A comprehensive method for determination of fatty acids in the initial oral biofilm (pellicle)

Abstract

The acquired pellicle is a tenacious organic layer covering the surface of teeth, protecting the underlying dental hard tissues. Lipids account for about one quarter of the pellicle's dry weight and are assumed to be of considerable importance for their protective properties. Nevertheless, only preliminary information is available about the nature of lipids in the pellicle. Gas chromatography coupled with electron impact ionization mass spectrometry was used to establish a convenient analytical protocol in order to obtain a qualitative and quantitative characterization of a wide range of FAs (C(12)-C(22)). In situ biofilm formation was performed on bovine enamel slabs mounted on individual splints carried by 10 subjects. A modified Folch extraction procedure was adopted to extract the lipids from the detached pellicle, followed by transesterification to fatty acid methyl esters using methanol and concentrated hydrochloric acid. Tridecanoic and nonadecanoic acid were used as internal standards suitable and reliable for robust, precise and accurate measurements. The present study demonstrates, for the first time, a procedure based on a combination of innovative specimen generation and convenient sample preparation with sensitive GC-MS analysis for the determination of the fatty acid profile of the initial oral biofilm.

Fig. 1.

GC/MS chromatogram of a pellicle sample (formation time 30 min) acquired in SIM mode. The sample was separated through a thermally stable Select FAME capillary column (50 m × 0.25 mm ID, 0.25 µm film thickness). The GC oven program started at 50°C (hold time 5 min) and was increased to 260°C (hold time 8 min) at a ramp rate of 6.5°C/min. Characteristic fragment ions (m/z 74, m/z 87, m/z 81, and m/z 79) were monitored throughout the run.

Fig. 2.

FA composition of pellicle sample (formation time 30 min). Values represent the means ± SD of 10 subjects expressed as percent of the investigated FAs. This profile seems to be characteristic for the biological structure of the pellicle.

Fig. 3.

Amount of total investigated FAs of the 30 min pellicle of the 10 study subjects (A–J). Values vary from 680 to 1600 ng per cm² pellicle formation surface, illustrating interindividual differences (dashed line marks the mean amount of total fatty acids of the 10 subjects).