Biomedical and forensic applications of combined catalytic hydrogenation-stable isotope ratio analysis

Abstract

Studies of biological molecules such as fatty acids and the steroid hormones have the potential to benefit enormously from stable carbon isotope ratio measurements of individual molecules. In their natural form, however, the body's molecules interact too readily with laboratory equipment designed to separate them for accurate measurements to be made. Some methods overcome this problem by adding carbon to the target molecule, but this can irreversibly overprint the carbon source 'signal'. Hydropyrolysis is a newly-applied catalytic technique that delicately strips molecules of their functional groups but retains their carbon skeletons and stereochemistries intact, allowing precise determination of the carbon source. By solving analytical problems, the new technique is increasing the ability of scientists to pinpoint molecular indicators of disease, elucidate metabolic pathways and recognise administered substances in forensic investigations.

Keywords: fatty acids; hydropyrolysis; stable isotopes; steroids.

Figure 1.

Strategies to make biological molecules such as fatty acids and steroids amenable to stable isotope ratio analysis by GC-C-IRMS. a) A common steroid molecule (cholanoic acid) with a functional group (in red) that causes it to interact too readily with the separation equipment. A successful isotope ratio analysis must measure the carbon isotope ratio of the carbon skeleton (in black). b) One approach is to modify the functional groups by attaching small molecules (blue) but this adds carbon. c) Another approach is to remove functional groups by catalytic reactions but this has previously removed parts of the carbon skeleton. d) The hydropyrolysis approach removes functional groups but retains the carbon skeleton intact for stable isotope analysis.

Figure 2.

Schematic of the hydropyrolysis equipment.

Figure 3.

GC-C-IRMS traces of n-octadecanoic acid and its hydropyrolysis product, n-octadecane, displaying a marked increase in chromatographic performance (Sephton et al. 2005a).

Figure 4.

GC-C-IRMS traces of (a) cholanic acid and (b) its hydropyrolysis product 5b cholane displaying an increase in chromatographic performance.

Figure 5.

GC-C-IRMS traces of (a) 5α cholestanol and (b) its hydropyrolysis product 5α cholestane displaying an increase in chromatographic performance (Sephton et al. 2005b).

Figure 6.

GC-C-IRMS traces of (a) cholesterol and (b) its hydropyrolysis products (mainly 5b and 5a cholestane) (Sephton et al. 2005b).

Figure 7.

The effect of partial conversion on the carbon isotopic composition of hydropyrolysis products (Sephton et al. 2005b).