Analyst software version 1

Detection employed a Qtrap 5500 mass spectrometer (AB Sciex, Concord, ON, Canada) equipped with a TurboIon Spray™ source. Data acquisition and integration were performed using Applied Biosystems Analyst software version 1.6.3. MS parameters optimized for each analyte and IS by infusion of their respective standards using a syringe pump at a flow rate of 20 μL/min. The declustering potentials (V) and collision energies (eV) were as follows: VAN 100, 20; MER 140, 33; VPA −180, −10; MER-D6 110, 21, and VPA-D6 −190, −12. VAN, MER, and MER-D6 were detected by positive ion ESI and the transitions (m/z) for MRM were VAN 725.5 → 144.2, MER 384.3 → 113.9, and MER-D6 390.3 → 147.3. VPA and VPA-D6 were detected by negative ion ESI and the transitions (m/z) for MRM were VPA 143.3→143.3 and VPA-D6 149.3→149.3 (Figure 3).

The plasma VD2 concentrations were detected by a LC-MS/MS method, using an AB SCIEX TRIPLE QUAD™ 4500MD LC-MS/MS System (ABsciex, Toronto, Canada). Briefly, 10 μL of internal standard was added to 100 μL of plasma samples and then mixed with 300 μL of extraction solution. The supernatant was collected after vortex and centrifugation, subsequently injected for LC-MS/MS analysis. Calibrators and quality controls were prepared based on the same procedure.
Chromatographic analysis was performed on the Jasper™ HPLC system equipped with a Gemini 3 μm C18 column. Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B consisted of acetonitrile with 0.1% formic acid. Thirty microliters of the sample solutions were injected into the LC system using a column temperature of 40°C and a flow rate of 0.8 mL/min. Mass spectrometry analysis was performed by multiple reaction monitoring in positive electrospray ionization mode. Optimized parameters for mass detection were as follows: curtain gas was 30 psi; collision gas was 6 psi; the temperature was 400°C; ion spray voltage was 5,500 V; ion source gas 1 was set at 30 psi; the duration was 5 min. All data were acquired and processed with Analyst Software, version 1.6.3 (Applied Biosystems).

4-PPanSH was performed on an LC-MS platform composed of an LC-20AD series HPLC (Shimadzu Corporation, Kyoto, Japan), connected with a QTrap 5,500 mass spectrometer (AB Sciex, Ontario, Canada) and TurboIonSpray™ source. The HPLC system is equipped with a binary pump, a degasser, a column department, and an auto sample manager. A Shim-pack GIST-HP C18 column (150 × 2.1 mm, 3 µm) was used to achieve the separations of 4-PPanSH at 40°C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The separation gradient program followed: 0.0–1.0 min, (5.0% B); 1.0–4.0 min, (5.0%–95.0% B); 4.0–6.0 min, (95.0% B); 6.0–6.1 min: (95.0%–5.0% B); 6.1–9.0 min (5.0% B). The samples (10 µl) were produced at a 0.4 ml/min flow rate.
The mass spectrometer was equipped with an electrospray ionization (ESI) source that worked in positive ion mode, with the following MS parameters: ion spray voltages: 4500 V; the source temperature: 450°C; and ion source gas: 1 40 psi; ion source gas: 2 40 psi; decluttering potentials: 70 V and collision energies: 4-PPanSH (20 eV), Car-d10 (30 eV). Transitions (m/z) for MRM were 4-PPanSH 359.1→216.2; Car-d10 247.1→204.2. Chromatographic peaks were processed by extracting the ion chromatogram of the analyses at theoretical m/z. Applied Biosystems Analyst software version 1.5 was used to perform data acquisition and integration.

Tofacitinib concentrations in mouse plasma were determined by a liquid chromatography tandem mass spectrometry (LC–MS/MS) method developed and validated for this study. The LC-MS/MS method was preformed using an AB-4500 Qtrap (Sciex, Concord, ON, Canada) mass spectrometer with electrospray ionization source interfaced with a Shimadzu high-performance LC system. Separation was performed on an XBridge C18 column (50 × 2.1 mm ID, 3.5 µm; Waters, Milford, MA, USA) at a flow rate of 0.4 mL/minute. The mobile phase consisted of A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). The gradient was 0.0-0.5 minutes, 2% B; 0.5-2.0 minutes, 2-95% B; 2.0-3.6 minutes, 95% B; and 3.6-4.1 minutes, 95-2% B. The mass spectrometer was operated in positive mode with multiple reaction monitoring for analysis. The multiple reaction monitoring transitions were m/z 313.1 > 173.1 for tofacitinib and 455.2 > 425.2 for the internal standard. The gas temperature was 500°C with an ionspray voltage of 5500 V, gas 1 and gas 2 of 30 psi, and curtain gas of 30 psi. Analyst Software (version 1.6) from Applied Biosystems (MDS SCIEX; Carlsbad, CA, USA) was used to control the LC-MS/MS system, as well as for data acquisition and processing. All pharmacokinetic parameters were estimated using non-compartmental analyses with Phoenix WinNonlin software (Certara, Princeton, NJ).

After overnight fasting, venous blood samples were collected from participants in the first and second trimesters. The FPG and serum lipids, including cholesterol (CHOL), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were directly detected by an automatic biochemical analyzer (CI16200, Abbott, Abbott Park, IL, USA) in the first trimester. The serum levels of BCAAs (Val, Ile, and Leu) were detected by liquid chromatography-mass spectrometry on an Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to a QTRAP^® 4500 mass spectrometer (AB SCIEX, Foster City, CA, USA) in both the first and second trimesters. The mobile phase was 80% acetonitrile (acetonitrile/water) containing 0.1% formic acid. The detail settings are as follows: speed of the pump, 140 μL/min × 0.2 min to 30 μL/min × 1.0 min to 300 μL/min × 0.2 min; ESI source, positive mode; curtain gas, 14 psi; nebulizer gas, 40 psi; auxiliary gas 45 psi; ion spray voltage, 5500 V; source temperature, 580°C; declustering potential energy, 35 V; collision energy, 30 eV. BCAAs were measured by a neutral loss scan of 102 Da (scan range 125–340 Da) and multiple reaction monitoring. Applied Biosystems Analyst software (version 1.6) was used to control the system and to process the data.