Tsq quantum ultra

The quantities of EAs were determined by liquid chromatography coupled with a tandem mass spectrometry (LC/MS/MS) (TSQ Quantum Ultra, ThermoFisher Scientific, Villebon sur Yvette, France) at Qualtech laboratory (Vandœuvre, France).
After preparation, diets were analyzed four times and the mean EA concentrations were 2.36 and 5.05 mg/kg, for diets with ergot dose 1 and 2, respectively. The most abundant alkaloid was ergotamine, followed by ergosine, ergocristine and their corresponding-inine epimers (Table 4). The amount of the-ine isomers was approximately two-thirds of total alkaloids.
Contamination with other mycotoxins was investigated. Deoxynivalenol was naturally present in the wheat (19 μg/kg) and corn (171 μg/kg) but at an insignificant rate. Other mycotoxins, including DON acetylated, nivalenol, T2, HT-2, zearalenone and fumonisin were below the detection limit.

Aflatoxins in milled maize and peanuts were extracted with previously described method (Warth et al., 2012 (link)). One gram of each sample was placed into a 5 ml polypropylene tube and extracted with 5 ml acetonitrile/water (84/16, v/v). The samples were mixed for 1 min on vortex, and then sonicated for 60 min. After centrifugation, 1 ml of supernatant was transferred into new tubes and 1 ml of hexane was added. Following a 10 min extraction, 500 μl of the upper (organic) layer was sampled and used for LC-ESI-MS aflatoxin analysis with a Thermo Surveyor plus HPLC system coupled to a TSQ Quantum Ultra mass spectrometer (Thermo Scientific, CA, USA) (Warth et al., 2012 (link)). The mobile phase consisted of eluent A (MeOH) and eluent B (water containing 5 mM ammonium acetate and 0.05% formic acid); the flow rate for the analysis was 0.3 mL/min. The linear gradient program was as follows: 0–1 min, 20% A; 1–4 min, 20–100% A; 4–5 min, 100% A; 5–5.5 min, 100%–20% A; 5.5–7 min, 20% A, giving a total analysis time of 7 min. Authentic reference standard aflatoxin compounds (AFB1, AFB2, AFG1, and AFG2) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and used as standards for identification and quantification.

LC–MS/MS investigations were carried out employing a Thermo Scientific Vanquish LC system connected to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Scientific, Orlando, FL, USA) equipped with a heated electrospray ionisation source (H-ESI). Xcalibur 4.4 Software (Thermo Scientific) was used for method setup, data processing and reporting. The mass spectrometer was operated with a H-ESI interface handled in both positive and negative ionization modes and was utilized for the multiple reaction monitoring. Instrument settings were: positive ion: 3600 v, negative ion: 3400 v, sheath gas 0.3 mL/min, aux gas 0.3 mL/min, ion transfer tube temp 345 °C, vaporizer temp 350 °C.
Chromatographic fractionation was accomplished on a Thermo Hypersil GOLD C18 column (1.9 µm, 100 × 2.1 mm) at 30 °C, and versatile stage comprised 0.1% formic corrosive and water for dissolvable A and acetonitrile for dissolvable B at a stream rate of 0.3 mL/min. The gradient elution conditions of mobile phase B are as follows: 0–0.5 min: 5%, 0.5–2 min: 5–8%, 2–2.1 min: 40%, 2.1–4 min: 40–50%, 4–6 min: 50–60%, 6–6.1 min: 60–70%, 6.1–8 min: 70–80%, 8–8.1 min: 80–5%, 8.1–10 min: 5%. The sample size for analysis was 3 μL.

To perform the flow injection measurements, a LC-20 AD pump equipped with a low-pressure gradient unit and the standard mixer (Shimadzu Benelux, 's-Hertogenbosch, The Netherlands) delivered the mobile phase at a flow rate of 20 µL/min. The mobile phase consisted of ACN/water/NH₄FA pH 3.7 (45/54/1, v/v/v). A HTC PAL (CTC Analytics, Switzerland) with a 10 µL loop was used to inject test solutions into the solvent flow. The loop was overfilled three times. The same TSQ Quantum Ultra triple-quadrupole mass spectrometer with IonMax HESI2 interface (Thermo Scientific, Erembodegem-Aalst, Belgium) was used in SRM mode. The source settings were as follows: vaporizer temp. off, capillary temp. 300 °C, sheath gas press. 10 a.u., auxiliary gas press. 1 a.u., sweep gas press. 1 a.u. The collision gas was argon at 1.5 mTorr. The spray voltage was set to 3 kV and the tube lens to 110 V. The following transitions (respective collision energies in parentheses) were continuously measured for the run time of 2 min: for Caffeine m/z 195->83 (30 eV), 195->110 (30), 195->138 (30); for AFB1 m/z 313->241 (37 eV), 331->270 (29).

All commercial reagents and solvents were used as received without further purification unless otherwise indicated. Synthetic compounds 3a–d and 4a–l were directly used for reaction without further purification and characterization. Melting points were recorded on an SGW X-4 microscope melting point apparatus (Shanghai Tech Instrument Co., Ltd., Shanghai, China). Infrared spectra (IR) were performed on NICOLET iS10 sepectrometer (Shimazu Co., Ltd., Kyoto, Japan). NMR spectra were recorded on a Bruker Avance 500MHz spectrometer (Bruker Co., Ltd., Zurich, Switzerland) at room temperature with tetramethylsilane (TMS) as an internal standard and CDCl₃ or DMSO-d₆ as solvents. Mass spectra (MS) were obtained by LCMS-IT-TOF spectrometer (Shimadzu Co., Ltd., Kyoto, Japan) or TSQ Quantum Ultra (Thermo Scientific Co., Ltd., Madison, WI, USA). Elemental analysis for C, H, O, and N were carried out with Elementar VarioMICRO Cube analyzer (Elementar, Frankfurt, Germany).