G6460 triple quadrupole mass spectrometer

A 1290 Infinity HPLC pump system (Agilent Technologies, Santa Clara, CA, USA) with an Agilent G4226A autosampler was coupled to an Agilent G6460 triple quadrupole mass spectrometer equipped with the electrospray jet stream interface (ESI). In order to perform MS and MS/MS analyses in full scan (mass range m/z 50–500) and in product ion mode, the acquisition parameters were optimized by infusion at a flow rate of 10 μL·min⁻¹ of a solution of fosetyl in water without additives (10 ng·μL⁻¹). Nitrogen was used as a nebulizing and collisional gas. The fragmentor potential was optimized in order to maximize the parent ion intensities, and, by operating in product scan mode, the collision energy (CE) was optimized (Table 1). Finally, all the analyses were carried out by LC-MS/MS in MRM mode, acquiring diagnostic product ions from the chosen precursor to obtain high specificity and sensitivity. Three main fragments 81, 63, and 79 m/z were formed from the precursor m/z 109; the ion m/z 81 was chosen as a quantifier for the definitive MRM analyses. MassHunter Software was used for the acquisition and the elaboration of the data set. Figure 2 shows MS/MS spectrum obtained with the electrical parameters in Table 1.

The RapidFire 365 (Agilent Technology) high-throughput system (RF) was coupled to a G-6460 triple quadrupole mass spectrometer (Agilent) operated in electrospray negative-ion mode. A type C cartridge was used for sample trapping and elution. The RapidFire method employs a solid phase extraction (SPE) sample cleanup step directly coupled to MS detection.
Samples were aspirated for 600 ms, followed by 4000 ms loading and washing with mobile phase A of 98% ddH2O+2%ACN+TFA0.01% at a flow rate of 1.5 mL/min. A fixed loop of 40 μL samples was loaded onto the cartridge. Samples were then eluted for 5000 ms with mobile phase B of 80% CAN + 20% ddH2O + TFA 0.01% at a flow rate of 1.25 mL/min, followed by reequilibrating the cartridge with mobile phase A at 0.7 mL/min for 500 ms.
MS parameters: Gas Temp: 200; Drying Gas: 9; Nebulizer: 40; Sheath Gas Temp: 400; Seath Gas Flow: 12; VCap: 3500; Nozzle Voltage: 300; Delta EMV: 400
MRM transitions for tubulin peptide substrat and reaction products were m/z 858.3 → 669.5, m/z 939.8 → 719.8, respectively. The dwell time for each transition was 4 ms. Peak areas were integrated, and the areas under curves are converted into the amount of substrate remaining and the product formed using a substrate and reaction product calibration curve.