An (UHPLC-MS/MS) Method for Toxicity and Safety Level Assessment of Oxygen Heterocyclic Compounds and Terpene in Cold-Pressed Grapefruit Essential Oils

Authors: ZAHIRUDDIN, SULTAN - University Of Mississippi; WANG, YAN-HONG - University Of Mississippi; KOTHAPALLI, HARI BABU - University Of Mississippi; AVULA, BHARATHI - University Of Mississippi; CHITTIBOYINA, AMAR GOPAL - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi

Submitted to: ACS Omega
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2025
Publication Date: 7/2/2025
Citation: Zahiruddin, S., Wang, Y. H., Kothapalli, H. B., Avula, B., Chittiboyina, A. G., & Khan, I. A. (2025). An ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for toxicity and safety level assessment of oxygen heterocyclic compounds and terpene in cold-pressed grapefruit essential oils. ACS Omega, 10, 29654–29664. https://doi.org/10.1021/acsomega.5c03454.
DOI: https://doi.org/10.1021/acsomega.5c03454

Interpretive Summary: Grapefruit (Citrus × paradisi), a citrus fruit known for its flavor and juiciness, comes in several varieties, including white, pink, and red grapefruits. Grapefruit essential oils (GEOs) are used for their aromatic properties, but also for various medicinal and therapeutic purposes. GEOs are complex mixtures of volatile and nonvolatile components containing several types of compounds, including monoterpenes, sesquiterpenes, polymethoxyflavones (PMFs), coumarins (CMNs), and furanocoumarins (FCMNs).  However, some of these compounds may be toxic to human health, such as ingestion of excessive amounts of CMNs, or the interaction of FCMNs with UVA rays and with certain OTCs and prescription drugs. This study compared different varieties of grapefruit essential oils (GEOs) and commercial grapefruit essential oils CGEOs). This will be the first report for simultaneous analysis of FCMNs, CMNs, PMFs, and NKT in different varieties and commercial samples of essential oil of grapefruit by using UHPLC-MS/MS, and additionally, this method will help to determine the reduction and elimination of oxygen heterocyclic compounds in raw materials as well as finished products.

Technical Abstract: Oxygen heterocyclic compounds (OHCs) like polymethoxyflavones (PMFs), coumarins (CMNs), furanocoumarins (FCMNs), and terpenes like nootkatone (NKT) are key secondary metabolites in grapefruit essential oils (GEOs). While these compounds exhibit a plethora of promising bioactivities (e.g., antioxidant, antiallergic, anti-inflammatory, and antibacterial), potential adverse effects, such as CMN toxicity at high concentrations and FCMN phototoxicity exacerbated by UVA exposure and interactions with certain over-the-counter medications, warrant a sensitive and selective analytical method for determining their levels in GEO-based products intended for topical and oral use. To address these analytical needs, a rapid 16 min ultrahighperformance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous identification and quantification of 25 OHCs (three PMFs, seven CMNs, and 15 FCMNs) and NKT in cold-pressed GEOs. The method exhibited excellent linearity (R2 > 0.99 for all analytes), low limits of detection (LODs, 6 × 10-8 to 1.5 × 10-6 mg g-1 ), and limits of quantification (LOQs, 2 × 10-7 to 5 × 10-6 mg g-1 ). Intra- and interday precision (% RSD) ranged from 1.43 to 3.59% and from 2.16 to 6.90%, respectively. The resulting quantitative data will be essential for establishing safe levels of OHCs in cold-pressed GEOs, thereby minimizing phototoxicity and drug interaction risks. Furthermore, this validated method can also be applied to various GEO-based products to ensure the quality of raw materials and inform the reduction or elimination of specific OHCs.