Xdb c18 guard column

The protocols in this section were approved by the Ethics Committee of Zhongshan Hospital, Xiamen University. The methods employed were performed in accordance with the approved guidelines. After acquiring informed consent, human saliva was collected from individual volunteers (two males and two females; Minimum age: 22; maximum age: 28) in the morning prior to oral cleaning. The collected saliva samples were centrifuged at 12,000 rpm for 20 min. The supernatant was collected and filtered through the 0.45 mm membrane filter to remove any debris. HBAMP was added into the human saliva to achieve the final concentration of 500 μg/ml. The saliva with HBAMP was incubated at 37 °C. At 0, 5, 20 and 60 min, 1 ml samples were taken from the total sample and served in the specific sample bottles, the machine will automatically extract samples from the bottles to detect. The samples were analysed by reversed-phase HPLC using Zorbax Eclipse XDB-C18 analytical column (150 × 4.6 mm, Agilent Technologies, Inc., Santa Clara, CA, USA) protected by a XDB-C18 guard column (4 × 4 mm). For the elution of HBAMP, a flow rate of 1.2 mL/min and a linear gradient from 88:12 to 65:35 (0.1% TFA in water: 0.1% TFA in acetonitrile) for 10 min were employed. Total run time of HPLC-UV (215 nm) analysis was 15 min and the injection volume was set at 40 μL.

Profiling of tissue/serum metabolites was performed on a RRLC 1260 system (Agilent Technologies, Santa Clara, CA, USA) coupled to a QTOF 6520 detector (Agilent Technologies, Santa Clara, CA, USA), equipped with an electrospray source operating in full scan mode, from 50 to 1000 Da for both positive and negative ionization modes. The gas temperature was set at 350 °C, gas flow of 12 L/min, capillary voltage at 3.5 kV, and fragmentor voltage at 120 V. Two reference masses were used to maintain the mass accuracy during analysis: m/z 121.050873 and m/z 922.009798 in positive mode, and m/z 112.985587 and m/z 980.016375 in negative mode. Sample aliquots of 10 mL were injected on a Sb-Aq column (100 × 2.1 mm, particle size 1.8 mm, Agilent Technologies, Santa Clara, CA, USA), protected by a XDB-C18 guard column (5 × 2.1 mm, particle size 1.8 mm, Agilent Technologies) and heated at 40 °C. The gradient mobile phase consisted of 0.2% acetic acid (v:v in water) (A) and acetonitrile (B). The flow rate was set at 0.3 mL/min. The initial condition was set as 98% phase A and 2% phase B, and the gradient changes as follows: from 2% to 95% phase B in 7 min, 95% phase B for 3 min, and equilibration with 2% phase B for 3 min. The autosampler was kept at 4 °C. Profiling data were treated as described below.