Spectrofluorometric determination of alkoxy-substituted benzylimidazolidinones in biological fluids.

Sensitive and specific spectrofluorometric assays were developed for the determination of d, 1-4-(3,4-dimethoxybenzyl)-2-imidazolidinone and d,1-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone in blood and urine. Each compound is selectively extracted from buffered blood or urine and oxidized with alkaline permanganate to its respective fluorescent benzoic acid derivative, which is quantitated in acetic acid in ethanol (1:99). The method was applied to the determination of blood levels and urinary excretion in the dog following the administration of single intravenous and oral doses of each compound.

High-pressure liquid chromatographic determination of amoxicillin in urine.

A rapid and specific high-pressure liquid chromatographic (HPLC) assay was developed for the simultaneous determination of amoxicillin and its pencilloic acid metabolite in urine. The two compounds, assayed directly in urine or after dilution with water-methanol (85:15), are separated by reversed-phase chromatography and quantitated spectrofluorometrically following postcolumn derivatization with fluorescamine. Linear calibration curves were measured in the ranges of 25-250 and 50-400 ng injected for amoxicillin and the penicilloic acid metabolite, respectively. The sensitivity limit of the assay is 2.5-5.0 microgram/ml of urine for amoxicillin and the penicilloic acid metabolite. Urine samples (0-8 hr) taken from six subjects following single 250-mg po doses and assayed by HPLC showed ranges of cumulative percent of the dose excreted as amoxicillin and the penicilloic acid metabolite (reported as amoxicillin equivalents) of 50.2-68.0, and 21.6-30.0%, respectively. An excellent correlation (r = 0.985) was demonstrated for the measurement of amoxicillin concentrations by the HPLC and microbiological assays.

Determination of nitroimidazoles in biological fluids by differential pulse polarography.

A sensitive differential pulse polarographic assay was developed for the determination of metronidazole, ornidazole, and 2-nitro-1H-imidazole-1-(3-methoxy-2-propanol) in plasma. The compounds are selectively extracted into ethyl acetate from a protein-free filtrate of plasma, buffered to pH 7.0 +/- 0.2. The residue of the ethyl acetate extract is dissolved in 0.1 N NaOH and analyzed by differential pulse polarography for the reduction of the nitro group at approximately -0.600 v versus the saturated calomel electrode. The overall recovery from plasma was about 55 +/- 3.0% (SD) for the three compounds investigated. A TLC step after the ethyl acetate extraction may also be included to ensure specificity. This step reduced the overall recovery to approximately 45%. The sensitivity limit of detection from plasma using a 2-ml sample is 0.1 mug/ml. The assay may also be employed for the analysis of urine. The urine is adjusted to pH 7.0 +/- 0.2 and extracted with ethyl acetate, and the residue is analyzed as described for plasma. The assay was applied to the determination of ornidazole in blood and urine in the dog following 10 mg/kg po.

Spectrofluorometric determination of the antibiotic lasalocid in blood.

A spectrofluorometric assay was developed for the determination of the antibiotic lasalocid in dog blood, based on the intrinsic fluorescence of the compound in ethyl acetate. The assay can measure "total" levels of drug and any metabolites present. The specificity of the assay was verified by TLC separation of the dog blood extract, which indicated the presence of only intact drug. The overall recovery (+/- SD) of lasalocid was 62.0 +/- 3.6% in the concentration range of 1.0-10 mug of compound/ml of dog blood. The sensitivity of the assay is 0.5 mug/ml. The assay was applied to the determination of blood levels of lasalocid in the dog following the intravenous administration of a 5-mg/kg dose.

Determination of the anti-tumor agent, 10-chloro-5-(2-dimethylaminoethyl)-7h-indolo[2,3-c] quinoline-6(5h)-one. HCl in blood or plasma by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the determination of 10-chloro-5-(2-dimethylaminoethyl)-7H-indolo [2,3-C] quinolin-6(5H) one [I] in blood or plasma with an overall recovery of 100.3 +/- 9.1% and a limit of quantitation of 1.0 ng per ml of blood or plasma. the assay was used to determine blood concentrations of the drug in the rat following oral administration by intubation of a 1.17-mg dose of [I] . HCl.

Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy.

A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses.