Lipophilic benzamide and anilide derivatives as high-performance liquid chromatography internal standards: application to sirolimus (rapamycin) determination.

Finding a suitable internal standard in reversed-phase high-performance liquid chromatography is often difficult. A general approach for selecting and synthesizing the proper internal standard is presented and applied to a validated method for quantitation of sirolimus in several biological matrices. A series of fifteen N-alkylbenzamides, N-alkyltoluamides and N-alkanoylanilines with a log P range of 3.51 to 6.68 were synthesized as internal standards; N-undecyl-o-toluamide was evaluated most extensively. Sirolimus quantitation involves a simple sample clean-up procedure followed by isocratic chromatography on a heated C18 analytical column with an 70% methanol-water mobile phase and ultraviolet detection at 278 nm. This method was linear from 2.5 to 200 ng with a limit of quantitation of 2.5 ng using a 1-ml blood sample. Sirolimus recovery was above 72.1%. The intra-day and inter-day coefficients of variation were less than 11.7%. Several drugs and sirolimus metabolites do not interfere with the analysis. This method was used to measure sirolimus in blood from rats, rabbits and humans.

Reversed-phase high-performance liquid chromatographic determination of taxol in mouse plasma.

A simple and selective high-performance liquid chromatographic technique was developed for the determination of taxol in mouse plasma. This technique employs a single extraction step followed by isocratic chromatography on a C18 analytical column and ultraviolet detection at 227 nm, and is suitable for the analysis of microsamples (50 microliters plasma). A series of aromatic and aliphatic benzamides were synthesized as internal standards; N-octylbenzamide was evaluated most extensively. The calibration curve is linear within the range 0.15-10 nmol/ml. The intra-day and inter-day coefficients of variation were below 7%. The specificity of the assay was confirmed by comparing the chromatographic properties of samples with authentic taxol, using a diode-array detector. The assay was used to conduct pharmacokinetic studies of taxol in mice after administration in different formulations.

Probenecid disposition by parallel Michaelis-Menten and dose-dependent pseudo-first-order processes.

Re-evaluation of published data on the urinary excretion of probenecid [4-(dipropylamino)sulfonylbenzoic acid, 1] and its metabolites as a function of orally administered dose has revealed that the elimination process is comprised of five parallel pathways. Excretion of the major metabolite, the acyl glucuronide 2 (35-45% of dose), follows Michaelis-Menten kinetics. The three oxidized metabolites, the mono-N-propyl, carboxylic acid, and secondary alcohol derivatives 3, 4, and 5, respectively, (totaling 30% of dose), each adhere to pseudo-first-order kinetics in which the elimination rate constant, as a result of product inhibition, is a function of the administered dose. Four to 13% of the dose is eliminated unchanged in apparent first-order fashion. A mathematical relationship between elimination half-life and dose, under conditions of product inhibition, is derived. Computer simulation of the elimination process confirms the experimental observation that the fraction of dose excreted in the form of each metabolite remains relatively constant over the dose range of 0.5 to 2 g. This study is believed to represent the first application of parallel Michaelis-Menten and dose-dependent pseudo-first-order processes to drug disposition. It demonstrates that the observation of constant proportions of excreted metabolites over a relatively wide range of doses does not provide evidence that the elimination process remains first-order.

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. III. Effects of physical properties of the solvent systems and operating temperature on the distribution of two-phase solvent systems.

Statistical studies were made to correlate the hydrodynamic behavior of two-phase solvent system in counter-current chromatography (CCC) to their physical properties including interfacial tension, viscosity, and the difference in density of the two phases. Settling time measured under unit gravity provided a reliable numerical index for the hydrodynamic behavior of the solvent systems in a centrifugal force field. Viscosity and settling time were strongly correlated (correlation coefficient, r = +0.88) while interfacial tension (r = -0.65) and phase density difference (r = -0.45) showed moderate and weak correlation, respectively. Studies of the effect of temperature on settling time as well as a preliminary apparatus operated at higher temperature show that raising the temperature will improve the performance of high-speed CCC.

Renal tubular transport and nephrotoxicity of DDA.

Since DDA [bis(p-chlorophenyl)acetic acid] has been shown to be transported and concentrated by the renal proximal tubule, this metabolite of DDT has been postulated to be a potential nephrotoxic agent. The present study explored the renal transport of DDA in the isolated, perfused rat kidney and the effects of DDA on renal function. When DDA (0.6 microM) was present in a dextran perfusate which eliminated DDA-colloid binding, the DDA/inulin clearance ratio was congruent to 0.05; however, some metabolism of DDA was apparent. During these studies, DDA had no effect on the glomerular filtration rate and the fractional reabsorption of Na, K or H2O. To determine the concentration of DDA which would produce an effect on renal cellular function, studies were performed with renal cortical slices. DDA at media concentrations greater than or equal to 0.1 mM were needed to produce significant alterations in tetraethylammonium transport, tissue oxygen consumption and intracellular electrolyte composition; however, no effect was demonstrated on Na-K-ATPase activity although DDA did affect Mg-ATPase activity. In conclusion, DDA at a 0.6 microM perfusate concentration undergoes net tubular reabsorption and metabolism without affecting the function of the perfused kidney. Only high concentrations of DDA were shown to produce alterations in cellular function.

Renal transport and renal metabolism of 2, 4, 5-trichlorophenoxyacetate by the chicken.

In vitro and in vivo renal tubular transport of 2,4,5-trichlorophenoxyacetate (2,4,5-T), a hormone-type herbicide, was studied in the chicken. Renal cortical slices incubated with 14C-labeled 2,4,5-T demonstrated a slice-to-medium ratio of the 14C-label of 26 after 2 hr of incubation. This accumulation was inhibited significantly by probenecid, phenol red, dinitrophenol and iodoacetamide. In vitro metabolism of 2,4,5-T was apparent in the renal slice studies; at least two metabolites were found by electrophoretic analysis. Even though chicken liver slices were able to accumulate 2,4,5-T, the accumulated herbicide was not metabolized. In addition, chicken kidney slices metabolized 2,4,5-T to a much greater extent than did rat or rabbit kidney slices, which produced only negligible amounts of metabolites. When [14C]-2,4,5-T was infused into the renal portal system of the chicken, the 14C-label originally associated with 2,4,5-T was shown to be secreted by the renal tubule. Probenecid inhibited the transport of the 14C-label without affecting the simultaneous transport of tetra-ethylammonium, an organic cation transported by the renal proximal tubule. At the present infusion rate of 2,4,5-T (2.9 X 10(-10) mol/min), the excreted label was associated primarily with an acidic metabolite(s). It can be concluded that the chicken transports 2,4,5-T by a probenecid sensitive mechanism; however, this transport appears to be associated with the metabolism of the herbicide.

Effect of coadministered salicylamide on terbutaline metabolism in rats.

Concomitant oral administration of salicylamide (200 mg/kg) and 3H-terbutaline (1 mg/kg) to rats with ligated bile ducts decreased absorption of terbutaline from the gut from 73 to 56% as measured by urinary excretion of radioactivity in 48 hr. No increase in the fraction of terbutaline excreted unchanged was observed, suggesting that salicylamide does not substantially inhibit the conjugation of terbutaline with glucuronic acid. An increase in the fraction of terbutaline excreted unchanged observed in normal animals may result from enhanced excretion of terbutaline glucuronide into bile rather than from inhibition of conjugation.

Urinary excretion of probenecid and its metabolites in humans as a function of dose.

A GLC assay was used to study the excretion of probenecid and its metabolites in the urine of human subjects following oral doses of 0.5, 1, and 2 g. From 75 to 88% of the dose was found in the urine. The major metabolite, probenecid acyl glucuronide, accounted for 34-47% of the dose. Approximately equal amounts (10-15%) of the mono-N-propyl, secondary alcohol, and carboxylic acid metabolites were excreted in the unconjugated from with only traces in the conjugated form. The primary alcohol metabolite was not found in measurable amounts. The terminal half-lives for excretion of all metabolites were in the range of 4-6 hr, were independent of dose, and were limited by their rates of formation. A prolonged time course of excretion of the metabolites, particularly at higher doses, suggests that probenecid, being poorly soluble in water, precipitates from solution in the GI tract, forming a depot of drug from which absorption is dissolution rate limited. The urinary excretion of unchanged probenecid, which accounts for 4-13% of the dose, is dependent on both the pH and flow rate of urine.

Determination of dicumarol metabolites in bile of rats.

After intravenous administration of dicumarol-14C to rats, the bile excreted over the next 24 hr contained from 32 to 46% of the administered radioactivity. At least three primary metabolites and a small amount of unchanged dicumarol were present in the bile. Over 91% of the primary metabolites was converted to dicumarol and 7-hydroxydicumarol by hydrolysis with beta-glucuronidase. Some primary metabolites were hydrolyzed simply by acidification to pH 3 or by treatment under the acidic conditions utilized in the enzymatic hydrolysis. The three primary metabolites contain carboxylic acid groups, as indicated by their electrophoretic mobility-pH profiles, and some are simple glucuronides of dicumarol and 7-hydroxydicumarol. The possibility that others are derivatives of these compounds in which a coumarin lactone ring is opened cannot be ruled out. When the metabolites released by either acidification or enzymatic hydrolysis were chromatographed in n-butanol-3 M ammonia, artifacts were produced, presumably as a result of decomposition of 7-hydroxydicumarol. The question is raised whether a previously reported metabolite (B055) is an artifact.