −80°C for 6 months. Repeated injection of monoHER from aliquots of 7.2 nmol g⁻¹ placed on the sample tray at 4°C for 24 h showed a decrease in the concentration of 30.3%. Analyzing sample duplicates in a mirror image sequence could compensate for the influence of this gradual decrease. The small sample volume allowed one to measure monoHER in the hearts of mice.     

Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection

We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.     

Determination of quinapril and quinaprilat by high-performance liquid chromatography with radiochemical detection,coupled to liquid scintillation counting spectrometry

Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase high-performance liquid chromatographic procedure with radiochemical detection, coupled to liquid scintillation counting spectrometry. Quinapril and quinaprilat were measured in perfusate and urine after pretreatment with acetonitrile and subsequent centrifugation. Perfusate ultrafiltrate was used as collected. Two quinapril diketopiperazine metabolites, PD 109488 and PD 113413, were separated chromatographically from quinapril, quinaprilat, and from each other. Assay performance for quinapril and quinaprilat was assessed by examining precision and accuracy of the assay over four days. Using a 100-μl sample volume, the limit of quantitation for both ³H-quinapril and ³H-quinaprilat (sp. act. ≈ 2.0 μCi/μg) was 1 ng/ml.     

Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.     

Determination of monoamine oxidase B activity by high-performance liquid chromatography with electrochemical detection

A sensitive high-performance liquid chromatography method with electrochemical detection for measuring monoamine oxidase B activity in blood platelets is described. Dopamine is used as substrate and is incubated with isolated platelets and aldehyde dehydrogenase to convert dihydroxyphenylacetaldehyde to dihydroxyphenylacetic acid (DOPAC). The acid and the added internal standard hydrocaffeic acid are separated from dopamine and the incubation mixture by extraction with 5 ml of ethyl acetate-toluene (5:1, v/v). The organic phase is evaporated under nitrogen stream and the residue dissolved in 0.1 M critic acid. Dihydroxyphenylacetic acid and the internal standard dihydrocaffeic acid are then separated on the Eurosphere 100-C₁₈ 5 μm column. The mobile phase used was a mixture of sodium acetate, citric acid, and acetonitrile at pH 2.5. The standard curve was linear from 125 pg to 10 ng. Absolute recovery of DOPAC was 85±3.8% and of hydrocaffeic acid 87±4.1%. The method presented is sensitive (detection limit 8.0 pg of DOPAC injected) and reproducible (coefficient of variation 0.4-1%) with good accuracy (94–98%).     

Determination of aromatic compounds by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection.

This paper describes a novel high-performance liquid chromatographic (HPLC) method for the determination of aromatic compounds with peroxyoxalate chemiluminescence (PO-CL ) detection following on-line UV irradiation. Aromatic compounds were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide, which was determined via PO-CL detection using a mixture of bis(2,4,6-trichlorophenyl)oxalate (aryloxalate) and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine (fluorophore) as a post-column CL reagent. Generation of hydrogen peroxide from aromatic compounds was confirmed using a flow injection analysis (FIA) system incorporating an enzyme column reactor immobilized with catalase. The conditions for UV irradiation were optimized using benzene and monosubstituted benzenes (phenol, benzaldehyde, nitrobenzene and N,N-dimethylaniline) by an HPLC system to evaluate the analytical performance of the proposed system. The detection limits for benzene and monosubstituted benzenes were in the range 2.1-124 pmol/injection at signal:noise (S:N) ratio = 3. Monocyclic and polycyclic hydrocarbons were also employed to investigate their CL properties. The possibility of PO-CL detection for a wide variety of aromatic compounds was shown for the first time.     

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.     

Determination of tryptophan and metabolites in rat brain and pineal tissue by reversed-phase high-performance liquid chromatography with electrochemical detection

Tryptophan and many of its indole metabolites were separated using reversed-phase high-performance liquid chromatography (HPLC) and determined using electrochemical detection. This was accomplished isocratically using an acetate—citric acid eluent with various amounts of methanol. Brain and pineal tissue was analyzed for several tryptophan metabolites. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the HPLC column. Detection limits in the low picogram range were found for those indoles separated.     

Determination of Δ-tetrahydrocannabinol levels in brain tissue using high-performance liquid chromatography with electrochemical detection

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive component of cannabis. To assist in investigating the mechanism(s) of action of Δ⁹-THC, a convenient method for determining its levels in brain tissue is required. We now describe a method for determining nanogram quantities of Δ⁹-THC in rat brain tissue. The method employs solvent extraction with methanol-hexane-ethyl acetate, followed by analysis using liquid chromatography with electrochemical detection. Overall recoveries were greater than 80%. The relationship between the peak-height ratio for processed standards extracted in the presence of tissue (Δ⁹-THC/internal standard) and the amount of Δ⁹-THC added was shown to be linear within the range of concentrations examined. Quantitative measurements of Δ⁹-THC in different brain regions following the intravenous administration of Δ⁹-THC are presented as examples of the applications of this method.     

Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction

An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.6 mm I.D. Kromasil C8 5 microm column was performed using a mobile phase of water-acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 microg/mL with a 50-microL sample size. The method was successfully applied to in vitro pharmacokinetic-pharmacodynamic studies.     

Determination of carbohydrates in urine by high-performance liquid chromatography and optical activity detection

Improvements in a detector for liquid chromatography based on optical activity of the components have led to a detectability of 100 ng. This allows the simultaneous determination of six naturally occurring carbohydrates in 100-μl samples of human urine, which is injected directly except for a simple deionization step. The reproducibility and reliability of this method should allow better insight into the relation between urinary sugars and physiological conditions.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection

Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with flourescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limitof 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999±0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.     

Assay of brain aldehyde dehydrogenase activity using high-performance liquid chromatography with electrochemical detection

A method has been developed for assay of aldehyde dehydrogenase (ALDH) in brain tissue or in other tissues containing low ALDH-activity. The aldehyde of dopamine was used as the substrate, and the 3,4-dihydroxyphenylacetic acid formed was measured using high-performance liquid chromatography (HPLC) with electrochemical detection. The aldehyde was prepared enzymatically by incubating dopamine with a monoamine-oxidase preparation from rat liver mitochondria in the presence of Na+-bisulfite in 10 mM K+-phosphate buffer (pH 7.5). Rat brain homogenates were incubated in 50 mM Na+-pyrophosphate buffer (pH 8.8) containing 0.5 mM NAD+ and 5 microM aldehyde. The reaction was terminated with perchloric acid containing Na+-bisulfite to trap excess of the aldehyde. The acid supernatants were injected on a reverse-phase HPLC column and elution was performed with citrate buffer, pH 2.50. The method permits assay with 1-10 mg of brain tissue with an overall precision of 3%. The assay rate was 5-6 samples per hour.     

Determination of plasma tocopherols by high-performance liquid chromatography with coulometric detection

An HPLC procedure has been developed for tocopherol determination with coulometric detection in human serum samples. Eluent optimization and foreign peak identification (bilirubin) by mass spectrometry are described. An extraction procedure gave yields around 98% with 1.3% coefficient of variation, and the calibration ranged from 0.1 to 200 mg/l with a correlation coefficient of 0.999. The detection limit achieved for vitamin E was 60 pg (3 ng/ml).     

Determination of maprotiline in plasma by high-performance liquid chromatography with chemiluminescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of maprotiline, an antidepressant, in plasma. After a single-step extraction from plasma (100 μl) with n-hexaneisoamylalcohol (19:1, v/v), the drug and desipramine (internal standard) are converted into their chemiluminescent derivatives by reaction with 6-isothiocyanatobenzo[g]phthalazine-1,4(2H,3H)-dione, a new chemiluminescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSKgel ODS-80, using isocratic elution with acetonitrile-100 mM acetate buffer (pH 3.2), and produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline medium. The detection limit for maprotiline added to plasma is 0.36 pmol (0.1 ng)/ml plasma (1.5 fmol on column), at a signal-to-noise ratio of 3.     

Determination of bestatin in serum by high-performance liquid chromatography with fluorescence detection

A simple method for the determination of bestatin and its major metabolite in man, p-hydroxybestatin, in human serum was investigated; the method employs high-performance liquid chromatography with fluorescence detection. Bestatin and p-hydroxybestatin are oxidized to phenylacetaldehyde and p-hydroxyphenylacetaldehyde, respectively, with periodate, which are then converted into fluorescent compounds with 4,5-dimethoxy-1,2-diaminobenzene. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18. The detection limits of bestatin and p-hydroxybestatin are 0.2 and 0.4 μg/ml serum, respectively. This method permits the precise determination of bestatin in serum (20 μl) from patients administered bestatin. p-Hydroxybestatin in serum can not be measured by this method because of its low concentration (less than the detection limit).