Reliable and easy-to-use LC-MS/MS-method for simultaneous determination of the antihypertensives metoprolol, amlodipine, canrenone and hydrochlorothiazide in patients with therapy-refractory arterial hypertension

TY - JOUR

T1 - Reliable and easy-to-use LC-MS/MS-method for simultaneous determination of the antihypertensives metoprolol, amlodipine, canrenone and hydrochlorothiazide in patients with therapy-refractory arterial hypertension

AU - Johannsen, Jan-Ole

AU - Reuter, Hannes

AU - Hoffmann, Fabian

AU - Blaich, Cornelia

AU - Wiesen, Martin H J

AU - Streichert, Thomas

AU - Müller, Carsten

N1 - Copyright © 2018 Elsevier B.V. All rights reserved.

PY - 2019/2/5

Y1 - 2019/2/5

N2 - BACKGROUND: Therapy-refractory arterial hypertension is defined as a blood pressure (BP) in a subset of patients who fail to achieve BP control despite a three-drug regimen (including a diuretic). Various factors have impact on loss of therapy response. Drug-drug-interactions (DDIs) may cause altered pharmacokinetics (PK) of antihypertensive drugs. Upregulation of activity and expression of cytochrome P450 (CYP) enzymes can result in decreased plasma drug levels. Besides these PK considerations a significant problem could be nonadherence to drug therapy. In this regard Therapeutic Drug Monitoring (TDM) is a useful tool for detecting nonadherence. Therefore a LC-MS/MS-method for determination of Metoprolol (MET), Amlodipine (AML), Canrenone (CAN) and Hydrochlorothiazide (HCT) was developed.METHODS: An UHPLC-MS/MS method was developed and validated for simultaneous determination of MET, AML, CAN and HCT in plasma matrix. Extraction of serum samples consisted of simple protein precipitation using acetonitrile. Stable isotope labeled analogues for each antihypertensive were obtained for internal standardization and quantitative analysis ([2H7]-MET, ([13C6]-AML, [2H4]-CAN, [13C6]-HCT). Calibrators and quality controls were prepared in plasma matrix of normal individuals. Sample preparation: protein precipitation with acetonitrile and addition of internal standard-mix.RESULTS: All analytes were eluted within a runtime of 2.5 min. Linearity experiments were demonstrated in plasma over following concentration ranges: MET: 5-750 μg/l, AML: 1-50 μg/l, CAN: 10-500 μg/l, HCT: 5-500 μg/l (R2 > 0.993). Chromatographic separation was achieved using a C18 column (50 × 2.1 mm, 1.9 μm particle size) and an isocratic elution. LC-MS/MS analyses were performed on a triple quadrupole mass spectrometer using positive and negative electrospray ionization in selected reaction monitoring (SRM) mode. Ion transitions monitored for quantitation were m/z 268.2 → 74.1 for MET, m/z 409.1 → 238.0 for AML, m/z 341.2 → 91.0 for CAN and m/z 296.0 → 205.1 for HCT. For all analytes, inter- and intra-day precision (CV, %) varied between 1.7 and 14.0 and inter- and intra-day accuracy values ranged from -2.5 to 7.1%. The lower limits of detection and quantification were: 0.08 and 0.23; 0.05 and 0.15; 2.82 and 8.54; and 0.02 and 0.05 μg/l for MET, AML, CAN and HCT, respectively. Results of stability experiments were within the required range of +/- 15%.CONCLUSIONS: Although the level of recommendation of TDM of antihypertensive drugs in patients with refractory hypertension is not yet established, the present LC-MS/MS-method can serve as an effective tool for detection of PK-alterations/nonadherence and may help to monitor antihypertensive pharmacotherapy.

AB - BACKGROUND: Therapy-refractory arterial hypertension is defined as a blood pressure (BP) in a subset of patients who fail to achieve BP control despite a three-drug regimen (including a diuretic). Various factors have impact on loss of therapy response. Drug-drug-interactions (DDIs) may cause altered pharmacokinetics (PK) of antihypertensive drugs. Upregulation of activity and expression of cytochrome P450 (CYP) enzymes can result in decreased plasma drug levels. Besides these PK considerations a significant problem could be nonadherence to drug therapy. In this regard Therapeutic Drug Monitoring (TDM) is a useful tool for detecting nonadherence. Therefore a LC-MS/MS-method for determination of Metoprolol (MET), Amlodipine (AML), Canrenone (CAN) and Hydrochlorothiazide (HCT) was developed.METHODS: An UHPLC-MS/MS method was developed and validated for simultaneous determination of MET, AML, CAN and HCT in plasma matrix. Extraction of serum samples consisted of simple protein precipitation using acetonitrile. Stable isotope labeled analogues for each antihypertensive were obtained for internal standardization and quantitative analysis ([2H7]-MET, ([13C6]-AML, [2H4]-CAN, [13C6]-HCT). Calibrators and quality controls were prepared in plasma matrix of normal individuals. Sample preparation: protein precipitation with acetonitrile and addition of internal standard-mix.RESULTS: All analytes were eluted within a runtime of 2.5 min. Linearity experiments were demonstrated in plasma over following concentration ranges: MET: 5-750 μg/l, AML: 1-50 μg/l, CAN: 10-500 μg/l, HCT: 5-500 μg/l (R2 > 0.993). Chromatographic separation was achieved using a C18 column (50 × 2.1 mm, 1.9 μm particle size) and an isocratic elution. LC-MS/MS analyses were performed on a triple quadrupole mass spectrometer using positive and negative electrospray ionization in selected reaction monitoring (SRM) mode. Ion transitions monitored for quantitation were m/z 268.2 → 74.1 for MET, m/z 409.1 → 238.0 for AML, m/z 341.2 → 91.0 for CAN and m/z 296.0 → 205.1 for HCT. For all analytes, inter- and intra-day precision (CV, %) varied between 1.7 and 14.0 and inter- and intra-day accuracy values ranged from -2.5 to 7.1%. The lower limits of detection and quantification were: 0.08 and 0.23; 0.05 and 0.15; 2.82 and 8.54; and 0.02 and 0.05 μg/l for MET, AML, CAN and HCT, respectively. Results of stability experiments were within the required range of +/- 15%.CONCLUSIONS: Although the level of recommendation of TDM of antihypertensive drugs in patients with refractory hypertension is not yet established, the present LC-MS/MS-method can serve as an effective tool for detection of PK-alterations/nonadherence and may help to monitor antihypertensive pharmacotherapy.

KW - Amlodipine

KW - Antihypertensive Agents

KW - Canrenone

KW - Carbon Isotopes

KW - Chromatography, High Pressure Liquid

KW - Deuterium

KW - Drug Monitoring

KW - Drug Resistance

KW - Humans

KW - Hydrochlorothiazide

KW - Hypertension

KW - Limit of Detection

KW - Male

KW - Metoprolol

KW - Middle Aged

KW - Reproducibility of Results

KW - Tandem Mass Spectrometry

KW - Case Reports

KW - Journal Article

KW - Validation Studies

U2 - 10.1016/j.jpba.2018.11.002

DO - 10.1016/j.jpba.2018.11.002

M3 - SCORING: Journal article

C2 - 30439665

VL - 164

SP - 373

EP - 381

JO - J PHARMACEUT BIOMED

JF - J PHARMACEUT BIOMED

SN - 0731-7085

ER -