(Des-Gly10,D-Ala6,Pro-NHEt9)-LHRH

(Des-Gly10, D-Ala6, Pro-NHEt9)-LHRH as internal standard for leuprorelin acetate^[1]
(1) Add 10 μL 200 ng/mL (Des-Gly10,D-Ala6,Pro-NHEt9)-LHRH solution to 100 μL plasma sample.
(2) Add ice-cold acetonitrile to solution in (1) in two batches (500 μL each time).
(3) Stir at 2500 rpm for 10 min.
(4) Centrifugation at 12500 rpm (4°C) for 10min.
(5) Transfer the supernatant into a centrifuge tube, dried under nitrogen gas at 35°C, and finally add 100 μL 0.1% formic acid in water/acetonitrile: 90/10 (v/v) solution.
(6) The Thermo Scientific Vanquish UPLC system is connected to the Thermo Scientific Orbitrap Explorer 480 mass spectrometer. Using the Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) separation at a temperature of 40°C.
(7) The mobile phase A is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid in acetonitrile solution. The gradient starts at 95% mobile phase A, decreases to 10% over 5 min, and then remains constant for 0.5 min. At 6 min, the column returned to 95% mobile phase A and is rebalanced for 2 min. The flow rate was 0.3 mL/min and the injection volume is 20 μL.
(8) The sample is ionized by 3500V electric spray. In positive-ion mode, m/z 605.3275 is leuprolide acetate, and m/z 584.3052 is the internal standard. Data are analyzed by Xcalibur software (version 4.4).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.