GC-MS Analysis of Garlic Breath . . . . . . .

Preparation of Garlic Bread

Garlic bread samples each weighing 170g contained 35g of garlic butter. The butter had a concentration of garlic (either control fresh cloves or Dr Sakai garlic) at a concentration of 21%. All samples were held in a refrigerator at 4ºC until used. The bread was baked for 12 minutes at an oven temperature of 180ºC immediately prior to consumption.

Pre-Concentration of Breath Samples for GC-MS Analysis

Two non smoking male subjects, each weighing approximately 8Okg consumed one garlic baguette containing the control garlic at approximately 11am after refraining from any garlic products for the previous 24 hours (but with no other modifications to the normal diet). Before consumption of the garlic bread a breath sample was taken and used as a blank. A breath sample was taken directly after the consumption of the garlic bread (0 hours), then at 3 hours and 5 hours after consumption. A 3.4mm ID Unpacked glass injector insert was tightly packed with 75mg of Tenax TA, with a plug of glass wool at either end. The weight of packing was determined by weighing the pre column before and after packing. The subject held a Teflon tube in the oral cavity, and whilst inhaling through the nose and exhaling via the mouth, 100 ml of breath was drawn slowly through the pre concentration tube. The pre concentration tube was instantly detached from the Teflon tubing and placed into the GC/MS injection port and thermally desorbed at 250ºC. Prior to re-use the trap was baked at 2500C for 30 minutes. Blank runs of the reconditioned trap proved that there was no carryover between samples.

The same experiment, with the same controls (cross-over design) was repeated several days later to avoid any carry-over, using garlic baguettes containing Dr Sakai garlic instead of the control.

Direct Sampling for P-FPD
The P-FPD detector did not require a pre-concentration stage. An aliquot of breath was withdrawn from the subject’s mouth via Teflon tube in the oral cavity. A direct injection of 0.5 ml was then introduced into the system. As with the pre concentration method a blank breath sample was taken immediately prior to consumption of the garlic bread. Immediately after consumption of the garlic bread the first sample was taken (0 hours) with subsequent samples taken every 15 minutes up to and including 60 minutes. This analysis was only preformed on the control garlic bread by subject one.

GC/MS Operating Parameters
The GC/MS was a Saturn 4D ion trap mass spectrometer supplied by Varian (Varian Associates Inc. CA) with Varian Star 3400 CX GC.

Column:	30 M x 0.25 mm i.d. x 0.25 μm film thickness DB5-MS (J&W Scientific)
Carrier:	Helium 30 ml/min
Column Oven: (Cooled with liquid CO2 )	-10°C for 3 min, 10°C/min to 300°C
Injector:	50°C for 0.5 min; 300°C/min to 250°C, hold for run.
Transfer Line:	260°C
Injector Mode:	Splitless injection for first 5 min of run
Mass Range:	45 - 100
Scan Time:	1 sec
Mult. Delay:	0.1 min
Peak Threshold:	1 count
Ion Mode:	Electron Impact
Ion Trap:	260°C

Table 1: Instrumental Conditions for Mass Spectrometer

Compounds were tentatively identified by comparison to reference spectra via the mass spectral database. Compounds tentatively identified were confirmed by comparing their mass spectra and GC retention times to those of authentic compounds analysed in the laboratory under identical conditions. Quantitation was performed using the Q ion for methyl mercaptan (m\z 47) and peak area for all other compounds.