Jasper lc system

The LC–MS/MS analysis utilized a Jasper LC system (Paolo Alto, USA) and an AB Sciex Triple Quad 4500 MD with electrospray ionization. The column used was the Venusil CAs column, 100 mm × 2.1 mm, 5 μm, from Bonna-Agela Technologies, Tianjin, China. Eppendorf Centrifuge 5430R with low-temperature and high-speed was used. MNs (1 mg/mL in methanol, Lot: C-110), 3-MT (1 mg/mL in methanol, Lot: M − 171), (±)-Metanephrine-d3 hydrochloride (100 μg/mL in methanol, Lot: M − 148), (±)-Normetanephrine-d3 hydrochloride (100 μg/mL in methanol, Lot: N-068), and 3-Methoxytyramine-d4 (100 μg/mL in methanol, Lot: M − 172) were purchased from Cerilliant (Texas, USA). HPLC grade methanol and acetonitrile were from Merk, USA. Formic acid was from Thermo Fisher Scientific (Waltham, MA, USA), and ammonium formate was from Sigma (St. Louis, MO, USA). Deionized water was purified using a Millipore Synergy UV water purification system (Billerica, MA, USA). 24-h Urine samples from apparently healthy volunteers were collected in clean containers containing 5 g of boric acid, and 1 mL samples were stored at −80 °C until analysis.

1-MNA was detected by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Briefly, cells were seeded in 10-cm plates, incubated for 24 h, and treated with different concentration of vanillin for 48 h. The cells were then harvested and washed with PBS twice. Next, 50% of the cells were fixed with methanol for LC-MS/MS, and the other 50% was used to extract total protein for quantification. A total of 5 µl of the samples was injected into an Eclipse XDB-C18 column (4.6×150 mm, 5 µm, Agilent Technologies, Inc.), which was connected to Jasper™ LC system (AB Sciex). The isocratic mobile phase, a mixture of 0.1% formic acid and methanol mixture (v/v), was delivered at a flow rate of 1 ml/min into the mass spectrometer electrospray ionization chamber. Quantitation was achieved by MS/MS Detection in positive ion mode for 1-MNA and internal standard (N-MNA-d4, cat. no. M321172, Tan Mo Quality Inspection Technology Co., Ltd.). Detection of the ions was performed in the multiple reaction monitoring mode. The retention times of 1-MNA and N-MNA-d4 were 1.31 and 1.65 min, respectively. The experiments were independent and conducted a minimum of three times.