HPLC-electrochemical Detection Research Articles

Effects of serotonin depletion by p-chlorophenylalanine, p-chloroamphetamine or 5,7-dihydroxytryptamine on central dopaminergic neurons: focus on tuberoinfundibular dopaminergic neurons and serum prolactin.

Three serotonin (5-HT) neurotoxins, p-chlorophenylalanine (PCPA, 125 and 250 mg/kg, i.p.), p-chloroamphetamine (PCA, 10 mg/kg, i.p.) and 5,7-dihydroxytryptamine (5,7-DHT, 200 microg/rat, i.c.v.) were used to examine whether depletion of central 5-HT has an effect on central dopaminergic (DA) neuronal activities or on prolactin (PRL) secretion. Adult ovariectomized Sprague-Dawley rats primed with estrogen (polyestradiol phosphate, 0.1 mg/rat, s.c.) were treated with one of three neurotoxins and then decapitated in the morning after 3-7 days. Blood sample and brain tissues were collected. The acute effect of PCA (from 30 to 180 min) was also determined. The concentrations of 5-HT, DA and their metabolites, 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid, in the median eminence, striatum and nucleus accumbens were determined by HPLC-electrochemical detection. All three toxins significantly depleted central 5-HT stores by 11-20%. Except for PCPA, neither PCA nor 5,7-DHT had any significant effect on basal DA neuronal activities or PRL secretion. PCA also exhibited an acute effect on the release and reuptake of 5-HT and DA. In summary, depletion of central 5-HT stores to a significant extent for 3-7 days did not seem to affect basal DA neuronal activity and PRL secretion.

Repair of mtDNA in Vertebrates

The author's research is supported by National Institutes of Health grants GM29681 and ES04068.

Analysis of 3-Nitrotyrosine in Biological Fluids and Protein Hydrolyzates by High-Performance Liquid Chromatography Using a Postseparation, On-line Reduction Column and Electrochemical Detection: Results with Various Nitrating Agents

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence forin vivoformation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significancein vivohas not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-μl injection). We have applied this method to comparein vitrothe ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholino-sydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO−) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.

Modulatory effects of type-C natriuretic peptide on sympathetic cotransmission in the rat isolated tail artery.

1. Rat type-C natriuretic peptide (CNP) has been studied for its effects on the neurogenically induced overflow of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine (ADO) and noradrenaline (NA) in endothelium-free segments of rat isolated tail artery. The overflow of each was evoked by electrical field stimulation (EFS) of 0.5 ms pulses at 8 Hz for 3 min and the amount of ATP, ADP, AMP and ADO was quantified by high-performance liquid chromatography (HPLC)-fluorescent detection, while the amount of NA was quantified by HPLC-electrochemical detection. 2. Type-C natriuretic peptide (100 nmol/L) was found to cause a significant reduction of the overflow of all adenine purines and NA. However, at lower concentrations (1 and 10 nmol/L), CNP caused a significant reduction of the overflow of NA but did not change ATP overflow. 3. The overflow of ADP, AMP and ADO was significantly reduced by either concentration of CNP, so that the ratio ATP:ADP was diminished from 1:2 in controls to 1:1 after 1 nmol/L CNP and to 1:1.2 after 10 nmol/L CNP. 4. The production of inorganic phosphate (Pi) in response to the exogenous application of ATP was significantly reduced by 1, 10 or 100 nmol/L CNP. 5. Type-C natriuretic peptide exerts neuromodulatory effects on the neurogenically induced release of the cotransmitters ATP and NA in rat tail artery, consisting of an inhibition of the release of both ATP and NA. This effect is accompanied by inhibition of the breakdown of ATP by ecto-ATPases. Either effect results in apparent CNP-induced differential modulation of the overflow of the cotransmitters ATP and NA.

Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO −). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO − generated from Angeli’s salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO − and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C 1 8 Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 m M KH 2PO 4–Na 2HPO 4 buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml −1. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.

Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection

The analysis of morphine in biological fluids is of vital interest in monitoring opiate abuse and in drug abuse research. Although methods for analysis of morphine and its metabolites are well established, studies are still being carried out to improve sample preparation procedures as well as detection levels of morphine in biological samples. In this study, morphine-specific immunosorbents were developed to concentrate morphine prior to HPLC analysis. Urine (0.1 ml) was diluted 10-fold with phosphate-buffered saline, pH 7.4 (PBS), loaded onto a solid-phase immunoextraction column and washed with 15 ml PBS followed by elution with 2 ml of elution buffer (40% ethanol in PBS, pH 4). The eluted fraction was analysed for morphine by HPLC–electrochemical detection using a cyanopropyl (CN) analytical column with 25% acetonitrile in phosphate buffer–sodium lauryl sulphate, pH 2.4 as the mobile phase. Duration of the extraction procedure was approximately 40 min. Calibration graphs were linear from 100 ngml−1 to 500 ngml−1 in urine. The inter-assay R.S.D. was <10% and the recovery of morphine from urine was >98%. Immunocolumns demonstrated remarkably high specificity towards morphine showing minimal binding with other opiate metabolites such as codeine, normorphine, norcodeine, morphine-3-glucuronide, morphine-6-glucuronide.

Facts and artifacts in the measurement of oxidative base damage to DNA

This short survey is aimed at critically evaluating the main available methods for measuring oxidative base damage within cellular DNA. Emphasis is placed on separative methods which are currently widely applied. These mostly concern high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with sensitive detection techniques such as electrochemistry (EC) and mass spectrometry (MS). In addition, the comparison is extended to 32P-postlabeling methods, immunoassays and measurement of two main classes of oxidative DNA damage within isolated cells. It may be concluded that the HPLC-electrochemical detection (ECD) method, even if restricted to the measurement of only a few electroactive oxidized bases and nucleosides, is the simplest and safest available method at the moment. In contrast, the more versatile GC-MS method, which requires a HPLC pre-purification step in order to prevent artifactual oxidation of overwhelming normal bases to occur during derivatization, is more tedious and its sensitivity may be questionable. Alternative simpler procedures of background prevention for the GC-MS assay, which, however, remain to be validated, include low-temperature for derivatization and addition of antioxidants to the silylating reagents. Interestingly, similar levels of 8-oxo-7,8-dihydroguanine were found in cellular DNA using HPLC-ECD, HPLC-MS/MS and HPLC/32P-postlabeling methods. However, it should be noted that the level of cellular 8-oxodGuo, thus determined, is on average basis 10-fold higher than that was inferred for more indirect measurement involving the use of DNA repair enzymes with methods on isolated cells. Further efforts should be made to resolve this apparent discrepancy. In addition, the question of the biological validation of the non-invasive measurement of oxidized bases and nucleosides in urine is addressed.

Glutathione and neonatal lung disease

Glutathione (GSH) was measured using HPLC-electrochemical detection in bronchoalveolar lavage fluid from 28 neonates for up to 21 days after birth. GSH levels varied from 0.1–11.2 μmol 1 −1 (with a geometric mean concentration of 1.3 μmol l −1). GSH in epithelial lining fluid was estimated using the urea dilution method at 15.0 μmol l −1 (range 0.5–196 μmol l −1), which is significantly lower than observed in adult subjects. There was an L shaped relationship between GSH and the two markers of oxygen therapy, oxygen index and FiO 2. The lowest GSH levels were associated with the group of infants with the most severe airways problems who required high oxygen.

Analysis and Pharmacokinetics of Apomorphine in Rat Brain by Microdialysis Coupled with Microbore HPLC Electrochemical Detection

The feasibility of an on-line microdialysis coupled with a sensitive microbore high performance liquid chromatography with electrochemical detection (HPLC-ED) system for the direct analysis of apomorphine was investigated. A microdialysis probe was inserted into the right striatum of male Sprague-Dawley rats, which had been administered apomorphine (10 mg/kg, i.v.).

Evaluation of Plasma Low Molecular Weight Antioxidant Capacity by Cyclic Voltammetry

The low molecular weight antioxidants (LMWA) of biological samples include many compounds and contribute to the total antioxidant capacity of the system. They act as direct chemical scavengers neutralizing, reactive oxygen-derived species (ROS), and contribute to the ability of the sample to cope with oxidative stress. We propose cyclic voltammetry (CV) as a new method for evaluating the antioxidant capacity of plasma-LMWA and the severity of oxidative stress exerted on the plasma. It is based on the reducing properties of these molecules. CV has been proven to be a simple, sensitive and reliable method. Its tracing does not change during storage of frozen plasma for up to six months. We analyzed the CV tracings by the oxidation potential E 1 2 , and the current heights Ia of its anodic wave(s). E 1 2 indicates the specific component of the LMWA and its ability to donate electron(s); Ia indicates the concentration of this component. Two anodic waves have been identified in human plasma, at E 1 2 = 420 ± 25 and 920 ± 25 mV. Ascorbate (AA) and urate (UA) were identified as major LMWA components of the first anodic wave, and were confirmed by HPLC-electrochemical detection. Ia was shown to depend linearly on the concentration of either of these LMWA, both in buffer and in plasma. Oxidative stress exerted by exposure to peroxyl radicals, copper ions and ionizing irradiation caused marked changes in the CV tracing. These changes represent corresponding alterations particularly in Ia, rather than in E 1 2 . The Ia and E 1 2 values reflect the antioxidant capacity of the plasma, while the change of Ia value represents the severity of the oxidative stress induced. Copyright © 1996 Elsevier Science Inc.

α-Melanocyte-Stimulating Hormone (α-MSH) and Melanin-Concentrating Hormone (MCH) Modify Monoaminergic Levels in the Preoptic Area of the Rat

Gonzalez, M. I., V. Kalia, D. R. Hole and C. A. Wilson. α-Melanocyte-stimulating hormone (α-MSH) and melanin-concentrating hormone (MCH) modify monoaminergic levels in the preoptic area of the rat. Peptides 18(3) 387–392, 1997.—The effect of perfusion of melanin-concentrating hormone (MCH) or α-melanocyte-stimulating hormone (α-MSH) (100 ng/μl) in the ventromedial nucleus (VMN) or medial preoptic area (MPOA) on monoaminergic levels of female rats was measured using microdialysis and HPLC-electrochemical detection. In the MPOA, α-MSH raised 5-HIAA concentration, whereas MCH reduced both 5-HT and 5-HIAA. Neither peptide had any effect in the VMN. The opposite effects of the peptides on the serotonergic system might be responsible for their antagonistic or opposite actions previously reported on several CNS functions. Dopamine may mediate the similar effects of the two peptides, because α-MSH inhibits dopaminergic release in the MPOA (but not VMN) and MCH tends to follow the same pattern.

Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea-pig vas deferens.

1. Experiments were carried out to quantify the stimulation-evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve-smooth muscle preparation of the guinea-pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC-fluorescence detection and catecholamines by HPLC-electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS-evoked overflow of NA and the purines from the guniea-pig vas deferens preparation was examined after treatment with the neuronal amine-uptake inhibitors desipramine and cocaine, the alpha 1-adrenoceptor agonist methoxamine, the alpha 1-adrenoceptor antagonist prazosin, the alpha 2-adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline-depleting drug reserpine and the adrenergic neuron-blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non-neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon-ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation-dependent metabolism of ATP.

HPLC-Electrochemical detection with graphite-poly (tetrafluoroethylene) electrode Determination of the fungicides thiram and disulfiram

The suitability of composite graphite-poly(tetrafluoroethylene) (Teflon) electrodes as amperometric indicator electrodes in HPLC detection is demonstrated. The determination of the fungicides thiram and disulfiram in the presence of ziram has been chosen as an analytical problem. The optimization of working conditions, such as the choice of the organic solvent used in the mobile phase as well as its percentage, the potential applied to the composite electrode, and the time elapsed between mixing the carbamates and the injection, has been accomplished by using the wall-jet flow-cell configuration. The effect of the acetonitrile percentage used in the mobile phase on the retention of thiram, disulfiram, ziram and phenol was evaluated. Resolution up to the baseline can be achieved with 45% acetonitrile. The sensitivity of the determination of thiram and disulfiram in the presence of a constant concentration of ziram is slightly better when using a wall-jet cell; however, the background current is higher, as well as the baseline noise and the time necessary to achieve stabilization of the baseline before the injection. Lower limits of detection for both fungicides, as well as a better repeatability, were obtained when using a thin-layer flow cell configuration. As an application, the determination of thiram in spiked apple samples, at a level of 0.5 mg thiram kg −1 apple, has been carried out with a mean recovery of 97 ± 3% for a significance level of 0.05.

Crude protein and supplemental dietary tryptophan effects on growth and tissue neurotransmitter levels in the broiler chicken.

Indian River male broiler chickens growing from 7 to 28 d of age were fed on diets containing 120, 210 and 300 g crude protein/kg diet and 0, 1.67 or 16.7 g added tryptophan (TRP)/kg diet. The hypothesis tested was that crude protein levels and TRP would affect both growth and neurotransmitter metabolism. Heart, brain and pancreatic neurotransmitter (noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA)) concentrations were determined by HPLC separation and electrochemical detection. Malate dehydrogenase (2-oxoglutarate decarboxylating) (NADP+) (MDH(NADP+); EC 1.1.1.40), isocitrate dehydrogenase (NADP+) (ICD(NADP+); EC 1.1.1.42) and aspartate aminotransferase (AAT; EC 2.6.1.1) activities were also measured. Supplemental TRP decreased growth and feed intake. Increasing dietary crude protein decreased MDH(NADP+), but increased (ICD(NADP+) and AAT activities. Additional dietary TRP decreased MDH(NADP+) activity, but had no effect on other enzyme activities. Cardiac NA concentrations were directly related to dietary crude protein levels while pancreatic levels were inversely related. An increase in dietary crude protein decreased both brain NA and DA. Supplemental dietary TRP increased both 5-HIAA and 5-HT. Changes in feed intake caused by different levels of both dietary crude protein and TRP are accompanied by altered levels of neurotransmitters. The present study indicates that much larger amounts of TRP are required to make simultaneous changes in feed intake and neurotransmitters.

A Rapid Quantitative Analysis of the β-Blocker Timolol in Human Urine by HPLC-Electrochemical Detection

Abstract A High Performance Liquid Chromatographic method with amperometric detection has been developed for the determination of the β-blocker 1-[(1, 1-dimethylethyl) amino]-3-[[4-(4-morpholinyl)-1, 2, 5-thiadiazol-3-yl] oxy], timolol. The chromatographic separation was performed using a μ-Bondapak C18 column and a mobile phase of acetonitrile:water (30:70), containing 5mM KH2PO4/K2HPO4, pH 6.5 pumped at a flow rate of 1.3 mL/min. The amperometric detector equipped with a glassy carbon electrode was operated at +1000 mV. The method was applied to the determination of timolol in urine samples obtained from a hypertensive patient under medical treatment with the pharmaceutical formulation Blocadren 10 mg (timolol 10 mg). Using a simple liquid-liquid extraction procedure, a good recovery (93.25±2.5) and separation from the urine matrix is achieved. A good reproducibility, linearity and accuracy are obtained, and the quantitation limit of 10 ng/mL, allows the method to be applied to doping analysis in human ...

High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissues using electrochemical detection

This paper describes a sensitive HPLC-electrochemical detection analytical method for determining the concentration of the intravenous anesthetic, propofol, in human or rat plasma or serum and a variety of rat tissues. Internal standard and drug are extracted from serum or plasma and other tissues with pentane. 2,6- tert.-Butylmethylphenol is used as internal standard. It includes a novel steam distillation procedure for separating the highly lipophilic propofol from skin and fat. The plasma/serum assay has a precision of 1–4% (C.V.) in the range 10 ng/ml to 1 μg/ml and permits the assay of 5 ng/ml from 0.1 ml of plasma/serum. The tissue procedure allows the estimation of 50 ng/g in 0.1 g of tissue for most of the major organs with less than 2% (C.V.) precision. This assay was used to measure propofol concentrations in plasma/serum and tissue samples in support of a project to develop a physiological pharmacokinetic model for propofol in the rat.

Regional differences of endogenous ATP release in rabbit arteries

The releases of endogenous ATP from ear, femoral, renal and pulmonary arteries of rabbit were compared. Adenyl purines, such as ATP, ADP, AMP and adenosine, and norepinephrine (NE) were quantified by HPLC-fluorescence detection and HPLC-electrochemical detection, respectively. In all four blood vessels, electrical stimulation (ES) at 16 Hz significantly increased overflow of adenyl purine and NE. ATP was the main largest among adenyl purine released; release of adenosine was very small. The rank order of amounts of total purine released by ES was ear artery > renal artery = femoral artery ⪢ pulmonary artery. There was no significant difference among the amounts of NE release induced by ES from these four arteries. ES-induced purine release was notably reduced by denudation of endothelium and prazosin at 1 μM. Methoxamine, α 1-adrenoceptor agonist, also produced release of adenyl purines in the four arteries. The rank order of amount of total purine released was ear artery > renal artery = femoral artery ⪢ pulmonary artery. These suggest that the sources of ATP released by sympathetic nerve stimulation, which seems to be α 1-adrenoceptors on endothelial cells, not distributed homogeneously in the vasculature.

Comparison of two different inductively coupled plasma mass spectrometric procedures and high-performance liquid chromatography with electrochemical detection in the determination of iodine in urine

A comparison between two ICP-MS methods and HPLC with electrochemical detection (ED) for the determination of iodine is described. The first ICP-MS method involves 10-fold dilution with water using Rh as internal standard and the calibration graph was obtained with a synthetic urine. In the second ICP-MS method the sample preparation provided a 5-fold dilution with water using In as internal standard and adding nitric acid and the calibration graph was obtained by making additions to a pool of human urine samples. In the HPLC method, the urine was previously purified from organic substances through an SPE C18 column and the final portion was collected. The chromatographic column used was a C18 reversed-phase column and a silver working electrode was used versus a Pd/Pt reference electrode for ED. The calibration graph was obtained with a synthetic urine. The results show that the two ICP-MS methods are comparable in terms of analytical parameters such as precision and limit of detection. Good agreement between both ICP-MS procedures and HPLC–ED was also obtained, although the limit of detection was higher in HPLC–ED and the linear dynamic range was wider in both ICP-MS methods.

Sensitive detection of 8-hydroxy-2'deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissues.

Oxidative damage from reactive oxygen species including free radicals has been considered to play a vital role in many degenerative diseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh8dG) in tissue DNA has been used as a benchmark for oxidative DNA damage. We report here an ultrasensitive 32P-postlabeling method to detect and quantitate Oh8dG in DNA and have determined basal levels of Oh8dG in rat tissues. The method is comprised of DNA digestion to 3'-monophosphates, 5'-32P-labeling, conversion to 5'-monophosphates and separation by a 2-directional PEI-cellulose TLC (D1 = 1.5 M formic acid; and D2 = 0.6 M ammonium formate, pH 6.0). Under these conditions, all radioactive contaminants were either removed from the chromatogram (normal nucleotides and 32Pi) or remained at the origin (ATP and other contaminants), while Oh8dG migrated in the middle of the chromatogram. Calf thymus DNA incubated with ascorbic acid and H202 produced predominantly one spot under the chromatography conditions used; a chromatographically identical spot was also detected in untreated DNA, but at a much lower level (125 +/- 40 Oh8dG/10(6) nucleotides). A chromatographically identical spot was also found in dGp incubated with ascorbic acid and H202, but not with dAp, dCp or dTp. When applied to rat tissue DNA, the assay readily permitted detection of Oh8dG in the liver, lung, kidney, heart, brain, spleen, intestines and mammary epithelial cells of 3-month old female Sprague-Dawley rats. The tissue Oh8dG levels were found in the range of 87 +/- 29 to 133 +/- 49 per 10(6) nucleotides, with liver and heart being the highest and kidney and brain the lowest. These values are in the vicinity to those found by gas chromatography/mass spectrometry but 10-50 times higher than those reported by HPLC-electrochemical detection. Because of its high sensitivity (<1 Oh8dG per 10(5-6) nucleotides) to detect Oh8dG using nanogram quantity of DNA digest, the 32P-postlabeling method is likely to be valuable in quantitating Oh8dG in human tissue biopsies.