Multi-mycotoxin analysis using dried blood spots and dried serum spots

Anal Bioanal Chem DOI 10.1007/s00216-017-0279-9 RESEARCH PAPER Multi-mycotoxin analysis using dried blood spots and dried serum spots Bernd Osteresch 1 & Susana Viegas 2,3 & Benedikt Cramer 1 & Hans-Ulrich Humpf 1 Received: 9 December 2016 / Revised: 8 February 2017 / Accepted: 24 February 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract In this study, a rapid multi-mycotoxin approach was developed for biomonitoring and quantification of 27 important mycotoxins and mycotoxin metabolites in human blood samples. HPLC-MS/MS detection was used for the analysis of dried serum spots (DSS) and dried blood spots (DBS). Detection of aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), trichothecenes (deoxynivalenol, DON; DON-3-glucoronic acid, DON-3-GlcA; T-2; HT-2; and HT-2-4-GlcA), fumonisin B1 (FB1), ochratoxins (OTA and its thermal degradation product 2’R-OTA; OTα; 10hydroxychratoxin A, 10-OH-OTA), citrinin (CIT and its urinary metabolite dihydrocitrinone, DH-CIT), zearalenone and zearalanone (ZEN, ZAN), altenuene (ALT), alternariols (AOH; alternariol monomethyl ether, AME), enniatins (EnA, EnA1, EnB, EnB1) and beauvericin (Bea) was validated for two matrices, serum (DSS), and whole blood (DBS). HPLC-MS/MS analysis showed signal suppression as well as signal enhancement due to matrix effects. However, for most analytes LOQs in the lower pg/mL range and excellent recovery rate were achieved using matrix-matched calibration. Besides validation of the method, the analyte stability in DBS and DSS was also investigated. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0279-9) contains supplementary material, which is available to authorized users. * Hans-Ulrich Humpf 1 Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstr. 45, 48149 Münster, Germany 2 Environment and Health RG, Lisbon School of Health Technology, Polytechnic Institute of Lisbon, Av. D. João II, Lote 4.69.01, Parque das Nações, 1990-096 Lisbon, Portugal 3 Centro de Investigação em Saúde Pública, Escola Nacional de Saúde Pública, Universidade NOVA de Lisboa, Avenida Padre Cruz, 1600-560 Lisbon, Portugal Stability is a main issue for some analytes when the dried samples are stored under common conditions at room temperature. Nevertheless, the developed method was applied to DBS samples of a German cohort (n = 50). Besides positive findings of OTA and 2’R-OTA, all samples were positive for EnB. This methodical study establishes a validated multi-mycotoxin approach for the detection of 27 mycotoxins and metabolites in dried blood/serum spots based on a fast sample preparation followed by sensitive HPLC-MS/MS analysis. Keywords Biomonitoring . Dried blood spot . Dried serum spot . HPLC-MS/MS . Mass spectrometry . Mycotoxin Abbreviations 2’R-OTA 2’R-Ochratoxin A 10-OH-OTA 10-Hydroxyochratoxin A AFB1 Aflatoxin B1 AFB2 Aflatoxin B2 AFG1 Aflatoxin G1 AFG2 Aflatoxin G2 AFM1 Aflatoxin M1 ALT Altenuene AME Alternariol monomethyl ether AOH Alternariol BEA Beauvericin DBS Dried blood spot DH-CIT Dihydrocitrinone DON Deoxynivalenol DON-3-GlcA DON-3-glucuronide DSS Dried serum spot EnA Enniatin A EnA1 Enniatin A1 EnB Enniatin B Enniatin B1 EnB1 B. Osteresch et al. FB1 HT-2 HT-2-4-GlcA OTA OTα sMRM T-2 ZAN ZEN Fumonisin B1 HT-2 toxin HT-2-toxin-4-glucuronide Ochratoxin A Ochratoxin α Scheduled multiple reaction monitoring T-2 toxin Zearalanone Zearalenone Introduction Mycotoxins are toxic secondary metabolites produced by molds contaminating food during production, shipping, processing, or storage [1]. In order to protect consumers’ health maximum levels for food and feed have been set by regulatory authorities [2]. Regulatory attempts are usually based on food contamination and consumption data. However, this approach does not take individual exposure due to personal, regional as well as cultural divergences into account [3]. Biomarker-based approaches are more and more used to assess dietary exposure, as they allow to analyze physiological samples like urine or blood for each test person individually [4]. Various sample preparation and quantification techniques are described for mycotoxins and their metabolites in several physiological samples [5–9]. Main challenges in biomonitoring-based methods are usually low analyte concentrations and matrix interferences during analysis. Recently developed dilute-and-shoot approaches for urine attempt to remove the majority of matrix compounds by chromatographic separation and take advantage of highly sensitive mass spectrometers [10, 11]. For blood analysis, dried blood spots (DBS) currently undergo a comeback concerning medical or forensic issues mainly due to improved detection limits and therefore leading to new fields of application [12, 13]. For example, DBS are suitable for extensive biomonitoring studies of environmental contaminants in humans or animals [14]. Particularly, for animal studies the application of DBS is an effective improvement concerning sample collection as only limited blood volumes are often available for analysis due to the low body weight of small animals [15]. Advantages of DBS compared to conventional blood collection are the minimally invasive sampling, simple sample preparation, easier storage, and shipping as well as the small volume required [16]. Samples can be collected on filter paper cards by heel, ear, or finger pricking as well as by spotting a known blood volume from ampoules after conventional vain puncture [17, 18]. Even if DBS are the standardized basis for medical tests, dried serum spots (DSS) are an additional opportunity for immunological tests and other blood counts. Likewise to DBS, DSS take advantage of simplified storage and shipment conditions [19, 20]. Recently, a DBS method for the detection of ochratoxin A in dried blood spots using HPLC-MS/MS has been published including the optimization of various basic parameters such as spotting volume and hematocrit which did not have a strong influence [21, 22]. Thus, the application of DBS for mycotoxin analysis showed positive findings in all samples for OTA as well as 2’R-OTA in the blood of coffee drinkers [21]. Here, we present the further development of this method by the incorporation of 27 relevant mycotoxins and metabolites and its application to serum and blood samples. Compounds monitored were aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), Alternaria toxins (ALT, AME, AOH), citrinin (CIT), and its metabolite dihydrocitrinone (DH-CIT). Furthermore, trichothecenes as deoxynivalenol (DON), deoxynivalenol-3-glucuronide (DON3-GlcA), T-2-toxin (T-2), HT-2-toxin (HT-2), HT-2-toxin4-glucoronide (HT-2-4-GlcA) have been incorporated. In addition, the structurally related cyclic hexadepsipeptides beauvericin (BEA) and enniatins (EnA, EnA1, EnB, EnB1) are included. Lastly, fumonisin B1 (FB1), ochratoxin A, and its thermal degradation product 2’R-ochratoxin A as well as (...truncated)