Development and evaluation of a multiple-plate fraction collector for sample processing: application to radioprofiling in drug metabolism studies.

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-µL fractions (within 4.1% CV) and 250-µL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.

Equine metabolism of buspirone studied by high-performance liquid chromatography/mass spectrometry.

The metabolism and urinary excretion of a 100 mg dose of the non-sedating anxiolytic drug buspirone was examined using high-performance liquid chromatography/electrospray ionization mass spectrometry in the positive ion mode. In addition to a significant proportion of unchanged buspirone we were able to detect three major metabolite classes. These were identified as monohydroxy, dihydroxy and dihydroxymethoxy products. Detection of the metabolites and the parent drug was possible in all the urine samples collected (1-12 h) post-administration.

Metabolism of the antianxiety drug buspirone in the rat.

The metabolism of an orally administered, 10-mg single dose of the antianxiety drug buspirone was studied in the rat. Samples of bile and urine were collected for 6 hr and were treated with beta-glucuronidase/arylsulfatase. The deconjugated metabolites were isolated and purified by HPLC. Structural analysis was carried out by combined gas chromatography/electron impact mass spectrometry as their trimethylsilyl derivatives and by 1H-NMR spectroscopy. Structures of the metabolites were further confirmed by co-elution on HPLC with authentic standards when possible. In addition to the already known metabolites 5-hydroxy-buspirone and 1-pyrimidinylpiperazine, seven major metabolites were unambiguously identified together with unchanged drug. Ten minor metabolites were partially characterized. Hydroxylation alpha to the glutaramidyl carbon at the 6'-position on the bicyclo ring system, hydroxylation on the pyrimidine aromatic ring, and N-dealkylation of the butyl side chain were observed as major routes of metabolism. Minor routes of metabolism observed were: 3'-hydroxylation on the bicyclo ring system and formation of the methylated catechol derivatives. The identified metabolites accounted for greater than 90% of the total metabolites excreted in the rat bile and urine samples.

Metabolism of the antianxiety drug buspirone in human subjects.

The metabolism of an oral dose (20 mg) of the antianxiety drug buspirone labeled with 14C/15N was studied in human subjects. 15N was incorporated in the molecule to facilitate structural characterization of the metabolites by mass spectrometry. Urine samples were collected at intervals up to 24 hr and analyzed for radioactivity. Cumulative urinary excretion accounted for 50% of the dose in 24 hr. The urine was hydrolyzed with beta-glucuronidase/arylsulfatase and the deconjugated metabolites were isolated and purified by HPLC. The purified metabolites were identified by GC/MS, 1H-NMR, and comparison with authentic standards when available. Seven metabolites of buspirone were identified unambiguously, together with unchanged drug. Hydroxylation alpha to the glutarimidyl carbonyl at the 6'-position on the spiro ring system, hydroxylation at the 5-position on the pyrimidine ring, and N-dealkylation of the butyl-substituted side chain were major routes of metabolism. The identified metabolites accounted for 88% of the total radioactivity in the urine. A scheme for metabolism of buspirone in human subjects has been proposed.

Simultaneous quantitation of buspirone and 1-(2-pyrimidinyl)piperazine in human plasma and urine by capillary gas chromatography-mass spectrometry.

Buspirone and a buspirone metabolite, 1-(2-pyrimidinyl)piperazine (1-PP), are extracted from matrix using C18 extraction columns. The metabolite and its internal standard (d4-1-PP) are derivatized with pentafluorobenzoyl chloride to the corresponding amides. The 1-PP derivatives, buspirone and the buspirone internal standard (5-fluorobuspirone) are co-chromatographed. Chromatography and detection are performed using capillary gas chromatography with a fused-silica column and selected-ion monitoring-mass spectrometry. Linear range of the standard curves in plasma is 0.1-14 ng/ml for buspirone and 0.2-25 ng/ml for 1-PP with lower limits of quantitation of 0.1 and 0.2 ng/ml, respectively. In urine the linear range of the standard curves is 0.2-14 ng/ml for buspirone and 8-500 ng/ml for 1-PP with lower limits of quantitation of 0.2 and 8.0 ng/ml, respectively. Intra-assay accuracies were within 14% for buspirone and 1-PP in plasma and urine. Intra-assay precision was within 12% for both compounds in both matrices.