ABSTRACT
PURPOSE: As nonadherence to antihypertensive drugs (AHDs) can increase the risk of cardiovascular events, hospitalization, and higher costs, there is a need for a reliable, objective, and easy method to assess nonadherence in patients. The dried blood spot (DBS) sampling method used to measure drug concentrations meets these requirements. For detecting nonadherence, identification is more important than quantification. Owing to their use in clinical practice, it is important to measure multiple AHDs with a single method. Therefore, we developed and validated a single DBS method for 17 commonly used AHDs and 4 active metabolites using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHODS: Analytical validation of the DBS assay was performed in accordance with the guidelines on bioanalytical method validation of the European Medicines Agency and US Food and Drug Administration as well as the International Association of Therapeutic Drug Monitoring and Clinical Toxicology guidelines. RESULTS: We validated 12 of the 17 AHDs according to the European Medicines Agency and Food and Drug Administration requirements for bioanalytical method validation. Eleven AHDs were validated for both identification and quantification of drug concentrations, whereas nifedipine was only validated for identification. However, 5 of the 17 AHDs were excluded due to suboptimal validation results. Lercanidipine was excluded due to nonlinearity, and all 4 AHDs measured in the negative mode of UHPLC-MS/MS were not in accordance with one or more of the acceptance criteria and were therefore excluded. CONCLUSIONS: The described method accurately measured AHDs in DBS and can be used to determine nonadherence in patients. However, method validation revealed a challenging balance between analytical limitations and clinical needs when analyzing multiple drugs using the same method.
Subject(s)
Antihypertensive Agents , Tandem Mass Spectrometry , Chromatography, High Pressure Liquid/methods , Dried Blood Spot Testing/methods , Drug Monitoring/methods , Humans , Reproducibility of Results , Tandem Mass Spectrometry/methodsABSTRACT
Dried blood spot (DBS) microsampling has several advantages over venous blood sampling. In a clinical validation study of tacrolimus microsampling it was noted that tacrolimus DBS concentrations ([Tac]DBS) were systematically higher than tacrolimus whole-blood concentrations ([Tac]WB). This observation was explored by investigating the effect of using freeze-dried standards (STFD) for [Tac]DBS measurement. For all experiments, both non-frozen whole-blood samples and whole-blood samples that were frozen and thawed (to simulate freeze-drying) of 10 patients were analyzed. Multiple tacrolimus concentrations were measured: 1) [Tac]WB, 2) [Tac]DBS, where 15 µL was volumetrically applied to a pre-punched DBS disk, and 3) [Tac]DBS, where 50 µL was applied before a 6 mm DBS disk was punched from the card. All tacrolimus concentrations were determined independently using STFD and standards made of non-frozen blood spiked with tacrolimus (STSP). In both non-frozen and frozen and thawed whole-blood samples, [Tac]WB measured with STFD appeared similar to [Tac]WB measured with STSP (Ratios 1.061 and 1.077, respectively). In non-frozen samples, the median ratio between the [Tac]DBS measured with STFD, and [Tac]WB measured with STFD (the reference method), was 1.396. When blood was volumetrically applied to the DBS card (to eliminate the effect of the spreading over the filter paper), this ratio was 1.009. In conclusion, when using DBS microsampling to quantify concentrations of analytes, one should be aware that using the commercially available freeze-dried blood samples for the preparation of standards may affect the spreading of blood on the filter-paper, leading to a systematic error in the results.
Subject(s)
Dried Blood Spot Testing , Tacrolimus , Dried Blood Spot Testing/methods , Drug Monitoring/methods , Freeze Drying , Humans , Research DesignABSTRACT
Endothelin receptor antagonists (ERAs) such as, ambrisentan, macitentan and sitaxentan are primarily used for the treatment of pulmonary arterial hypertension. Considering the rise in endothelin in pre-eclampsia, ERAs may also be useful in its treatment. To evaluate the pharmacokinetics of ERAs, a rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated to determine the concentration of ambrisentan, macitentan and sitaxentan in human plasma. Plasma samples were treated with methanol to induce protein precipitation. A chromatographic separation was performed on a C18 column using a gradient of methanol-water containing 0.1% formic acid and 0.013% ammonium acetate and a flow rate of 0.5 ml/min. Multiple reaction monitoring was used for quantification. This method was validated in a linear range of 20.28-2028 µg/l for ambrisentan, 4.052-405.2 µg/l for macitentan and 205.4-10 270 µg/l for sitaxentan. The method was successfully validated according to US Food and Drug Administration guidelines to determine the concentrations of macitentan, ambrisentan and sitaxentan in human plasma. This method is now being used for study samples and clinical patient samples.
Subject(s)
Chromatography, High Pressure Liquid/methods , Isoxazoles/blood , Phenylpropionates/blood , Pyridazines/blood , Pyrimidines/blood , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Thiophenes/blood , Antihypertensive Agents/blood , Antihypertensive Agents/chemistry , Humans , Isoxazoles/chemistry , Linear Models , Phenylpropionates/chemistry , Pyridazines/chemistry , Pyrimidines/chemistry , Reproducibility of Results , Sensitivity and Specificity , Sulfonamides/chemistry , Thiophenes/chemistryABSTRACT
In this paper we present an FDA validated method to analyze ten antiarrhythmic drugs (atenolol, bisoprolol, carvedilol, diltiazem, flecainide, lidocaine, metoprolol, propranolol, sotalol and verapamil). A simple and fast sample preparation protocol with protein precipitation followed by ultra performance liquid chromatography (UPLC) for chromatographic separation and mass spectrometric detection applying electrospray ionization (ESI+) and selected reaction monitoring mode (MS/MS) was used. Only 50⯵l plasma sample is needed for the simultaneous quantification of all compounds within a 5â¯min run-to-run analysis time. Sotalol-D6, carvedilol-D5 and verapamil-D6 were used as internal standards. The method was validated according to the FDA guidelines. Correlation coefficients were higher than 0.998 for all compounds. Intra- and interday accuracies were within 15 CV(%) for all analytes. The method is currently successfully applied for routine analysis in our hospital.