Turboionspray

Plasma samples were analyzed for DTG using a validated analytical method [9 ]. DTG was extracted from human plasma by protein precipitation using acetonitrile containing [¹⁵ N ²H₇]-DTG as an internal standard. Extracts were analyzed by liquid chromatography–tandem mass spectroscopy using a TurboIonSpray® (AB Sciex, Framingham, MA, USA) interface with positive ion multiple reaction monitoring. The lower limit of the assay was 5 ng ml^–1 or 20 ng ml^–1 depending on the study, with a within- and between- run precision of ≤8.0% and ≤7.5%, respectively.

The liquid chromatography with tandem mass spectrometry system for deucravacitinib and moxifloxacin consisted of an API 4000 with the TurboIonSpray (AB Sciex LLC; Framingham, Massachusetts) interface operated in the positive ion mode. Quantitation was performed by selected reaction monitoring (m/z 426.2 → m/z 358.2 for deucravacitinib; m/z 431.2 → m/z 359.2 for BMT‐143848‐03; m/z 402.2 → m/z 358.3 for moxifloxacin; m/z 406.2 → m/z 362.2 for moxifloxacin‐d₄).
Linear regression analysis was conducted using a weighting of 1/x². The calibration ranges were 0.5 ng/mL (lower limit of quantitation) to 500 ng/mL (upper limit of quantitation) for deucravacitinib and 25.0 ng/mL (lower limit of quantitation) to 5000 ng/mL (upper limit of quantitation) for moxifloxacin.
Assessment of assay performance for deucravacitinib indicated between‐run precision of ≤0.6% coefficient of variation, within‐run precision of ≤2.6% coefficient of variation, and accuracy of ±4.8% mean percent deviation from nominal concentration. The assay performance was also assessed for moxifloxacin (≤1.8%, ≤2.2%, and ±4.0%, respectively).

A2E was synthesized in the dark using a commercial microwave synthesizer “Microwave 300” (Anton Paar GmbH, Austria). To scale up A2E synthesis⁴⁵ all-trans retinal (100 mg equivalent to 352 µmol) and ethanolamine (9.5 µL; 155 µmol) were added in ethanol (3 mL) in the presence of acetic acid (9.3 µL; 155 µmol) using stochiometric ratios as described.²⁹ (link) Further, large scale purification was achieved using liquid partitioning⁴⁶ (link) to obtain HPLC pure A2E.
Chromatography separation and peak monitoring were achieved using an ultrahigh performance liquid chromatography (UPLC; Thermo Accela; Thermo Electron Corp., Waltham, MA, USA) system with a quaternary pump connected to an online degasser and photodiode array detector (PDA). The UPLC system was coupled with a tandem mass spectrometer (4000 Q-Trap; AB Sciex Biosystems, Foster City, CA, USA). ChromQuest software version 4.1 (San Jose, CA, USA) was used to control all parameters of UPLC. For analytical separations, an Xterra RP 18 column was used (4.6 × 150 mm with 5 µm particle size; Waters, Milford, MA, USA). Ionization of analytes was accomplished by electrospray ionization (ESI; TurboIon Spray; ABSciex) operating in the positive ion mode followed by tandem mass spectrometric analysis. Data acquisition and integration were performed by Analyst 1.5.2 software (ABS Biosystems, Foster City CA, USA).

Chromatographic separation was performed using an ExonLC system (AB Sciex, Foster City, CA, USA). The chromatographic column was a Waters Acquity UHPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) with an operating temperature of 35 °C. The experiment was performed with 0.1% formic acid aqueous solution (v/v) as mobile phase A and acetonitrile solution (without formic acid) as mobile phase B. The following gradient program was used: 0–5min 3%–8% B, 5–11min 8%–30% B, 11–20min 30%–80% B, 20–21min 80%–95% B, 21–27min 95% B. 27–27.5min 95%–3% B, 27.5–32min 3% B. The sample injection volumes were all 2 μL. A 5600 Q-TOF mass spectrometer equipped with an electrospray ionization source (Turbo Ionspray) (AB Sciex, Foster City, CA, USA) was used to provide high-resolution detection in this experiment. MS detection work in positive and negative ion modes. Summary of mass spectrometer parameters: Gas 1 and Gas 2, 45 psi; Curtain gas, 35 lbs. Heat block temperature:550; ion spray voltage negative −4.5 kV and negative 5.5 kV; declustering potential,50 v; collision energy,35 v; collision energy spread (CES) of 15 V.