Agilent 1290 6460

Entecavir (ETV) provided by Meilun Biologic Co., Ltd. (Dalian, China) is a substrate of ENT1 and was applied to determine the transport activity of ENT1. The quantification of ETV in cell lysates was detected by an Agilent 1290-6460 liquid chromatography-mass spectrometer with a triple quadrupole mass spectrometer (Agilent, Santa Clara, CA) with the method established in our laboratory before.
The accumulation of DAC was detected using LC-MS by Agilent 1290-6460. RCC cells were incubated in HBSS containing 500 μM DAC or ENT1 inhibitor, NBTI (Sigma), at 37 °C for 3 min after rinsed 3 times with pre-warmed HBSS. 1 ng/mL loratadine (Aladdin) was used as internal standard. Chromatographic conditions of DAC detection was same to 5-mC as described in 2.10.
Mass spectrometry conditions: ESI⁺, ion source temperature: 140 °C, desolvation temperature: 350 °C, capillary voltage: 3.5 kV; cone voltage: 500 V, gasification gas flow: 5 L/min, spray pressure: 45 psi, sheath gas temperature was 350 °C, the sheath gas flow rate was 11 L/min.
Quantitative ion pair: DAC m/z 251→135, fragmentation voltage and collision energy were 100 V and 4 V, respectively; loratadine (IS) m/z 383→337, fragmentation voltage and collision energy were 170 V and 20 V, respectively.

Prior to analysis, all leaf samples were dried to a constant weight at 80°C for 48 h and then ground into a fine powder. The NSC concentrations, including free low molecular weight soluble sugars (SS, including glucose, fructose, and sucrose) and starch, were analyzed using an Agilent 1290/6460 liquid chromatography system and tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, United States) with a Waters XBridge^TM BEH Amide 2.5 um 2.1 × 50 mm XP column (Waters, Milford, MA, United States). Approximately 20 mg of plant powder samples were extracted with 2 ml of ethanol (80%, v/v) at 80°C for 30 min., and then centrifuged (10,000 g for 10 min.). Subsequent to three extraction processes, the supernatant was utilized for the determination of SS, and the residue was dried with nitrogen for 24 h to dislodge any ethanol. The starch was initially hydrolyzed with diastase (Tokyo Chemical Industry, Japan) (60°C for 10 min.) and then analyzed using the same method as for the determination of SS. We added SS and starch concentrations to obtain NSC concentrations. All sugar and starch concentrations in these tissue samples were expressed as per unit of weight (mg g^-1).

The detection of phenolic compounds using UPLC-QQQ-MS analysis (model Agilent 1290–6460, Agilent Corporation, Santa Clara, CA, USA) was carried out by an Agilent 1290 HPLC system equipped with a Zorbax Eclipse Plus-C18 (100 × 4.6 mm, 1.8 µm, Agilent, Santa Clara, CA, USA) column at 35 °C. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in water (B). The gradient elution was run as follows: from 10% to 45% A over 8 min, from 45% to 80% A over 8 min, from 80% to 100% A over 4 min, keep 100% A over 3 min, from 100% to 0 A over 3 min. The sample injection volume was 5 µL. The flow rate was delivered at 0.3 mL/min under a gradient program.
The chromatograph was coupled to a triple quadrupole mass spectrometer with an ESI interface. The ESI source worked in negative ion and multiple reaction monitoring modes.