The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.

Rapid methods are described for determination of flumequine (FLU) residues in muscle and plasma of farm-raised channel catfish (Ictalurus punctatus). FLU residues were extracted from tissues with an acidified methanol solution, and extracts were cleaned up on C18 solid-phase extraction cartridges. FLU concentrations were determined by liquid chromatography (LC)using a C18 analytical column and fluorescence detection (excitation, 325 nm; emission, 360 nm). Mean recoveries of FLU from fortified muscle were 87–94% at 5 levels ranging from 10 to 160 ppb (5 replicates per level). FLU recoveries from fortified plasma were 92–97% at 5 levels ranging from 20 to 320 ppb. Limits of detection (signal-to-noise ratio, 3:1)for the method as described were 3 and 6 ppb for muscle and plasma, respectively. Relative standard deviations (RSDs) for recoveries were ≤12%. Live catfish were dosed with 14C-labeled or unlabeled FLU to generate incurred residues. Recoveries of 14C residues throughout extraction and cleanup were 90 and 94% for muscle and plasma, respectively. RSDs for incurred FLU at 2 levels in muscle and plasma ranged from 2 to 6%. The identity of FLU in incurred tissues was confirmed by LC/mass spectrometry.

The present study was undertaken in order to determine whether 18O-labeled sterols could be used in place of 14C-sterols in clinical studies of cholesterol metabolism. (3 beta-18OH)Cholesterol and (3 beta-18OH)sitosterol were simply and inexpensively synthesized and precisely and accurately quantified by gas chromatography/mass spectrometry. 18O-Sterols added to fecal homogenate and saponified were completely recovered. However, in a series of validation studies in humans, the fecal recoveries of orally administered (18O)cholesterol and (18O)sitosterol were significantly lower than the recoveries of 14C-sterols given simultaneously. We found that the losses were largely limited to the coprostanol and ethylcoprostanol fecal metabolites. In vitro fecal incubations of 18O-sterols and unlabeled water or of unlabeled sterols with H2(18)O indicated that the losses occurred during fecal bacterial metabolism and were likely due to 3 beta-oxygen exchange with the oxygen of water, possibly via a 3-ketosteroid intermediate. These data indicate that (18O)cholesterol and (18O)sitosterol are invalid tracers for the measurement of human cholesterol metabolism by methods based on fecal sterol recovery.

Chemical noise limits mass spectrometric detection of chloramphenicol (CAP) with electron capture ionization at low resolution, and makes CAP identification at concentrations of 5 parts per billion (ppb) difficult. Increasing the resolution from 1000 to 3500, however, was sufficient to separate the analyte signals from the noise signals, and resulted in a 100 times higher analytical sensitivity. The introduction of sweep gas in the ion source decreased the scattering of the quantitative results on average by a factor of 7, and thereby improved the precision of the analyses to an acceptable level (CV < 10%). Under such conditions, CAP residues of 1.5 and 2.1 ppb in shrimp as determined by electron capture gas chromatography/mass spectrometry can readily be identified by monitoring four diagnostic ions.

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