Lc 20ad uhplc system

All blood samples were stored at 4°C and centrifuged (10 min at 3,000 × g) within 8 h after sampling, and the plasma was separated and stored in clean polypropylene cryogenic vials at −80°C until analysis. The blood concentration of various drugs was measured through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The recoveries of each drug after different times of circulation were calculated based on the blood concentration at 2 min.
Intra- and inter-assay means were within 15% of the target range value. For tigecycline, the linear calibration range was 0.07–8 ug/ml. For cefoperazone, the linear calibration range was 2.67–320 ug/ml. For sulbactam, the linear calibration range was 0.93–112 ug/ml. For teicoplanin, the linear calibration range was 1.6–192 ug/ml. For caspofungin, the linear calibration range was 0.53–64 ug/ml. For meropenem, the linear calibration range was 1–120 ug/ml. For voriconazole, the linear calibration range was 0.27–32 ug/ml. For micafungin, the linear calibration range was 0.54–64 ug/ml. For polymyxin B, the linear calibration range was 0.93–112 ug/ml.
Drug concentrations were measured using a Shimadzu (Kyoto, Japan) LC-20AD UHPLC system interfaced with a Shimadzu LCMS-8040 triple quadrupole mass spectrometer (MS/MS). Data acquisition and quantitative analysis were carried out using Shimadzu LabSolutions software.

Oligonucleotide primers were purchased from Eurofins Genomics. Other chemicals were purchased from Wako Chemical Ltd., and Kanto Chemical Co. Inc., (Tokyo, Japan). The LCMS data were obtained using a compact microTOF-MS (Bruker) attached to an LC-20AD UHPLC system (Shimadzu) with a COSMOSIL 2.5C18-MS-II column (2 mm i.d. × 75 mm; Nacalai Tesque, Inc.). Analytical HPLC was performed on a Shimadzu LC20-AD HPLC system, using a Thermo Scientific Hypersil GOLD analytical column (4.6 ×250 mm, 5 μm). 3-Oxo-glucose and 3”-oxo-puerarin were synthesized according to the published method^{17 (link)}.

The standard enzyme reaction of NvfI was performed in 50 mM Tris-HCl buffer (pH 7.5), containing 100 μM 2, 2 mM α-KG, 2 mM ascorbate, 200 μM FeSO₄, and 10 μM NvfI, for 8 h at 30 °C. The reaction was quenched by adding an equivalent volume of methanol. The samples were centrifuged and clarified with a 0.22 μm filter, and the reaction products were analyzed by a compact microTOF-MS (Bruker) attached to an LC-20AD UHPLC system (Shimadzu). Isocratic elution was performed with 58% of CH₃CN/H₂O solution both containing 0.1% formic acid. All measurements were conducted in triplicate.