Api 3200

For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100 mm (Waters^®) was fitted in an Agilent 1290 Infinity Series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst^® version 1.5 software package (SCIEX, Framingham, Massachusetts).
To achieve isocratic chromatographic separation, a flow gradient of 200 μL min^-1 was established with a 45% of mobile phase A and a 55% of mobile phase B, which corresponded to extraction solutions A and B, respectively, as well as a volume of injection of 20 μL and a column oven temperature of 35 °C.
A multiple reaction monitoring (MRM) scan type was used for acquiring and visualizing LC-MS/MS data. The source temperature was 450°C, and the pressure was 40 psi for nebulizer (GS1), 20 psi for turbo ion (GS2), 20 psi for curtain gas and 10 psi for collision gas. The ionization was performed by electrospray, and the ion spray voltage was 5000 V. Table 1 lists the monitored ions masses.

After five days of 3D co-culture, different concentrations of prepared OPs (CPF, MT, and PT), MUS, DXM, and EtOH were added to the medium inlet of the neurovascular endothelial lane (blood lane). After 24 hours, the solution was removed from the blood lane and stored at −20°C. The residual concentrations of the OPs were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using a triple quadrupole mass spectrometer coupled with a Shimadzu Prominence High Performance LC (HPLC) system (API 3200, AB Sciex).

The content of dopamine were performed by a high-performance liquid chromatography system (LC-30A, Shimadzu, Japan) coupled with tandem mass spectrometer (API 3200, AB Sciex, USA).The synchronized worms were washed with M9 buffer to remove bacteria, harvested in 500 µl 0.2 M HClO₄, and then sonicated and centrifuged at 12000 rpm for 15 min. The supernatants were prepared for quantification of dopamine.

An HPLC system coupled to an AB Sciex API 3200 tandem mass spectrometer with turbo ion spray was used to rapamycin levels in the blood and liver. Rapa and ASCO (internal standard) obtained from LC Laboratories (Woburn, MA) were used in the quantification of Rapa. Approximately 100 ml of blood or 100 mg of liver samples and 0.1 mL of calibrator were mixed by sonification with 10 µL of 0.5 µg/mL ASCO and mixed with a solution containing 0.1% formic acid and 10 mM ammonium formate dissolved in 95% HPLC grade methanol. Supernatants, which were obtained by centrifugation at 15,000 g for 5 min at 23°C, were then injected into the LC/MS/MS. The ratio of the peak area of Rapa to that of the internal standard ASCO (response ratio) for each unknown sample was compared against a standard curve using different concentrations of Rapa standard. The concentration of Rapa was expressed as ng/ml for blood and pg/mg of liver tissue protein.

LC-MS analysis was performed on Waters UPLC-H-Class system (Waters, Milford, MA, USA) connected to AB Sciex API 3200 by ESI probe (AB Sciex, Framingham, MA, USA) on a Waters BEH C18 column (2.1 mm i.d. × 50 mm, 1.7 μm) with elution of acetonitrile/0.05% aqueous formic acid linear gradient system (acetonitrile: 5 to 100% in 4 min at 0.5 mL min⁻¹). HR-ESI-TOF-MS was measured by Waters VION IMS QTof system. JEOL JMS-T100GCV was used for HR-EI-TOF-MS analysis (JEOL, Tokyo, Japan). Teledyne ISCO CombiFlash Companion (Teledyne ISCO, Lincoln, NE, USA), and RediSep Rf Gold silica column or RediSep Rf Gold HP C18 were used for MPLC. Preparative HPLC was performed using Waters 600E pump system with Senshu Pak Pegasil ODS column (20 mm i.d. × 250 mm or 10 mm i.d. × 250 mm, 5 μm). The NMR data were obtained at 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR on JEOL JNM-ECA-500 spectrometer (JEOL, Tokyo, Japan). Chemical shifts (in ppm) were referenced based on the residual undeuterated solvent.

Analysis of nucleotides was performed on API 3200 and QTrap 3200 (AB Sciex) spectrometers with electrospray ionization (ESI) in negative-ion mode. Samples were analysed using direct infusion with a syringe pump (4.6 mm diameter) at flowrates of 10–100 μl/min. An ion spray voltage (IS) of −4500 V, curtain gas (CUR) of 25 psi, high collision gas (CAD), and ion source gas 1 (GS1) of 20 psi were applied. Other parameters were optimized for each compound; typical values included a declustering potential (DP) in the range of −30 to −50 V, entrance potential (EP) in the range of −3 to −10 V, and collision energy (CE) in the range of −30 to −55 V.