Understanding the dipyrone oxidation allying electrochemical and computational approaches.

Electroanalytical methodology by boron-doped diamond electrode (BDDE) associated to the square-wave voltammetry (SWV) for the determination of hydrolyzed dipyrone (DIP) in commercial formulations, raw natural waters and in human urine was developed. Through cyclic voltammetry (CV), it was shown that the oxidation of the DIP on the BDDE was irreversible with diffusional control. Computational studies suggested that the oxidation mechanism of DIP occurred with participation of two electrons and one proton. The analytical curves were obtained for concentrations of DIP ranging from 1.0 × 10-6 to 6.5 × 10-5 mol L-1 (r = 0.9994). The values of detection limit (LOD) and quantification limit (LOQ) of DIP were calculated from SWV and found to be 2.6 × 10-7 mol L-1 and 8.8 × 10-7 mol L-1. The methodology was effectively applied to real samples with the values of calculated recoveries varying between 91.0% and 117.3% and validated by iodometric titration experiments whose values were between 93.3% and 106.9%. The proposed methodology with BDDE represents an alternative tool and it has advantageous, such as very easy handling, low cost, no need for modification, low detection limit. Furthermore, it can be used for the routine analysis of DIP in different real samples.

Bilirubin oxidation derived from oxidative stress is associated with disease severity of atopic dermatitis in adults.

BACKGROUND: Bilirubin is an essential antioxidant. Its oxidative metabolites, biopyrrins, are sensitive urinary markers of oxidative stress. Multiple studies suggest that oxidative stress affects the pathogenesis of skin diseases such as atopic dermatitis (AD). AIM: To examine oxidative stress-induced bilirubin oxidation and its association with AD pathogenesis in adults. METHODS: In total, 11 patients with AD and 7 healthy controls (HCs) were enrolled. Bilirubin oxidation profiles in the combined urine of the patients and that of the HCs were examined using high-performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry. The concentrations of urinary biopyrrins and serum biomarkers for AD disease severity, such as IgE and thymus and activation-regulated chemokine (TARC)/CCL17, were measured by ELISA to determine correlations between urinary biopyrrins and serum biomarkers. Local bilirubin oxidation in AD skin lesions was assessed by immunohistochemical analyses using two antibodies against bilirubin. RESULTS: Levels of dipyrrole-monopyrrole-aldehyde, a novel urinary biopyrrin, were higher in patients with AD than in HCs, and increased with disease severity based on the SCORing Atopic Dermatitis (SCORAD) objective scoring system. Additionally, urinary biopyrrin levels correlated significantly with serum IgE and TARC/CCL17 levels. Furthermore, immunohistochemical analyses revealed that biopyrrins were strongly expressed in both infiltrating and resident cells in AD lesions. However, bilirubin was expressed at low levels in the lesions, suggesting that bilirubin oxidation is augmented in AD lesions. CONCLUSIONS: Bilirubin oxidation derived from oxidative stress in the skin lesions can be associated with disease severity of AD.

Dynamics of Oxidative Stress Evoked by Myocardial Ischemia Reperfusion After Off-Pump Coronary Artery Bypass Grafting Elucidated by Bilirubin Oxidation.

BACKGROUND: Revascularization therapy relieves myocardial ischemia, but can also result in ischemia-reperfusion injury caused by oxidative stress. However, the biokinetics of oxidative stress after myocardial ischemia-reperfusion are uncertain. This study aimed to evaluate the dynamics of oxidative stress after off-pump coronary artery bypass grafting (OPCAB) by measuring urinary biopyrrin levels. Biopyrrin is an oxidative metabolite of bilirubin thought to reflect oxidative stress, along with reactive nitrogen species (RNS).Methods and Results:The study included 18 patients who underwent OPCAB; patients were divided into effort angina pectoris (EAP; n=11) and unstable angina pectoris (UAP; n=7). Urinary biopyrrin and RNS levels were measured during the perioperative period (≤48 h after surgery). Biopyrrin levels transiently increased 4-12 h post-surgery (early phase), followed by a prolonged increase approximately 24-32 h post-surgery (late phase). The delayed increase in biopyrrin tended to be higher in patients with UAP, with a simultaneous increase in RNS. The patients in the UAP group had generally high pulmonary capillary wedge pressure (PCWP), although the cardiac index was within a normal range during the delay phase. CONCLUSIONS: The dynamics of biopyrrin levels revealed a biphasic pattern of oxidative stress after OPCAB. Delayed production of oxidative stress may be influenced by preoperative severity of myocardial ischemia and delayed RNS production.

Gender and functional CYP2C and NAT2 polymorphisms determine the metabolic profile of metamizole.

Metamizole is a pain-killer drug that has been banned in some countries because of its toxicity, but it is still used in many countries due to its effective analgesic and antispasmodic properties. Although large variability in the biodisposition and adverse effects of metamizole are known, factors underlying this variability are poorly understood. We analyzed the urinary recovery of metabolites, as well as the association of these profiles with genetic and non-genetic factors, in a group of 362 healthy individuals. Gender and functional polymorphisms are strongly related to metabolic profiles. N-demethylation of the active metabolite MAA is diminished in carriers of the CYP2C19*2 allele and in NAT2-slow acetylators. Acetylation of the secondary metabolite AA is decreased in men, in drinkers and in NAT2-slow acetylators with a differential effect of NAT2*5 and NAT2*6 alleles. The formylation of MAA is diminished in older subjects and in carriers of defect CYP2C9 and CYP2C19 alleles. Two novel arachidonoyl metabolites were identified for the first time in humans. Women and NAT2-slow acetylators show higher concentrations, whereas the presence of the rapid CYP2C19*17 allele is associated with lower concentrations of these metabolites. All genetic associations show a gene-dose effect. We identified for the first time genetic and non-genetic factors related to the oxidative metabolism of analgesic drug metamizole, as well as new active metabolites in humans. The phenotypic and genetic factors identified in this study have a potential application as biomarkers of metamizole biotransformation and toxicity.

Effects of lifestyle factors on urinary oxidative stress and serum antioxidant markers in pregnant Japanese women: A cohort study.

Oxidative stress plays a major pathological role in pregnancy-related complications. Although oxidative stress is induced by exogenous toxins in association with a poor lifestyle in normal subjects, there is little information on the factors altering oxidative stress and antioxidant levels during pregnancy. The purpose of this study was to determine the relationship between lifestyle factors and oxidative stress/antioxidant levels during each trimester and 1-month postpartum. This prospective cohort study followed 54 healthy women through pregnancy; first, second, and third trimester and 1-month postpartum. Participants were administered a questionnaire on characteristics and lifestyle factors. Morning blood and urine samples were obtained to measure urinary biopyrrins and serum coenzyme Q10 (CoQ10) levels. The levels of urinary biopyrrins and serum CoQ10 increased significantly throughout pregnancy, with peak values registered during the third trimester. Higher biopyrrin levels were significantly associated with non-consumption of morning meal during the first trimester, smoking during the third trimester and 1-month postpartum, alcohol consumption during the third trimester, high food-based polyunsaturated fatty acid intake during the third trimester, and poor mental health scores during the first and third trimesters. Higher CoQ10 levels were significantly associated with no smoking during pregnancy and at 1-month postpartum, and with a high frequency of exercise during the third trimester and 1-month postpartum. Thus, pregnancy represents a state of oxidative stress, which can be counterbalanced by increased levels of antioxidants, such as CoQ10. We speculate that certain lifestyle choices such as avoiding smoking can reduce oxidative stress and increase antioxidant levels during pregnancy.

Elevated urinary biopyrrin excretion and oxidative bilirubin metabolism during 24-hour ultramarathon running.

The oxidative metabolite of bilirubin, biopyrrin, is considered a useful candidate marker of oxidative stress in vivo. The present study examines whether urinary biopyrrin excretion is elevated and how general laboratory parameters are changed by long-duration running such as that involved in ultramarathons. Fifteen volunteer runners (12 males and 3 females; aged 44 +/- 9 years; means +/- SD) provided written informed consent to participate in this study. The 24-h experimental run was not a race against time but rather to determine the effects of running around a track for 24 h without sleep and sufficient rest. Blood and urine samples were obtained before (0 h), during (16 h), and immediately after (24 h) running for 24 h. All of the participants completed the run. The mean (+/- SD) distance run was 162.6 +/- 18.3 km. Mean urinary biopyrrin excretion values at 0, 16, and 24 h were 1.23 +/- 0.73, 2.55 +/- 0.95, and 4.00 +/- 1.50 U/g creatinine, respectively. Urinary biopyrrin excretion was positively and significantly correlated with the serum bilirubin concentration (p<0.05) and distance run (p<0.05). These results suggest that urinary biopyrrin excretion could be a useful marker of oxygen stress incurred during a 24-h ultramarathon.

Urinary excretion of oxidative metabolites of bilirubin in subjects with Gilbert syndrome.

BACKGROUND AND AIM: Bilirubin is a potent endogenous antioxidant substance. Recent data suggest a direct relationship exists between urinary excretion of biopyrrins, a novel group of bilirubin oxidative metabolites, and severity of oxidative stress. The aim of this study was to evaluate urinary excretion of biopyrrins in subjects with Gilbert syndrome. METHODS: The study included patients with Gilbert syndrome (n = 33) and healthy blood donors (n = 25). In all subjects complete biochemical tests were conducted along with analysis of urinary excretion of biopyrrins. Linear and logistic regression analyses were used for multiple adjustments of possible confounders/modifiers. RESULTS: As expected, high serum bilirubin levels were found in the Gilbert syndrome group as compared to controls (27.8 +/- 9.7 vs 9.9 +/- 3.0 micromol/L, P < 0.001). In contrast, urinary levels of biopyrrins were substantially lower in the Gilbert syndrome group as compared to normobilirubinemic control subjects (19.9 +/- 26.0 vs 90.2 +/- 139.1 U/g urinary creatinine, P < 0.001). The Gilbert syndrome group also had very low prevalence odds ratios for urinary biopyrrins above the median of the control values even after adjustment for possibly confounding factors (odds ratio 0.18, 95% confidence interval 0.33-0.94; P = 0.042). CONCLUSIONS: An inverse relationship was demonstrated between serum bilirubin level and urinary excretion of biopyrrins, which is presumably due to antioxidative effects of elevated serum bilirubin levels in Gilbert syndrome.

Bilirubin oxidation provoked by nitric oxide radicals predicts the progression of acute cardiac allograft rejection.

Bilirubin, a strong intrinsic antioxidant, quenches free radicals produced under inflammatory conditions. The oxidized bilirubin metabolites, i.e. biopyrrins, are immediately excreted into urine and can indicate the intensity of oxidation in vivo. Our preliminary studies suggested the involvement of reactive nitrogen species (RNS) in generation of biopyrrins. However, little is known about biological significance of bilirubin oxidation by RNS. Here, we analyzed the correlation between bilirubin oxidation and nitric oxide (NO) radicals during rat acute cardiac allograft rejection. In allograft recipients, urinary biopyrrins steeply increased on day 3 prior to the increase in myocardial tissue damage marker, serum troponin-T. In contrast, no significant changes in urinary biopyrrins were evident in recipients of isografts or cyclosporine-A treated allografts. Urinary nitrotyrosine, a marker of oxidation by NO radicals also increased on day 3, while administration of a NO synthase inhibitor, N(G)-monomethyl-L-arginine apparently diminished the elevation of urinary biopyrrins as well as nitrotyrosine. Immunohistochemistry revealed enhanced local expression of heme oxygenase-1, biopyrrins and nitrotyrosine in allografts in accordance with the cellular infiltrates, suggesting that changes in urinary biopyrrins reflect the bilirubin oxidation in grafts undergoing rejection. These results indicate that locally evoked bilirubin oxidation by NO radicals can predict the progression of rejection.

[Clinical biochemical evaluation of central fatigue with 24-hour continuous exercise].

OBJECTIVE: The present study was conducted to clarify the effects of ultra-marathon (ultra long-term aerobic exercise in which people run long distances) on the brain; examine the issue of central fatigue; verify the serotonin hypothesis of exercise-induced brain fatigue, and ascertain relationships between central fatigue and oxidative stress. METHODS: Subjects consisted of 15 individuals (12 men, 3 women) who ran continuously for 24 h. Mean age was 44 +/- 9 years (range, 31 approximately 64 years). Blood tests were conducted: (1) before starting to run (around 09:00); (2) 16h after starting (02:00 the next day); and (3) just after the finish (around 10:00 the next day) to measure the serum levels of serotonin, melatonin, free tryptophan (f-Tp) and free fatty acid. At the same time, urine samples were collected to measure levels of urinary biopyrrins (BPn). Subjective symptoms were investigated using the Japanese version of the Profile of Mood States (POMS) instrument. RESULTS: (1) Participants ran a mean (+/- SD) distance of 162.6 +/- 18.3 km. (2) There were not marked changes in serum serotonin levels. Serum melatonin levels at 3 time points were 3.4 +/- 0.6 pg/ml, 57.2 +/- 15.2pg/ml and 7.8 +/- 8.9pg/ml, respectively(p < 0.01 before start vs. 16h after start). Serum f-Trp levels at the 3 time points were 5.4 +/- 0.9 nmol/ml, 9.7 +/- 2.1 nmol/ml and 11.5 +/- 4.9 nmol/ml, respectively (p< 0.05 before start vs. just before finish). Free fatty acid levels were 0.42 +/- 0.10 nmol/ml, 1.26 +/- 0.11 nmol/ml and 1.39 +/- 0.23 nmol/ml, respectively (p < 0.01 before start vs. 16 hours after start) (p < 0.05 before start vs. just after finish). (3) Urinary BPn levels increased with time, from 1.2 +/- 0.7 nmol/ml to 2.6 +/- 1.0 nmol/ml to 4.0 +/- 1.5 nmol/ml, respectively (p < 0.01 before the start vs. 16 hours after the start). (4) In terms of POMS scores, fatigue score (Factor F) increased, but vitality score (Factor V) was high at all time points and did not demonstrate any marked changes. Scores for anger and hostility were low (Iceberg profile-type: convex type). Urinary BPn levels were correlated significantly with both serum f-Trp level and Factor F:(y = 8.41x + 2.5, r = 0.708, n = 42) and (y = 2.82x + 5.9, r = 0.568, n = 42), respectively. Urinary BPn thus reflected the degree of subjective fatigue with a high level of sensitivity. CONCLUSIONS: The present results suggest that running continuously for 24h induces brain fatigue and that oxidative stress may be involved.

Effort-reward imbalance and physical health among Japanese workers in a recently downsized corporation.

OBJECTIVES: The present study investigated the effort-reward imbalance (ERI) model-related biological markers of disease risks in Japanese workers of a recently downsized manufacturing corporation. METHODS: A total of 441 workers was examined to find whether situational effort-reward imbalance or personal overcommitment was associated with hematological and biochemical measurements, serum cortisol, and urine biopyrrins as oxidative metabolites of antioxidant bilirubin. RESULTS: The effort-reward imbalance was positively associated with the values of red blood cells, hemoglobin, hematocrit, triglycerides and glutamic pyruvic transaminase (GPT), and negatively correlated with the high-density lipoprotein cholesterol level. Overcommitment was positively related to the values of hematocrit and glucose levels, but negatively associated with the total protein level. The relationships between effort-reward imbalance and GPT level, and that between overcommitment and glucose level, persisted when potential confounders were adjusted for. The ERI model was not significantly related to either cortisol or biopyrrins levels. CONCLUSIONS: The ERI model seems to have an impact on the physical health of the downsized Japanese workers, although the results are mixed and are different from those in workers of Western countries.

Associations between oxidative stress levels and total duration of engagement in jobs with exposure to fly ash among workers at municipal solid waste incinerators.

The fly ash from municipal solid waste incinerators (MSWIs) is known to contain heavy metals, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polyaromatic hydrocarbons (PAHs), and other organic materials. Heavy metals, PCDDs, PCDFs and PAHs reportedly cause oxidative stress in vitro and in vivo. In this study, we have measured the blood and urinary levels of several oxidative stress markers in MSWI workers and discuss herein whether the duration of engagement in jobs with exposure to MSWI fly ash is associated with these levels. The subjects were 81 male workers (mean age 42.7 years) from four MSWIs in the same city. Job history was determined from each subject and jobs were categorized according to the possibility of exposure to fly ash. The subjects were classified into four groups: long duration of engagement in jobs with exposure to fly ash, short duration of engagement in jobs with exposure to fly ash, engagement in jobs with limited exposure to fly ash and control. Blood and urine specimens were obtained from the subjects in the morning before breakfast. The levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in the urine and leukocytes were measured as markers of oxidative DNA damage. Blood malondialdehyde and lipid peroxide levels and the level of total urinary biopyrrins were also measured as markers of systemic oxidative stress. The mean levels of all markers were compared among the four groups. There was a significant trend showing that the level of urinary 8-OH-dG rose with increased duration of engagement in jobs with exposure to MSWI fly ash (P<0.05). Considering this result, we speculate that certain chemicals in fly ash might have induced oxidative stress in the study subjects.

Bioavailability of two metamizole (dipyrone) solutions as single doses of 2 g versus metamizole capsules.

OBJECTIVE: The aim of this study was to investigate the extent and the rate of absorption of metamizole, appearing in blood as methylaminoantipyrine (MAA), from a new oral solution and a parenteral solution administered by the oral route relative to capsules. METHODS: An open, randomized, 3 single-dose (2 g metamizole), crossover study with intervals of 7 days between periods was performed in 19 male and female healthy volunteers (age 22 - 45 years, body weight 49 - 88 kg, body height 156 - 189 cm). Metamizole metabolites were measured with an HPLC technique. The test formulations were considered bioequivalent with the reference formulation if the 90% confidence limits of the AUC0-->infinity and Cmax ratios and the tmax differences were within the range of 80 - 125%. RESULTS: The 90% confidence limits of the comparisons between capsules (reference) and oral solution, capsules (reference) and ampoules, and ampoules (reference) and oral solution were 98.5 - 117.8, 99.5 - 132.6 and 81.3 - 105.8 for AUC0-->infinity 98.7 - 119, 101.7 to 129.2, and 82.1 - 104.8 for Cmax, and 84.4 to 115.6, 100 - 105.6 and 70.3 - 100 for tmax, respectively. CONCLUSION: The oral solution was bioequivalent to capsules with regard both to the extent and the rate of MAA absorption. Metamizole as oral solution was bioequivalent to reference ampoules in the extent of MAA absorption, but absorption rate was faster. Ampoules showed a higher MAA bioavailability than capsules.

In vitro monitoring sub-nanogram amounts analgin in human urine by its inhibitory of the luminol-periodate chemiluminescence reaction using reagent immobilization release technique.

A selective and sensitive as well as rapid chemiluminescence (CL) flow sensor for the determination of analgin is described. The analytical reagents involved in chemiluminescence reaction, luminol and periodate, were both immobilized on an anion-exchange column. The CL signals produced by the reaction between luminol and periodate, which were eluted from the column through water injection, were decreased in the presence of analgin. Analgin was sensed by measuring the decrement of CL intensity, and which was observed linear over the logarithm of analgin concentration range of 0.1 to 50.0 ng mL(-1), and the limit of detection was 0.04 ng mL(-1) (3ó). At a flow rate of 2.0 mL min(-1), including sampling and washing, the detection could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The proposed procedure was applied successfully in the monitoring of analgin in human urine samples without any pre-treatment process. It was found that the analgin concentration reached its maximum after being orally administrated for 4 h, and the analgin metabolism ratio in 10 h was 9.28% in the body of volunteers. The flow sensor offered reagentless procedures and remarkable stability in determination of analgin, and could be easily reused over 80 h.

Death due to baclofen and dipyrone ingestion.

A fatal suicidal intoxication with unusual drugs is reported. A 56-year-old man was found dead in his house; near by the corpse several empty drugs boxes were found. An autopsy was performed and the biological fluids were submitted to a full toxicological work-up. The analytical results supported the hypothesis of a death due to the acute baclofen (4-amino-3-(p-chlorophenyl)butyric acid) and dipyrone (sodium [N-(1,5-dimethyl-3-oxo-2-phenylpyrazolin-4-yl)-N-methylamino] methanesulfonate) intoxication.

Impairment of the metabolism of dipyrone in asymptomatic carriers of the hepatitis-B virus does not occur in rapid acetylators.

OBJECTIVE: We previously found that, compared with healthy subjects. asymptomatic hepatitis-B virus (HBV) carriers displaying slow acetylator phenotype demonstrate a significant prolongation of the elimination half-life of 4-methylaminoantipyrine (MAA) and a decrease in the clearance of formation of 4-aminoantipyrine (AA) and 4-formylaminoantipyrine (FAA). However, the formation of 4-acetylaminoantipyrine (AAA) was unchanged. The present study was designed to examine the effect of the asymptomatic HBV carrier state on the metabolism of dipyrone. as a model drug, in rapid acetylators. METHODS: The plasma and urine concentrations of the metabolites of dipyrone were measured in eight asymptomatic HBV carriers and eight healthy subjects who had normal liver function tests, all displaying the rapid acetylation phenotype and genotype, after the administration of a 1.0-g oral dose of dipyrone. RESULTS: The following pharmacokinetic parameters were evaluated: peak plasma concentration, time to peak plasma concentration, elimination rate constant, area under the plasma concentration-time curve (0-->infinity), amount excreted (0-->infinity), renal and non-renal clearances for MAA and the clearances of formation for AA, FAA and AAA. No significant differences were found between the two subject groups. CONCLUSION: The effect of hepatic viral carrier state on drug metabolism may vary according to metabolic pathways and genetic polymorphism.

Moderate alcohol consumption reduces urinary 8-hydroxydeoxyguanosine by inducing of uric acid.

Recent studies suggest that moderate alcohol consumption is associated with a low risk of cancer, coronary heart disease, and other diseases. Most of these diseases are considered to be related to the action of reactive oxygen species (ROS) at certain stages of disease progression. However, considerable evidence exists indicating that ethanol generates ROS in vivo. Thus, the reduced risk of disease as a result of alcohol consumption seems to contradict evidence suggesting the induction of ROS by ethanol. In the present study, we investigated whether oxidative stress was induced in moderate alcohol drinkers. We measured the total urinary biopyrrins and 8-hydroxydeoxyguanosine (8-OHdG) levels as a systemic oxidative stress marker and an oxidative DNA damage marker, respectively. Serum uric acid was also measured as an alcohol-induced antioxidant. We compared total urinary biopyrrins and 8-OHdG levels among groups with different alcohol habits. The results showed that total biopyrrins levels increased with the amount of alcohol consumed, but that the level of 8-OHdG significantly decreased with the amount of alcohol consumed. The decrease in 8-OHdG levels seemed to be associated with increasing levels of uric acid. Judging from the increasing level of total biopyrrins, alcohol may induce ROS. ROS may then cause cell damage in liver, as suggested by the positive correlation between the total biopyrrins levels and the serum GOT, GPT, and gammaGTP levels. However, since ROS may be more effectively counteracted by uric acid in organs other than the liver, DNA damage may be suppressed rather than induced. Accordingly, moderate alcohol consumption seems to have the overall effect of reducing DNA damage, as shown by the decrease in urinary 8-OHdG levels observed in our study.

Chemiluminescence analysis of menadione sodium bisulfite and analgin in pharmaceutical preparations and biological fluids.

A novel chemiluminescence (CL) flow system for two sulfite-containing drugs, namely, menadione sodium bisulfite (MSB) and analgin is described. It is based on the weak chemiluminescence induced by the oxidation of sulfite group in drugs with dissolved oxygen in the presence of acidic Rh6G. Tween 80 surfactant micelles showed a strong enhancement effect on this weak chemiluminescence. For MSB analysis, online conversion of MSB in alkaline medium into sodium bisulfite was necessary, whereas analgin could be determined directly. The proposed method allowed the measurement of 0.05-50 microg/ml(-1) MSB and 0.05-10 microg/ml(-1) analgin. The limits of detection (3sigma) were 0.01 microg/ml(-1) MSB and 0.003 microg/ml(-1) analgin. The method was applied satisfactorily to pharmaceutical preparations as well as biological fluids.

Determination of aminopyrine, dipyrone and its metabolites in urine by high-performance liquid chromatography.

A readily applicable and accurate isocratic high-performance liquid chromatography method for the detection of aminopyrine, dipyrone and its metabolites in urine is described. Parent drugs and four metabolites were chloroform-extracted from 1 ml of urine after addition of the internal standard isopropylaminoantipyrine and alkalinization. The organic phase was evaporated to dryness, and the residue was reconstituted in the mobile phase, which was injected onto a Spherisorb ODS 5 microns particle-size column (250 x 4.6 mm) using as mobile phase water, methanol, triethylamine, and acetic acid. The column eluent was monitored by ultraviolet absorption at 254 nm. Excellent linearity (r > 0.99) was obtained in the range 1-150 micrograms/ml urine, either for parent drugs and metabolites. This method offers a sensitive assay for aminopyrine, dipyrone (widely consumed in some countries) and its metabolites. After oral administration and collection of 24-h urine, this method allows the in vivo study of aminopyrine metabolism, which reflects liver function.