Api 2000 lc ms ms system

All reagents were purchased from commercial sources (e.g., Sigma) and directly used unless stated otherwise. Solvents were purified by the most used methods. All solutions and buffers were prepared with using metal-free water that was passed through a Millipore-Q ultrapurification system. Elementary analysis was carried out on a Vario EL III elementary analysis instrument. UV-Vis spectra were recorded on an analytik jena Specord 210 spectrophotometer. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury 400 spectrometer at 400 and 100 MHz, respectively. Electrospray ionization mass spectra (ESI-MS) were acquired on an Applied Biosystems API 2000 LC/MS/MS system.

Glutathione stability was assessed using an assay adapted from a recently reported method27 (link). 250 μL of a 40 mM DMSO stock solution of each compound was added to L-glutathione (15.4 mg, 5 mol equiv.) suspended in 250 μL of DMSO. The resulting mixture was placed in a 37 °C shaker. 10 μL aliquots were removed and quenched in 990 μL of water (containing 0.5% formic acid) at various time points, including at t = 0 min, for analysis by ESI-LC-MS. All ESI-LC-MS analyses were collected on an API2000 LC/MS/MS System (Applied Biosystems) equipped with a turbo-ion spray ESI probe interfaced with a Prominence UFLC (Shimadzu) equipped with a reverse phase BDS Hypersil C18 50 × 2.1 mm column, particle size 3 μm (Thermo Scientific). HPLC/LCMS UV absorption was monitored at 254 nm and 210 nm. Both the compound and the glutathione adduct were identified by MS. Area of the UV peak was recorded for each time point.

The API2000 LC-MS/MS system (Applied Biosystems, Foster City, CA, USA) was used to further analyze the molecular weight of synthetic Hf1a-NH₂ through electrospray ionization mass spectrometry (ESI MS). The sample was dissolved with 15% ACN in 85% milliQ water and chromatographically separated after loaded (0.2 μL) at a flow rate of 0.2 mL/min using solvent B of 50% methanol in water with nebulizing gas flow at 1.5 L/min. Mass data were then acquired via measurement of ion intensity.