1290 infinity 2 series uhplc system

An ultrahigh-performance liquid chromatograph was implemented on a 1290 Infinity II series UHPLC System (Agilent Technologies). The analytical column was a Waters ACQUITY HSS T3 column (100 × 2.1 mm, 1.8 μm) from Waters Co. (USA) with a temperature of 40°C. (A) 0.1% acetic acid aqueous solution and (B) methanol constituted the two parts of the mobile phase at the following gradient elution procedures (0–1 min, 70–70% A; 1–6 min, 70–5% A; 6–10 min, 5–5% A; 10–10.5 min, 5–70% A; 10.5–14 min, 70–70% A). The flow rate was controlled at 0.3 mL/min, and the temperature of the sample tray was set at 4°C. The injection volume was 1 μL.

The concentrations of ¹³C₄SA in plasma and tissues were determined using a 1290 Infinity II series UHPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled with an AB Sciex Qtrap 6500+ mass spectrometer (Concord, Canada). A Waters Atlantis Premier BEH C₁₈ AX column (2.1 × 100 mm, 1.7 μm particle size) was used, and the column temperature was maintained at 30 °C. The gradient mode consisted of mobile phases A (0.9% formic acid in water) and B (0.9% formic acid in ACN) as follows: 0–1.5 min (0%–0% B), 1.5–4.0 min (5%–30% B), 4.0–4.5 min (30%–30% B), 4.5–5.0 min (30%–0% B), and 5.0–5.5 min (0%–0% B). The flow rate was 0.3 mL/min. Meanwhile, 5-μL prepared samples were injected for analysis. The multiple reaction monitoring mode with negative electrospray ionization was used to quantify ¹³C₄SA and IS. The source temperature and ion spray voltage were 350 °C and −4500 kV, respectively. The ion source gas 1 flow pressure was 50 psig, and the gas 2 flow setting was 50 psig. The curtain gas pressure was 40 psig, and collision gas is medium. The optimized mass parameters for ¹³C₄SA and CAD₄ are listed in Table 1.

To quantify HSYA in the CDNVs, a targeted metabolomic assay was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC‒MS/MS). Briefly, samples were prepared by transferring a 10-µL aliquot of each individual sample to an EP tube and adding 990 µL of methanol. After vortexing and incubation at − 20 °C, the samples were centrifuged, and a 50-µL aliquot of the clear supernatant was transferred to an auto-sampler vial for LC‒MS/MS analysis. Stock solutions were prepared by dissolving or diluting each standard (HSYA) to a final concentration of 1 mM. A mixed working standard solution was produced by transferring a 100-µL aliquot of each of the stock solutions to a 10-mL flask; a series of calibration standard solutions were then prepared by stepwise dilution of this mixed standard solution. UHPLC separation was performed using an Agilent 1290 Infinity II series UHPLC System, and an Agilent 6495 triple quadrupole mass spectrometer was used for assay development. The MRM parameters for each target analyte were optimized, and calibration curves were generated using the least-squares method with 1/x weighting. The limits of detection and limits of quantitation were determined using signal-to-noise ratios, according to the US FDA guidelines for bioanalytical method validation.