Page 64 - Clinical-Medical Solution for Clinical Chemistry

Application No.C142$
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MRM parameter:
Q3 Q3 Ret. Time CE CE
# Name Polarity Q1
Qualifier 1 Qualifier 2 (min) Qualifier 1 Qualifier 1
1 Meldonium + 147.20 58.25 59.25 8.18 -27 -18
2 Etilefrine + 182.30 135.25 91.25 5.34 -20 -27
3 Norfenefrine + 154.20 91.25 65.25 6.01 -21 -35
4 Octopamine + 154.20 91.25 119.20 6.00 -21 -15
5 Oxilofrine + 182.30 149.25 105.25 5.69 -20 -22
6 Synefrine + 168.20 135.20 107.25 5.87 -20 -31
7 Meldonium-d3 + 150.20 62.25 60.25 8.18 -18 -30
8 Etilefrine sulphate + 262.20 164.15 5.19 -19
9 Synefrine sulphate + 248.20 150.25 135.20 5.68 -15 -30
10 Norfenefrine sulphate + 234.20 136.20 91.20 5.62 -18 -35
11 Etilefrine sulphate_neg - 260.20 180.20 121.10 5.19 18 39
12 Oxilofrine sulphate_neg - 260.20 77.10 178.20 5.49 26 12
13 Synefrine sulphate_neg - 246.20 148.20 106.10 5.70 20 30
14 Norfenefrine sulphate_neg - 232.20 152.20 121.15 5.69 17 36
15 Octopamine sulphate_neg - 232.20 134.15 107.10 5.81 22 30
#7 : Internal Standard
#8 ʙ 15 : Confirmation of Sulpho-conjugate
Compound list including MRM transitions for unchanged parent drug molecules and corresponding sulfonated
metabolites. Rapid polarity switching was used during the analysis to confirm peak identification.

Trap Analysis

Trap column
Waste
2 1 2 1
Pump A
Pump C
3 6 3 6
4 5 45
Analytical
column

Pump B

Fig. 2 Flow Diagram of 2D-HILIC System

Diluted urine samples were injected directly onto the
FCV (1-2) FCV (1-6)
2D HILIC system using a HILIC trapping column for B Conc. (%) Flow (mL/min)
clean-up and pre-concentration followed by an 100 B Conc.
effective HILIC analytical separation. 0.3
Pump A/B Flow
50 0.2
0.1
Pump C Flow
0 4.0 8.0 12.0 16.0 18.0
Fig. 3 Flow Rate and Gradient Program

Q Sample Preparation of Urine Sample
1. Centrifuge urine samples at 3,000 rpm for 10 min at room temperature.
2. Transfer 60 μL supernatant to new tube and add 10 μL IS solution (*) and 140 μL acetonitrile, mix the solution by
vortex mixing.
3. Centrifuge at 13,000 rpm for 5 min.
4. Transfer 180 μL supernatant to vial.
(*) Meldonium-d3 in 200 mM Ammonium Acetate