Development of a Sensitive and Reliable UHPLC-MS/MS Method for the Determination of Multiple Urinary Biomarkers of Mycotoxin Exposure

Abstract

A variety of mycotoxins from different sources frequently contaminate farm products, presenting a potential toxicological concern for animals and human. Mycotoxin exposure has been the focus of attention for governments around the world. To date, biomarkers are used to monitor mycotoxin exposure and promote new understanding of their role in chronic diseases. The goal of this research was to develop and validate a sensitive UHPLC-MS/MS method using isotopically-labeled internal standards suitable for accurate determination of 18 mycotoxin biomarkers, including fumonisins, ochratoxins, Alternaria and emerging Fusarium mycotoxins (fumonisin B1, B2, and B3, hydrolyzed fumonisin B1 and B2, ochratoxin A, B, and alpha, alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid, beauvericin, enniatin A, A1, B, and B1) in human urine. After enzymatic digestion with β-glucuronidase, human urine samples were cleaned up using HLB solid phase extraction cartridges prior to instrument analysis. The multi-mycotoxin and analyte-specific method was validated in-house, providing satisfactory results. The method provided good linearity in the tested concentration range (from LOQ up to 25–500 ng/mL for different analytes), with R2 from 0.997 to 0.999. The limits of quantitation varied from 0.0002 to 0.5 ng/mL for all analytes in urine. The recoveries for spiked samples were between 74.0% and 133%, with intra-day precision of 0.5%–8.7% and inter-day precision of 2.4%–13.4%. This method was applied to 60 urine samples collected from healthy volunteers in Beijing, and 10 biomarkers were found. At least one biomarker was found in all but one of the samples. The high sensitivity and accuracy of this method make it practical for human biomonitoring and mycotoxin exposure assessment.

Biomonitoring of Enniatin B1 and Its Phase I Metabolites in Human Urine: First Large-Scale Study

“…Differences showed among the mentioned studies could be due to the limited sampling. A more recent work, conducted by Liu et al [ 22 ], analyzed 60 urine sample from the Chinese population, reporting the absence of Enn B1 (LOQ = 0.0002 ng/mL). The variation between the Chinese population urinary patterns of Enn B1 and the obtained results could be explained either by dietary habits or the quality of foodstuffs.…”

Determination of Urinary Mycotoxin Biomarkers Using a Sensitive Online Solid Phase Extraction-UHPLC-MS/MS Method

“…For trapping and pre-concentration of the analytes while removing interfering matrix compounds, an Oasis ® HLB column (2.1 × 20 mm, 5 µm; Waters GmbH) was proven to be suitable. The Oasis HLB material has generally been shown to be able to bind mycotoxins in physiological matrices [12][13][14]30]. However, not all Oasis materials are available for online SPE applications.…”

“…Certain multi-mycotoxin methods using more laborious sample preparation such as conventional SPE combined with immunoaffinity chromatography (IAC) [14] or liquid-liquid extraction (LLE) [30] as well as a QuEChERS-based procedure (Quick, Easy, Cheap, Effective, Rugged, Safe) [16] provided a lower sensitivity for some analytes than the presented online SPE clean-up. Nevertheless, the sole use of Oasis ® Prime HLB SPE columns implied considerably lower LODs [12,13]. Besides the DaS approach [8,10], the analysis of ALT, AME and AOH was only covered by a few methods for multi-mycotoxin analysis in human urine samples.…”

“…This procedure has the major drawback of a reduced sensitivity due to the dilution step. More laborious approaches such as solid phase extraction (SPE), immunoaffinity chromatography (IAC), or the use of dried urine spots (DUS) facilitate analyte enrichment as well as the separation of interfering matrix compounds, resulting in lower limits of detection [11][12][13][14][15]. IAC clean-up in particular enables the specific retention of the targeted analytes, but the high selectivity makes it complex and often unsuitable for multi-mycotoxin analysis [7].…”

Development of a multi-mycotoxin LC-MS/MS method for the determination of biomarkers in pig urine

“…According to the best knowledge of the author, this important step has never been fully optimized before. In recent times, β-glucuronidases were only quantified for human urine samples (Liu et al 2020). In the case of pig urine, enzymatic hydrolysis was carried out but only in three recent studies: β-glucuronidases from Helix Pomatia (Gambacorta et al 2013(Gambacorta et al , 2019Brezina et al 2014) and E. coli (Nagl et al 2014).…”