Beh 1.7 μm

A self-fabricated nano-reverse phase C18 column (15 cm, 75 μm i.d., BEH 1.7 μm, 130Å, Waters) was used for FA separation. A Waters nanoAcquity UPLC system was coupled to a Thermo Scientific Orbitrap Elite mass spectrometer for all LC-MS/MS analyses. Mobile phase A was water with 0.1% formic acid, and mobile phase B was ACN with 0.1% formic acid. The flow rate was 0.3 μL/min with 2 μL injection volume for each experiment. The following gradient was used (time, % mobile phase B) unless otherwise specified: (0 min, 10%), (5 min, 10%), (85 min, 90%), (104 min, 90%), (105 min, 10%), (120 min, 10%). The following MS parameters were used for all data acquisition. Samples were ionized in positive ion mode with a spray voltage of 1.9 kV. S-lens RF level was set to be 65, and capillary temperature was set to be 275 °C. Full MS scans were acquired at m/z 300–700 with resolving power of 30 K (at m/z 400). Maximum injection time of 100 ms, automatic gain control (AGC) target value of 1e6, and 1 microscan were used for full MS scans. Top 3 data-dependent MS/MS analysis was performed at a resolving power of 30 K (at m/z 400) with HCD normalized collision energy of 30 and fixed first mass at m/z 100.

Serum (20 μl) from each group was processed by adding 3 volume of acetonitrile to precipitate protein and the supernatant was subjected to liquid chromatography-tandem mass spectrometry (LC-MS). All analyses were performed using an ACQUITY UPLC system, coupled to an API-6500 mass spectrometer. The analytes were separated using a reverse phase C18 column (Waters, BEH 1.7 μm, 100 Å, 2.1 × 50 mm) at 50 °C and the mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid with a flow rate of 0.8 ml/min. The flow was sprayed to API-6500 mass spectrometer at electrospray ionization (ESI) positive mode in unit resolution and source temperature of 550 °C. Multiple reaction monitoring (MRM) was used to monitor the transition of protonated parent ion 425.1 Da to the product ion 305.0 Da for compound Ro-31-8425, transition of 237.1-194.4 Da for internal standard carbamazepine. The limit of quantification (LOQ) of Ro-31-8425 in the mouse serum was 0.1 ng/ml. The method was linear in the range of 0.1–250 ng/ml with a coefficient of correlation greater than 0.994 in the mouse serum.