Quattro premier xe tandem mass spectrometer

A Waters Acquity UPLC apparatus paired to a Quattro Premier XE Tandem Mass Spectrometer (Waters, Milford, MA, USA) was utilized for the identification and quantification of the analytes. Data acquisition and processing utilities included the use of the MassLynx™ (V. 4.1) and QuanLynx^® (V. 4.1) software (Micromass, Manchester, UK). The column used to separate the analytes of interest was a Symmetry C18 column (150 mm × 2.1 mm i.d. 5 μm particle size) with a guard column (10 mm × 2.1 mm i.d.) of the same material (Waters, Zellik, Belgium). The chromatographic conditions set were similar to those of Ediage et al. [44 (link)]. Mobile phase A contained acetic acid/methanol/water (1/5/94, v/v/v) and 5 mM ammonium acetate (0.385 g/L), and mobile phase B contained acetic acid/water/methanol (1/2/97, v/v/v) and 5 mM ammonium acetate (0.385 g/L). With a sample injection volume set at 10 μL, the total analytical run time was 28 min with a pressure that varied between 0 and 5000 psi. The mass spectrometer was operated using selected reaction monitoring (SRM) channels in positive electrospray ionization (ESI+) mode. Further details on the mycotoxin transitions are reported by De Boevre et al. [45 (link)] and Monbaliu et al. [46 (link)]. For the identification of the targeted mycotoxins, the criteria of the Commission Regulation 657/2002/EC [47 (link)] were followed.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of PAs of the plant fractions were conducted based on a protocol described by Cheng et al. (2011 (link)). Prior to analysis, 10 μl of each fraction or sub-fraction in DMSO was diluted with 1 ml of water and transferred to an HPLC vial. Analysis was conducted on an Acquity UPLC system coupled to a Quattro Premier XE tandem mass spectrometer (Waters, Milford, MA, USA) operated in positive electrospray mode. Separation of the PAs was accomplished on a BEH C18 150 × 2.1 mm, 1.7 μm, UPLC column (Waters) by using an acetonitrile/water/6.5 mM ammonia gradient, from 0 to 50% acetonitrile in 12 min. Column temperature was set at 50 °C and the flow was at 400 μl/min. The PAs were quantified using external standard calibration prepared from a blank extract spiked with PA standards (range: 0–500 ng/ml). The limit of quantification for individual PAs in the fractions was approx. 0.5 μg/g dry plant material.
The five fractions were all found to contain PAs. The majority of the PAs were detected in the CHCl₃ fraction (67%, which is equal to 0.24 mM PAs) (Fig. S3). The n-BuOH fraction contained 24%, the H₂O fraction 5% and the EtOAc fraction 3% and the n-hexane fraction 0.2% of the total PA content (Fig. S3). The WFT survival rates on the fractions were not correlated with their PA content (r = 0.03, n = 5, NS).

Urine samples were collected on site. Early morning saliva samples were obtained using a Salivette® (Sarstedt AG & Co. Nümbrecht, Germany) swap which was provided during the visit together with a return envelope. Participants were requested to obtain saliva immediately after awakening, between 06.00 and 09.00 a.m. and prior to having breakfast and to return the sample by postal mailing.
Urine and saliva were stored at -80 °C. Both samples were analysed for cortisol and cortisone. 0.1 mL of urine or 0.1 mL of saliva was used to assess cortisol and cortisone concentrations, using an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Internal standards (13C3 labeled cortisol and cortisone) were added to the samples. Samples were extracted using supported liquid extraction (Isolute, Biotage, Uppsala, Sweden) and analysed by LC-MS/MS [Quattro Premier XE tandem mass spectrometer (Waters Corp., Milford, Massachusetts, USA)]. Lower limit of quantitation was 1.0 nmol/L for cortisol and 0.5 nmol/L for cortisone. The intra-coefficients of variation (CV%) for cortisol were 7 and 4% at a level of 3 and >5 nmol/L, respectively, and for cortisone <5% at all levels >2.8 nmol/L. The inter-CV% was <11% for both cortisol and cortisone.