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.

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.

Elevating the use of sugar-free chewing gum in Germany: cost saving and caries prevention.

OBJECTIVE: To analyse the lifetime monetary and health related effects of the consumption of sugar-free chewing gum. MATERIAL AND METHODS: Using a Markov model we assumed that the German consumption of sugar-free chewing gum (111 gums/year) could be elevated to the level of Finland (202 gums/year). The calculation was based on a model patient representing the development of oral health from the age of 12 to 74 years and clinical data on the effectiveness of chewing sugar-free gum. Lifetime and yearly costs for the 'Finland-scenario' were determined and compared with the actual German expenses of the statutory health insurance companies for dental health. RESULTS: The actual total lifetime expenditures of the statutory health insurance companies are 17,199.96€ per capita and would be 12,188.94€ in the scenario with elevated consumption of sugar-free chewing gum in Germany. Thus, 5011.02€ per capita could be saved in a lifetime and 80.82€ per year. CONCLUSIONS: On a national scale, the elevation of the consumption of sugar-free chewing gum in Germany to the level of Finland would lead to a considerable benefit for cost saving and oral health for the statutory health insurance companies.

Residual rivaroxaban exposure after discontinuation of anticoagulant therapy in patients undergoing cardiac catheterization.

PURPOSE: Patients treated with direct oral anticoagulants (DOACs) frequently undergo interventional procedures requiring temporary discontinuation of anticoagulant therapy. Little is known about remaining peri-procedural exposure to rivaroxaban in real-world patients. METHODS: Fifty-six patients with rivaroxaban treatment and scheduled cardiac catheterization were included in this prospective, observational, and single-center study. Rivaroxaban concentrations were determined by LC-MS/MS and a chromogenic anti-Xa assay. Population pharmacokinetic modeling was carried out on LC-MS/MS concentration data using NONMEM software, and results were applied to Monte Carlo simulations to predict appropriate rivaroxaban discontinuation intervals. RESULTS: Rivaroxaban concentrations ranged from

A global approach to assess the economic benefits of increased consumption of sugar-free chewing gum.

PURPOSE: To analyze the influence of increasing the average consumption of sugar-free gum (SFG) in 25 industrialized countries on dental expenditures due to caries by the national health care systems. It was assumed that large cost savings were possible, because the regular consumption of SFG significantly reduces the relative risk of caries and therefore, improves dental health, which reduces expenditures on dental treatments. METHODS: A budget impact analysis (BIA) was performed to model the decrease in the relative risk of caries and the subsequent cost savings for dental care. Annual consumption of SFG, dental expenditures due to caries, chewing frequencies by age groups and the relative risk reduction for caries due to the consumption of SFG were identified and used as model parameters. Three different scenarios for the increase in the number of SFG were calculated. Besides overall results for all countries together, analyses were conducted for countries grouped by regions and the Human Development Index (HDI). RESULTS: For the entity of all 25 analyzed countries together, possible annual cost savings range from US$805.77 M in the scenario with the lowest increase of SFG consumption up to US$18,248 billion in the scenario with the biggest increase of SFG consumption. Europe and the USA show potential cost savings of US$1,061 billion and US$2,071 billion per year, respectively, if all chewers increase their consumption of SFG by 1 piece per day. The analysis showed the potential cost savings in dental expenditures due to caries that can be achieved by only slightly increasing the consumption of SFG. The regular consumption of SFG cannot replace good dental hygiene like tooth brushing, but can have a significant impact on dental health, which can lead to increased cost savings for health care systems worldwide. CLINICAL SIGNIFICANCE: Based on the fact that a regular consumption of sugar-free chewing gum has the beneficial effect of reducing caries prevalence, an increased consumption may not only lead to improved dental health but significant cost savings in expenditures for dental treatment worldwide.

Reliable and Easy-To-Use Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Analysis of Fluconazole, Isavuconazole, Itraconazole, Hydroxy-Itraconazole, Posaconazole, and Voriconazole in Human Plasma and Serum.

BACKGROUND: A fast and easy-to-use liquid chromatography-tandem mass spectrometry method for the determination and quantification of 6 triazoles [fluconazole (FLZ), isavuconazole (ISZ), itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), posaconazole (PSZ), and voriconazole (VRZ)] in human plasma and serum was developed and validated for therapeutic drug monitoring. METHODS: Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. Isotope-labeled analogues for each analyte were used as internal standards. Chromatographic separation was achieved using a 50 × 2.1 mm, 1.9 µm polar Hypersil Gold C18 column and mobile phase consisting of 0.1% formic acid/acetonitrile (45%/55%, vol/vol) at a flow rate of 340 µL/min. The triazoles were simultaneously detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring mode with positive heated electrospray ionization within a single runtime of t = 3.00 minutes. RESULTS: Linearity of all azole concentration ranges was verified by the Mandel test and demonstrated for all azoles. All calibration curves were linear and fitted using least squares regression with a weighting factor of the reciprocal concentration. Limits of detection (µg/L/L) were FLZ, 9.3; ISZ, 0.3; ITZ, 0.6; OH-ITZ, 8.6; PSZ, 3.4; and VRZ, 2.1. The lower limits of quantitation (µg/L/liter) were FLZ, 28.3; ISZ, 1.0; ITZ, 1.7; OH-ITZ, 26.2; PSZ, 10.3; and VRZ, 6.3. Intraday and interday precisions ranged from 0.6% to 6.6% for all azoles. Intraday and interday accuracies (%bias) of all analytes were within 10.5%. In addition, we report on a 29-year-old white woman (94 kg body weight) with a history of acute myeloid leukemia who underwent stem cell transplantation. Because of diagnosis of aspergillus pneumonia, antifungal pharmacotherapy was initiated with different application modes and dosages of ISZ, and plasma concentrations were monitored over a time period of 6 months. CONCLUSIONS: A precise and highly sensitive liquid chromatography-tandem mass spectrometry method was developed that enables quantification of triazoles in plasma and serum matrix across therapeutically relevant concentration ranges. It was successfully implemented in our therapeutic drug monitoring routine service and is suitable for routine monitoring of antifungal therapy and in severely ill patients.

Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS.

BACKGROUND: Assessment of the anticoagulant activity of direct oral anticoagulants (DOACs) is justified in special clinical situations. Here, we evaluated two independent extraction methods and developed a multi-analyte ultra-high performance liquid chromatography tandem mass (UHPLC-MS/MS) method for the quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. METHODS: Routine extraction based on protein precipitation with acetonitrile and subsequent centrifugation was compared to sample clean-up using commercial paramagnetic micro-particles and subsequent magnetic depletion. Stable isotope-labeled analogs of all analytes were employed as internal standards. The method was validated according to international guidelines in terms of linearity, precision, trueness, sensitivity, recovery and matrix effects. The performances of both extraction methods were assessed in clinical samples obtained from patients treated with either apixaban or rivaroxaban. Additionally, we report on a patient with nonadherence to rivaroxaban treatment and fulminant pulmonary embolism. RESULTS: The method was linear from 2 to 500 ng/mL for all analytes, and quantification of DOACs was established within a run time of 2.0 min. Based on MS/MS analyte responses, relative matrix effects were better controlled for dabigatran after extraction with paramagnetic micro-particles. Internal standards fully compensated for recovery and matrix effects in all assays, yielding equivalent results for both methods. Apixaban and rivaroxaban concentrations determined in clinical samples after extraction with both methods were in good agreement (R2=0.990). CONCLUSIONS: A rapid and accurate multi-component UHPLC-MS/MS method for the quantification of four DOACs in human plasma was established. Paramagnetic micro-particles appear suitable for clean-up of plasma samples for LC-MS/MS-based therapeutic drug monitoring purposes.

The Direct Factor Xa Inhibitor Rivaroxaban Passes Into Human Breast Milk.

Thromboembolic disorders frequently require antithrombotic treatment during pregnancy and lactation. Vitamin K antagonists and heparins are the treatment options of choice in breastfeeding women. Factors including the route of administration, discomfort during treatment, and fetal and neonatal safety affect women's choices about anticoagulant therapy. Direct-acting oral anticoagulants (DOACs) have emerged as alternatives to these agents and may offer advantages compared with vitamin K antagonists. As breastfeeding women were excluded from clinical trials evaluating DOACs, no safety and efficacy data are available for these special patients and, crucially, estimates for infant exposure are lacking. Therefore, the manufacturer recommends against using DOACs during the lactation period. We present the case of a patient who stopped breastfeeding owing to a diagnosis of postpartum cardiomyopathy. Anticoagulation with enoxaparin that commenced after the diagnosis of postpartum pulmonary embolism was switched to rivaroxaban. At that time, breast milk samples were collected and rivaroxaban concentrations were determined by liquid chromatography tandem-mass spectrometry. Rivaroxaban appears in human breast milk in comparatively small amounts; its safety has not been determined.

Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry.

BACKGROUND: The non-vitamin K antagonist oral anticoagulants (NOACs) apixaban, dabigatran, and rivaroxaban are being administered in fixed doses without routine monitoring of anticoagulant activities. Despite this key advantage over vitamin K antagonists (VKAs), assessment of anticoagulant intensities is required in various clinical circumstances. We developed a multi-analyte approach for mass spectrometric analysis of NOACs in human plasma. METHODS: Plasma samples were precipitated with acetonitrile. Separation was achieved by liquid chromatography using a C18 column and a gradient elution within a run time of 2.5 min. Positive electrospray ionization was used and ion transitions monitored by a triple quadrupole mass spectrometer. Stable-isotope-labeled analogues of analytes were employed as internal standards for quantitative analysis. Certified external quality control samples were obtained for external validation. RESULTS: For all analytes, linearity could be demonstrated over the concentration range of 1-500 µg/L (R2>0.999), and the calculated limits of quantification were <1 µg/L. Results for inter- and intra-day assay precision and trueness were obtained using internal quality control samples and remained within the acceptance criterion of ±15%. External quality control samples were measured at the specified nominal values with inter- and intra-day precisions <14%. Matrix effects were fully compensated by co-eluting internal standards, which in turn did not relevantly influence ionization efficiency. CONCLUSIONS: The method enables rapid and reliable simultaneous determination of NOAC concentrations in human plasma. It was successfully introduced into clinical practice; a case with rivaroxaban overdose is presented to exemplify the method's applicability.