2.3 Sample Preparation and LC-MS Protein precipitation of serum samples (10 µL) and serum standards

(10 µL) was performed in 96-well Strata Impact 2 ml filtration plates (Phenomenex, Torrance, CA). To each well was added 490 µL acetonitrile:water:formic acid (85:14.8:0.2 v/v) containing citrulline+5 stable isotope as internal standard (IS). This was followed by the addition of 10 µL of serum. After mixing gently, the plate was covered, allowed to stand Sapitinib clinical trial for 5 minutes, and the filtrate was collected under vacuum. The 96-well collection plate was loaded into the Acquity (Waters, Corp., Milford, MA) sample manager and the sample (3 µL) was injected onto the analytical column. The high-performance liquid chromatography (HPLC) system was a Waters Acquity series (Waters) equipped with a sample manager, binary pump, in-line degasser, and a column thermostat. The mass spectrometer was a Quattro Premier equipped with an electrospray ionization probe (Waters).

Analytical separation was optimally achieved on a Phenomenex 1.7 µm KinetexDiol analytical column [50 × 2.1 mm (i.d.)]. FA was separated using a linear binary gradient in hydrophilic interaction liquid chromatography (HILIC) mode (Mobile phase A: acetonitrile containing 0.1 % formic selleck screening library acid, 0.2 % acetic acid and 0.005 % trifluoroacetic acid; Mobile phase B: water containing 0.1 % formic acid, 0.2 % acetic acid and 0.005 % trifluoroacetic acid). Initially the flow rate was 0.4 mL/min. The gradient was increased from 10 to 80 % B in the first 2.3 minutes and held at 80 % B for 0.2 minutes while the flow check rate was increased to 0.6 mL/min. The gradient was returned to 10 % B over 1 minute. The total run time was 5.0 minutes. Detection of 5-13C, 4,4,5,5-2H-citrulline

(citrulline+5) and FA was achieved following electrospray ionization interfaced to a Quattro Premier triple quadrupole mass spectrometer (Waters). Positive ions for FA and citrulline+5 were generated using a cone voltage of 22 and 18 V, KU55933 ic50 respectively. Product ions were generated using argon collision-induced disassociation at collision energy of 10 eV while maintaining a collision cell pressure of 2.8 × 10−3 torr. Detection was achieved in the multiple-reaction-monitoring (MRM) mode using the precursor → product ions, m/z180.2 → 162 and 181 → 164, for FA and citrulline+5, respectively. Citrulline+5 (5 µM) served as the internal standard. Matrix ion effects were evaluated using the post-column infusion technique, which has been described elsewhere [14]. Separate citrulline+5 (10 µM) and FA (10 µM) solutions were prepared in acetonitrile containing 20 % water. These were infused in separate experiments at a rate of 10 µL/min and mixed with column eluent during an injection of extracted serum. Analytical recovery and inter-day precision were evaluated using quality control standards prepared from a separated stock solution of FA.