1. Analysis of urine by (1)H-nuclear magnetic resonance (NMR) spectroscopy is used to detect biochemical disturbances predictive of toxicological changes. Recent studies, using (1)H-NMR spectroscopy have suggested that Alderley Park rats can be classified as hippuric acid (HA) or m-(hydroxyphenyl)propionic acid (m-HPPA) excretors. Evidence exists for the role of intestinal microflora in the excretion of aromatic phenolic compounds including HA and m-HPPA. 2. We sought to investigate whether intestinal microflora contribute to the difference in excretion. Urinary HA and m-HPPA levels were monitored to characterize excretion over time. The effect of intestinal microflora on the (1)H-NMR spectrum was also investigated using antibiotics to sterilize the intestine. Finally, the levels of m-HPPA and phenylpropionic acid (a precursor for HA) were analysed in the caecum and colon (entire tissue, including contents). 3. Characterization confirmed the presence of HA and m-HPPA excretors; enquiries revealed that the rats were obtained from two floors within a barriered breeding unit. Housing the rats from the two floors together for 21 days resulted in comparable levels of HA and m-HPPA excretion demonstrating that the profiles are not stable. 4. Following antibiotic treatment, HA and m-HPPA excretion decreased, indicating that intestinal microflora contribute to the excretion of these compounds. Finally, m-HPPA levels were higher in the colon of rats that excreted m-HPPA whilst PPA was increased in the caecum and colon of rats that excreted HA. 5. These results demonstrate that the observed difference in HA/m-HPPA excretion is due to differences in the intestinal microflora.

Fast and accurate analytical methods are essential to keep pace with sample libraries produced from combinational chemistry and high-throughput biological screening. Many laboratories now use a combination of ionization techniques for the characterization of these samples, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and photoionization (PI). Data are shown here from the analysis of a compound collection plate containing a variety of sample structures. ESI will normally analyze around 80% of these samples, necessitating a source change to analyze a further 10%. In this work, we have developed a new combined ESI-APCI source (ESCi) for use in on-line HPLC applications. The combined source allows alternate on-line ESI and APCI scans with polarity switching within a single analysis. The ESCi source has been designed to be a simple replacement for the existing mass spectrometer interfaces. Each ionization method is optimized independently using separate tuning parameters. Instrument electronics can readily switch between the two ionization methods and polarities within normal interscan time periods. The new source has reduced the analysis time of sample plates by eliminating the need for a source hardware change, source optimization, and repeat analyses.

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Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging.

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