Beh phenyl column

The chemical constitutions of PNS and PNE were quantitatively determined using a Waters ACQUITY-UPLC CLASS system (Waters Corp., USA) coupled with an ACQUITY UPLC BEH phenyl column (150 mm × 2.1 mm, 1.7 μm) maintained at 45°C. Elution was performed with a mobile phase of water (A) and Acetonitrile (B) under a gradient program: 0-10 min, 19% B; 10-15 min, 19-35% B; 15-20 min, 35-38% B. The flow rate was 0.4 mL/min, and the injection volume was 2 μL. The analytes were monitored at the UV wavelength of 205 nm. Prior to the next injection, the column was washed with 100% B for 2 min and then equilibrated with the initial mobile phase for 3 min. Notogisenoside R1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, and ginsenoside Rd (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pufei De Biotech Co., Ltd. (China). Acetonitrile was purchased from RCI Labscan Limited (Thailand) of HPLC grade. Milli-Q water was prepared using a Milli-Q system (Millipore, USA).

The analysis was carried out with an Acquity UPLC system (Waters, Milford, MA, USA) consisting of an auto-sampler, a binary pump equipped with a 10 µL Loop (partial Loop injection mode) and a BEH PHENYL column (2.1 mm × 100 mm, 1.7 µm; WATERS, Waxford, Ireland). The solvents used were (A) water + 0.1% (v/v) formic acid and (B) acetonitrile + 0.1% (v/v) formic acid at a constant flow rate of 0.3 ${\mathrm{m}\mathrm{L}·\mathrm{m}\mathrm{i}\mathrm{n}}^{-1}$ . The elution gradient (for 113 min) was 100% A, gradually decreasing until reaching 10% A and 90% B, to move from normal conditions (100% A) one minute later to re-equilibrate the column. MS detection was performed on a Q-ToF quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF™, Waters, Milford, MA, USA) equipped with an electrospray ionization source (ESI). The sample acquisition mode was in negative ionic polarity, analysis mode in sensitivity and normal dynamic range, in a mass range of 50 to 1200 Da, sweep conditions of 0.5 ${\mathrm{s}}^{-1}$ , a centroid data format, a collision energy of 6 V and a cone voltage of 40 V.

Brown glass vials with polypropylene PTFE vial caps were used to ship reconstituted, undiluted aliquots of MCLR vendor samples to EPA in Las Vegas, Nevada (EPA, NV) for validation of our standards measurements. Samples were shipped from the EPA, NC to the EPA, NV overnight on ice.
The seven MCLR vendor samples were also quantified by HPLC-MS/MS using an Agilent 1290 Infinity II HPLC coupled to an Agilent 6495 Triple Quadrupole MS with positive ESI. The HPLC was equipped with a BEH phenyl column (2.1 × 100 mm, 1.7 μm, Waters Corp, Milford, MA, USA) at 35 °C and mobile phase running at 300 μL/min consisting of (A) 0.05% acetic acid in 95:5 DI water-methanol and (B) 0.05% acetic acid in 95:5 methanol-DI water. All samples received were diluted accordingly such that two concentrations (5 and 50 pg/μL) from each vendor were used for the analysis. A 20-μL diluted sample was injected and eluted with the following gradient: initial condition of 35% B ramped to 90% B over 5 min and held for 1.5 min, then back to initial condition over 3.5 min followed by a 3 min post time equilibration prior to the next run. The MS acquisition was performed in positive polarity using the ²⁺ precursor ion 498.3 with product ions 135.2, 105.1 and 103.1. An external five-point calibration curve was prepared using NRC Canada CRM and quantified using product ion 135.2 peak area.