Histamine Quantification in Hdc−/− Mouse Feces

Fecal specimens were weighed and dissolved in 200 μL of 50/50 PBS/methanol by vortexing and ultrasound disruption for 4 minutes with 30-second intervals. After spinning for 5 minutes, 14,000 × g at 4°C, the supernatant fluids were size-fractionated with Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff, 3 kDa; Millipore, Temecula, CA). The histamine quantities in the collected supernatant fluids were determined by liquid chromatography-mass spectrometry using selected reaction monitoring (SRM).

For histamine quantification, 30 μL of each sample was dried in a SpeedVac for 2 hours. The dried samples were mixed with 30 μL of 20 ng/mL histamine-d4 (histamine-α,α,β,β-d4; CDN Isotopes, Pointe-Claire, Canada) solution by vortexing for 1 minute. The prepared samples were loaded into a Shimadzu (Kyoto, Japan) SIL-20ACxr autosampler and separation was achieved using a Shimadzu Nexera-XR HPLC system. Samples (5 μL) were loaded onto a Phenomenex (Torrance, CA) 1 mm × 50 mm phenylhexyl reversed phased column equipped with a Phenomenex phenylhexyl 4 mm × 2 mm guard column. The aqueous mobile phase (A) consisted of water/acetonitrile/formic acid/perfluoroheptanoic acid (99.3:0.5:0.1:0.1 v/v/v/v), and the organic mobile phase (B) consisted of acetonitrile/formic acid (99.9:0.1 v/v). Column flow was set at 80 μL/minute. The elution gradient was optimized as follows: started from 20% B for 0.5 minute and increased to 70% B over 5.5 minutes; ramp to 80% B after 0.1 minute and hold for 1 minute; ramp back to 20% B over 6 seconds and maintained at 20% for a total chromatographic run time of 12 minutes to re-equilibrate.

SRM was performed on a Sciex (Framingham, MA) 6500 QTRAP mass spectrometer equipped with a Turbo V ion source. The mass spectrometer was operated in the positive ion mode under the following conditions: curtain gas of 20 psi; collision gas, High; spray voltage, 4.5 kV; ion source gas 1, 20 psi; ion source gas 20, 2 psi; interface heater temperature, 175°C; Q1 and Q3 resolution, unit; scan time, 100 milliseconds; de-clustering potential, 100 V; entrance potential, 8 V; and collision exit potential, 10 V. The instrument was calibrated by using Sciex PPG calibration standard and tuned to the manufacturer's specifications. SRM transitions monitored for histamine were 112 → 95 (20 eV) and 112 → 68 (30 eV). For histamine-d4, the SRM transitions 116 → 99 (20 eV) and 116 → 72 (30 eV) were monitored. Data were acquired with Analyst Software version 1.6.2 (AB Sciex LP, Concord, ON, Canada) and quantification performed using Multiquant Software version 3.0.1 (AB Sciex LP).