Liquid chromatographic determination of nalidixic acid in pharmaceutical preparations

A simple, rapid, and environmentally safe liquid chromatographic (LC) method was developed for the qualitative and quantitative determination of pharmaceutical preparations of nalidixic acid. The new method was applied to commercial preparations of tablets and a suspension of nalidixic acid and found to be satisfactory for both quantitative and qualitative determinations. Previous LC methods either used chloroform to extract, which we were trying to eliminate, or used a mobile phase of about pH 2.5, which will destroy the column coating. The LC system for the new method uses sulfanilic acid as internal standard, a mu-Bondapak C18 column, and a mobile phase of methanol, 0.0045M dibasic potassium phosphate, and 0.0072M hexadecyltrimethylammonium bromide. The detection wavelength is 254 nm. The sample is dissolved in methanol, and an aliquot is injected through a 20 microL injection loop. Average recoveries ranged from 99.4 to 101.3%.     

Determination of pyridonecarboxylic acids in plasma by reverse-phase high-performance liquid chromatography

A simple qualitative and quantitative determination for pyridonecarboxylic acids including nalidixic acid (NA), oxolinic acid (OA) and pipemidic acid (PPA) in chicken plasma was carried out by microbiological, spectrophotometric, thin-layer chromatographic (TLC) and reverse-phase high-performance liquid chromatographic (HPLC) methods. As a test organism for bacteriological bioassay, Bacillus subtilis ATCC-6633 was the most sensitive of seven organisms investigated. Using the cup and the disc methods, a standard curve was obtained by determining the relationship between various drug concentrations and the diameter of the inhibition zone. The three drugs had two strong UV absorbance wavelengths (257 and 330 nm) on spectrophotometry. TLC analysis using a silica gel 60 F254 plate was investigated, and a solution of methanol:chloroform:acetic acid (3:1:1, v/v/v) was found to be the most suitable solvent for separation. The minimum concentration of drug detectable by this method was 0.5 microgram/ml for NA, 0.075 microgram/ml for OA and 0.39 microgram/ml for PPA. For HPLC analysis, a solution of acetonitrile:0.2 M phosphoric acid (1:1, v/v) was superior, and simultaneous determination of all three drugs was possible under the HPLC conditions used. The lowest measurable amount of drug in chicken plasma was 0.01 microgram/g. Recovery from extracts spiked with each drug at a known concentration was close to 100% for NA and OA, but only about 50% for PPA.     

Biotransformation of trithiozine. III - Isolation and identification of further metabolites in rat urine

The metabolism of a new antisecretory and antiulcer drug, trithiozine (I.S.F. 2001, T), was studied in 4 hr rat urine samples after i.p. administration. After extraction at pH 7 with chloroform, the urine was either incubated with beta-glucuronidase or acidified to pH 2 and subsequently extracted with chloroform. The organic layers were evaporated to dryness and the residues used for TLC analysis. The neutral extracts revealed five spots, not present in control rat urine, corresponding to the unchanged drug T and to four metabolites. Two of the metabolites had been previously identified as the 4-(3,4,5-trimethoxybenzoyl)tetrahydro-1,4-oxazine (TBO) and the 4-(3,4,5-trimethoxythiobenzoyl)tetrahydro-1,4-oxazine-S-oxide (TO). Three other metabolites were found in the extracts after beta-glucuronidase incubation. TLC, U.V. and M.S. data were consistent with the structure 4-(3,5-dimethoxy-4-hydroxythiobenzoyl)tetrahydro-1,4-oxazine (HT), the corresponding S-oxide (HO) and the 4-(3,5-dimethoxy-4-hydroxybenzoyl)tetrahydro-1,4-oxazine (HBO). The acidic extracts revealed two spots structurally identified as the 3,4,5-trimethoxybenzoic acid (TBA) and the previous HBO. On the basis of present knowledge, a possible metabolic pathway of T is reported, consisting in a rapid metabolic oxidation on the sulfur atom and a slower demethylation on the para methoxy group. The presence of TBA is indicative of subsequent enzymatic hydrolysis of TBO. The intense and long-lasting inhibitory effect of T on gastric secretion is tentatively correlated with the pharmacological activities of some of its metabolites.     

Gas-chromatographic analysis for 1,2:5,6-dianhydrogalactitol, an anti-neoplastic agent, in plasma

We describe a gas chromatographic method for measuring submicrogram quantities of dianhydrogalactitol, a promising antineoplastic agent (currently undergoing clinical trials in humans), in plasma. The drug is first extracted from blood plasma by saturating the aqueous phase with solid potassium carbonate and extracting with isopropanol/chloroform (9/1 by vol). It is then converted to the corresponding n-butaneboronic ester by reaction at room temperature with butaneboronic acid and chromatographed on an SE-30 (3%) column, with flame ionization detection. Practicality of the method for monitoring drug distribution was demonstrated by administering dianhydrogalactitol in therapeutic doses to a dog and monitoring its concentrations in blood for the next 2 h.     

Method of purification affects some interfacial properties of pulmonary surfactant proteins B and C and their mixtures with dipalmitoylphosphatidylcholine

Two methods were employed for preparation of lipid extracts from porcine lung surfactant. Pulmonary surfactant proteins SP-B and SP-C were isolated from the extracts using gel-exclusion chromatography on LH-60 with chloroform:methanol acidified with hydrochloric acid. Monolayers of pure SP-B or SP-C isolated from butanol lipid extracts spread at the air-water interface showed larger molecular areas than those determined in films of SP-B or SP-C isolated from chloroform surfactant extracts. Aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) supplemented with 2.5 and 5.0 wt% of SP-B or SP-C obtained from butanol extracts adsorbed faster to the air-water interface than their counterparts reconstituted with proteins isolated from chloroform extracts. Surface pressure-area characteristics of spread monolayers of DPPC plus SP-B or SP-C did not depend on the method of isolation of the proteins. The diagrams of the mean molecular areas vs. composition for the monolayers of DPPC plus SP-B or SP-C showed positive deviations from the additivity rule, independently of the procedure used for preparation of lipid extract surfactant. Matrix-assisted laser desorption/ionization spectrometry of the proteins isolated from different extraction solvents was consistent with some differences in the chemical compositions of SP-Bs. Butylation of SP-B during extraction of surfactant pellet with butanol may account for the differences observed in the molecular masses of SP-Bs isolated by the two different extraction protocols. The study suggests that the method of purification of SP-B and SP-C may modify their ability to enhance the adsorption rates of DPPC/protein mixtures, and this may be relevant to the formulation of protein-supplemented lipids for exogenous treatment of pulmonary surfactant insufficiency.     

Microanalysis of tryptophan metabolites

In tryptophan metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been reported to show a carcinogenic action to mice bladder and the relation of the metabolites to human bladder cancer has been discussed. We developed methods for the fluorometric assay of these compounds and showed that the excretion of 3-hydroxyanthranilic acid increased in the patients with bladder cancer. We also devised methods for the fluorometric assay of glucuronide and sulfate of 3-hydroxyanthranilic acid and showed that the minor excretion of these conjugated forms was shown in humans. The distribution of these compounds was also studied and the obtained data suggests that 3-hydroxykynurenine has affinity for the pancreas. We then developed methods for the determination of other metabolites of tryptophan. A fluorescence reaction with UV irradiation was found and applied to the determination. This method is the most sensitive to kynurenic acid but can be applied to kynurenine, nicotinamide and quinolinic acid. Furthermore, this methods also applied to the determination of some medicines, e.g. indomethacin, isoniazid, nalidixic acid, nicorandil and disodium cromoglicate in the serum or urine. We further devised other methods for the determination of xanthurenic acid and 5-hydroxyindoles.     

Probenecid inhibits the renal clearance and renal glucuronidation of nalidixic acid. A pilot experiment

The aim of this pilot study was to demonstrate the possible inhibitory effect of probenecid on the renal glucuronidation and on the renal clearance of nalidixic acid in a human volunteer. Under acidic urine conditions, hardly any nalidixic acid is excreted unchanged (0.2%). It is excreted as acyl glucuronide (53.4%), 7-hydroxymethylnalidixic acid (10.0%), the latter's acyl glucuronide 30.9%, and 7-carboxynalidixic acid (4.2%). Under probenecid co-medication the renal glucuronidation of nalidixic acid is reduced from 53% to 16%; the renal clearance of both nalidixic acid and 7-hydroxymethylnalidixic acid are reduced (p < 0.001); the intrinsic t1/2 of the metabolite 7-hydroxymethylnalidixic acid increased from 0.48 h to 4.24 h. The amount of acyl glucuronidation of 7-hydroxymethylnalidixic acid was not altered. The in vitro protein binding of both acyl glucuronides was increased, while no effect on the unconjugated compounds was seen. Nalidixic acid had no effect on the maximal renal excretion rate of probenecid acyl glucuronide.     

Resistance to nalidixic acid. A misconception due to underdosage

The clinical impression of inordinate selection of resistant mutants to nalidixic acid cannot be substantiated on close scrutiny when sensitive infections are treated at a full dosage of 4 gm/day. When 27 consecutive patients were treated with 4 gm of nalidixic acid per day, resistance developed in the bacteriuric population in only 7%. Moreover, resistance in the fecal reservoir was surprisingly minimal and much less than that reported for sulfonamides, tetracyclines, and ampicillin. The observation is important because multiply-resistant Enterobacteriaceae maintain their sensitivity to nalidixic acid since extrachromosomal R-factor resistance to nalidixic acid has never been demonstrated and cannot be transferred from one organism to another. In vitro data on 100 sensitive strains of Enterobacteriaceae show that the developmental of resistance to nalidixic acid is inversely proportional to the concentration of nalidixic acid regardless of whether the inoculum size is 10(5) or 10(8) bacteria per milliliter. Underdosage (less than 4 gm/day) with nalidixic acid is the probable cause of excessive resistance.     

Antibacterial activity of ofloxacin in urine for 4 days after a single oral dose of 400 mg

Antibacterial activity of ofloxacin in urine after a single oral dose of 400 mg was evaluated in ten healthy female volunteers. Urine was collected over six periods, i.e., 0-6 h, 6-12 h, 12-24 h, 24-48 h, 48-72 h, and 72-96 h postdose. Ofloxacin levels were assayed in all samples using a microbiological method and HPLC. Urinary ofloxacin MICs were determined for five bacterial strains recovered from urine, two E. coli strains of which one was susceptible and the other resistant to nalidixic acid (Nal-A), one Klebsiella pneumoniae resistant to nalidixic acid (Nal-B), one Staphylococcus saprophyticus strain, and one Enterococcus faecalis strain; MICs were 0.06, 0.25, 1, 0.25, and 2 mg/L, respectively. Mean urinary ofloxacin levels by the microbiological method during the six collection periods were 193.3 +/- 30.3, 138.1 +/- 31, 53.2 +/- 7.3, 8.3 +/- 0.8, 1.4 +/- 0.2, and 0.6 +/- 0.1 mg/L, respectively. HPLC provided similar results: 216.7 +/- 31.6, 130.7 +/- 20.5, 56.5 +/- 7.1, 8.3 +/- 0.9, 1.5 +/- 0.3, and 0.5 +/- 0.05 mg/L, respectively. Mean urinary ofloxacin excretion over 96 h was 67.4 +/- 3.6% of the dose by the microbiological method was 72.5 +/- 2.5% of the dose by HPLC. On the first day, bacteriostatic activity of urine against enterobacteria exceeded 32 and was greater than 8192 for the nalidixic acid-susceptible E. coli strain; on the next day, overall values were equal or greater than 8 for the nalidixic acid-resistant E. coli and K. pneumoniae strains. Bacteriostatic activity was equal to or greater than 32 for the S. saprophyticus strain during the first two days and equal to 8 on the first day and 4 on the second day for the E. faecalis strain.     

Spectrofluorometric determination of substituted tetrahydrocarbazoles by a methylene blue sensitized photolytic reaction

A spectrofluorometric method was developed for the determination of 6-chloro-9-[2-(2-methyl-5-pyridyl)ethyl]-1,2,3,4-tetrahydrocarbazole-2-methanol hydrochloride and its carboxylic acid analog in blood and urine. It involves extraction of both compounds at neutral pH, either from blood into ethyl acetate (the residue of which is dissolved in either) or from urine directly into ether. Both the alcohol and the acid are separated from each other by selective extraction into acid or base, respectively, and then reextracted into either from the respective aqueous medium by appropriate pH adjustment. The residues of the ether extracts containing the compounds are dissolved separately in 0.25 N NH4OH. Methylene blue is added to all samples, which are then exposed to UV energy for 15 min to produce the fluorophores. The fluorescence of the solutions is read at 370 nm, with excitation at 340 nm. The linear range of quantitation of both compounds is 0.02-10 mug/each/ml of final solution. The method was applied to the determination of blood levels and urinary excretion of the alcohol and its acid metabolite in a dog.     

Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.     

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Pharmacokinetics of methyl palmoxirate, an inhibitor of beta-oxidation, in rats and humans

Recent studies from our laboratory have shown that methyl palmoxirate (MEP), an inhibitor of mitochondrial beta-oxidation of long chain fatty acids, can be used to increase incorporation of radiolabeled palmitic acid into brain lipids and reduce beta-oxidation of the fatty acid. Thus, MEP allows the use of carbon labeled palmitate for studying brain lipid metabolism in animals and humans by quantitative autoradiography or positron emission tomography (PET). As it is essential to pretreat human subjects with an acute dose of MEP prior to intravenous injection of [1-11C]palmitate for PET scanning, this study was undertaken to determine the plasma elimination half-life of MEP in rats and human subjects and to provide insight about the drug's absorption and metabolism. A gas chromatographic method was developed to measure MEP in body fluids. Following oral administration of MEP to rats (2.5 and 10 mg/kg) and to humans, the unmetabolized drug could not be detected in plasma or urine (sensitivity of detection was 1 ng). However, when MEP was injected intravenously (10 mg/kg) in rats, a peak initial concentration could be measured in plasma (7.7 microg/mL), the clearance of the drug from plasma was rapid (t1/2 = 0.6 min), which indicates that MEP readily enters tissue lipid pools or is metabolized like long-chain fatty acids. As no adverse experience occured in the 11 human subjects studied, oral administration of a single dose of MEP was safe under the conditions of this study and may be used to increase the incorporation of positron labeled palmitic acid for studying brain lipid metabolism in vivo by PET.     

Three-step purification of glucocorticoid receptors from rat liver

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography--mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 microgram/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

The metabolism of the hypolipidaemic drug sultosilic acid in rat, dog and man

1. Single oral doses of the hypolipidaemic drug [35S]sultosilic acid to rats (40 mg/kg), dogs (40 mg/kg) and man (7 mg/kg) were well absorbed. During three days, means of 59.2%, 58.8% and 61.8% in urine and 37.7%, 31.9% and 19.7% in faeces, were excreted by these species respectively. Most of the dose was excreted during the first 24 h. 2. Peak plasma levels of 35S were generally reached during 1-2 h after oral doses in rats (12 micrograms equiv./ml), dogs (45 micrograms equiv./ml) and two human subjects (15.2 and 10.3 micrograms equiv./ml). In humans, peak plasma levels of unchanged drug (at 1-1.5 h) were 10.5 and 6.3 micrograms/ml. Plasma concentrations of 35S increased almost proportionately to dose in rats following oral doses of 400 and 1200 mg/kg, although in dogs, concentrations were similar at these two dose levels but several times higher than at 40 mg/kg. 3. Tissue concn. of 35S were generally higher in rats than in dogs. Highest concn. occurred at 3 h in rats and 1 h in dogs. Apart from those in the liver and kidneys, tissue concn. were appreciably lower than the corresponding plasma levels. 4. The major radioactive component in dog urine was sultosilic acid. Rat and human urine contained sultosilic acid and also two more polar major metabolites. In male and female rat urine, the proportions of these excretory products differed and the proportions in male rat urine were similar to those in human urine. Sultosilic acid was also the only component detected in dog plasma, whereas rat and human plasma also contained the two urine metabolites. Dog bile contained a conjugate of sultosilic acid. 5. The two metabolites have been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as products resulting from oxidation of the methyl in the p-toluenesulphonyl group. The structures assigned are the corresponding carboxylic acid and the hydroxymethyl derivatives.     

High-performance liquid chromatographic determination of human serum albumin in plasma and urine by post-column fluorescence enhancement detection using 8-anilino-1-naphthalenesulfonic acid

A method for the quantitative determination of human serum albumin in plasma and urine by post-column high-performance liquid chromatography with fluorescence detection is presented. Human serum albumin in plasma and urine is separated by gel-permeation column chromatography, which is followed by fluorescence detection. The detection uses the fluorescence enhancement observed when 8-anilino-1-naphthalenesulfonic acid binds to human serum albumin. The method is rapid to perform and is highly sensitive (detection limit is 2.5 ng/injection volume at a signal-to-noise ratio of three.     

Measurement of nitrite and nitrate in plasma, serum and urine of humans by high-performance liquid chromatography, the Griess assay, chemiluminescence and gas chromatography-mass spectrometry: interferences by biogenic amines and N(G)-nitro-L-arginine analogs

In this paper, the HPLC method for the measurement of nitrite and nitrate in serum of humans newly reported by E1 Menyawi et al. is discussed, especially in regard to the extremely low nitrate levels measured in serum of healthy humans. From the discussion, it is concluded that: (1) Biogenic amines at physiological concentrations do not significantly interfere with the batch Griess assay. (2) The HPLC method of E1 Menyawi et al. does not reveal accurate levels for serum nitrate. (3) In serum and plasma of healthy humans, nitrate ranges within 15-70 microM. (4) Exogenous NG-nitro-L-arginine analogs can interfere with the measurement of nitrate in human plasma and urine by the batch Griess assay, chemiluminescence and GC-MS; interferences can be effectively eliminated by solid-phase extraction on cation-exchangers.     

Determination and confirmation of identities of flumequine and nalidixic, oxolinic, and piromidic acids in salmon and shrimp

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.     

Metabolism of haloperidol to pyridinium species in patients receiving high doses intravenously: is HPTP an intermediate?

The metabolism of haloperidol (HP) to the potentially neurotoxic pyridinium species, HPP+ and RHPP+, has been demonstrated in humans. In vitro studies in microsomes harvested from various animal species indicate that the tetrahydropyridines, HPTP and RHPTP, could be intermediates in this pathway. However, this has not yet been demonstrated in vivo in humans. In this study, plasma and urine collected from eight critically ill patients treated with high doses of intravenous HP were analyzed for HPTP and RHPTP using HPLC with electrochemical detection. However, neither HPTP nor RHPTP were detected despite plasma concentrations of HP and RHP higher than any previously reported. HPP+ and RHPP+ were both present in the urine in high concentrations and accounted for 1.1 +/- 0.5% and 5.3 +/- 3.6%, respectively, of the administered dose of HP. The apparent elimination half-lives of HPP+ and RHPP+ were 67.3 +/- 11.0 hr and 63.3 +/- 11.6 hr, respectively. The absence of HPTP and RHPTP in plasma and urine suggests that in humans these tetrahydropyridines either are insignificant intermediates in the metabolism of HP in vivo or are present only transiently at their site of formation and are not released into the circulation.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.