Uhplc system

AEA, 2-AG, AA, OEA, and PEA were extracted, purified, and quantified in serum by stable isotope dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) as previously described [22 (link),23 (link),24 (link)]. LC-MS/MS analyses were conducted on a Sciex (Framingham, MA, USA) QTRAP^® 6500+ mass spectrometer coupled with a Shimadzu (Kyoto, Japan) UHPLC system. Liquid chromatographic separation was obtained using 5 μL injections of samples onto a Kinetex 2.6 μm C18 (100 × 2.1 mm) column from Phenomenex (Torrance, CA, USA). The autosampler was set at 4 °C, and the column was maintained at 40 °C during the entire analysis. Endocannabinoids were detected in positive ion mode using electron spray ionization (ESI) and a multiple reaction monitoring (MRM) mode of acquisition, using d₄-AEA as internal standard (IS). The IonDrive^TM Turbo V source temperature was set at 450 °C with an ion spray voltage of 4000 V. The curtain gas was set at 30.0 psi. The nebulizer gas (Gas 1) was set to 40 psi, and the turbo heater gas (Gas 2) was set to 40 psi. The dwell time was set to 30 ms. The collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) for the monitored transitions are listed in Table 1. The LC-MS/MS chromatogram is presented in Figure 1. The serum levels of AEA, 2-AG, OEA, PEA, and AA were measured in duplicate against standard curves.

To evaluate the levels of MPS and MP after infusion at each blood collection time, sera were prepared and analyzed in duplicate. To 100 µL of serum, 30 ng (10 μL of a methanolic solution at 30 µg/mL) of the internal standard hydrocortisone succinate (HC) and budesonide (BUD) were added before each analysis. Afterwards, samples were extracted with 6 mL of ethyl acetate, evaporate to dryness with filtrated air at 30 °C using Eppendorf Concentrator 5301 (Eppendorf AG, Hamburg, Germany) and re-dissolved in 100 μL methanol. The chromatography was performed under reverse phase conditions using a Shimadzu (Kyoto, Japan) UHPLC System as described in more details in the Supplementary materials. Results are presented as the mean ± STDEV. The large number of data points allowed for a detailed data analysis and NCAanalysis using Phoenix ® WinNonlin ® www.certara.com (Certa USA, Inc., Princeton, NJ, USA).

Following the incubation period, 25 g maize grains were collected from the test samples and ground to a fine powder under aseptic conditions and suspended in 250 mL solution of acetonitrile and water (v/v, 6:4). The blend was mixed for 15 min at 140–160 rpm and the supernatant was collected by centrifugation at 6000 rpm for 5 min. The supernatant was subjected to clean-up with immunoaffinity columns of DON and ZEA in according to the instructions of the manufacturer (Vicam, Waters business, USA). The quantification DON and ZEA were done as per our previous reported methodology (Mudili et al., 2014 (link); Kalagatur et al., 2015 (link)) using UHPLC system (Nexera, Shimadzu, Japan). The quantification of DON and ZEA was deducted from their respective standard calibration curve and concentration was expressed in μg/g.

Chromatographic analysis was performed using a UHPLC system (Shimadzu, Kyoto, Japan) consisting of an LC-30AD binary pump, an autosampler (Model SIL-30SD), an online degasser (DGU-20A5R), and a temperature controller for columns (CTO-30A). Separation was carried out on an extended C₁₈ Column (2.1 mm × 100 mm, 1.8 μm; Agilent, Palo Alto, CA, USA) at 30 °C and the flow rate was 0.3 mL/min. The optimal mobile phase consisted of A (HCOOH/H₂O, 0.1:100, v/v) and B (C₂H₃N). The optimized UHPLC elution conditions were as follows 0–2 min, 3–15% B; 2–7 min, 15–20% B;7–8 min, 20% B; 8–9 min, 20–30% B; 9–13 min, 30–32% B; 13–21 min; 32–54% B; 21–23 min, 54–100% B; 23–27 min, 100–3% B; and 27–28 min, 3% B. The injection volume was 2 μL.

Synthesis of the analogue peptide was performed according to the solid phase approach using standard Fmoc methodology, with a Biotage Initiator+Alstra (Uppsala, Sweden) automated microwave synthesizer (for detailed conditions, see Supporting Information ).
The quantification of BRP2 in buffalo ricotta digesta and BRF2 was performed on a Nexera UHPLC system coupled online to an LCMS-8050 mass spectrometer (Shimadzu, Kyoto, Japan), equipped with an ESI source operated in positive mode. MS/MS analysis was conducted in selected reaction monitoring (SRM), employing the synthetic peptide as an external standard. Stock solution was prepared in water, the calibration curve was obtained in a concentration range of 0.1-125 μg L⁻¹ with eight concentration levels, and triplicate injection of each level was run. Peak areas of BRP2 were plotted against the corresponding concentrations. Linear regression was used to generate the calibration curve (y = 0.0004x–1.5321) with R² values being ≥0.9998 (see Supporting Information ).

The UHPLC–MS/MS system is composed of a Shimadzu UHPLC system equipped with a LC-30AD binary pump, an on-line degasser (DGU-20A5R), an autosampler (Model SIL-30SD), a column temperature controller compartment (CTO-30A), and a 5500 triple quadtandem mass spectrometer (AB Sciex, Concord, ON, USA) with an electrospray ionization (ESI) source. The separation of the analytes was achieved on a Waters BEH-C₁₈ column (100 mm × 2.1 mm, 1.7 μm).