Analysis of Volatile Bacterial Metabolites by Gas Chromatography–Mass Spectrometry -

Analysis of Volatile Bacterial Metabolites by Gas Chromatography–Mass Spectrometry

Jun 1, 2006
By: William L. Wood, Daniel J. Higbee, Melanie Gooldy, Scott Glogowski, Rich Fitzpatrick, Richard J. Karalus, Troy D. Wood, David J. Mangino
LCGC Asia Pacific
Volume 2, Issue 9

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A method for the identification of key volatile organic compound (VOC) markers associated with infection by Neisseria meningitidis bacteria by gas chromatography–mass spectrometry (GC–MS) was developed. Headspace samples of bacterial VOCs were trapped on triple-sorbent bed tubes and then thermally desorbed into a laboratory GC–MS system for separation. Identification was carried out by comparison of GC retention time and electron ionization mass spectra to the National Institute of Standards and Technology (NIST) database. Further confirmation was obtained by GC–MS of known standard chemicals. A total of 75 VOCs were detected, five of which can be considered key VOC markers for Neisseria meningitidis. These peaks were identified as 1,2-dimethylcyclopropane, 2-methylpropanal, methacrolein, N-2-dimethyl-1-propanamine, and 3-methylbutanal by the NIST database.

Figure 1

Due to the high potential for natural or intentional introduction of foreign infectious agents into the United States, tools to rapidly identify disease threats have become increasingly valuable. Recently, there has been growing interest in breath analysis. This technique is ideal for on-site analysis because it is noninvasive, rapid (compounds of interest are already in gas phase), and simpler than measurements in complex biological matrices (1–3). Breath samples from both animals and humans have been used to identify metabolic end products such as hydrogen, methane, volatile fatty acids, and other volatile organic compounds (VOCs) (4). By monitoring concentration changes of VOCs, either depleted from inhaled air (by degradation and excretion in the body) or added to exhaled breath as products of metabolism, one can determine the presence of an infection, a particular disease state, or a recent exposure to a drug or environmental pollutant (1,2,5).

Figure 2

To date, over 200 organic compounds have been identified in human breath (6). These compounds include hydrocarbons, alcohols, ketones, and aldehydes (1,2,7–9). The most abundant VOCs in breath are isoprene, acetone, ethanol, methanol, and other alcohols (1). Of these 200 compounds, only a few have been linked to a particular disease state. For example, breath pentane has been shown to increase in a number of diseases including breast cancer (10), heart transplant rejection (11), acute myocardial infarction (11,12), schizophrenia (11), and rheumatoid arthritis (11). Acetone has been long known to be a key metabolic byproduct in patients with diabetes (2,13) and congestive heart failure (14). Other studies have shown that an increase in concentration of dimethyl sulfide, mercaptans, and fatty acids occurs in patients with cirrhosis (2), isoprene concentration decreases in patients with heart failure (8), and dimethyl- and trimethylamine concentrations increase in patients with renal disease (2).

Figure 3

There have been numerous breath-analysis studies focused on the identification of disease states. However, there are surprisingly few reports in the area of bacterial infection identification. Khaled and colleagues (15) used the urea (¹³ C) breath test to detect Helicobacter pylori infection in humans. This same test was used to detect Helicobacter pylori infection in squirrel monkeys (16) and cats (4). Gas chromatography (GC) has proved an extremely valuable tool for identifying VOCs associated with bacterial infection in dental research (17–19). For that reason, we undertook the present study to identify key VOCs associated with infection by the Gram-negative bacteria Neisseria meningitidis (N. meningitidis) using gas chromatography–mass spectrometry (GC–MS). N. meningitidis is the major cause of both meningitis and sepsis, two diseases which have particularly high mortality rates in children and young adults (20); it infects humans exclusively (21) and is transmitted from person to person during close contact via airborne salivary droplets. The overall frequency of asymptomatic carriers is ~ 10% in the general population (22), with invasive infection rates between 0.3 and 7.1 per 100,000 inhabitants in Europe (23).

Experimental

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