Scherzo sm c18

All extracts (10 μL injection volume) were analyzed on 1290 UPLC chromatograph (Agilent Technologies, Santa Clara, CA) using 3.0 μm, mixed-mode Scherzo SM-C18, 2 × 150 mm column (Imtakt, Portland, OR) and 1.8 μm Zorbax Eclipse octadecyl 2 × 200 mm column coupled to Agilent 6550 iFunnel Q-TOF mass spectrometer in positive and negative ESI in the mass range 100–1000 m/z. Additional details including LC-MS settings are provided in SI.

The 1260 infinity LC system (Agilent Technologies, Palo Alto, CA, USA) was used for the separation of ET and ETA. To select a suitable separation column for the analyte, four types of columns with different fixed surfaces were tested. The columns tested included X bridge C18 (50 × 2.1 mm I.D., 3.5 μm, Waters Corporation, Milford, MA, USA), Scherzo SM-C18 (30 × 2.0 mm I.D., 3.0 μm, Imtakt, Portland, OR, USA), Hypurity Aquastar (50 mm × 2.1 mm I.D., 5.0 μm, Thermo Fisher Scientific, Waltham, MA, USA) and Hypercarb (50.0 mm × 2.1 mm, 5.0 μm, Thermo Fisher Scientific, Waltham, MA, USA). For appropriate separation of the analyte, the column temperature was optimized and maintained for the entire analysis time.
The mobile phase compositions and the ratios of acid, buffer, and organic solvents were optimized. The composition of the optimized mobile phase included 0.05% formic acid in deionized water for the solvent A and acetonitrile: methanol (50:50, v:v) for organic solvent B. The gradient elution included 15% B for 0.4 min, 85% B for 3 min to 5 min, and re-equilibration to 15%B for 7.5 min (Table 1). The analysis time was 7.5 min and the injection volume was 5 μL. The auto-sampler temperature was maintained at 4 °C to prevent analyte degradation. The Sciex software (version 1.5.1, Sciex, Toronto, ON, Canada) was used for data collection.

For quantification of the intracellular histamine, each cell fraction from peritoneal lavage and peripheral blood was fixed with Cellcover (AL Anacyte Laboratories UG, Hamburg, Germany) and sorted by FACS Aria Fusion. Histamine level was quantified with a modified protocol from the previously reported methods using TSQ Vantage AM mass spectrometer (Thermo Fisher Scientific, Waltham, MA)^{41 (link),42 (link)}. Reversed-phase Scherzo SM-C18 (3 mm × 100 mm, 3 μm, Imtakt Corp. Japan) was used for the analytical column.