Development and Validation of HPLC-MS/MS Method for Quantitative Determination of Metoprolol in Blood Plasma of Patients

INTRODUCTION: Metoprolol is a selective β1-adrenoblocker without intrinsic sympathomimetic activity. The effectiveness of metoprolol has been proven in numerous clinical studies in the treatment of arterial hypertension, stable angina, myocardial infarction, chronic heart failure. To improve the efficiency and safety of the therapy, it is advisable to carry out therapeutic drug monitoring of metoprolol, which requires a sensitive method for its quantitative determination. AIM: To develop, validate and test a method for the determination of metoprolol in human plasma using high performance liquid chromatography (HPLC) with tandem mass spectrometric detection (MS/MS). MATERIALS AND METHODS: The work was performed on Ultimate 3000 TSQ Fortis HPLC-MS/MS (Thermo Fisher, USA). For sample preparation, acetonitrile was used with fexofenadine hydrochloride at a concentration of 10 ng/ml as an internal standard, which was added to plasma samples in 3:1 ratio. The volume of the injected sample was 5 μl. Separation was performed on UCT Selectra C18 4.6 mm × 100 mm, 3 um, 100 A column with a similar pre-column at 35°C, in gradient elution mode in proportion of 0.1% formic acid solution/acetonitrile: 0 min — 80%/20%, 0.1 min — 45%/55%, 5 min — 10%/90%, 10 min — 80%/20%, at flow rate of 300 µl/min. Detection was performed in positive electrospray ionization mode, electrospray voltage 4000 V, sheath gas 50 arb, auxiliary gas 10 arb, purge gas 1 arb, evaporator temperature 350°C, ion transport tube temperature 300°C, using the multiple reaction monitoring mode (MRM) at argon flow rate 2 mTorr, 268 m/z → 115.5 m/z, Collision Energy 18 V, Tube lens 95 V, 268 m/z → 191 m/z, Collision Energy 17 V, Tube lens 95 V. The blood plasma of healthy volunteers served as matrix. RESULTS: The analytical range of the technique was 2–1000 ng/ml. The developed technique was tested on a patient with arterial hypertension. During the analysis, an equilibrium concentration of metoprolol of 12.0 ng/ml was detected in the patients blood plasma (previously, the patient took metoprolol tartrate at a dose of 12.5 mg twice a day for a week), and in 2 hours after taking the drug — 31.0 ng/ml. CONCLUSION: A method for the quantitative determination of metoprolol in human blood plasma using HPLC-MS/MS has been developed, validated and tested.