Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials.

Combining HPLC separations with an isotope-ratio mass spectrometric (IRMS) detection produces a device capable of measuring very low alterations in 13C abundance from analyte species that cannot be volatilized. Examples are presented showing proteins, carbohydrates, and nucleotides that are eluted from varying types of HPLC columns (reversed-phase, normal-phase, ion-exchange and size-exclusion). This wide range of chromatographic methods enables the analysis of compounds never before amenable to IRMS techniques and may lead to the development of many new assays.

Labeling DNA with stable isotopes: economical and practical considerations.

Labeling DNA with stable isotopes to measure cell proliferation can be a technique as effective as 3H-thymidine labeling without the limitations imposed by using radioisotopes. Here, we investigated the relative efficiency of four nonradioactive precursors to DNA: [1-13C]-glycine, [1,2-13C2]-glycine, [U-13C]-glucose, and [U-13C, 15N]-thymidine. The efficiency of incorporation for each of these labeled precursors in HEP G2 cells in culture has been studied. When considering the actual costs of in vivo experiments in which large doses of labeled material are needed, economical constraints may play an important role in defining a practical method. Therefore, the economics of this process were also considered. Using the enrichment per dollar for whichever nucleoside had the highest incorporation in a given experiment, glycine is about five times more economical as a label than thymidine and eight times more economical than glucose in these cells.

Flow injection with chemical reaction interface-isotope ratio mass spectrometry: an alternative to off-line combustion for detecting low levels of enriched 13C in mass balance studies.

We have evaluated the potential of flow injection chemical reaction interface isotope-ratio mass spectrometry to replace radioactive labeling techniques in material balance studies. A sample is flow injected and transmitted through a desolvation system followed by combustion to form 13CO2 with a microwave-powered chemical reaction interface. We can detect trace amounts of a 13C-labeled drug (3'-azido-3'-deoxythymidine, AZT) in urine or feces. Our ability to quantify less than 100 ng/mL of excess 13C (approximately 1 microgram/mL of 13C-labeled AZT) from a sample equivalent to 10 microL of urine is superior to previous detection limits for 13C in urine that use off-line combustion methods. Parallel studies using 14C-labeled AZT showed that our stable isotope method provides comparable percent excretion data for urine and feces. These results support previous findings that mass balance studies could be carried out with isotope-ratio mass spectrometer, here using doses as low as 1-2 mg/kg.

Electrospray tandem mass spectrometry for analysis of native muramic acid in whole bacterial cell hydrolysates.

Muramic acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature. This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized muramic acid in bacterial hydrolysates. Fungal hydrolysates were used as negative controls. The only processing used was hydrolysis in sulfuric acid followed by extraction with an organic base (N,N-dioctylmethylamine) to remove the acid prior to ESI MS/MS analysis. Compared with pure muramic acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable. In contrast, product ion spectra of m/z 252 were identical among pure muramic acid, Gram positive bacteria, and Gram negative bacteria. However, no characteristic product ion spectrum was manifested from m/z 252 in fungal samples. This allowed ready, visual differentiation of bacteria and fungi. Multiple reaction monitoring (MRM) following muramic acid fragmentations (m/z 252-->144 and m/z 252-->126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine (m/z 196-->44). ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of muramic acid in whole bacterial cell hydrolysates.

Determination of carbohydrate profiles of Bacillus anthracis and Bacillus cereus including identification of O-methyl methylpentoses by using gas chromatography-mass spectrometry.

Bacillus anthracis and Bacillus cereus are closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically. It is well known that vegetative and spore forms of bacilli are quite distinct both morphologically and chemically, but spore-specific chemical markers allowing these species to be distinguished have not been previously described. By using gas chromatography-mass spectrometry, vegetative cells and spores of the two species were shown to exhibit distinct carbohydrate profiles. Profiles of vegetative B. anthracis typically contained high levels of galactose but did not contain galactosamine, whereas B. cereus contained galactosamine and generally low levels of galactose. Spore cultures exhibited unique carbohydrate profiles compared with those of vegetative cultures. B. anthracis spore profiles contained rhamnose alone, whereas B. cereus spore profiles contained rhamnose and fucose. Additionally, two spore-specific O-methylated methylpentoses were discovered. Both B. anthracis and B. cereus spores contained 3-O-methyl rhamnose, whereas B. cereus spores also contained 2-O-methyl rhamnose. Carbohydrate profiling is demonstrated to be a powerful tool for differentiating the two closely related species. Differentiation does not depend on whether organisms are in the vegetative or spore stage of growth.