Triple quad 4500 system

Mass spectrometric analysis of amino acids (AS) and acylcarnitines (AC) was performed using a SCIEX Triple Quad 4500 System (AB SCIEX, Darmstadt, Germany) with Turbo Ion Spray Source (TIS) in combination with a HTC Pal autosampler and a Shimadzu UFLC system for flow injection analysis (FIA) according to a validated protocol [35 (link)]. Briefly, 10 µL serum was diluted 1:10 with methanol. After centrifugation, 10 µL of the supernatant was diluted with 100 µL of methanol-containing isotope-labeled standards (Chromsystems Germany). Samples were evaporated at 70 °C for 40 min and derivatized using 60 µL of 3 n butanolic-HCL (Chromsystems, Germany) at 65 °C. After evaporation the samples were reconstituted with 150 µL of the mobile phase (1/1 v/v methanol/water) analysed with a SCIEX 4500 quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM). Concentrations of 26 AAs, 34 ACs, and free carnitine were quantified using ChemoView™ 1.4.2 software (AB SCIEX, Darmstadt, Germany).

LC–MS/MS analysis was performed with a Triple Quad 4500 system (AB Sciex, Foster City, CA) equipped with an ESI source and an LC-20A series high-performance LC system (Shimadzu Corp., Kyoto, Japan). The column used was a 150 mm × 2.1 mm i.d., 3 μm, InertSustain C18 (GL Sciences Inc., Tokyo, Japan). The mobile phase was a binary gradient of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) programmed as follows: at 0 min, 30% B; at 8 min, 80% B; and at 10 min, 80% B. The total run time was 15 min, which included 5 min of equilibration. The flow rate was set at 0.2 mL/min. Chromatographic separation was achieved at 40 °C. The injection volume was 2 μL. The ESI source was operated at 300 °C in the positive ionization mode. Other MS parameters were as follows: curtain gas, 40 psi; ion spray voltage, 5500 V; nebulizer gas (GS1), 30 psi; turbo heater gas (GS2), 80 psi; and collision-activated dissociation gas, 7 (arbitrary units). The multiple reaction monitoring transitions of CTV were 403 [M+H]⁺ to 315 (collision energy, 9 eV) and 403 [M+H]⁺ to 139 (collision energy, 31 eV). The retention time of the CTV in the sample solution (8.8 min) was consistent with that of the analytical standard. The standard of CTV was purchased from FERMENTEK Ltd. (Jerusalem, Israel).

UPLC-MS/MS assay was performed to determine drug content in biosamples such as plasma and tissues by using an AB Sciex Triple QuadTM 4500 system (AB SCIEX, USA) connected with Shimadzu LC-30AD via electrospray ionization (ESI) interface. The mobile phase was methanol (A) and 0.1 formic acid water (B) with the following gradient elution: 90%–55% (0–0.5 min); 55%–35% (0.5–3.5 min); 35%–10% (3.5–6.5 min), column temperature 40 °C, flow rate 0.4 mL/min, injection volume 5 μL. Mass spectrometry using electrospray ion source ESI, detection mode is positive ion mode, scanning mode is multiple reaction monitoring (MRM), meloxicam m/z:352.3 →115.2, piroxicam m/z: 332.1 → 94.8.