Determination of d-limonene in adipose tissue by gas chromatography-mass spectrometry

Abstract

We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0-2529 ng d-limonene per 0.1g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7-9.6%) and accuracy (%difference of -2.7 to 3.8%) and between-day precision (R.S.D. 6.0-10.7%) and accuracy (%difference of 1.8-2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.

Figure 1

Mass spectra of d-limonene (a) and perillyl aldehyde (b).

Figure 2

Representative chromatograms of unextracted standard, blank pork fat sample, and extracted standard; a. Unextracted standard of d-limonene (2,529 ng spiked) and perillyl aldehyde (5,790 ng spiked); b. Extracted blank pork fat sample; c. Extracted standard d-limonene (2,529 ng spiked) and perillyl aldehyde (5,790 ng spiked) from the pork fat.

Figure 3

Representative chromatograms of the initial biopsy and the post-intervention biopsy collected from a Mediterranean lemonade feeding study; a. Biopsy sample collected 6 hr after consumption of 40oz of Mediterranean lemonade. d-Limonene level was determined to be 42.7 ng in 0.123 g of fat; b. Biopsy sample collected after 4 weeks of daily Mediterranean lemonade consumption. d-Limonene level was determined to be 447 ng in 0.061g of fat.

Figure 4

d-Limonene levels quantified by GC/MS in study participant fat biopsies collected from a 4-week Mediterranean-style lemonade feeding trial (500-600mg d-limonene daily).