Cortecs t3 column

Targeted, quantitative analysis of in vitro hepatocyte metabolic stability and plasma protein binding assay samples was conducted on a Waters Xevo-TQS micro mass spectrometer (Waters, Milford, MA, USA) ultra-high-performance liquid chromatography tandem mass spectrometry system (UPLC-MS/MS), modified with a PFAS analysis kit. Samples were analyzed in negative ion mode with UniSpray ionization. A CORTECS T3 column (Waters CORTECS, 3 × 100 mm, particle size 2.7 µm) was used for chromatographic separation with a flow rate of 0.6 mL/min and a temperature of 55 °C. Gradient elution was used with eluent A (95:5 H₂O/ACN) and eluent B (95:5 ACN/H₂O) with 2.5 mM of ammonium acetate. The gradient is available in Table S11. Additional details for MS parameters are found in Table S12 and monitored transitions are found in Table S13.

High-performance liquid chromatography (HPLC) grade solvents and LC–MS modifiers were purchased from Sigma-Aldrich (St. Louis, MO, USA). Detection and quantification were achieved by ultra-performance liquid chromatography—tandem mass spectrometry (UPLC-MS/MS) utilizing a Thermo Scientific Vanquish UPLC with a Thermo Scientific Altis triple quadrupole mass spectrometer, heated electrospray ionization (HESI-II) in positive ion mode (3500 V). 50 µl of sample was mixed with 200 µl of acetonitrile (ACN), vortexed for 5 min and then centrifuged at 4 °C, 17,000 g for 15 min. The supernatant was transferred to an LC–MS vial for analysis. Injection volume was 1 µl. A Waters Cortecs T3 column, 2.1 × 100 mm, 1.6 µm column was maintained at 35 °C. Solvent A: H₂O with 0.1% formic acid (FA) and Solvent B: ACN with 0.1% FA. The flow rate was 250 µl/min, the gradient was 25% B at 0 min for 0.25 min, increasing to 65% B at 5 min, further increasing to 90% B at 5.5 min, remained 90% B until 7.5 min, then decreased to 25% B at 8 min. The total running time was 10 min. Samples were analyzed in triplicates. Quantitation of ¹⁴NAT and ¹⁵NAT were based on multiple reaction monitoring (MRM) transitions m/z, 170.062 → 115.042 and 171.062 → 115.042, respectively. The result was based on the percentage ratio of ¹⁵NAT/(¹⁴NAT + ¹⁵NAT).

Chromatographic separation was carried out at 30°C on a Waters CORTECS T3 column (2.1 mm × 100 mm, 1.6 μm). The mobile phase consisted of 0.1% formic acetonitrile (A) and 0.1% formic acid water (B) using an elution gradient of 2–19% A at 0–2 min, 19–42% A at 2–11.5 min, 42–55% A at 11.5–15 min, 55–65% A at 15–16.5 min, 65–75% A at 16.5–18 min, 75–100% A at 18–22.5 min, and 100–2% A at 22.5–24 min. The sample volume injected was 3 μL, and the flow rate was 0.4 mL/min.
The conditions of the ESI source were as follows: ESI in positive mode; capillary voltage, 800 V; fragmentor, 20 V; sampling frequency, 10 Hz. The QDA analysis worked using full scan mode, and the mass range was set at m/z 450–1200.

Identification analysis of metabolites in bio-samples was performed as described previously [5 (link)] using a Dionex Ultimate 3000 UHPLC system combined with a Q-Exactive Focus Orbitrap mass spectrometer (Thermo Fischer Scientific, MA, USA) fitted with a CORTECS T3 column (2.1 × 150 mm, 2.7 μm; Waters, MA, USA). An electrospray ionization (ESI) source in both negative- and positive-ionization mode was used. Data recording and processing were carried out using the Xcalibur software (version 2.2, Thermo Fischer Scientific, MA, USA) and Compound Discover software (version 3.2, Thermo Fischer Scientific, MA, USA).
A total of 100 μL plasma, bile, urine and feces homogenate samples were deproteinized with 500 μL, 500 μL, 500 μL and 900 μL of acetonitrile, respectively. After vortex-mixing and centrifugation at 14,000 rpm for 10 min twice, 5 μL of supernatant was injected into the UHPLC–HRMS system for qualitative analysis.

The sample analyses were performed using a Shimadzu LC‐20AD HPLC system coupled with an ABSCIEX 5500 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. Chromatographic separation was performed with a Waters CORTECS T3 column (100 × 2.1 mm, 2.7 µm). The column temperature was kept at 35°C. The mobile phase was composed of water containing 0.1% formic acid (A) and ACN containing 0.1% formic acid (B). The gradient program was set as follows: 0 min 5% B, 5 min 60% B, 5.5 min 90% B, 5.5 ~ 6.5 min 90% B, and 6.6 ~ 8 min 5% B. The flow rate was set at 0.3 ml/min, and the injection volume was 2 µl.
The MS was operated in positive ion mode with sMRM. The optimized MS parameters for each antimicrobial are summarized in Table 1. The parameters of the source conditions were as follows: ion spray (IS) voltage: 4500 V; curtain gas: 20 psi; nebulizer gas (GS1): 50 psi; auxiliary gas (GS2): 50 psi; and source temperature: 500°C. Data acquisition was performed by Analyst 1.6.3 software. The peak integration and data analysis were processed by MultiQuant software (version 3.0.2).

Four different substrates of slimming health foods were selected to validate the accuracy of the AuNPs-LFIA. The spiked concentrations were chosen according to the sensitivity of samples of different substrates. The spiked scalar was the furosemide concentration corresponding to 0.25, 0.5, 1 and 2 times the cut-off value. A total of 16 spiked samples were tested. Each sample was combined with 4 mL methanol, mixed on a vortex mixer for 2 min, and then centrifuged at 4000× g for 3 min. Half of the supernatant was diluted 5 times with PB and tested with test strips, and the other half was diluted 5 times with methanol and passed through a 0.22 μm filter membrane, then tested with LC-MS/MS. Each sample was tested three times (n = 3).
Chromatographic separation of LC-MS/MS was performed on a Waters CORTECS T3 column (2.1 × 100 mm, 2.7 μm). The column temperature was 30 °C. Mobile phase A was an aqueous solution containing 0.1% formic acid, and phase B was an acetonitrile solution containing 0.1% formic acid, which was used for gradient elution. The sample volume was 1 µL.

The chromatographic separation was carried out on a CORTECS T3 column (150 × 2.1 mm 120 Å, 2.7 $\mathsf{\mu }$ m, Waters). The flow rate was set at 0.6 mL/min, and the column temperature was maintained at 55 ${}^{\circ }$ C. The mobile phase was composed of solvent A (ultrapure water MilliQ containing 0.1% formic acid) and solvent B (LC-MS grade acetonitrile acidified 0.1% formic acid). The chromatographic method consisted of the following linear gradient: 5% B from 0 to 0.5 min. The concentration of B was then increased to 80% from 0.5 to 27 min. Compounds were analyzed based on data from UV absorption at a wavelength range set at 195–600 nm, and mass spectra with a mass scan range was set at 50–1800 m/z. Electrospray ionization (ESI) was performed in positive and negative ion modes. The ion source parameters in negative ion mode were capillary voltage set at 3.0 kV, nebulizer at 0.7 bar, dry gas at 6.0 L/min, and dry temperature at 200 ${}^{\circ }$ C. Data acquisition and processing were performed using DataAnalysis 4.3 (Bruker Daltonik GmbH, Bremen, Germany). All analytes, including phenolic compounds, were identified based on previously literature data and based on data from the library Compound Crawler Bruker Program, DataAnalysis 4.3 (Bruker Daltonik GmbH, Bremen, Germany).