Microdialysis sampling with on-line microbore HPLC for the determination of tirapazamine and its reduced metabolites in rats.

An on-line microdialysis microbore HPLC method is described for the determination of the bioreductive anti-tumor agent, tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide, SR4233, WIN59075, Tirazone, TPZ) and its two major reduced metabolites, 3-amino-1,2,4-benzotriazine-1-N-oxide (SR4317) and 3-amino-1,2,4-benzotriazine (SR4330). Detection limits of 0.003 microM, 0.005 microM and 0.007 microM were obtained for tirapazamine, SR4317 and SR4330, respectively. Linear ranges of 0.011-20 microM, 0.017-20 microM and 0.025-20 microM for tirapazamine, SR4317 and SR4330 permitted quantitative analysis of all three compounds in microdialysis samples. Typical intra-day reproducibilities (n = 7) of 4.1% (tirapazamine), 6.6% (SR4317), 9.9% (SR4317), and 1.8% (tirapazamine), 2.4% (SR4317) and 2.6% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. Inter-day reproducibilities (n = 5) of 3.4% (tirapazamine), 1.8% (SR4317), 4.5% (SR4330) and 2.5% (tirapazamine), 2.5% (SR4317) and 1.7% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. The use of an on-line microdialysis HPLC system, permitted the determination of tirapazamine, SR4317 and SR4330 in blood and muscle tissue of rats with a high temporal resolution of sampling. The pharmacokinetics of tirapazamine and its metabolites were studied in the muscle and blood of rats previously administered an intraperitoneal dose of tirapazamine.

[An improved method for microdialysis study of noradrenaline level in rat myocardium]. / Usovershenstvovannaia metodika mikrodializnogo issledovaniia soderzhaniia noradrenalina v miokarde krysy.

A level of myocardial norepinephrine (NE) is considered as one of the meaningful parameters in the estimation of myocardium functioning. Long lasting changes of myocardial NE appear to be not only a sequence of pathologic processes in myocardium, but could also be a factor responsible for some diseases. An improved microdialysis sampling technique with HPLC-ED analysis was developed to measure in vivo NE content in a rat myocardial interstitium. Linear polyacrylonitryl 5 mm probes were flushed with methanol while being immersed in water. In vitro NE recovery with the flow rate of perfusate was found at the level 67.9 + 0.4%. Probes were implanted into the rat myocardium under the general anaesthesia. Myocardial dialysate was collected during 20 min into plastic vials, containing 10 microliters of 0.1 M perchloric acid. Low noise, high sensitivity dual electrochemical detection was used for the determination of NE amount in the samples. Overall sensitivity threshold was found about 0.3 pg of NE in 20 microliters. Steady state concentration of NE in myocardial dialysate was found 54 + 7 pg/ml. 20 min before cardiac arrest the concentration of NE in myocardial dialysate was found to be increased 4-5 times. 40 min after cardiac arrest further increase in NE content was registered up to 1-4 ng per ml of perfusate. It is suggested that the method will be useful for routine study of myocardial NE.

Determination of bupivacaine and three of its metabolites in rat urine by capillary electrophoresis.

A capillary electrophoretic (CE) method for the analysis of urinary extracts of the local anesthetic, bupivacaine, and its three main metabolites, desbutylbupivacaine, 3'-hydroxybupivacaine, and 4'-hydroxybupivacaine, in rat urine has been developed. The limits of detection were 0.22 microM for desbutylbupivacaine and bupivacaine, 0.15 microM for 3'-hydroxybupivacaine, and 0.16 microM for 4'-hydroxybupivacaine. The linear range was from 0.7 microM to 16.8 microM for all four compounds. Migration time and peak height reproducibilities, and extraction efficiencies were determined for all four compounds. Peak height reproducibilities (n = 5) for the overall method were improved through the use of prilocaine as an internal standard. Peak height reproducibilities were 5.6% RSD for desbutylbupivacaine and bupivacaine, and 9.9% RSD for 3'-hydroxybupivacaine and 4'-hydroxybupivacaine. Migration time reproducibilities (n = 5) were 2.4% for all compounds. Urine samples were collected from rats administered therapeutic doses of bupivacaine and extracted using a solid-phase extraction method (SPE). Separation of bupivacaine and its metabolites was achieved in 15 min.

[Diazepam metabolism by multiple forms of cytochrome P-450 in rat liver microsomes]. / Metabolizm diazepama mnozhestvennymi formami tsitokhroma P-450 mikrosom pecheni krys.

The synthesis of pharmacologically active diazepam metabolites (oxazepam, 4-hydroxydiazepam, N-demethyldiazepam) in liver microsomes of intact and phenobarbital-, 3-methylcholanthrene- and dexamethasone-induced male and female Wistar rats as well as in a reconstituted system with isolated forms of cytochrome P-450 (P-450a, P-450b, P-450c, P-450d and P-450k according to the Ryan nomenclature) was studied. Marked sex-dependent differences in the rates of diazepam metabolism in liver microsomes of intact and induced animals were revealed. The changes in the spectrum of diazepam metabolites in liver microsomes of induced rats (as compared to control animals) were revealed. In a reconstituted system only phenobarbital-induced cytochromes P-450b and P-450k were found to be active participants of diazepam N-demethylation; none of the isoenzymes tested were shown to be involved in diazepam hydroxylation.

[Determination of diazepam and its metabolites in the blood by microcolumn high-performance liquid chromatography]. / Opredelenie diazepama i ego metabolitov v krovi metodom mikrokolonochnoi vysokoéffektivnoi zhidkostnoi khromatografii.

A rapid, sensitive and specific quantitative method of simultaneous determination of diazepam and its pharmacologically active metabolites in biological fluids by means of high-performance liquid chromatography on a liquid chromatograph "Milichrom" was used for pharmacokinetic studies in the "mother-placenta-fetus" system.