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Detection of the Cytotoxic Penitrems A-F in Cheese from the European Single Market (EU) by HPLC-MS/MS Svetlana Kalinina, Annika Jagels, Sebastian Hickert, Lucas Maciel Mauriz Marques, Benedikt Cramer, and Hans-Ulrich Humpf J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06001 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018
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Journal of Agricultural and Food Chemistry
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Detection of the Cytotoxic Penitrems A-F in Cheese from the European Single
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Market (EU) by HPLC-MS/MS
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Svetlana A. Kalinina†,‡, Annika Jagels†, Sebastian Hickert†,‡, Lucas M. Mauriz Marques§,
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Benedikt Cramer†, Hans-Ulrich Humpf*†, ‡
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†
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48149 Münster, Germany.
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‡
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§
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Preto, University of São Paulo, Ribeirão Preto, CEP 14049-900, Brazil.
Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45,
NRW Graduate School of Chemistry, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany.
Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirão
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*Corresponding author (Tel: +49 251 33391; Fax: +49 251 83 33396; E-mail:
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[email protected])
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ABSTRACT
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Penitrems are fungal indole diterpene derived tremorgenic secondary metabolites, which
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are mainly produced by Penicillium spp. Several cases of intoxications with penitrems and
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subsequent occurrences of penitrem A in foodstuff underline the need for reliable
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quantitation methods for the detection of these mycotoxins in food. In this study, a simple
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and fast HPLC-MS/MS method for the quantitative analysis of penitrems A-F in cheese was
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developed. Therefore, penitrems A-F were isolated from Penicillium crustosum as analytical
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reference standards. The analysis of 60 cheese samples from the European single market
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(EU) revealed the occurrence of penitrem A in 10% of the analyzed samples with an average
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concentration of 28.4 μg/kg and a maximum concentration of 429 μg/kg. In addition to
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penitrem A, other members of the group of penitrems namely penitrems B, C, D, E, and F,
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were for the first time quantitatively detected in food samples, although in lower
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concentrations and with lower incidence in comparison to penitrem A. Moreover, we report
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cytotoxic effects of all penitrems on two cell lines (HepG2, CCF-STTG1). This clearly
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underlines their relevance and the importance to analyze food samples in order to get
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insights into the human exposure towards these mycotoxins.
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Keywords: penitrems, mass spectrometry, liquid chromatography, cheese.
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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Penitrems are toxic secondary metabolites produced mainly by Penicillium species.1
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Penitrem A, 1 (Figure 1), being a paxilline-like, 8 (Figure 1) indole diterpene, was first isolated
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by Wilson et al.3 from Penicillium cyclopium.2 Its absolute configuration as well as the
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structures of the other members of the group of penitrems B, C, D, E, and F, 2-6 (Figure 1)
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were described later in 1983. In 2003, a new member of the penitrems was isolated by
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Gonzalez et al.4 and named penitrem G, 7 (Figure 1). Intoxications with penitrems have been
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well documented in animals and humans. Clinical signs of poisoning comprise severe
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generalized muscle tremors accompanied by incoordination, seizures, muscle fasciculations,
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and generalized convulsions, eventually resulting in death.5-8 In 2005, the ingestion of food
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contaminated with 1 was shown to cause tremor syndromes in humans.9 A comprehensive
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toxicity profile was also well described and the tremorgenic dose of 1 after oral
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administration (PO) to mice was found to be 0.5 mg/kg body weight (bw). The LD50 in mice
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was reported to be 10 mg/kg bw (PO), whereas the intraperitoneal (IP) LD50 is 1.1 mg/kg.10
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Chicken showed neurological syndromes after administration of 5 mg/kg bw (PO) followed
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by mortality at higher concentrations.11 In lower doses, 1 exhibits high insecticidal activity
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against the insects Spodoptera frugipera and Heliothis zea.12 Insecticidal activities were also
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reported for penitrems A-D, and F.4 Notably, the chlorinated penitrems A, C and F exhibit the
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highest acute toxicities. The halogen atom was proposed to be important for the lethality of
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treated insects, whereas the epoxy group seems to be responsible for their delayed
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mortality. In general, relative toxicity ranged in the order 1 > 6 > 3 > 2 > 4.4
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Analytical approaches to detect penitrems in food, feed, and physiological samples were
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focused on 1 in the past. For instance, Braselton and Rumler13 analyzed the stomach
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contents of dogs, which exhibited neurological disease by gas chromatography coupled with 3 ACS Paragon Plus Environment
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a tandem mass spectrometer (GC-MS/MS), revealing the presence of 1. Penicillium toxins,
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including 1, were further analyzed in food and feed by using liquid chromatography coupled
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to tandem mass spectrometry (LC-MS/MS). The limit of detection (LOD) for 1 was 5 μg/kg.14
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In 2005, Tor et al.15 elaborated a rapid LC-MS/MS method for the detection of 1 in serum
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and urine samples. Furthermore, an LC-MS/MS based multi-mycotoxin method including 1
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with an LOD of 5 μg/kg was reported by Sulyok et al.16
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For the quantitative determination of penitrems B-F, only semi-quantitative methods have
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been applied so far.17 Analytical studies have been limited, mostly due to the lack of
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reference standards. Up to date, there is only one publication regarding the presence of
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penitrems A-F in food waste of private households in a concentration range of 30-
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7500 μg/kg.17 In addition, penitrems A-D were detected in organ tissues of poisoned dogs in
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a semi-quantitative matter.18 A quantitative method for the simultaneous determination of
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all penitrems A-F has not been described so far.
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In our previous study, we reported the optimal incubation parameters for the production of
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penitrem A-F in high concentrations applying various abiotic factors and stress factors.19 In
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this study, all penitrems A-F were isolated in high yield and high purity. It was further shown
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that penitrems were also produced by P. crustosum on a cheese medium in high
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concentrations and cheese might thus be a food potentially susceptible to contamination
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with penitrems. Consequently, the objectives of the presented study were the development
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of a robust HPLC-MS/MS method for the quantitative determination of penitrems A-F and its
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application to the analysis of cheese samples purchased from the EU market. In addition,
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cytotoxic effects on two different cell lines were investigated to get further insights into the
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toxicity of penitrems A-F.
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MATERIALS AND METHODS
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Chemicals and Reagents
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If not otherwise mentioned all solvents were of gradient grade and purchased from VWR
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(Darmstadt, Germany). HPLC-MS grade acetonitrile was purchased from Fisher Scientific
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(Schwerte, Germany). ASTM type 1 water was obtained with a Purelab Flex 2 system from
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Veolia Water Technologies (Celle, Germany). Formic acid was from Merck (Darmstadt,
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Germany). Penitrems A-F (purity≥96%) were isolated and spectroscopically characterized as
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reported in our previous study.19
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Penitrems A-F were each dissolved in MeCN/H2O (80:20, v/v), exact concentration was
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determined using molar absorptivity values obtained on a V-750 series UV spectrometer
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(Jasco, Gross-Umstadt, Germany). Obtained stock solutions were used for the calibration.
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Calibration
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Due to the lack of isotopically labeled standards for penitrems, matrix-matched calibration
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was used for quantitation. The matrix-matched blank solution was obtained by combining
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three different blank extracts of the cheese samples. The extraction was carried out after
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mixing and homogenizing of the cheese samples. Solutions of the penitrem A-F were
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evaporated under a gentle nitrogen stream at 40 °C and subsequently re-dissolved in 1 mL of
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blank cheese matrix extract. Eleven calibration points ranging from 2-202 ng/mL of
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penitrems (A-F) were prepared in blank cheese matrix extract using a solvent mixture of
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MeCN/H2O (80:20, v/v). Solvent-calibration was prepared in the same concentration range
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as in the matrix-matched calibration. The signal suppression/enhancement factor (SSE) was
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determined using the equation: 5 ACS Paragon Plus Environment
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Slope
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SSE % = 100 x
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Samples
matrix-matched calibration Slopesolvent calibration
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Different types of cheese (firm, soft, hard, processed, supplemented with nuts, fruits, fungal
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culture) were purchased in several European countries (Germany, United Kingdom, the
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Netherlands and Belgium). After homogenization a part of each sample was transferred to
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50 mL polypropylene tubes and stored at -22 °C until sample preparation.
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Sample preparation
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5.0 g (± 0.05 g) of cheese was placed into a 50 mL plastic tube and the exact sample weights
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were determined. 10 mL of the extraction solvent (MeCN/H2O (80:20, v/v)) were added and
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the mixture was vortexed for 30 s. Extraction was carried out on a laboratory shaker for 1 h
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at 250 rpm. Subsequently, the samples were centrifuged (3.000 × g, 5 min) and approx. 1 mL
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of the supernatant was passed through a 16 mm, 0.45 µm membrane filter (Phenomenex,
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Aschaffenburg, Germany). The filtered extracts were stored at -22 °C until HPLC-MS/MS
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analysis. All samples were analyzed in triplicate.
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HPLC-MS/MS parameters
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A QTrap 5500 mass spectrometer (SCIEX, Darmstadt, Germany) equipped with an LC system
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(Shimadzu, Kyoto, Japan) was employed for the analysis using electrospray ionization in the
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positive mode. The source temperature was 500 °C, curtain gas was set to 35 psi, the ion
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source gases 1 and 2 to 35 and 45 psi, respectively. The ion spray voltage was 4500 V and the
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collision gas was set to “high”. A dwell time of 40 ms was used for all multiple reaction
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monitoring (MRM) transitions. The separation of penitrems was performed on a column 150
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mm x 2.0 mm i.d., 3 µm, Reprosil Gold RP-18, with a 5 mm x 2 mm i.d. guard column of the 6 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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same material (Dr. Maisch GmbH, Ammerbuch, Germany) using a binary gradient consisting
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of MeCN (solvent A) and H2O (solvent B), both containing 0.1% formic acid. The column oven
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was operated at 40 °C and the flow rate was 350 µL/min. Twenty µL of the sample solution
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were injected. Starting conditions of the gradient were: 0-6 min, 40-100% A; 6-10 min held
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at 100% A, followed by re-equilibrating the column under starting conditions (40% of A) for 4
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minutes.
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Method performance
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The limits of detection (LODs) and the limits of quantitation (LOQs) were based on a signal-
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to-noise ratio (S/N) equal 3 for LOD and equal 10 for LOQ. Therefore cheese matrix obtained
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from a non-contaminated sample was spiked with penitrems down to concentrations with
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the respective S/N ratios. Recovery rates for each analyte were examined by spiking 5 g of
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blank cheese at three different levels within the calibration range (50, 100, 150 μg/kg - 100,
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200, 300 ng/g) in triplicate, followed by the sample preparation described above.
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Quantitative determination of penitrems in cheese samples
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To analyze the obtained results, Analyst software, ver. 1.6.2, (SCIEX, Darmstadt, Germany)
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was employed. Calibration curves were generated by plotting the peak area against the
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concentration for each penitrem. Each calibration solution was measured in duplicate and
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the peak areas were averaged. The slope and intercept of these curves were used to
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calculate the concentrations of each penitrem. The sample weight was taken into account
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and the results were not corrected by recovery rates. Results are based on three
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independent measurements and given as mean ± standard deviation.
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Cytotoxicity
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For cytotoxicity assays, 3.0 mg of 1, 2.0 mg of 5 and 1.0 mg of penitrems B, C, D and F, were
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dissolved in MeOH. These stock solutions were diluted to the respective concentrations with
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serum free culture medium. Human liver cancer cells (Hep G2, ACC 188) (DSMZ,
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Braunschweig, Germany) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM)
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supplemented with 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES
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buffer), 100 µg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glutamine and 10% (v/v) fetal
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calf serum (FCS) using standardized culture conditions (37 °C, 5% CO2, saturated humidified
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atmosphere). Human astrocyte cells (CCF-STTG1) (ATCC, Manassas, VA) were cultivated in
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Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 100 µg/mL
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streptomycin, 100 U/mL penicillin, 2 mM L-glutamine and 10% (v/v) FCS. Twice a week both
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culture media were changed and the cells were subcultivated after trypsination when a
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microscopic confluence was at the level of 80%. For the evaluation of cytotoxic effects of
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penitrems on both cell lines, Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Tokyo, Japan)
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was used. The assay was performed according to the manufacturer’s instructions and our
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previous studies.20-22 Both cell lines were treated with penitrems A-F (1 µM – 50 µM) and
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incubated for 48 h. After toxin application, media were replaced by 100 µL/well of the
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tenfold diluted water-soluble tetrazolium dye solution WST-8 (2-(2-methoxy-4-nitrophenyl)-
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3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
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incubation for 70 min at 37 °C. The viability of the cells was assessed by their ability to
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reduce WST-8 dye to a water-soluble formazan, which was analyzed by measuring the
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formazan absorbance at a wavelength of 450 nm with a TECAN infinite 200 PRO microplate
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reader (Tecan, Salzburg, Austria). Absorption values were subtracted by the average
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absorption of six cell-free blank wells. The results for toxin-exposed cells were normalized to
monosodium salt) followed by
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the values of a solvent control (1% MeOH). Tests were repeated in triplicate for each cell line
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from three independent passages (n≥9). The data are presented as the mean ± standard
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deviation (SD). The IC50 values were calculated by log-linear regression and significance
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indicated refers to the significance level as compared to the solvent treated control (1%
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MeOH) calculated with the OriginPro 2016G (64-bit) Sr2 b9.3.2.303 (SF8T5-3089-7901139)
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(OriginLab Corporation, Northampton, USA). Obtained data were evaluated by analysis of
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variance (ANOVA) and Student’s t test, * statistically significant (p ≤ 0.01), ** statistically
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highly significant (p ≤ 0.001).
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RESULTS AND DISCUSSION
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Method development and performance
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A simple and fast HPLC-MS/MS method for the quantitative determination of penitrems A-F
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in cheese was developed. The penitrems were analyzed in the positive ion electrospray
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ionization mode, which had the best relative signal intensity. The separation of penitrems
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was achieved by HPLC using a C18 column with a binary gradient consisting of MeCN/H2O. A
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chromatogram displaying the recorded multiple reactions monitoring (MRM) transitions and
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the observed typical elution order of penitrems A-F is shown in Figure 2. The retention times
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were as follows: 5, 7.1 min; 1, 7.8 min; 4, 8.6 min; 2, 8.8 min; 3, 9.1 min; 6, 9.3 min. The
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signal intensities of penitrems A and E are much higher than for any other penitrems, due to
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enhanced ionization, which could be explained by the presence of an additional hydroxy
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group at C15 of the cyclobutyl unit. The MRM transitions used to quantitate the analytes
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included m/z 600 → 524 for 5, m/z 634 → 558 for 1, m/z 568 → 550 for 4, m/z 584→ 566 for
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2, m/z 602 → 532 for 3 and m/z 618 → 530 for 6 (Figure 2). For quantitation, matrix-
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matched calibration was used, whereas signal enhancement was observed for all penitrems 9 ACS Paragon Plus Environment
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in cheese matrix. Signal enhancement was: 1, 239%; 2, 228%; 3, 453%; 4, 377%; 5, 348%; 6,
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455%. Further method characteristics are given in Table 1. The limits of detection (LODs) and
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the limits of quantitation (LOQs) were determined by dissolving the reference standards in a
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blank cheese matrix extract based on a signal-to-noise ratio (S/N) equal 3 for the LOD and
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S/N 10 for the LOQ in a working range 2-202 ng/mL (Table 1). Recovery rates were
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determined by spiking 5 g of blank cheese in triplicate (n=3) at three different levels
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(50, 100, 150 ng/mL). These spiked samples were worked up analogously to the samples.
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The lowest recovery rates at all spiking levels were detected for penitrems E and F with 57%
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and 74% respectively. Recoveries for all other penitrems A, B, C, D were found to be close to
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100%. Recoveries at three different spiking levels within the calibration range are
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summarized in Table 2.
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Screening of penitrems in cheese products
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The analysis of 60 cheese samples from the EU market by application of a newly developed
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HPLC-MS/MS method revealed the occurrence of 1 in 10% of the analyzed samples with a
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minimum concentration of 1.2 μg/kg and a maximum concentration of 429 μg/kg (Table 3).
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Furthermore, five of those samples were also contaminated with 5. Moreover, two of the
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samples that were found to contain penitrem A additionally contained penitrems B, C, and D
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with a maximum concentration of 13.5 μg/kg, 4.4 μg/kg and 15.4 μg/kg, respectively.
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Penitrem F was detected in one of the six positive samples only, but below LOQ
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(Table 3, Figure 3). In general, penitrems in dairy products can originate either from indirect
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contamination, because of the ingestion of contaminated feed by dairy animals; or direct
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contamination, which appears due to accidental or intentional moulds growing. The
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predominant flora found on cheese belongs to the genus Penicillium, which is known to be a
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producer of penitrems, demonstrating a high stability in cheese.24,25 In the context of the 10 ACS Paragon Plus Environment
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penitrem production direct contamination and therefore accidental growth of moulds is the
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major source. Four out of six positive samples contained nuts with various consistencies.
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Samples N2 and N3 contained whole walnuts placed on the cheese surface. In a cheese
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supplemented with hazel nuts, a variety of penitrems A-F was observed, which is typical for
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P. crustosum. Interestingly, the hard cheese supplemented with pistachios was found to be
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positive for penitrems A and E. Relying on these data it can be hypothesized that
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contaminated nuts could be a frequent source of mould. It is assumed that in these cases
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cheese played the role of medium or substrate for the cultivation of penitrems-producing
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moulds. The results of cultivating P. crustosum on cheese medium in our previous study
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confirm these findings.19 Also, we suggest that different Penicillium species could be involved
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in penitrem production regarding the tested samples. In processed cheese with walnuts
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(sample N3), only penitrem A in a high concentration of 429 μg/kg was detected. Recently, it
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was reported that P. melanoconidium is able to produce only penitrem A up to mg/kg scale,
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but no other penitrems.26 However, positive samples were found not only among cheese
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supplemented with nuts. For instance, in a creamy blue cheese (N3), 1 (1.2 μg/kg) and 5
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(
