Development and validation of a gas chromatography-mass spectrometry method to analyze octanoate enrichments at low concentrations in human plasma

Abstract

A new method for accurately analyzing octanoate enrichment in plasma was developed and validated. Samples were derivatized directly in plasma by transesterification with isobutanol and were analyzed by gas chromatography-mass spectrometry (GC-MS). This method was developed to analyze the precursor enrichment in a stable isotope tracer protocol. Glyceryl tri[1,2,3,4-¹³C₄] octanoate, a stable isotope-labeled medium-chain triglyceride (MCT), was orally administered in combination with (1) exclusively MCT or (2) a combination of protein, carbohydrates, and MCT to investigate the metabolic route of oral MCT under various conditions. Accurate analysis of octanoate enrichment in plasma at concentrations as low as 0.43 μM (lower limit of quantification, LLOQ) was performed. This is an improvement of about twenty times for the LLOQ for analysis of the enrichment of octanoate when compared with the gold-standard method for fatty acid analysis (methyl esterification). Moreover, we found that' with this gold-standard method, study samples were easily contaminated with (unlabeled) octanoate from other sources, leading to biased, incorrect results. The precision and linearity obtained using the new method were good (coefficient of variation intraday < 9.1%, interday < 9.3%, R² of the calibration curve > 0.99). The sensitivity was sufficient for analyzing samples obtained using the stable isotope protocol. This new method is more sensitive than methyl esterification and it minimizes the risk of contamination. Graphical abstract.

Keywords: Elongation; Mass spectrometry; Medium-chain triglycerides; Stable isotopes.

Graphical abstract

Fig. 1

Schematic representation of the administration of the tracer and the unlabeled glyceryl trioctanoate. Both supplementations were isocaloric but differed in composition (100 vs 30 energy% from MCT). In this example, the tracer-tracee ratio percent (TTR%) of the glyceryl trioctanoate administered was 1.5 TTR% for the MCT-only supplementation and 5.1 TTR% for the mixed nutrient supplementation. The timeline below indicates the duration of the protocol (360 min) and the arrows indicate blood sampling

Fig. 2

Chromatogram of pool plasma acquired in the single ion monitoring (SIM) mode for m/z value 127.1. The octanoate peak is indicated with an asterisk (*). One of the components in the chromatogram (RT 8.9 min) was identified with a high probability as the isobutyl derivate of fumaric acid. The others could not be identified through the NIST library

Fig. 3

Mass spectra. a Obtained after derivatization of octanoate by isobutylation. Mass fragment 145 results from a McLafferty + 1 rearrangement (M-55, loss of C₄H₇), while mass fragment 127 results from loss of the derivate group C₄H₉O (M-73). In these fragments, all isotope-labeled positions of 1,2,3,4-¹³C₄-octanoate are preserved. b Obtained after the derivatization of octanoate by methylation

Fig. 4

Calibration curve for the enrichment of 1,2,3,4-¹³C₄-octanoate. The peak area for 1,2,3,4-¹³C₄-octanoate was divided by the peak area for unlabeled octanoate for the calibration standards to obtain the experimental tracer-tracee ratio (TTR). These results are plotted against the theoretical TTR% (amount of tracer, divided by the amount of tracee, multiplied by 100) of the standards. The theoretical value of the slope is 0.01

Fig. 5

Analysis results obtained with the stable isotope protocol. a Enrichment results for 1,2,3,4-¹³C₄-octanoate in plasma. Results obtained during MCT-only supplementation were marked by circles; the results obtained during the mixed nutrients supplementation were marked by crosses. Different shades were used to indicate different subjects. b Comparison between the average theoretical TTR% values of the different supplemental nutrition (calculated from the TTR% value of the administered glyceryl trioctanoate) and the analysis result (TTR% of 1,2,3,4-¹³C₄-octanoate in plasma). The range of the data was represented by the error bars