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Determination of β-blockers in Plasma using Cleanert Micro 96-well Plate PEP and analysis by LC-MS/MS

Application Introduction

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Introduction

Metoprolol, oxprenolol and propranolol are β-receptor antagonists used to treat arrhythmia, prevent second heart attack (secondary prevention) and treat hypertension under certain conditions. β-receptor antagonists non-competitively block the endogenous catecholamine adrenaline and the norepinephrine in sympathetic nervous system, especially β-adrenergic receptors, whereas some drugs block all β-adrenergic receptors, others do so competitively.


 

 Metoprolol

Oxprenolol

Propranolol


 

Figure 1 Structures of Metoprolol, Oxprenolol and Oxprenolol

Experimental

Sample preparation

High concentration (50 ng/mL): Add 100 ng/mL standard dissolve in Methanol: H2O =1:9 with plasma (1/1, v/v). Low concentration (5 ng/mL): Add 10 ng/mL standard dissolve in Methanol: H2O =1:9 with plasma (1/1, v/v). Blank: Add Methanol: H2O =1:9 with plasma (1/1, v/v).

SPE Procedure with Positive Pressure SPE-M96

Condition: 200 μL Methanol;200 μL Water;

Load: 200 μL plama;

Wash: 200 μL 5% Methanol in Water;

Elution: 50 μL 2% Formic acid in Acetonitrile: Isopropyl alcohol=4:6;

Reconstitute : Add 150 μL Water with elution to 200 μL.

Instrumentation

Column: Unisol C18 ( 2.1x150mm, 5μm, 100 Å);

Flow rate: 300 μL/min;

Temperature: 30 °C;

Injection: 10 mL;

Mobile phase: A-0.1% formic acid in waterB-0.1% formic acid in Acetonitrile;

Scanning mode: Positive; Scan type: MRM; IonSpray Voltage: 5000 V; Curtain Gas: 15 Psi

Ion Source Gas 1: 50 Psi; Ion Source Gas 2: 40 Psi; Temperature: 500 °C.

Table 1 Gradient condition used for separation

Total Time(min)

Flow Rate(µl/min)

A (%)

B (%)

0

300

78

22

10

300

78

22

Table 2 Quantifier and qualifier MRM transitions and retention times for the compounds

Compound

Retention time

Q1

Q3

DP

CE

Metoprolol

2.5

268.3

116.1*

115

26

191.0

25

Oxprenolol

4.1

260.1

183. 1*

100

24

157.1

29

Propranolol

7.7

266.1

116.1*

105

24

225.1

19


 

Results and Discussion

Figure 2 MRM spectra of standard solution(5 ng/mL)

Figure 3 MRM spectra of blank plasma

Figure 4 MRM spectra of spiked plasma

Table 3 The recovery of compounds (n=8)

Concentration

Metoprolol          Average recovery (RSD)

Oxprenolol        Average recovery(RSD)

Propranolol   Average recovery (RSD)

50 ng/mL

108.7% (RSD=3.3%)

103.1% (RSD=5.1%)

106.0% (RSD=4.4%)

5 ng/mL

107.7% (RSD=4.5%)

99.0% (RSD=4.5%)

94.2% (RSD=8.4%)

Conclusion

This experiment uses Cleanert® Micro Plate PEP to extract metoprolol, oxprenolol and propranolol from plasma and elute them in micro volume. The eluent will reach the same detection limit without the need for nitrogen concentration. The recovery rate and RSD value are both good and suitable for relevant testing.

Ordering Information

Product   Name

Specification/Package

Catalog   No.

Cleanert® Micro Plate PEP

5 mg,1 mL/well,2/PK

PE00501-MW

96-well   Positive pressure device

Compatible   with 96-well plate

SPE-M96

Collection   Plate

1mL/well,   square well

96SP2036-2

Unisol C18

2.1x150mm,5 μm,100 Å

UO950502-2



  • Cleanert PEP

    Cleanert PEP is made of polydivinylbenzene on which the surface is functionalized with vinyl pyrrolidone. The material has a balanced hydrophilic and hydrophobic property and can be used in the entire pH range of 1-14.

  • Unisol Technology and Column Products

    Bonna-Agela's unique sol-gel process technology generates an uniform surface that produces an universal column applicable to several kinds of HPLC applications.

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