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A rapid and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound, trans-3-[6-(methylthio)-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid, [I], in plasma. The assay involves acetonitrile protein precipitation followed by the analysis of an aliquot of the protein-free fraction by reversed-phase HPLC with fluorescence detection (excitation at 245 nm, with emission greater than 418 nm). The overall recovery of [I] from plasma was 103 +/- 10%. The sensitivity limit of the assay was 0.125 microgram/ml of plasma. The analogous compound, trans-3-[6-[(1-methylethyl)thio]-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid, [II], is used as the internal standard. The assay was used to monitor the plasma concentration--time fall-off profile of [I] in the dog and in man. The stability of [I] was demonstrated in dog plasma on long-term storage for up to 180 days at -17 degrees C and -70 degrees C.

A rapid and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiarrhythmic quinidine analog, 7'-trifluoromethyldihydrocinchonidine.2HCl ([I].2HCl) in plasma and urine. The overall recovery of [I] from plasma was 86 +/- 9% with a sensitivity limit of detection of 0.2 microgram/ml. The assay involves extraction of [I] into benzene--methylene chloride (9:1) from plasma or urine made alkaline with 0.1 N sodium hydroxide (pH 13) and saturated sodium chloride, the residue of which is dissolved in methylene chloride, an aliquot of which is analyzed by HPLC using adsorption chromatography on silica gel with UV detection at 254 nm. The mobile phase composed of methylene chloride--methanol--conc. ammonium hydroxide (95.5:4:0.5) yields baseline resolution of quinidine used as the internal (reference) standard, compound [I] and dihydroquinidine, a common contaminant in quinidine. The assay was applied to the analysis of plasma and urine samples taken from a dog administered a single 20 mg/kg dose via intravenous and oral routes. The stability of [I] in human plasma for up to 37 days of storage at -17 degrees C was also demonstrated.

The compound 3-phenoxy-N-methylmorphinan (I) is under development as an analgesic agent. Studies on the biotransformation of the drug in the rat and in the dog have shown that I is extensively metabolized by N-demethylation to yield the nor-analogue (I-A), by para-hydroxylation of the 3-phenoxy ring to yield the phenolic analogue (I-B), cleavage of the ether linkage to yield levorphanol (I-C), and its N-demethylation to yield nor-levorphanol (I-D). The presence of these four metabolites (two of which, I-B and I-C, have analgesic potential) in addition to the parent drug, necessitated the development of sensitive and specific assays for their quantitation in plasma. This was accomplished by the development of (a) a high-performance liquid chromatographic assay using UV detection to obtain a qualitative/semi-quantitative profile of the metabolites present in plasma; (b) a gas chromatographic--nitrogen--phosphorus-specific detection method for the determination of the parent drug (I) for pre-clinical drug evaluation; and (c) a sensitive and specific gas chromatographic--positive chemical ionization mass spectrometric assay for eventual clinical evaluation for the determination of I and a key metabolite levorphanol (I-C). This report presents some preliminary pharmacokinetic data on I and I-C in the dog obtained during pre-clinical development.

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]- benzodiazepine (I), and its four metabolites in plasma. The assay involves extraction into diethyl ether--methylene chloride (7:3) from plasma buffered to pH 9 and subsequent analysis by reversed-phase HPLC with ultraviolet detection at 254 nm. The overall recovery of I from dog plasma was 94.5 +/- 7.1% and greater than 89.0% for its metabolites. The sensitivity limit of the assay was 50 ng/ml of plasma for all compounds. The HPLC assay was used to determine plasma concentrations of I and its metabolites from selected samples taken from an oral toxicity study in the dog.

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene (I) and its phenolic metabolite, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- methylethenyl]phenol (II) in plasma. The assay for both compounds involves precipitation of the plasma proteins with acetonitrile, followed by extraction of the entire mixture into methyl tert.-butyl ether and subsequent analysis by reversed-phase HPLC. The overall recovery of I and II was 96.9 +/- 5.3 and 96.2 +/- 5.8% for dog plasma, 80.0 +/- 4.0 and 93.5 +/- 5.0% for human plasma and 97.4 +/- 2.9 and 97.6 +/- 9.6% for rat plasma, respectively. The sensitivity limit is 20 ng/ml of plasma for both compounds I and II using UV detection at 280 nm. The HPLC assay was used in studies in the dog and in the rat.

A method is described for measuring a trimethyl prostaglandin E2 analog, TM-PGE2, in human plasma. Trideuterated and monofluorinated analogs of TM-PGE2 and added to plasma as internal standard and carrier, respectively. The plasma is adjusted to pH 3.0 and is extracted with a mixture of benzene-dichloromethane (9:1). The residue, following removal of the extracting solvent, is reacted consecutively with pentafluorobenzyl bromide and bistrimethylsilyltrifluoroacetamide. The excess derivatizing reagents are removed by evaporation, and an aliquot of the reconstituted residue is analyzed by capillary column gas chromatography using methane as the carrier gas. A quadrupole mass spectrometer is set to monitor in the gas chromatographic effluent the (M - C7H2F5)-fragment ion of TM-PGE2 (m/e 449) and trideuterated TM-PGE2 (m/e 452) generated by methane negative chemical ionization. Quantitation of unknowns is based on a comparison of the m/e 449 to m/e 452 ion ratio in each unknown to that obtained from the analysis of control plasma spiked with known amounts of TM-PGE2 and fixed amounts of internal standard and carrier. The sensitivity limit of the assay is approximately 100 pg ml-1, which is equivalent to 1 pg injected. The assay was used to measure the concentration of TM-PGE2 in the plasma of two subjects following a single 10 micrograms kg-1 oral dose of the drug.