Micellar electrokinetic chromatographic method for the dabrafenib determination in biological samples.

Two different micellar electrokinetic chromatographic methods to determine dabrafenib in urine and serum, both using borate buffer (pH 9.2, 20 mM) and SDS as separation electrolyte, are developed and validated. The analyses were carried out in a fused-silica capillary of 75 µm of internal diameter and total length of 47 and 37 cm for urine and serum determination, respectively. The detection of the target compound was performed at 227 nm in urine samples and at 251 nm in serum samples. The linearity range was from 1 to 21 mg/L of dabrafenib in urine and from 2 to 40 mg/L in serum. In all cases, inter- and intraday RSDs were <4%. Sample preparation of serum samples consists of an only step of 1:1 dilution with water before its injection in the electrophoretic system. These simple, sensitive, accurate, and cost-effective methods can be used in routine clinical practice to monitor dabrafenib concentrations in urine and serum of metastatic melanoma skin cancer patients.

[Experimental study of pinostrobine oxime biotransformation].

We have experimentally studied pathways of elimination of an oximized derivative of phytoflavonoid pinostrobine by HPLC/mass spectrometry. Four potential metabolites of pinostrobine oxime have been found and there was an attempt to determine their molecular structures on the basis of their fragmentation under positive electrospray ionization conditions. It is established that pinostrobine oxime is removed from the organism mainly unchanged and also in the form of glucuronated derivative.

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry.

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.

Disposition and metabolism of [14C]brasofensine in rats, monkeys, and humans.

Brasofensine is an inhibitor of the synaptic dopamine transporter. These studies were conducted to characterize the pharmacokinetics, absolute bioavailability, disposition, and metabolism of brasofensine after i.v. and/or p.o. administrations of [(14)C]brasofensine in rats (1.5 mg/kg i.v., 4 mg/kg p.o.) and monkeys (4 mg i.v., 12 mg p.o.) and humans (50 mg p.o.). Brasofensine was rapidly absorbed after p.o. administration in rats and monkeys, with peak plasma concentrations occurring 0.5 to 1 h but 3 to 8 h for brasofensine in humans. Plasma terminal elimination half-lives were approximately 2 h in rats, approximately 4 h in monkeys, and approximately 24 h in humans. Total body clearance and steady-state volume of distribution values were 199 ml/min/kg and 24 l/kg, respectively, in the rat and 32 ml/min/kg and 46 l/kg, respectively, in the monkey. Absolute bioavailability was 7% in rats and 0.8% in monkeys. After a single p.o. dose, urinary excretion of radioactivity accounted for 20% of the administered dose in rats, 70% in monkeys, and 86% in humans, with the remainder excreted into the feces. Brasofensine had extensive first-pass metabolism following p.o. administration in humans, monkeys, and rats. It primarily underwent O- and N-demethylation and isomerization. Some of the desmethyl metabolites were further converted to glucuronides. These primary metabolites and glucuronides of demethyl brasofensine (M1 and M2) were major circulating metabolites in humans and were also observed in rat and monkey plasma.

Studies on the metabolic fate of M17055, a novel diuretic (6). Assessment for drug-drug interactions of M17055 in metabolism, distribution and excretion.

1. The potential of M17055, a novel diuretic candidate, to affect the activities of human CYP enzymes, alter the plasma unbound fraction and compete with concomitant drugs in renal secretion as part of an assessment for drug-drug interactions in metabolism, distribution and excretion was investigated. 2. The effects of M17055 on the activities of human CYP1A2, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were considered negligible at clinically relevant concentrations. 3. The majority of M17055 (99%) was bound to human plasma proteins, but it is unlikely to alter the binding of other clinically relevant drugs. 4. The renal clearance of M17055 (corrected for the plasma unbound fraction in male rats) substantially exceeded the glomerular filtration rate and was markedly reduced by treatment with probenecid, suggesting that the renal excretion of M17055 is controlled predominantly by an active secretion mechanism. 5. The results show that M17055 is unlikely to cause or undergo significant pharmacokinetic interactions with concomitant drugs in metabolism and distribution. However, when it is administered simultaneously with certain organic anions, drug-drug interactions during kidney excretion may be possible.

Pharmacokinetics and pharmacodynamics of sibrafiban, an orally administered IIb/IIIa antagonist, in patients with acute coronary syndrome.

Sibrafiban is a double prodrug that is converted to the inactive single prodrug and to the active IIb/IIIa antagonist following oral administration. Pharmacokinetics (PK) and pharmacodynamics (PD) of oral sibrafiban and its metabolites were evaluated in patients postacute coronary syndrome receiving once- or twice-daily sibrafiban for up to 28 days at several dose levels. Mean peak concentrations of sibrafiban were < 5 ng/mL. Peak single prodrug concentrations occurred 1.7 +/- 1.0 (mean +/- SD) hours after sibrafiban dosing. Total apparent plasma clearance of the single prodrug was 40 +/- 15 L/h, and the elimination half-life was 2.3 +/- 0.8 hours. Mean values of the steady-state pharmacokinetics for total concentrations of the active drug over all doses were: time to peak plasma concentration, 5.0 +/- 1.7 hours; apparent clearance, 13.9 +/- 3.9 L/h; and half-life, 11.0 +/- 2.8 hours. Once-daily dosing resulted in high peak-trough excursions in active drug concentrations: trough concentrations were 21% +/- 6% of peak. Twice-daily dosing resulted in an AUC for the active drug on Day 28 that was 168% +/- 36% of that on Day 1, and steady-state trough concentrations were 54% +/- 10% of peak with sustained inhibition of platelet aggregation. Dose-adjusted steady-state active drug concentrations increased with increasing age and with decreasing renal function and body weight.

Disposition of methyl ethyl ketoxime in the rat after oral, intravenous and dermal administration.

1. The disposition of 14C-methyl ethyl ketoxime (MEKO) was determined in the male F344 rat following oral, intravenous (i.v.) and dermal administration. 2. Oral doses of 2.7, 27 and 270 mg/kg were primarily excreted as CO2 (71-49%) in decreasing percentage as the dose increased. Excretion in urine (13-26%) and as volatiles (5-18%) increased as the dose increased. Five to 6% of the dose remained in the major tissues after 72 h. 3. An i.v. dose of 2.7 mg/kg was also principally excreted as CO2 (48.8%) with excretion in urine and as expired volatiles accounting for 21.4 and 11.4%, respectively. About 7% of the administered radioactivity remained in the tissues after 72 h. 4. Following dermal administration, 13 and 26% of a 2.7 and 270 mg/kg dose, respectively, were absorbed. Volatilization from the dose site prior to placement in the metabolism cage may account for the low absorption. 5. MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.

Disposition of butanal oxime in rat following oral, intravenous and dermal administration.

1. The disposition of [1-14C]butanal oxime (BOX) was determined in the rat after oral, i.v. and dermal administration. 2. Oral doses of [14C]BOX (2 and 20 mg/kg) were predominantly excreted in the urine (> 42%) and converted to 14CO2 (> 30%) and about 10% of the dose remained in the tissues 72 h post-dosing. 3. Eight and 16% of a 2 and 20 mg/kg dermal dose of BOX, respectively, were absorbed, due in part to rapid volatilization from the surface of the skin. 4. Oral doses of BOX were transformed into several polar and/or anionic metabolites that include sulphate conjugates and a significant amount of thiocyanate. 5. The effect of inhibitors on the metabolism of BOX was investigated using 1-aminobenzotriazole (ABT; an inhibitor of diverse cytochrome P450s) and trans-1,2-dichloroethylene (DCE; an inhibitor of CYP2E1). No thiocyanate anion was detected in the urine of rat treated with DCE or ABT. ABT markedly increased the production of 14CO2 and excretion as volatile metabolites. DCE had no effect on 14CO2 excretion, but increased exhalation of radiolabel. ABT also effectively blocked the expression of toxic effects attributable to cyanide in rat given near-lethal doses of BOX. 6. The data are consistent with two distinct pathways of metabolism for BOX, (1) reduction to an imine, hydrolysis and subsequent conversion of butyraldehyde to 14CO2 and (2) CYP3A-catalysed dehydration of BOX to butyronitrile followed by CYP2E1-catalysed release of cyanide.

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.

99mTc-HMPAO as a tracer of cerebral blood flow in newborn infants.

Studies on the kinetics of 99mTc-D,L-hexamethylpropylene amine oxime (99mTc-HMPAO) in adults have shown that it is not an ideal tracer of CBF because it underestimates high-flow areas. Knowledge of the kinetics of the tracer is important in evaluating the studies. The kinetics of 99mTc-HMPAO in infants may be different from that in adults, therefore, we examined the cerebral uptake and retention of 99mTc-HMPAO in neonates and estimated the degree of brain-to-blood back diffusion by comparing corresponding 133Xe flow images and 99mTc-HMPAO distribution images. In addition, we measured the urinary excretion of 99mTc-HMPAO. Regional CBF was measured using a mobile brain-dedicated, fast-rotating, four-head multidetector system specially designed for neonatal studies. Tracers were 99mTc-HMPAO (4 MBq/kg) and 133Xe (500 MBq/kg). Cerebral uptake and leak-out of 99mTc-HMPAO were measured by a single scintillation crystal placed over the frontoparietal part of the infant's head. The cerebral retention of 99mTc-HMPAO was analyzed in 50 infants. The mean gestational age and birth weight (95% confidence interval) were 34.4 weeks (32.2-35.7) and 2,326 g (1,954-2,995), respectively. The cerebral uptake of 99mTc-HMPAO was examined in 16 of the 50 infants, and activity during 24 h was monitored in five. In 11 infants, corresponding 133Xe studies were performed. Urinary excretion was studied in 12 infants. The maximal activity in the brain was reached 90s after i.v. injection and was 104% (98-111) of the stable level, which was reached approximately 3 min after the injection. The decay corrected leakout of the tracer during the following 24 h was 1.0% (0.4-1.5) per hour. The cerebral retention was calculated at 6.8% (6.1-7.6), highest in the group of ictal studies and lowest in premature infants with intracranial hemorrhage. The mean value of the fixation/clearance ratio alpha was estimated at 3.4 (2.8-4.4). The mean urinary excretion over 24 h was 19.5% (11.4-27.7) and was significantly related to renal function as indicated by serum urea (p = 0.02 r2 = 0.55). A four-compartment model describing the kinetics of 99mTc-HMPAO is shown to be valid in neonates. The cerebral retention of the tracer is higher in neonates because of higher extraction and lower initial back diffusion from brain to blood. In linearizing 99mTc-HMPAO distribution images, a smaller correction is necessary, and we propose a value of the correction factor of 3.4. In this way, 99mTc-HMPAO is a more reliable tracer of the distribution of CBF in neonates compared with adults. The urinary excretion is significantly reduced compared with adults, and the radiation dose to the bladder wall is reduced. The effective dose is 0.3 mSv/MBq/kg.

Use of fresh and cryopreserved hepatocytes to study the metabolism of pesticides in food-producing animals and rats.

1. The possibility of using hepatocytes from food-producing animals in order to determine the metabolic routes of pesticides has been studied using a strobilurin fungicide (BAS 490 F). Hepatocytes suspensions were prepared from goat, pig, hen, and rat and the major metabolites were compared with those obtained in vivo. 2. The hepatocytes gave metabolite patterns matching qualitatively with in vivo results, but no good quantitative correlation was found. 3. A freezing and thawing method was developed using liquid nitrogen and a programmable freezer, which allows acceptable recoveries of functional cells as assessed by glutathione and cytochrome P450 contents, and phase I and II enzymatic activities (including 7-ethoxycoumarin-O-deethylase, ethoxyresorufin-O-deethylase, glutathione-S-transferase, and UDP-glucuronosyl transferase), with 60-70% viability. 4. The cells were damaged through freezing as indicated by the efflux of glutathione (40-60% of the intracellular content), but remained able to metabolize BAS 490 F, partially like fresh cells. A good qualitative but no quantitative matching of the metabolite patterns before and after cryopreservation was found, indicating that the metabolic activities are affected to variable extents during the freezing process. 5. The use of fresh and cryopreserved cells as models for metabolism and species comparison, and as a versatile tool to synthesize metabolites, is discussed.

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry.

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.

Naphthalene- and anthracene-2,3-dialdehyde as precolumn labelling reagents for primary amines using reversed- and normal-phase liquid chromatography with peroxyoxalate chemiluminescence detection.

Naphthalene-2,3-dialdehyde (NDA) and anthracene-2,3-dialdehyde (ADA) were applied as pre-column labelling reagents for the peroxyoxalate chemiluminescence detection of primary amines. The advantages of these labels are the selective derivatization reaction with primary amines and the good chemiluminescence properties. A serious disadvantage is the formation of cyanide-induced side-products which are major interferences in reversed-phase chromatography. For normal-phase chromatography, the excess of reagent was removed by adding a polar amine after derivatization, with subsequent extraction of the labelled analyte with an apolar solvent. The detection limit for NDA-labelled fluvoxamine, an anti-depressant, was in the low femtomole range in standard solutions and in urine samples. For ADA-labelled analytes difficulties were obtained with linearity in peroxyoxalate chemiluminescence detection, probably owing to oxidation of the derivative by hydrogen peroxide.

High-performance liquid chromatographic analysis of imidazolium and pyridinium oximes in plasma and urine.

Imidazolium oximes are useful in the treatment of organophosphorus agent poisoning. However, the extant methods for analyzing oximes in plasma and urine samples are not adequate. A unique high-performance liquid chromatographic assay method was developed for quantitating either imidazolium or pyridinium oximes. Plasma or urine samples can be injected directly onto the column after a centrifugation filtration step. This method demonstrates a different approach in the method development for quaternary ammonium compounds using non-end-capped reversed-phase columns and a mobile phase containing competing cations. In addition, a preliminary pharmacokinetic study of the imidazolium oxime in rabbits was carried out using this method.

Radiochemical purity of D,L-hexamethylpropylene amine oxime and analysis of urine-excreted HMPAO decomposition species.

The radiochemical purity of hexamethylproxypropylene amine oxime (HMPAO) was determined in 16 preparations using the three-strip method. Immediately post-formulation, 90.3% +/- 4.0% (mean +/- SD) of the activity was associated with the primary lipophilic complex having an Rf of 0.45 +/- 0.11. We recorded a significantly higher content of sodium pertechnetate Tc 99m in methylethyl ketone (MEK) (21.1% +/- 8.5%) than in saline (5.0% +/- 3.7%; P less than 0.001). To clarify this finding, we ran sequential chromatograms (t = 0, 1, 2, 3 h) and found that the primary complex steadily disappeared, with a rate constant of 0.31 h-1. These results suggest that there is a decomposition of the primary complex during chromatography in MEK that might be responsible for the larger fraction of sodium pertechnetate Tc 99m in MEK. Eluate composition might influence the Rf of the lipophilic complex and the rate of its in vitro decomposition. In another experimental setting, we investigated 99mTc-HMPAO decomposition species in urine after application of a suspension of labelled leukocytes by performing sequential chromatographic analyses in 11 patients. At 1 h after application, urinary activity reflected the presence of a secondary complex (84.8% +/- 9.2%) and sodium pertechnetate Tc 99m (15.2% +/- 9.2%). The values after 3 h were markedly different (91.4% +/- 4.8% and 8.6% +/- 4.8%; P less than 0.001). Thus, urinary activity mostly consisted of a secondary complex, increasing with time.

Conical precolumn as loading buffer for the main column.

A conical high-performance liquid chromatographic precolumn was developed to cope with the problems that arise during the processing of large volumes of biological samples. The shape of the column was designed so as to offer a large loading capacity at the front of the precolumn. The stainless-steel construction, which is pressure resistant up to 40 MPa, can be fully integrated into high-performance systems. In the present work, the precolumn arrangement was used in the assay of pamoic acid in human plasma and in the isolation of radioactive metabolites from pools of animal urine and of supernatants from liver homogenates. Apart from extremely polar compounds, which were not retained on the precolumn, recovery of metabolites was practically complete. Almost the same resolution was obtained with the equivalent of 900 ml of urine, purified and enriched on the precolumn, as with a 2-ml sample of the original urine. Likewise, the chromatographic metabolite pattern of 650 ml of supernatant from homogenized liver was similar to that of a deproteinized sample of 2 ml. It is suggested that the precolumn is usable for all chromatographic problems involving enrichment of small amounts of substances in large amounts of complex matrices.

Interindividual and interspecies variation in the metabolism of the hallucinogen 4-methoxyamphetamine.

1. The qualitative and quantitative aspects of the urinary elimination of orally administered 4-methoxy[14C]amphetamine have been examined in the rat and guinea-pig and in three volunteer human subjects, to determine interspecies and interindividual variations in disposition of the drug. 2. Both rat and guinea-pig excreted 70--80% of the administered dose(6 mg/kg) in the urine within 24 h, mainly as metabolites. 3. In the guinea-pig, the drug was metabolized by O-demethylation to give 4-hydroxyamphetamine, which was excreted free (4% dose) and conjugated (73%). No other metabolite was detected. 4. The rat metabolizes the drug both by O-dealkylation and by side-chain oxidation, the products being 4-hydroxyamphetamine (5% of dose free and 60% conjugated) and 1-(4'-methoxyphenyl)propan-2-one oxime (5% dose, free and conjugated). 5. In man the drug (dose 5 mg) is metabolized by O-demethylation and by side-chain oxidation. Marked intersubject variations were observed both in the array and quantitative aspects of metabolite excretion. Two subjects excreted mainly 4-hydroxyamphetamine (free and conjugated) together with smaller amounts of 1-(4'-methoxyphenyl)propan-2-one oxime and 4-hydroxynorephedrine. The third subject, however, who was previously known to exhibit a genetically determined defect in drug oxidation, was defective in O-dealkylation of 4-methoxyamphetamine, and the main excretion products were the unchanged drug together with products of side-chain oxidation, namely, 1-(4'-methoxyphenyl)propan-2-one oxime, 1-(4'-methoxyphenyl)propan-2-one and 4-methoxybenzoic acid. 6. Inter-individual differences in oxidative O-demethylation of the drug are discussed in relation to current theories on the aetiology of schizophrenia and reported fatalities arising from abuse of the drug.