Q advantage water purification system

5-(5-[2-(3-Aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl}amino)pyrazine-2-carbonitri le methanesulfonate hydrate [prexasertib methanesulfonate] and 5-({5-[2-(3-aminopropoxy-1,1,3,3-d₄)-6-methoxyphenyl]-1H-pyrazol-3-yl}amino)pyrazine-2-carbonitrile hydrochloride [prexasertib hydrochloride-d₄, internal standard (ISTD)] were supplied by Eli Lilly (Indianapolis, IN). Ammonium formate (for mass spectrometry, ≥99.0%) and albumin from bovine serum (lyophilized powder, ≥98% purity) were purchased from Millipore Sigma (St. Louis, MO). Formic acid (LC-MS/MS grade, 99.5% purity), acetonitrile (HPLC grade), and tert-butyl methyl ether (99% purity) were purchased from Fisher Scientific (Waltham, MA). Ringer’s Solution was purchased from Frey Scientific (Nashua, NH). CD-1 mouse plasma with sodium heparin as anticoagulant was purchased from BioreclamationIVT (Baltimore, MD). All water was prepared using Millipore Q-advantage water purification system (Temecula, CA). Captisol® used for dosing solution was purchased from Ligand (San Diego, CA).

The chemicals 5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]-purin-2-yl)pyrimidin-2-amine (GDC-0084; Figure 1a) and 5-(6,6-bis(methyl-d₃)-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]-purin-2-yl)pyrimidin-2-amine [GDC-0084-d₆, internal standard (ISTD); Figure 1b] were from Genentech (San Francisco, CA, USA). Ammonium formate (MS grade, ≥ 99.0%) was purchased from Millipore Sigma (St. Louis, MO, USA). Dimethyl sulfoxide (molecular biology grade), formic acid (LC-MS/MS grade, 99.5% purity), acetonitrile (ACN) (HPLC grade), and methanol (HPLC grade) were purchased from Fisher Scientific (Waltham, MA, USA). The Millipore Q-advantage water purification system (Temecula, CA, USA) was used to prepare the water used in all experiments. Human whole blood and human plasma, with K2-EDTA as an anticoagulant, and human CSF were purchased from BioIVT (Westbury, NY, USA).

5-({5-[2-(3-Aminopropoxy)-6-methoxyphenyl]-1H-pyrozol-3-yl}amino)pyrazine-2-carbonitrile methanesulfonate hydrate [prexasertib; Fig. 1a] and 5-({5-[2-(3-Aminopropoxy-1,1,3,3-d₄)-6-methoxyphenyl]-1H-pyrozol-3-yl}amino)pyrazine-2- carbonitrile hydrochloride [prexasertib-d₄, internal standard (ISTD); Fig. 1b] were supplied by Eli Lilly (Indianapolis, IN). Ammonium formate (for mass spectrometry, ≥99.0%) and albumin from bovine serum (lyophilized powder, ≥98% purity) were purchased from Millipore Sigma (St. Louis, MO). Formic acid (LC-MS/MS grade, 99.5% purity), acetonitrile (HPLC grade), and tert-butyl methyl ether (99% purity) were purchased from Fisher Scientific (Waltham, MA). Ringer’s Solution, an isotonic solution of sodium chloride, potassium chloride, calcium chloride, and sodium bicarbonate (to balance the pH) dissolved in water, was purchased from Frey Scientific (Nashua, NH). CD-1 mouse plasma with sodium heparin as anticoagulant was purchased from BioreclamationIVT (Baltimore, MD). All water was prepared using Millipore Q-advantage water purification system (Temecula, CA).