Selected reaction monitoring LC-MS determination of idoxifene and its pyrrolidinone metabolite in human plasma using robotic high-throughput, sequential sample injection.

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format. Method validation included determination of intra- and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy +/- 12.0% precision, 95.1 +/- 4.9%, and 90.3 +/- 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QC1 (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 +/- 14.4%, 91.1 +/- 13.0%, and 90.8 +/- 12.2%, respectively). The validated method was applied to the analysis of 613 human clinical plasma samples. An average run time of 23 s/sample (approximately 37 min/ 96-well plate or over 3,700 sample/day) was achieved. The successful validation presented indicates that rapid methods of analysis can efficiently and reliably contribute to the fast sample turnaround required for high sample number generating processes.

Microbial models of soil metabolism: biotransformations of danofloxacin.

Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine. Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock. This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin. Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites. Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification. Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed. Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin. The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring. At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures. Radiolabled [2-14C]danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation.

Putting mass spectrometry in the hands of the end user.

The ease of use of the newer liquid chromatography-mass spectrometry interfaces has made possible the automated acquisition of spectra from large batch queues of samples. This fact, combined with the realization that unit molecular mass determination was the only datum desired by a majority of drug discovery synthetic chemists, led us to develop open access mass spectrometry in the early 1990s. Open access spectrometers now scan over 100,000 samples per year from synthesis laboratories at Pfizer. Our experiences with this novel use of mass spectrometry in a large research facility are discussed and we detail some of the pitfalls we believe to be common to this approach. In addition, we offer some reflection on the cultural changes we have observed in our research environment since this experiment began.

Gas-phase ion-molecule chemistry of borate and boronate esters.

Borate esters B(OR)3 and boronate esters RB(OR)2 undergo ion-molecule reactions to yield both addition products (by an implied radiative emission mechanism), ligand exchange, and proton transfer products, in both positive and negative ion modes. Although an acidity for CH3B(OR)2 could not be determined, HOB(OR)2 has an acidity between acetaldehyde and nitromethane. In light of the negligible polar electron acceptor properties of the -B(OR)2 group, that functionality must therefore be one of the best resonance electron acceptor groups known, almost half again as effective as the nitro group.

Structures of gas-phase C2F 7 (+) ions.

In an ion cyclotron resonance spectrometer, less than 96% of the C7F 7 (+) cation formed on electron ionization of perfluorotoluene reacts with hexamethyldisilazane. In contrast, the C7F 7 (+) from perfluoronorbornadiene or perfluorobicyclo[3.2.O]hepta-2,6-diene is nonreactive with hexamethyldisilazane. Collision-induced dissociation results support this dichotomy, although the evidence is not as clear-cut. The reactive ion is assigned the benzyl structure and the nonreactive ion the tropyl structure, on the basis of analogy with the protio cases. By AM1 calculations, the perfluorobenzyl ion is 25 kcal/mol more stable than the perfluorotropyl ion, the opposite of the situation for the protio analogs (- 12 kcal/mol). Ab initio calculations at the 3-21G level agree with the semiempirical energy difference to within 0.4 kcal/mol; at the more appropriate 6-31G*/MP2 level, the perfluorobenzyl cation is 9.7 kcal/mol more stable than the perfluorotropyl cation.

A novel derivatization method for peptides to increase sensitivity and backbone fragmentation in liquid secondary-ion mass spectra.

Derivatization is used to increase both negative-ion sensitivity and positive-ion sequence information in the liquid secondary-ion mass spectra (LSIMS) of a series of peptides. The derivatization method involves acylation with pentafluorobenzoyl fluoride in a single-step reaction, and the reaction mixture is applied directly to the probe tip for analysis. Acylation takes place at the unprotected N-terminus, tyrosine, and lysine. The derivatives exhibit increased signal-to-noise ratio for [M-H]- ions, especially where there is not already an acidic amino acid residue in the peptide. In positive-ion LSIMS, the N-terminal group acts to retain the charge at the N-terminus, simplifying the fragmentation by producing N-terminal fragment ions. It also increases positive-ion fragmentation, sometimes very dramatically, making sequence determination more straightforward. The simplicity of the process, together with the enhancements it provides, make this a generally useful method for obtaining peptide structural information.