Infinitylab poroshell 120 ec c18 column

The water samples were collected and filtered by a 0.22 µm nylon syringe filter (ChromTech, Shanghai, China) and applied to HPLC analysis. The three preservatives were analyzed using an Agilent 1260 HPLC equipped with an InfinityLab PoroShell 120 EC-C18 column and monitored by a photodiode array detector at 254 nm (Agilent Technologies, Inc., Santa Clara, CA, USA). The solvents delivered by the analytical pump were acetonitrile (A) and water (5 mM KH₂PO₄) (B). Samples were eluted by 40/60 (A/B) with a flow rate of 1 mL/min. The recovery percentages for BA, HB and DHA were 96.2, 95.3 and 95.6, respectively. The detection limits for the preservatives were 0.1 mg/L.

Samples taken from the acceptor side of the diffusion cells were first neutralized with HCl and then analyzed for vanillin, vanillic acid, and guaiacol in a Waters H-Class UPLC system using a method described in the literature [28 (link)]. A gradient method with water (A) and acetonitrile (B), both with 10 mM formic acid, was used to separate the lignin monomers in an Agilent InfinityLab Poroshell 120 EC-C18 column (100 × 4.6 mm, 4 μm). The flow rate was 1.0 mL/min and the gradient for acetonitrile (B) was as follows: 0 min 3%, 12 min 15%, 15 min 15%, 20 min 80%, 20.1 min 90%, 25 min 90%, 25.1 min 3%, and 35 min 3%. The injection volume was 3 µL and the column temperature 50 °C. Detection was performed using a PDA detector at 280 nm with baseline correction at 350–400 nm.

Liquid chromatography-mass spectrometry (LC–MS) analysis were performed to validate whether the active compounds found by network pharmacology are actually existed in SH003. LC–MS analysis was conducted using a Waters TQD instrument. The reference component (four active compounds, 1.0 mg each) was weighed, dissolved in 1.0 mL of methanol to prepare a solution at a concentration of 1.0 mg/mL, then diluted. SH003 powder (10.0 mg) was weighed, sonicated in 1 mL of methanol for 10 min, and filtered through a 0.22 μm syringe filter. Chromatographic separation was achieved on an InfinityLab Poroshell 120 EC-C18 column (100 × 2.1 mm, 2.7 µm, Agilent) with a gradient elution profile using mobile phases A (0.1% Formic acid, 5 mM Ammonium Formate in Water) and B (0.1% Formic acid, 5 mM Ammonium Formate in Methanol). The gradient program commenced with 99% A at 0 min, transitioned to 20% A at 3 min, maintained this composition until 4 min, shifted to 1% A at 5 min, increased to 8% A at 8 min, returned to 99% A at 8.5 min, and persisted until the end of the 12-min analysis. The column temperature was maintained at 40 ℃, and the flow rate was set at 0.5 mL/min. Subsequent network pharmacology analysis was performed for the compounds validated via chromatography analysis.

Water samples were taken at 0 and 24 h of exposure from each treatment and stored at −20 °C in a dark environment until analysis. Due to the lengthy chromatographic method and large volume of solvent used per run, water samples were pooled from each replicate which resulted in one measurement per concentration. All samples were directly injected (10 µL) into an Agilent Infinity II (Agilent, Santa Clara, CA, USA) liquid chromatography instrument attached to a SCIEX Triple Quad 5500+ LC-MS/MS (SCIEX, Framingham, MA, USA). A Zorbax Diol (4.6 mm ID, 12.5 mm, 6 μm particle size) attached to an Agilent InfinityLab Poroshell 120 EC-C18 column (4.6 mm ID, 100 mm, 2.7 μm particle size) was used for separation of PFAS and each sample run with a ramping LC solvent gradient with methanol and nanopure water, each containing 10 mmol/L ammonium acetate. A flow rate of 0.4 mL/min was used, and the column was maintained at room temperature. Two multiple reaction monitoring (MRM) transitions were employed for each PFAS, one for quantitation and the other for confirmation of the PFAS (Table S2). The PFAS water concentrations are available in the Supplementary Materials (Table S3).

The extracts were
analyzed by high-performance
liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS)
on a Shimadzu LCMS-8060 triple quadrupole. The chromatographic method
for the Agilent InfinityLab Poroshell 120 EC-C18 column (3 ×
100 mm, 2.7 μm) fitted with a guard column (3 × 5 mm, 2.7
μm) was based on gradient elution with acetonitrile (CHROMASOLV,
Honeywell; LC–MS grade) and water (LiChrosolv, Merck; LC–MS
grade) as the mobile phases and was previously used for the separation
of similar solutes.^{35 (link)} The gradient program
began with 10% acetonitrile maintained for 0.5 min, succeeded by a
linear increase to 100% at 26 min mark; 100% acetonitrile was maintained
for 3 min, followed by rapid drop to 10% for column re-equilibration
for the next 6 min. In total, the gradient program took 35 min per
sample. Both mobile phases contained 0.1% formic acid, the total flow
rate was 300 μL min^–1, the injection volume
was 0.7 μL, and the column temperature was maintained at 25.0
°C. The retention times and precursor–product ion transitions
are listed in Table S1 in the Supporting
Information.

An Ultimate 3000 Series Ultra-High Performance Liquid System (UPLC, Thermo Scientific, Waltham, MA, USA) coupled with an InfinityLab Poroshell 120 EC-C₁₈ column (10 × 0.21 cm, 1.9 μm, Agilent Technologies, Palo Alto, CA, USA) was used to further purify the closed subfraction. The UPLC system was carried out using ultrapure water as eluent B and gradient of acetonitrile (containing 0.1% formic acid) as eluent A at a flow rate of 0.25 mL/min. The gradient for eluent A was 0–2.5 min, hold at 5%; 2.6–12.5 min, at 10%; 12.6–20 min, at 5%; 20–22 min, at 52.5%; 23–30 min, at 95.0%; and 31–35 min, at 5%. The peptide was automatically selected on basis of fragment information from the electrospray ionization mass spectrometry (ESI-MS) analysis which was performed on a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany) with mass range of 120–1800 m/z, full MS 35,000, ddMS² 17,500, and AGC target value of 1 e⁵. A De Novo™ software (Peak Studio 7.5, Bioinformatics Solutions, Inc., Waterloo, Ontario, Canada) was used to process the peptide sequence and molecular weight of the MS data [20 (link)].