Identification and quantification of thymol metabolites in plasma, liver and duodenal wall of broiler chickens using UHPLC‐ESI‐QTOF‐MS

Abstract

In the present study, we aimed to develop a method for thymol sulfate and thymol glucuronide determination in plasma, liver and duodenal wall of broiler chickens after feeding with a Thymus vulgaris essential oil at the different concentrations (0.01, 0.05 and 0.1% w/w). UHPLC coupled with accurate-mass QTOF-MS was used for identification and quantification of thymol metabolites. Novel Waters Oasis Prime HLB solid-phase extraction cartridges were applied to sample clean-up with extraction recoveries ranged from 85 to 92%. The presence of thymol glucuronide was confirmed by MS software according to molecular formula, score, mass error and double bond equivalent. In terms of validation, calibration curves of thymol sulfate were constructed in matrix samples with linearity from 3.91 to 250.0 ng/mL and correlation coefficients were within the range of 0.9979-0.9995. Limits of detection were 0.97, 0.29 and 0.63 ng/mL and limits of quantification were 3.23, 0.97 and 2.09 ng/mL for plasma, liver and duodenal wall, respectively. Intra-day and inter-day precision expressed as relative standard deviation were <4.35%. To highlight, thymol metabolites were directly detected for the first time in liver and duodenal wall and this method was shown to be successfully applicable for investigation of thymol metabolism in chickens after thyme essential oil ingestion.

Impact of dietary supplementation with olive and thyme phenols on alpha-tocopherol concentration in the muscle and liver of adult Wistar rats

“…Thymol sulphate and thymol glucuronide were the main metabolites related to the thyme detected in the liver, which are in agreement with the literature. 38,[63][64][65] Furthermore, p-cymene-diol glucuronide was detected in the T group (Table 2) and this is a good biomarker of thyme phenols, providing a good indication of thyme phenolic exposure similar to that observed in human plasma after the intake of a thyme phenol-enriched olive oil. 38 Similar to what was observed to a lesser extent in the muscle (see the next section), sexual dimorphism for some metabolites was noted, favouring the female gender (Table 2).…”

Thymol, thyme, and other plant sources: Health and potential uses

“…Nevertheless, these authors only studied total free thymol using gas chromatography–mass spectrometry (GC–MS), and no information about thymol metabolites could be obtained. Afterwards, in another study, thymol glucuronide and thymol sulfate were detected in plasma, liver, and duodenal wall of broiler chickens using liquid chromatography–mass spectrometry (LC–MS), but it was dependent on the administered concentration (Pisarčíková, Oceľová, Faix, Plachá, & Calderón, ). In addition, thymol may also pass to egg yolk samples (11.83 ng/g) from quails supplemented with thymol in their diets (80 mg thymol/bird per day; Fernandez et al, ).…”

Effect of Thymol Addition and Withdrawal on Some Blood Parameters, Antioxidative Defence System and Fatty Acid Profile in Rabbit Muscle

“…Thymol sulphate and thymol glucuronide are the main metabolites of thymol biotransformation, and so far, little is known about their bioactivity or whether these compounds are only inactive forms [ 3 ]. Pisarčíková et al [ 20 ] first detected thymol sulphate and thymol glucuronide in the liver and duodenal wall of broiler chickens after a sustained four-week administration of thyme essential oil, and according to Kohlert et al [ 32 ] and Rubió et al [ 9 ], thymol metabolites could be deconjugated to the parental compounds and, in this way, express their pharmacological properties. We have to bear in mind that, during biotransformation, plant compounds change their pharmacological properties, which usually differ from the properties of the parental compounds.…”

“…The Trolox equivalent capacity (TEAC) was determined in thymol and in the experimental feed according with the method described by Karamać et al [ 19 ] using the 2,2’ — Azinobis — (3 — Ethylbenzthiazolin — 6 — Sulfonic Acid (ABTS •+ ) decolorization assay. The results were expressed as mmol Trolox equivalents (TE) per g. Thymol evaporation in the feed was analysed every week during thymol application using High — Performance Liquid Chromatography HPLC according to the modified method of Pisarčíková et al [ 20 ]. Samples were analysed in triplicate and were relatively stable (0 d—151.89, 7 d—134.75 and 14 d—128.30 µg/g DM, respectively).…”