Uplc ms ms

The first morning urine samples were unfrozen and centrifuged at environmental temperature for 3–5 min at 5600× g prior to extraction. Then, the urine concentration was calculated by the creatinine percentage by a creatinine kit (Creatinine Respons KIT, Sigma-Aldrich) and further measured by spectrophotometry. Three milliliters of urine were mixed with 250 µL of sodium acetate buffer (1.4 M) at pH 5.0 and 40 µL of β-glucuronidase/arylsulfatase enzyme (Sigma-Aldrich), and was in-move incubated for 16 h in an oven at 37 °C. Oasis HLB Prime columns 1 cc (Waters) were conditioned with 100% methanol and then distilled water (Merck). The hydrolysate was later passed through them. The columns were cleaned twice with distilled water, and the contents were subsequently eluted with 3 mL of acetonitrile 100%. The eluate was evaporated under a gentle nitrogen stream (Thermo Scientific, MA, USA) at 45 °C and then reconstituted with 450 µL of acetonitrile and subsequently was filtered with a 0.22 µm Teflon syringe filter. The filtrate was received in amber vials and [13C]-labelled internal standards were added for further quantification by Ultra-High-Performance Liquid Chromatography with a mass spectrometric detection detector (UPLC-MS/MS; Shimadzu, Kyoto, Japan).

NOB and its metabolites in the rat plasma were identified using UPLC−MS/MS (Shimadzu, Kyoto, Japan) consisting of an LC-30AD and a triple quadrupole linear ion trap mass spectrometer (model QTRAP 4500) (AB SCIEX, Concord, Canada) based on multiple reaction monitoring (MRM). An Agilent Poroshell 120 PFP (4.6 × 150 mm, 2.7 μm) column was used in the detection. The measurement was performed according to the method previously reported by Zhang et al. (2020 (link)).

The solubility of various oils (castor oil, corn oil, mineral oil, olive oil, and soybean oil) and surfactants (Cremophor EL, Labrasol, Span 80, Span 85, and Tween 80) for methotrexate was evaluated to determine the suitable components for nanoemulsion containing methotrexate. Excess amounts (5 mg) of methotrexate were placed in capped vials with 1 mL of each oil or surfactant. Then, the mixture was vortex-mixed and kept in a shaking (70 opm) water bath at 25 °C for 72 h. After reaching equilibrium, the samples were centrifuged at 10,000× g for 10 min. The supernatant was taken and diluted with methanol to quantify the methotrexate; concentrations were determined by UPLC-MS/MS (Shimadzu Corp., Kyoto, Japan). The UPLC-MS/MS method used here is the same as the method in our previous study [25 (link)]. Brief analysis conditions are described in Section 2.6.2. Quantification of Methotrexate in Biological Samples. As a co-surfactant, ethanol was fixed without a separate solubility test by referring to previously reported studies [33 (link),34 (link),35 (link)]. In other words, optimization through solubility screening of oil and surfactant for methotrexate was the main focus for the preparation of methotrexate-loaded nanoemulsion in this study, and co-surfactant, which plays a relatively auxiliary role, was selected by referring to the previous studies.