An LC/MS/MS method for quantitation of chemopreventive sphingadienes in food products and biological samples

Abstract

Colorectal cancer (CRC) is a leading cause of cancer mortality. Diet has a significant influence on colon cancer risk. Identifying chemopreventive agents, dietary constituents, practices and/or diet supplements that promote gut health and reduce the incidence of intestinal neoplasias and CRC could significantly impact public health. Sphingadienes (SDs) are dietary sphingolipids found in plant-based food products. SDs are cytotoxic to colon cancer cells and exhibit chemopreventive properties. The aim of the present study was to develop a sensitive and robust ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantifying SDs in food products and biological samples. The assay was linear over a concentration range of 80nM to 50μM and was sensitive to a detection limit of 3.3nM. Post-extraction stability was 100% at 24h. SD content in soy oils was approximately 10nM. SDs were detected transiently in the plasma of adult mice 10min after gavage delivery of a 25mg/kg bolus and declined to baseline by 1h. SD uptake in the gut was maximal in the duodenum and peaked 1h after gavage delivery. Disappearance of SDs in the lower gastrointestinal tract suggests either rapid metabolism to yet unidentified products or potentially luminal export.

Keywords: Chemoprevention; Colon cancer; Mass spectrometry; Soy; Sphingadienes; Sphingolipids.

Figure 1. Optimization of SD detection

Panel 1A shows the structure of C18 sphinga-(4E,8Z)-diene (m/z 298.4), the SD used in the development of the assay. Panel 1B shows the mass spectrum of collision-induced dissociation fragmentation pattern obtained with C18 (4E, 8Z)-sphingadiene. Please note the precursor ion m/z of 298.4 for the parent. Loss of water yields a major product ion of m/z 280.4. For detection and quantification of SD, mass transition of 298.4 →280.4 was used. Panel 1C show the total ion chromatogram and extracted ion chromatograms for SD and C17-SO, which is used as internal standard for quantification.

Figure 2. Comparisons of matrix effects of different biological backgrounds

ME of plasma, tissue and soy homogenates were calculated as described in methods. Average matrix effects were 87.8 ± 10.0, 78.3 ± 14.6, 97.6 ± 17.5% in plasma, tissue and soy, respectively (Average of 6 replicates) and were similar across the different bioological matrices.

Figure 3. 13 point-calibration curve for SDs in PBS and plasma

The linearity of SD assay over the concentration ranges of 80 nM to 50 μM are shown. In panel A, the standard curve was created in PBS. Panel B shows the identical curve generated in human plasma matrix. Extraction procedure involves a simple liquid extraction with ethyl-acetate:isopropanol:water mixture followed by collection and drying down of the organic phase. Dried samples are simply reconstituted in methanol. Excellent linearity and similar slope and intercept was observed for both conditions, indicating that the matrix effects of plasma was negligible. The Inset shows the linearity of the assay over the lowest concentration ranges tested.

Figure 4. Post-extraction stability of SDs in plasma

To determine the post-extraction stability of SDs, analyzed samples were kept in 4°C for 24 hours and reinjected. Panel A shows the extracted ion chromatograph of SDs and C17-SO internal standards. Table shows the stability of SD compounds spiked in plasma and as shown, minimal loss is detected at 24 hrs.

Figure 5. SD concentrations in common soy-based food products

Panel A shows ranges of SDs quantified in soy oil products ranged from 4.7 to 17.4 μM. Each brand was analyzed in duplicate. Panel B shows ranges of SDs detected in tofu products.

Figure 6. Time course of SD absorption in the small intestine and colon sections

To determine the uptake characteristics along the small intestine and to locate major sites of absorption, intestinal segments were obtained from mice euthanized at specified times following oral gavage with 25 mg/kg SDs. Time-dependent SD uptake in the duodenum (panel A), jejunum (panel B), ileum (panel C), and colon (panel D) are shown. Results are mean and standard deviation of three mice per time point.

Figure 7. Time course of SD appearance in mice plasma

To determine oral bioavailability of SDs in vivo, C57/B6 mice were administered 25 mg/kg SDs by gavage, and 3 mice were sacrificed at each of the specified time points to obtain blood and tissue samples. A small but significant increase in plasma SD concentration was observed within 10 min following oral gavage and levels returned to baseline within an hour post gavage.