Radioimmunoassay for the testosterone 5 alpha-reductase inhibitor turosteride in biological fluids

An antiserum against turosteride (code name FCE 26073), a potent testosterone 5 alpha-reductase inhibitor, has been raised in rabbits by immunization with an immunogen produced by conjugation of a derivative of FCE 26073 (FCE 27424) to bovine serum albumin. The antiserum was able to distinguish FCE 26073 from its derivatives modified at the 17 beta position and from all the endogenous steroids tested. A radioimmunoassay for the determination of FCE 26073 in human plasma and urine was developed using this antiserum and tritium labeled turosteride. FCE 26073 was extracted from 50 microliters of plasma or 25 microliters of urine using ethyl-ether with a recovery greater than 90%. Using this procedure it was possible to achieve a final limit of quantitation of 142 pg/ml in plasma and 284 pg/ml in urine. The assay was validated in terms of reproducibility, accuracy and precision in the range 3.9-250 pg/50 microliters of plasma and 25 microliters of urine. The plasma concentration of FCE 26073 in a healthy male volunteer who received 0.2 mg of the drug was measured using the radioimmunoassay.     

Combined pharmacological and traction treatment in cases of painful syndromes of the cervical spine with degenerative and deformative changes

A simple, specific, and sensitive radioimmunoassay was developed for the determination of the diuretic bumetanide in plasma and urine. Antiserum to bumetanide was obtained from rabbits immunized with an immunogen prepared by covalently coupling the glycine conjugate of bumetanide to bovine serum albumin. Following extraction of the sample at pH 5.5 with ether, radioimmunoassay of the residue from the ether extract allows for the determination of bumetanide with a limit of sensitivity of about 1 ng/ml using 0.1 ml of plasma or urine. The specificity of the radioimmunoassay was established by comparison with specific radiometric and spectrofluorometric techniques. The pharmacokinetic profile of bumetanide in eight human subjects receiving single 2-mg oral doses of the drug was elucidated using the radioimmunoassay. The peak plasma levels ranged from 39 to 50 ng/ml at 1-4 hr after administration and declined with a mean apparent half-life of 1.17 hr. The mean plasma clearance rate was calculated to be 255 ml/min. During the first 24 hr, a mean of 43% of the bumetanide dose was excreted in the urine as intact drug.     

Dysregulation of the hypothalamo-pituitary axis in rheumatoid arthritis

OBJECTIVE: To study the dynamic response of the hypothalamo-pituitary- adrenal axis and of prolactin (PRL) pituitary secretion in rheumatoid arthritis (RA). METHODS: We performed a cortisol releasing hormone (CRH) provocation test followed by determination of adrenocorticotropin hormone (ACTH), beta-endorphin, and cortisol concentration, and then a thyrotropin releasing hormone (TRH) provocation test followed by assessment of PRL pituitary secretion in 10 patients with RA and 5 control subjects. All were women under 40 years of age. Hormone concentrations were assessed by radioimmunoassay. RESULTS: Basal PRL cortisol, and ACTH concentrations were similar in patients with RA and controls. We observed a dissociation between the pituitary secretion of beta-endorphin and of ACTH in response to CRH in RA. The ACTH peak and total ACTH production (area under the curve, AUC) were similar in the 2 groups. In contrast, basal beta-endorphin was increased in RA (12.6 +/- 1.41 vs 8.29 +/- 0.144 pg/ml), and the response upregulated (AUC: 83,080 +/- 12,000 vs 54,200 +/- 2400) after CRH compared to controls (p < 0.05). Cortisol adrenal response curve was blunted, but did not reach statistical significance. In contrast, the PRL response to TRH was increased at 120 and 150 min (3461 +/- 303 vs 1897 +/- 520 muIU/ml)(p < 0.01) in patients with RA, independent of disease activity. CONCLUSION: We observed upregulated pituitary PRL secretion in RA, and a dissociation of ACTH stress. The implication concerning the neuroendocrine system in the chronic immune response in RA is discussed.     

Radioimmunoassay of 8-arginine-vasopressin (antidiuretic hormone) in human plasma

A radioimmunoassay for 8-arginine-vasopressin (AVP) measurement in human plasma has been developed and evaluated, using a commercial preparation of an antibody of AVP. Detection limit of the assay was 0.4 pg. A simple acetone extraction procedure gave a recovery of 65% of added [125I]AVP. The overall sensitivity in the assay was 1.0 pg/ml when 2 ml plasma samples were extracted. The antigenic sites of the employed antibody seemed to be a combination of amino acid residues in the tripeptide tail and the pentapeptide ring. This can explain that the antibody was almost completely insensitive to chemically or enzymatically degraded AVP. The inter-assay coefficient of variation for the control plasma pools averaged 17%. A good correlation to plasma osmolalities above 290 has been found. AVP level in recumbent subjects (n = 8) with plasma osmolalities in the normal range was 2.8 +/- 1.0 pg/ml (mean +/- SD) and in ambulatory subjects (n = 10) on ad lib. water intake 4.5 +/- 1.9 pg/ml (mean /+- SD).     

Altered growth hormone and prolactin responsiveness to TRH in the infant rat

12-day-old female and male pups were killed 10 min after the injection of either saline or thyrotropin releasing hormone (TRH), and plasma growth hormone (GH) and prolactin (PRL) levels were measured by radioimmunoassay (RIA). At all doses used (0.15, 0.3, 0.6 and 1.5 mug/100 g b.w.i.p.), TRH induced a significant, although not dose-related, increase in plasma GH levels, but was effective in releasing PRL only at the greatest dose level (1.5 mug/100 g b.w.). The GH-releasing effect of TRH was even more evident in 12-day-old pups subjected to central sympathectomy of 6-hydroxydopamine (6-OHDA, 60 mug/10 mul intraventricular route) 1 week before; in these animals, TRH was ineffective in releasing PRL even at the greatest dose level (1.5 mug/100 g b.w.). In pups pretreated with 6-OHDA, the GH-lowering effect of insulin hypoglycemia or cold exposure was markedly reduced, while the PRL responses were unmodified. Baseline plasma PRL levels were markedly increased following 6-OHDA administration. It is proposed that in the infant rat the greater GH than PRL responsiveness to TRH, which opposed the pattern of response present in the adult animal, may be due to the existence of a 'physiologic' functional disconnection between the central nervous system (CNS) and the anterior pituitary (AP). Results obtained following central sympathectomy by 6-OHDA, which further disrupted CNS-AP links, substantiate this view.     

Hypofunction of the stress axis in Sj?gren's syndrome

OBJECTIVE: To examine the functional integrity of the hypothalamic-pituitary-adrenal (HPA) and thyroid axes in Sj?gren's syndrome (SS) via the assessment of basal and stimulated adrenocorticotropin (ACTH), cortisol, thyroid stimulating hormone (TSH), and prolactin levels. METHODS: Pituitary function of the HPA axis was assessed by determining the basal plasma levels of ACTH in the late afternoon, as well as the ACTH released to ovine corticotropin releasing hormone (oCRH) stimulation; adrenal function was assessed by measuring plasma cortisol levels in the late afternoon at baseline and after release of the endogenous ACTH during oCRH stimulation. Basal and thyrotropin releasing hormone (TRH) stimulated levels of TSH and prolactin were also assessed. Healthy volunteers were used as controls. RESULTS: Patients with SS, compared to controls, were characterized by significantly lower ACTH levels (pg/ml), (5.1 +/- 0.5 vs 11.4 +/- 1.5, respectively; p < 0.05) and cortisol levels (microg/ml), (2.4 +/- 0.6 vs 5.9 +/- 1.2, respectively; p < 0.05). Furthermore, a blunted pituitary and adrenal response to oCRH compared to controls was observed: peak plasma ACTH and cortisol levels for patients with SS were 46.2 +/- 5.4 pg/ml and 15.7 +/- 1.6 microg/ml, respectively, and for controls 61.5 +/- 3.8 and 19.6 +/- 0.7, respectively (p < 0.05). Basal TSH levels were significantly elevated in patients (1.3 +/- 0.3 microIU/ml vs 0.9 +/- 0.05 microIU/ml; p < 0.05). CONCLUSION: The above findings indicate hypoactivity of the HPA axis in patients with SS. Further studies are needed to definitively identify the locus of the defects and assess the significance of the pattern of the perturbations to the pathogenesis and expression of SS.     

Percutaneous penetration and metabolism of topical (14C)flutamide in men

This study was designed to determine the fate of the nonsteroid antiandrogen flutamide in men following a single 6-hr topical application of 5 mg 14C-labeled drug dissolved in 50% ethanol/50% propylene glycol. Analysis of 0-120 hr urine shows at least 16% of the applied flutamide is absorbed. Fifty-six percent of the dose is recovered from the site of application with cotton swabs moistened with 50% ethanol/50% propylene glycol. Flutamide plasma levels peak in 4 to 6 hr at about 1.3 ng/ml and then decline rapidly to about 0.08 ng/ml 24 hr after application. Only 13% of plasma 14C is associated with flutamide 6 hr after drug application. There are at least 10 plasma metabolites, of which 6 have been tentatively identified. These are alpha, alpha, alpha-trifluoro-4'-amino-m-acetotoluidide (A); alpha, alpha, alpha-trifluoro-4'-amino-2-methyl-m-lactotoluidide (B); alpha, alpha, alpha-trifluoro-4'-nitro-m-acetotoluidide (C); alpha, alpha, alpha-trifluoro-2-methyl-4'-nitro-m-lactotoluidide (D); alpha, alpha, alpha-trifluoro-4'-amino-2-methyl-m-propionotoluidide (E); and alpha, alpha, alpha-trifluoro-6-nitro-m-toluidine (F). (D) is the major plasma metabolite, and its concentration exceeds flutamide's between 8 and 24 hr after drug. All the plasma metabolites are found in 0-24 hr urine in minor amounts. An additional metabolite, alpha, alpha, alpha-trifluoro-amino-5-nitro-p-cresol (G), accounts for 27% of urine 14C.     

Effects of gamma-butyric acid on the release of thyrotropin-releasing hormone from the rat retina in vitro

Effects of gamma-butyric acid (GABA) on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of GABA, but not with bicuculline. The inhibitory effect of GABA on TRH release from the retina was blocked by adding bicuculline to the medium. The findings suggest that the GABAergic system inhibits TRH release from the rat retina in vitro.     

Effects of serotonin on the release of thyrotropin-releasing hormone from the rat retina in vitro

Effects of serotonin on the release of thyrotropin-releasing hormone (TRH) from the rat retina were studied in vitro. The retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of serotonin and enhanced with cyproheptadine. The inhibitory effect of serotonin on TRH release from the retina was blocked with the addition of cyproheptadine. The elution profile of methanol extract of the rat retina was identical to that of synthetic TRH. The findings suggest that the serotonergic system inhibits TRH release from the rat retina in vitro.     

Plasma prolactin responses to thyrotropin-releasing hormone in patients with breast cancer

Plasma human prolactin levels were measured by homologous radioimmunoassay in patients with primary breast cancer and in normal women of similar age. In normal controls mean (+/- SEM) basal plasma prolactin levels were 11.9 +/- 1.5 ng/ml and intravenous injection of synthetic thyrotropin-releasing hormone (TRH), 500 mug, caused a significant rise in plasma prolactin in all subjects examined with a maximum response of 52.6 +/- 3.3 ng/ml (mean +/- SEM). Markedly high plasma prolactin levels and exaggerated plasma prolactin responses to TRH were demonstrated in some patients with breast cancer. However, mean basal plasma prolactin levels and mean plasma prolactin increments following TRH in patients with breast cancer did not differ significantly from those in normal subjects. Plasma prolactin responses to TRH were slightly blunted during the administration of androgen in patients with breast cancer. These results suggest that some of the patients with primary breast cancer have abnormal prolactin secretion.     

Determination of N,N',N"-triethylenthiophosphoramide in biological samples using capillary gas chromatography

A sensitive assay for the determination of N,N',N"-triethylenthiophosphoramide (thioTEPA) in microvolumes of human plasma and urine has been developed. ThioTEPA was analysed using gas chromatography with selective nitrogen-phosphorus detection, after extraction with ethyl acetate from the biological matrix. Diphenylamine is the internal standard. The limit of quantitation was 0.1 ng/ml, using only 100 microl of sample; recoveries ranged between 85 and 100% and both accuracy and precision were less than 10%. Using a flame ionisation nitrogen-phosphorus detector, the assay was not linear over the concentration range of 2-1000 ng/ml for plasma and 10-1000 ng/ml for urine. Linearity was accomplished in the range of 1-1000 ng/ml for plasma and urine when a thermionic nitrogen/phosphorous detector was used. The stability of thioTEPA in plasma proved to be satisfactory over a period of 3 months, when kept at -20 degrees C, whereas it was stable in urine for at least 1 month at -80 degrees C. ThioTEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay.     

Perinatal hypoxic-ischemic brain injury

A highly sensitive radioimmunoassay for the measurement of plasma prostaglandins A and B, expressed in equivalents of PGA1, is described. This method was used for the measurement of prostaglandins A and B (PGA/B) in 23 healthy volunteers and 25 hypertensive patients. The PGA/B concentration in peripheral venous plasma of 23 healthy normotensive subjects is 115 +/- 15 pg/ml. The repeated measurement of the same plasma samples kept frozen for 60 days at -20 degrees C shows mean 194% increase of PGA/B concentration. The major site of synthesis of PGA/B seems to be the kidney. However in two patients PGA/B concentration in arterial blood was greater than in venous blood suggesting the possibility of cardio-pulmonary synthesis. The major site of inactivation is the hepatic circulation, as PGA/B concentration in hepatal venous blood is by 30% lower than in vena caval blood. The arterial concentration is 3% lower than venous PGA/B demonstrating very low pulmonary inactivation. Therefore the prostaglandins of the A and B series may represent a "circulating hormone". The plasmatic PGA/B is significantly increased in reno-vascular and essential hypertension.     

Clinical and molecular genetic study in seven Japanese families with spinocerebellar ataxia type 6

This study examined the effect of thyrotrophin-releasing hormone (TRH) administration on thermoregulation in the newborn. Twin lambs were either delivered near-term by caesarean section or born vaginally at term. Colonic temperature, O2 consumption, CO2 production, breathing and heart rates, plus plasma thyroid hormone and nonesterified fatty acid (NEFA) concentrations and thermogenic activity (i.e. GDP binding) of brown adipose tissue (BAT) were measured. In caesarean section delivered lambs colonic temperature decreased rapidly after birth, a response that was greater in the group designated for TRH treatment, in which colonic temperature fell to below 36.0 degrees C at 80 min of life, prior to TRH administration. At this age colonic temperature had been restored to a mean of 38.70 degrees C in controls. TRH had no influence on the composition or thermogenic activity of BAT. The incidence of shivering was not influenced by TRH, but treated lambs maintained a higher rate of O2 consumption and ventilation compared with controls after colonic temperature had been restored to 38.56 degrees C. TRH appeared to promote fat oxidation as O2 consumption remained unchanged and CO2 production declined by a greater rate in treated lambs, resulting in a lower respiratory quotient compared to controls. Heart rate and plasma concentrations of NEFA increased following TRH administration although this did not result in values greater than controls. Normothermic lambs born vaginally had BAT with a greater thermogenic activity, higher plasma thyroid hormone and NEFA concentrations compared with caesarean section delivered lambs, but a thermogenic response was not observed to TRH despite a rise in thyroid hormone concentrations. In conclusion, TRH can improve thermoregulation, an effect that could be linked to an increase in fat oxidation.     

Genotoxicity of ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine: induction of SCE in human lymphocytes and mutagenicity in Salmonella typhimurium TA 100

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and -10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and +/-2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at -80 degrees C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Effect of preinduction of heat-shock proteins on acetic acid-induced small intestinal lesions in rats

Bowel dysfunction such as irritable bowel syndrome caused by stress is well described. Previous reports suggest that stress is known to cause the release of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine, corticotropin-releasing factor, and thyrotropin-releasing hormone (TRH). However, the role played by these neurohormonal mediators in bowel dysfunction under stress conditions is not well known. We investigated the influence of water-immersion stress or TRH administration on the expression of 60-kDa, 72-kDa, and 90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa by Western blot and immunohistochemical analyses. The cytoprotective function of preinduced HSPs on experimentally induced mucosal damage also was studied. In order to investigate the influence of preinduction of HSP60 on small intestinal damage, the small intestinal lumen was perfused with 1.5% acetic acid 1 ml/min for 15 min with or without pretreatment with water-immersion stress or TRH administration. Expression of HSP60 was significantly increased by water-immersion stress or TRH administration in the small intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of this protein showed the biphasic peak pattern after water-immersion stress or TRH administration. Each peak was observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRH administration. Immunohistochemical study also showed a significant increment of HSP60 in both the cytoplasm and nuclei of the small intestinal mucosal cells. No histopathologic alteration was observed in rat small intestinal mucosa after each treatment. Small intestinal damage caused by 1.5% acetic acid perfusion was not influenced by preinduction of HSP60. We demonstrated that water-immersion stress or TRH administration specifically induced HSP60, although preinduction of this protein did not show a cytoprotective function in the small intestinal mucosa.     

Determination of ivabradine and its N-demethylated metabolite in human plasma and urine, and in rat and dog plasma by a validated high-performance liquid chromatographic method with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method with native detection of fluorescence was developed and validated for the quantitation of ivabradine and its N-demethylated metabolite in plasma (rat, dog, human) and human urine. The procedure involves the use of an analogue as internal standard, solid-phase extraction on cyano cartridges, separation on a Nova-Pak C8 column and fluorescence detection. Calibration curves are linear in the concentration ranges from 0.5 to 100 ng/ml in plasma and 2.0 to 500 ng/ml in urine with a limit of quantitation set at 0.5 and 2.0 ng/ml in plasma and urine, respectively. The analysis of plasma and urine samples (spiked with the analytes at low, medium and high concentrations of the calibration range) demonstrates that both analytes can be measured with precision and accuracy within acceptable limits. Quality controls spiked with analyte concentrations up to 10000 ng/ml can also be analysed with excellent precision and accuracy after dilution of the samples. The parent drug and its metabolite are stable in plasma and urine after short-term storage (24 h at room temperature and after three freeze-thaw cycles) as well as after long-term storage at -20 degrees C (at least 6 months in animal plasma and 12 months in human plasma and urine). The method has been used to quantify both compounds in plasma and urine samples from clinical and non-clinical studies with ivabradine.     

Occupational respiratory allergic disease induced by Passiflora alata and Rhamnus purshiana

A group of 24 healthy young men were evaluated before and after serial suberythematous ultraviolet (UV) radiation: group I, control (no irradiation); groups II and III, 12 radiations in 4 weeks with two different spectra (both containing UV-B). Before the first and 2 days after the last exposure all the volunteers were given an intravenous injection of thyrotropin releasing hormone (TRH, protirelin 0.2 mg) and luteinizing hormone releasing hormone (LH-RH, gonadorelin 0.1 mg). The serum concentrations of TSH, follicle stimulating hormone, LH and prolactin were measured at 0, 20, 30, 45 and 60 min by radioimmunoassay. Neither basal nor stimulated levels of the pituitary hormones showed significant changes after UV radiation. The results showed that exposure to suberythematous doses of UV did not influence the regulation of pituitary hormones in these healthy individuals.     

Integrated analysis of diffusion and relaxation of water in blood

A fully validated gas chromatographic-tandem mass spectrometric (GC-MS-MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[2H3]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-microl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC-MS-MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0-1 microg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0-100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.     

Functional transplantation of the rat pituitary gland

OBJECTIVE: These studies evaluated the ability of transplanted pituitary cells to restore pituitary function in hypophysectomized rats. METHODS: The pituitary glands of neonatal Lewis rats were rapidly removed, enzymatically dispersed, and stereotactically introduced into the third ventricle of hypophysectomized adult male Lewis rats. Four weeks after implantation, plasma levels of anterior pituitary hormones in implanted animals were compared with those of sham-transplanted control animals. RESULTS: Plasma levels of prolactin, growth hormone, thyroid-stimulating hormone, and beta-endorphin were below the range of detection in 14 sham-operated animals. In implanted animals, restitution of serum prolactin occurred in 100% of the animals tested, with levels of 2.6 +/- 1.0 ng/ml (mean +/- standard error of the mean; normal, 2-4 ng/ml). Growth hormone was assayable in 71% of the animals, with a mean value of 29 +/- 13 ng/ml over all animals (normal, 1-100 ng/ml); thyroid-stimulating hormone was restored in 68%, with mean resting levels of 79 +/- 13 ng/ml (normal, 100-400 ng/ml); luteinizing hormone levels were found in 53%, with mean levels over all animals of 0.2 +/- 0.1 ng/ml (normal, 0.5-1.0 ng/ml); and beta-endorphin was restored in 45% to high resting levels of 163 +/- 31 pg/ml (normal, 20-30 pg/ml). A challenge with hypothalamic releasing factor and a cold stress test were performed on the animals that had received transplants. Positive hormone responses to both of these tests suggested sensitivity of the pituitary grafts to both endogenous and exogenous sources of stimulation. Histological sections of paraformaldehyde-fixed brains from implanted animals clearly demonstrated survival of clusters of grafted pituitary cells. Positive immunohistochemical staining for adrenocorticotropic hormone and thyroid-stimulating hormone was demonstrated in sections of the grafted tissue. CONCLUSION: These data suggest survival of neonatal pituitary transplants in the third ventricle of adult hypophysectomized rats with concomitant restoration of anterior pituitary hormone function.     

Methamphetamine-stimulated striatal dopamine release declines rapidly over time following microdialysis probe insertion

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.