Bio-Analytical SPE Method: Avatrombopag

Avatrombopag

Experiments	Results
Avatrombopag	Avatrombopag (Doptelet), is an orally administered, small molecule thrombopoietin receptor (c-Mpl) agonist that increases platelet number without increasing platelet activation, thereby decreasing the need for blood transfusions
Chemical structure
Molecular weight	649.65 g/mole
Dose, formulation and route of administration	20 mg tablets, Oral administration
LLOQ & ULOQ	1.0- 250 ng/ml

Chromatographic conditions

Experiments	Results
Mobile phase	A. 70:30 ACN:Methanol containing 0.1% Formic acid B.15 Mm Ammonium format containing 0.1% Formic acid.
Column	5 μm, GEMINI-NX C18 column (50 × 46mm),110 Å
Flow rate	0.8 mL/min Gradient start with 50% A to 65% A while peak elution
Column temp	40 0C

Mass spec conditions

Experiments	Results
Mass spec/td>	API-4000
AVT m/z	649.1 → 267
AVT D8 m/z	657.1 → 267
Ion source	Ion source
Mode	Positive ionization mode

Sample Preparation SPE, Affinity MAX 25mg, 1 cc cartridges

Experiments	Results
Sample pretreatment	Plasma (100 uL) + drug + IS- mix well. Add 650 uL Acetonitrile and shake well. Centrifuge. Take 400 ul supernatant and add 600 ul 10% Ammonia solution-Mix well
Condition	1 ml Methanol
Equilibration	1 ml 5% Ammonia solution
Sample Loading	Load supernatant sample
Washing	Wash-1 1 ml 5% Ammonia solution. Wash-2 1 ml water Wash-3 1 ml Methanol)
Elution	250 ul elution solution (1% formic acid in ACN) twice. Add 500 ul recovery solution (60:40, ACN: 15Mm Ammonium formate) and mix well. No evaporation needed.
Direct Injection	10 uL