O6-benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair.

Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent O6-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T)) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2. In tumor tissue, AGT depletion >86% of baseline was demonstrated at all doses tested. Residual tumor AGT activity, present 18 h after BG doses of 10-80 mg/m2, was eliminated at the 120 mg/m2 dose and is thus the BMD(T) of BG. BG pharmacokinetics are characterized by the rapid, dose-independent clearance of BG from plasma Metabolism of BG to its biologically active metabolite, 8-oxo-benzylguanine (8-oxo-BG), was found. The t(1/2) of 8-oxo-BG is longer than BG. Plasma concentrations of 8-oxo-BG well above 200 ng/ml 18 h after the end of the BG infusion were observed at the highest dose levels tested and appeared to correlate with depletion of AGT activity to undetectable levels in tumor tissue. AGT activity in peripheral blood mononuclear cells at baseline did not correlate with tumor tissue AGT activity. Depletion of AGT activity to undetectable levels in peripheral blood mononuclear cells occurred at lower doses and was not a reliable predictor for tumor tissue depletion. No serious side effects were observed with administration of BG alone or in combination with 13 mg/m2 1,3-bis(2-chloroethyl)-1-nitrosourea. This is the first clinical study in which biochemical analyses from pre- and posttreatment tumor biopsies have been used as an efficacy end point for the clinical development of an anticancer agent. From our tumor tissue biopsy data, we have established that a BG dose of 120 mg/m2 infused over 1 h should be used in Phase II clinical trials.

Phase I clinical and pharmacokinetic study of rebeccamycin analog NSC 655649 given daily for five consecutive days.

PURPOSE: Rebeccamycin analog (NSC 655649) is active against a variety of both solid and nonsolid tumor cell lines. We performed a phase I trial to determine the maximum-tolerated dose (MTD) of rebeccamycin analog when given on a daily x 5 schedule repeated every 3 weeks, characterize the toxicity profile using this schedule, observe patients for antitumor response, and determine the pharmacokinetics of the agent and pharmacodynamic interactions. PATIENTS AND METHODS: Thirty assessable patients received a total of 153 cycles according to the following dose escalation schema: 60, 80, 106, 141, and 188 mg/m(2)/d x 5 days. RESULTS: Grade 2 phlebitis occurred in all patients before the use of central venous access, placed at dose level 4 and higher. Dose-limiting toxicity (DLT), grade 4 neutropenia, occurred at 188 mg/m(2)/d x 5 days in both previously treated and chemotherapy-naive patients. Pharmacokinetic analysis revealed a three-compartmental model of drug elimination and a long terminal half-life (154 +/- 55 hours). The percentage drop in absolute neutrophil count correlates with the area under the curve infinity. The presence of a second peak during the elimination phase as well as a high concentration of NSC 655649 in biliary fluid compared with the corresponding plasma measurement (one patient) is suggestive of enterohepatic circulation. Two partial responses, two minor responses, and six prolonged (> 6 months) cases of stable disease were observed. Of these, three patients with gallbladder cancer and one patient with cholangiocarcinoma experienced either a minor response or a significant period of freedom from progression. CONCLUSION: The recommended phase II dose for NSC 665649 on a daily x 5 every 3 weeks schedule is 141 and 165 mg/m(2)/d for patients with prior and no prior therapy, respectively, with DLT being neutropenia. During this phase I trial, encouraging antitumor activity was been observed.

Furosemide kinetics in patients with hepatic cirrhosis with ascites.

Furosemide, 20 mg, was given intravenously as a bolus to seven patients with cirrhotic ascites and a 10-mg intravenous bolus dose was given to three normal subjects. Furosemide concentrations were measured by a specific high-performance liquid chromatographic analytic method. The median plasma elimination half-life (t1/2), volume of distribution at a steady state (VdSS), and VDarea of furosemide were 0.70 hr, 91 ml/kg, and 119 ml/kg in normal subjects. In the cirrhotic patients elimination t1/2 and Vd at steady state doubled and the Vdarea of furosemide was almost double that of the normal. There were no differences in plasma clearance or renal and nonrenal clearance between patients and controls, but five of the seven patients had lower renal clearances than controls. The water and sodium response to furosemide was directly related to the urinary furosemide excretion rate. We identified a subset of cirrhotic patients who responded poorly (125 ml/hr urinary output in the first 4 hr after furosemide compared to 300 ml/hr in the other patients and 400 ml/hr in the controls) to furosemide. These "poor responders" had the lowest renal clearance of furosemide and the lowest urinary furosemide excretion rates. Our data indicate that furosemide kinetics are altered in patients with cirrhotic ascites and lack of response in a subset of these patients is due to lack of delivery of furosemide to the renal site of its action.

Phase II and pharmacokinetic/pharmacodynamic trial of sequential topoisomerase I and II inhibition with topotecan and etoposide in advanced non-small-cell lung cancer.

PURPOSE: In vitro and in vivo preclinical models have demonstrated synergistic activity when topoisomerase I and II inhibitors are administered sequentially. Topoisomerase I inhibitors increase topoisomerase II levels and increase cell kill induced by topoisomerase II poisons. We evaluated this hypothesis in a cohort of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: A group of 19 patients with advanced NSCLC (70% adenocarcinoma) received topotecan at a dose of 0.85 mg/m2 per day as a continuous 72-h infusion from days 1 to 3. Etoposide was administered orally at a dose of 100 mg twice daily for 3 days on days 7-9 (schedule and dose derived from prior phase I trials). Total and lactone topotecan concentrations were measured at the end of the 72-h infusion. Blood samples were obtained immediately after each 72-h topotecan infusion in order to measure the mutational frequency at the hypoxanthine phosphoribosyl transferase (HPRT) locus in peripheral lymphocytes. RESULTS: A total of 55 cycles were administered. Toxicity was mainly hematologic with grade 4 neutropenia occurring in 7% of courses. Only one partial response and two stable diseases were observed. The 1-year survival rate was 33%. There was a statistically significant difference between steady-state lactone concentrations between cycle 1 and cycle 2 with decreasing concentrations with cycle 2 (P = 0.02). This was explained by a statistically significant increase in the clearance of topotecan lactone during cycle 2 (P = 0.03). Total but not lactone concentrations correlated with nadir WBC, ANC and platelet levels. Steady-state plasma lactone levels correlated with the mutational frequency at the HPRT locus (P = 0.06). In the one patient with a partial response a sixfold increase in HPRT mutational frequency was observed, which was not seen in patients with progressive disease. CONCLUSION: The combination of topotecan and etoposide in this schedule of administration has minimal activity in adenocarcinoma of the lung. This lack of activity may be due to the delay in administration of etoposide after the topotecan as studies have shown that the compensatory increase in topoisomerase II levels after treatment with topoisomerase I inhibitors is shortlived (<24 h). The HPRT mutational frequency results suggest that the lack of clinical response may be associated with failure to achieve sufficient cytotoxic dose as indicated by a lack of increase in mutational frequency in those patients with progressive disease. HPRT mutational frequency may correlate with plasma steady-state topotecan lactone levels. Future studies should be directed toward earlier administration of topoisomerase II inhibitors after topoisomerase I inhibition.

An enzymatic method for the determination of butyrobetaine via conversion to carnitine after isolation by high performance liquid chromatography.

An improved procedure for the determination of butyrobetaine [4-(N,N,N-trimethylammonio)butanoate] in plasma and tissue is described. Butyrobetaine was isolated by ion-exchange chromatography and high performance liquid chromatography. The isolation procedure was internally standardized with [3H]butyrobetaine. The recovery of butyrobetaine was greater than 90%. Following isolation butyrobetaine was enzymatically converted to carnitine using butyrobetaine hydroxylase and the resulting carnitine was assayed using carnitine acetyltransferase and [14C]acetylcoenzyme A. The conversion of butyrobetaine to carnitine and of carnitine to [14C]acetylcarnitine was greater than 98% as determined by high performance liquid chromatography. Using this method was analysed human sera (healthy controls) and tissues (autopsy) and found the following values: serum, 4.67 nmol/ml; kidney 17.6 nmol/g; liver, 26.5 nmol/g. The serum butyrobetaine values of twins suffering from carnitine deficiency were normal (3.78 and 3.87 nmol/ml), while the carnitine supplementation therapy caused an increase. Animal samples were analyzed and the values were 3-4 times higher than previously reported by others.

Atypical urinary opiate excretion pattern.

Heroin is rapidly metabolized in humans to 6-acetylmorphine (6-AM), which is further metabolized to morphine and morphine conjugates. Urinary 6-AM is the best diagnostic indicator of heroin abuse. This metabolite however, is usually present in urine at less than 3% of the concentration of urinary total morphine (MOR). We present two case studies of 43-year-old, apparently identical, male twins who displayed an atypical pattern of opiate metabolism. The subjects had a history of opiate abuse, and they are currently in a substance-abuse treatment program. Urine specimens submitted by these subjects for periodic clinical urine drug testing occasionally gave positive responses for opiates by enzyme immunoassay. These samples were then submitted for confirmation analysis using a mixed-mode solid-phase extraction sample preparation, trimethylsilyl derivatization, and capillary gas chromatography-electron impact-mass spectrometry confirmation analysis. These specimens contained as much as 2000 ng/mL of 6-AM with less than 350 ng/mL of MOR, which yielded 6-AM/MOR ratios as large as 1100%. Additional urine samples from these subjects that screened negative for opiates were also tested for the presence of 6-AM. Clinically significant concentrations of 6-AM were found in some of these samples.

Determination of plasma non-esterified fatty acids and triglyceride fatty acids by gas chromatography of their methyl esters after isolation by column chromatography on silica gel.

Non-esterified fatty acids (NEFA) and triglycerides were isolated from human plasma by column chromatography on silica gel. Eight principal fatty acids of each of these lipid classes were determined by gas chromatography of their methyl ester derivatives and quantified relative to multipoint standard curves. Within-day relative standard deviations for plasma non-esterified fatty acid and triglyceride fatty acid determinations were 2.4 and 3.2%, respectively. Day-to-day relative standard deviations for plasma non-esterified fatty acid and triglyceride fatty acid determinations were 1.4 and 1.1%, respectively. The total plasma concentration and the relative proportions of the eight non-esterified fatty acids determined by this method were significantly different from results obtained according to two generally accepted methods for direct plasma non-esterified fatty acid determination without a specific isolation step. These comparisons suggested that considerable fatty acid ester lipid hydrolysis occurred during these direct determination procedures, and that this hydrolysis resulted in 3-fold overestimation of plasma NEFA content by those methods. Measured levels of arachidonic acid are substantially overestimated by these direct determination methods in which non-esterified fatty acids are not isolated before derivatization.

High-performance liquid chromatographic separation of acylcarnitines following derivatization with 4'-bromophenacyl trifluoromethanesulfonate.

A high-performance liquid chromatographic method for the separation of acylcarnitines after derivatization with 4'-bromophenacyl trifluoromethanesulfonate is presented. Derivatization of acylcarnitines was achieved at room temperature within 10 min. Separation of the acylcarnitine 4'-bromophenacyl esters was accomplished by high-performance liquid chromatography using as the analytical column a Resolve-PAK 5-microns C18 radially compressed cartridge eluted with a tertiary gradient containing varying proportions of water, acetonitrile, tetrahydrofuran, triethylamine, potassium phosphate, and phosphoric acid. Acylcarnitine 4'-bromophenacyl esters were detected spectrophotometrically at 254 nm. Baseline separation was obtained for a standard mixture (5 nmol of each injected) containing carnitine, acetyl-, propionyl-, butyryl-, valeryl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl-, decanoyl-, lauroyl-, myristroyl-, palmitoyl-, and stearoylcarnitine. Nearly complete separation was obtained for a standard mixture containing butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyrylcarnitine. The method was applied to a normal human urine and then to this same urine spiked with the acylcarnitine standards. Urinary acylcarnitine profiles from patients having propionic acidemia, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency were performed. Urinary isovalerylcarnitine was quantified in the patient with isovaleric acidemia using heptanoylcarnitine as an internal standard.

Determination of free trimethyllysine in plasma and tissue specimens by high-performance liquid chromatography.

A method for the determination of 6-N-trimethyllysine in tissues and plasma is described. Trimethyllysine and the chemically analogous 6-N-triethyllysine (internal standard) were isolated from acid-soluble fractions of tissue homogenates or plasma by combined ion-exchange--ion-exclusion chromatography. Trimethyllysine and triethyllysine were separated from other sample constituents by reversed-phase ion-pair high-performance liquid chromatography, derivatized post-column by reaction with o-phthalicdicarboxaldehyde and 2-mercaptoethanol, and detected fluorometrically. Standard curves were linear over a sample concentration range of 0.5--4 nmol/ml. The detection limit corresponded with 25 pmol trimethyllysine injected into the chromatograph. The procedure was used for the determination of trimethyllysine in plasma, liver, kidney, and mixed skeletal muscle of rat.

Rapid high-performance liquid chromatography of 3-methylhistidine in human urine.

An internally standardized method for the determination of 3-methylhistidine in human urine is presented. This methylated amino acid and the chemically analogous internal standard 3-ethylhistidine were isolated from human urine specimens using small columns of cation-exchange resin. Quantification was accomplished by high-performance liquid chromatography using post-column derivatization with o-phthalicdicarboxaldehyde-2-mercaptoethanol followed by fluorometric detection. Sample-to-sample and day-to-day reproducibility were shown to have respective relative standard deviations of 2 and 5% for a human urine specimen containing 250 nmol/ml 3-methylhistidine when using 250 microliter urine per analysis. The chromatographic separation was evaluated in terms of various peak descriptors (capacity factor and retention time) and "Chromatographic Figures of Merit" (peak symmetry and chromatographic efficiency). The utility of the method was demonstrated by its successful application to 1000 human urine specimens.

Derivatization of carboxylic acids by reaction with 4'-bromophenacyl trifluoromethanesulfonate prior to determination by high-performance liquid chromatography.

The use of 4'-bromo-2-hydroxyacetophenone trifluoromethanesulfonate ester (4'-bromophenacyl triflate) in the preparation of carboxylic acid 4'-bromophenacyl ester derivatives for spectrophotometric detection in high-performance liquid chromatography is described. The reagent is prepared in 66% yield by the reaction of 4'-bromo-2-diazoacetophenone with trifluoromethanesulfonic acid in anhydrous sulfur dioxide and is stable for 3-6 months. Reactions of 10(-6) M carboxylate N,N-diisopropylethylammonium salts with this reagent in acetonitrile at room temperature proceed to completion in 1-5 min. Optimal rates of reaction are obtained with a 10-fold equivalent excess of alkylating agent and 5 equivalents of N,N-diisopropylethylamine present. The process has been applied successfully to mono-, di- and tricarboxylic and sterically hindered carboxylic acids.

Quantification of carnitine, acetylcarnitine, and total carnitine in tissues by high-performance liquid chromatography: the effect of exercise on carnitine homeostasis in man.

A method for the quantitative determination of carnitine, acetylcarnitine, and total carnitine in tissue was developed for application to clinical research and diagnosis. Human skeletal muscle and heart specimens (10-20 mg) were homogenized in 1 ml of water. Aliquots of the resulting homogenates (50 microliters) were extracted with 1.0 ml of acetonitrile:methanol (3:1) and the carnitine-related compounds were isolated using columns containing 300 mg of silica gel. Samples were then derivatized with 4'-bromophenacyl trifluoromethanesulfonate for spectrophotometric detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate for fluorescence detection and quantified by high-performance liquid chromatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl ester derivatives afforded a 500-fold increase in sensitivity when compared to derivatization with 4'-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute human muscle homogenates at quantities of 790 fmol of acetylcarnitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from subjects performing exercise at high work loads. The method permitted quantification of carnitine, acetylcarnitine, and total carnitine (sum of carnitine and all acylcarnitines) and demonstrated the specific redistribution of the carnitine pool from carnitine to acetylcarnitine with exercise above the lactate threshold. This HPLC method is facile, and provides a sensitive and specific approach for use in human biopsy specimens.

Improved high-performance liquid chromatographic method for the determination of 6-N,N,N-trimethyllysine in plasma and urine: biomedical application of chromatographic figures of merit and amine mobile phase modifiers.

An internally standardized method for the determination of 6-N,N,N-trimethyllysine in human plasma, human urine, rat plasma, rat urine and hydrolyzed rat urine is described. This methylated amino acid and the procedural internal standard 6-N,N,N-trimethyllysine were isolated from the sample matrices using short ion-exchange columns and detected following high-performance liquid chromatography using a postcolumn reaction (o-phthalic-dicarboxaldehyde-2-mercaptoethanol) and fluorometric detection. The reliable detection limit for 6-N,N,N-trimethyllysine was 0.2 nmol/ml in 200 microliters of human plasma. The chromatographic separation exploits the unique properties of a novel tertiary amine mobile phase modifier, 3-(N,N-dimethylamino)-1,2-propanediol. The capacity factor and "Chromatographic Figures of Merit" (including peak asymmetry and relative system efficiency) were calculated for the chromatographic peak representing 6-N,N,N-trimethyllysine in over 2200 injections made while evaluating 900 biological specimens.

Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.

Determination of 6-N-trimethyllysine in urine by high-performance liquid chromatography.

A method for determination of 6-N-trimethyllysine in urine is described. Trimethyllysine and the chemically analogous 6-N-triethyllysine internal standard were isolated from aqueous samples by microcolumn ion-exclusion chromatography. The specimens were derivatized by reaction with 1-fluoro-2,4-dinitrobenzene and reaction byproducts extracted by organic solvents. The trimethyllysine and internal standard derivatives were separated easily from other sample constituents by reversed-phase paired-ion high-performance liquid chromatography with spectrophotometric detection at 405 nm. Standard curves were linear over a sample concentration range of 10-150 nmol/ml; the detection limit corresponded with 0.1 nmol trimethyllysine injected into the chromatograph. The procedure was used for determination of trimethyllysine in human urine.

Method for isolation of non-esterified fatty acids and several other classes of plasma lipids by column chromatography on silica gel.

A method is described for isolation from human plasma of non-esterified fatty acids, cholesteryl esters, triglycerides, cholesterol and diglycerides, monoglycerides, and some phospholipids by extraction and silica gel column chromatography. All of these lipid classes except diglycerides and cholesterol were separated cleanly in seven elution steps. Diglycerides and cholesterol were isolated together. Recovery of model compounds which represent the most significant classes of plasma lipids during the column chromatographic step was nearly complete. The overall recovery of added heptadecanoic acid from plasma specimens was 81% after both sample isolation steps. The overall recovery of added synthetic pentadecanoic acid and heptadecanoic acid ester lipid homologues from plasma was 80-91% after both sample preparation steps. About 6 h are required for extraction and isolation in duplicate of these lipid classes from twenty plasma specimens. Alternatively, non-esterified fatty acids can be isolated from twenty plasma specimens in duplicate within 4 h by a variation of the full procedure.

A phase II and pharmacokinetic study of weekly 72-h topotecan infusion in patients with platinum-resistant and paclitaxel-resistant ovarian carcinoma.

BACKGROUND: As suggested by preclinical trials, prolonged administration of topotecan, a reversible inhibitor of topoisomerase-I, may have a therapeutic advantage. Following a phase I trial of weekly 72-h topotecan infusion, we performed a phase II trial utilizing this schedule in ovarian carcinoma. METHODS: Eligibility included platinum-/paclitaxel-resistant ovarian carcinoma, measurable disease, and adequate hematologic, renal, and hepatic function. A dose of 2.0 mg/m(2) of topotecan was administered as a 72-h infusion weekly via an ambulatory pump. Plasma topotecan concentrations were determined prior to and at the completion of each weekly course. RESULTS: Twenty-four patients were entered and 23 patients were evaluable for toxicity and response. Two hundred eighteen weekly courses of therapy were administered (median 7 weeks, range 4-46 weeks). Toxicity was mild with grade 3 leukopenia, neutropenia, and anemia occurring in 13, 13, and 17% of patients, respectively. Two of 23 patients (9.1%) (CI 1-28%) had partial responses of 2 and 3 months' duration and 6 had stable disease. Steady state plasma topotecan lactone concentrations were a median of 1.2 ng/ml (range 0.4-8.00 ng/ml) following the first week of infusion. Steady state topotecan lactone concentrations after the first week of infusion were highest in 2 patients with partial responses. Mean steady state plasma topotecan lactone concentrations after the first week of infusion were 4.6, 2.0, and 1.3 ng/ml for partial response, stable disease, and progressive disease, respectively. An analysis of variance of steady state plasma topotecan concentrations after the first week of infusion over all administered cycles demonstrated a significant difference in steady state plasma topotecan lactone concentrations between patients with partial response and stable disease and between partial response and no response (significant at the 0.05 level after adjustment for multiple comparisons). Controlling for cycle number, steady state topotecan lactone concentrations are significantly greater for patients with responding or stable disease than those with progressive disease (P = 0.0003) and have a lower bound of > or = 1.9 ng/ml (95% confidence level). CONCLUSION: Steady state topotecan lactone concentrations are associated with responding or stable disease in platinum- and paclitaxel-resistant ovarian cancer. Steady state topotecan concentrations could potentially be utilized to modify tumor exposure and response.

Determination of carnitine, butyrobetaine, and betaine as 4'-bromophenacyl ester derivatives by high-performance liquid chromatography.

A method for determination of carnitine, 4-(N,N,N-trimethylammonio)butanoate (butyrobetaine), and 2-(N,N,N-trimethylammonio)acetate (betaine) is described. These omega-trimethylammonio carboxylates and the chemically analogous internal standards 4-(N,N-dimethyl-N-propylammonio)-3-hydroxybutanoate or 6-(N,N,N-trimethylammonio)hexanoate were derivatized by reaction with 4'-bromophenacyl triflate in the presence of N,N-diisopropylethylamine. The trialkylammonio carboxylate 4'-bromophenacyl ester derivatives were separated from other sample constituents by reversed-phase ion-pair high-performance liquid chromatography with spectrophotometric detection at 254 nm. Standard curves were linear over a sample concentration range of 10-100 nmol/ml. Quantities of 2.5 nmol of omega-trialkylammonio acid derivatives injected into the chromatograph were detected with signal-to-noise ratios greater than 50.

Simultaneous determination of O6-benzylguanine and 8-oxo-O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of O6-benzylguanine (O6BG) in human plasma was modified to include the metabolite, O6-benzyl-8-oxo-guanine (8-oxo-O6BG). O6-(p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C18 base-deactivated reversed-phase column. Separation was accomplished by gradient elution with mobile phases consisting of acetonitrile and phosphate buffer, pH 3.60. Eluted compounds were observed with diode array detection at 288 nm (O6BG) and 292 nm (8-oxo-O6BG). Standard curves were linear from 12.5 ng/ml to 1000 ng/ml, with an average regression coefficient of 0.999 (n=5) for both compounds. The lowest limit of quantitation was 25 ng/ml, with a signal-to-noise ratio of 8:1. The within-day relative standard deviations for O6BG quality control samples (n=18) with concentrations of 735 ng/ml, 305 ng/ml and 38 ng/ml were 2.4%, 4.2% and 5.3%, respectively. The within-day relative standard deviations for 8-oxo-O6BG quality control samples (n=18) at concentrations of 735 ng/ml, 420 ng/ml and 42 ng/ml were 2.2%, 4.0% and 7.1%, respectively. The day-to-day relative standard deviations for the same control specimens were 3.1%, 4.8% and 7.1% for O6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine. O6-Benzyl-8-oxo-guanine was identified in patient plasma specimens by liquid chromatography-electrospray mass spectrometry by comparison with spectral data acquired from reference material.