E. Perlo

UHPLC–MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients

Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity(®) UPLC HSS T3 1.8μm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Adherence to antihypertensive therapy and therapeutic dosage of antihypertensive drugs

Adherence to antihypertensive therapy is critical to achieving adequate blood pressure control. About half of hypertensive patients do not take their drugs as directed and the physicians often underestimate this issue. Non-adherence has important public health economic implications (numbers of visits, diagnostic procedures, prescribed drugs) and, moreover, it results in increased morbidity and mortality rates. Poor adherence can have several patients and therapy related causes. Currently, multiple different direct and indirect methods to measure therapeutic adherence are available, but, in clinical practice, there is no cost-effective and simple one. Therapeutic drug monitoring (TDM), characterized by drug (or metabolites) concentration measurement in body fluids (blood or urine), is a cost-effective direct method to assess therapeutic adherence. Despite some limitations, TDM may decrease health costs, by reducing the number of visits and by identifying those patients who would undergo unnecessary invasive procedures. Moreover, TDM can be a new alternative method to identify patients with true resistant hypertension, improving the achievement of blood pressure control In this minor revision, we would assess poor therapeutic adherence in hypertensive population, analyzing the different direct and direct available methods, with emphasis on TDM.

UHPLC-MS/MS method with sample dilution to test therapeutic adherence through quantification of ten antihypertensive drugs in urine samples

Graphical abstract Figure. No caption available. HighlightsRobust UHPLC–MS/MS method to quantify a wide panel of antihypertensive drugs.Urine sampling allows immediate and non‐invasive testing.Fast sample preparation, suitable for rapid adherence‐screening purpose.The method has been tested on real‐life specimens with good results. Abstract Nowadays, hypertension represents an important health problem, particularly in developed countries. In some cases the standard therapeutic approaches are not able to reestablish the normal blood pressure values: this condition is called “resistant hypertension”. However, a fraction of cases of resistant hypertension are actually due to poor adherence to the prescribed therapy. Therapeutic Drug Monitoring could represent a direct and useful tool to correctly identify non‐compliant patients. Nevertheless, high throughput methods for the simultaneous monitoring of a wide panel of drugs in the same analysis are lacking and, furthermore, there is not a generally acknowledged “standard” matrix for this test (plasma or urine). In this work, we validated a UHPLC–MS/MS assay to quantify ten among the most used antihypertensive agents in urine samples, covering all the current classes: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Both standards and quality controls were prepared in urine matrix. Only 100 &mgr;L of each sample were added with 40 &mgr;L of internal standard and 860 &mgr;L of water:acetonitrile 90:10, acidified with 0.05% formic acid. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 &mgr;m 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra‐day and inter‐day precision fitted FDA guidelines for all analytes, while matrix effects resulted reproducible among different urine lots. Method performances were tested on urine samples from hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

NURSE-OBP: A NURSING STANDARDISED METHOD FOR MEASURING BLOOD PRESSURE

Objective: Objective. The role of the nurse in both diagnostic and therapeutic management of hypertensive patient is becoming more and more important; nevertheless until now there is not a nursing standardised method of blood pressure (BP) measurement. The aim of this study was to standardise a nursing method for measuring blood pressure (Nurse-OBP) and to compare it with the Ambulatory Office Blood Pressure. Figure. No caption available. Design and method: Design and Method. 118 treated non-diabetic hypertensive adults afferent to our Hypertension Unit were included in the study. All underwent an AOBP measurement with Omron HEM-907XL and the Nurse-OBP measurement, designed to be methodologically identical to the classical AOBP proposed by Meyers and colleagues but which takes place with the nurse staying in the same patients room during the BP measurement. All subjects were randomized into two groups, different according to the order in which the two method of measurement were performed to avoid eventual reduction of white coat effect mostly deriving from changes in patient behaviour, and finally, during the medical examination all underwent a traditional OBP measurement. All patients signed an informed consent and the study was approved by local ethical committee (CEI 652). Pearsons correlation coefficient was used to evaluate agreement between AOBP and Nurse-OBP measurements Results: Results. Mean Nurse-OBP values (132 ± 19.4/73.3 ± 12.9) were significantly lower than OBP ones (141.3 ± 18.8/84.7 ± 10.7). When AOBP and Nurse-OBP were compared, no statistically significant differences between the two methods were found. The Bland - Altman analysis showed how the presence of the nurse could cause a minimum rise of BP values, (mean SBP and DBP difference respectively of 3.57 ± 12.23 mmHg and 2.25 ± 7.26 mmHg), but clinically non significant; therefore the two modalities, AOBP and Nurse-OBP, can be considered as comparable. Furthermore no differences in terms of Nurse-OBP values were found when the two groups were compared. Conclusions: Conclusions. Our study proposes a standardised nursing method for measuring BP absolutely comparable to AOBP technique, that could have practical implications mostly when it is not possible to have a dedicated room where performing AOBP.

Therapeutic drug monitoring guided definition of adherence profiles in resistant hypertension and identification of predictors of poor adherence

Arterial hypertension is an important cardiovascular risk factor. A substantial proportion of patients show resistance to antihypertensive treatment but poor adherence to medication regimens is also a significant cause of treatment failure. In this context, therapeutic drug monitoring (TDM) could be useful. The objective of this study was to assess adherence to treatment in patients with resistant hypertension by TDM and to identify parameters that predict nonadherence.

[PP.25.08] THERAPEUTIC DRUG MONITORING IN RESISTANT HYPERTENSIVES PREVIOUSLY TREATED WITH INVASIVE APPROACHES

Objective: Renal denervation (RDN) and baroreflex activation therapy (BAT) are invasive therapeutic approaches for resistant hypertension (RH), indicated when antihypertensive drug therapy is inefficacious. Several reports have found an unsatisfactory response to these approaches; one of the factors called upon to account for the failure of these devices is the lack of therapeutic adherence. The aim of this study was to retrospectively evaluate the therapeutic adherence, using an objective assessment as the therapeutic drug monitoring (TDM), in a group of patients who have undergone invasive approach in previous years, either RDN or BAT, and correlate it with the answer to the invasive procedure. Design and method: We retrospectively analysed 12 patients with RH (9 females). Before performing invasive procedure, all patients were considered adherent according to the physician intuition and the answers given to the Morisky questionnaire. Serum TDM, by Ultra High Performance Liquid Cromatography (UHPLC) method, was done during a routine follow up visit, after at least one year from the last invasive approach. A clinical response to invasive approaches was considered as a reduction in 24 h Ambulatory Blood Pressure Monitoring (ABPM) systolic blood pressure (SBP) of at least 10 mmHg at 12 months from the procedure. Patients were considered adherents (AD) if all the prescribed drugs were found or only one was not found; patients were considered non-adherents (NAD) if none of the prescribed drugs were found. Results: 4 patients were AD and 8 NAD. In the whole sample a clinical response was found in 41% of the patients. Among AD the clinical response rate was 75%, whilst among NAD the clinical response rate dropped to 25%. No significant clinical and social differences were found between NAD and AD, but NAD had significant higher office SBP and 24 h ABPM SBP than AD (p < 0.05). Conclusions: Lack of therapeutic adherence is associated to a limited clinical efficacy of invasive approaches in apparent RH. Our data underlines the importance of adherence therapeutic evaluation before and after performing invasive procedures, whose effectiveness can be undermined by poor therapeutic adherence.

[OP.2B.04] DEVELOPMENT OF AN INNOVATIVE, HIGH THROUGHPUT AND COST-EFFECTIVE UHPLC-MS/MS METHOD FOR THE ASSESSMENT OF THERAPEUTIC ADHERENCE IN PATIENTS WITH “RESISTANT” HYPERTENSION

Objective: Nowadays hypertension affects a large fraction of global population. Moreover, 15–30% of patients show drug-resistance, needing the addition of more drugs; meanwhile, polytherapy often results in poor therapeutic adherence, classified as “pseudo-resistant” hypertension. In this context, Therapeutic Drug Monitoring (TDM) of antihypertensive drugs, which consists in the measurement of drug concentration in biological matrices, may help to discern problems in drug pharmacokinetics/pharmacodynamics from cases of poor therapeutic adherence, also considering that not-pharmacological alternative consists in invasive surgery. It has been hence validated an UHPLC-MS/MS method for simultaneous TDM on plasma samples of ten antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Design and method: This method has been validated according to FDA guidelines. 200 &mgr;l of sample, standard and quality control, added with 40 &mgr;l of internal standard working solution, undergo a simple protein precipitation protocol, followed by drying step, and resulting extracts are resuspended in water:acetonitrile 90:10 (v:v; +0.05% formic acid) and then analyzed through a Shimadzu NexeraX2® UHPLC system coupled with a LCMS-8050® tandem mass detector. The validated method was tested on real samples from patients with RH/pseudo-RH, all giving informed consent. Results: All analytical parameters of the method fitted FDA guidelines for all the analytes. 42 patients have been enrolled (SEAL study). TDM revealed that 55% of patients (n = 23) had detectable concentration of all prescribed drugs (considered fully adherent), 26% (n = 11) resulted partially non-adherent (only part of the prescribed drugs was detectable) and 19% (n = 8) were totally not-adherent (no drugs were detected). Through univariate/multivariate logistic regression, the diastolic pressure and the “white-coat” increase in heart rate resulted the best predictors of poor adherence. Through ROC curve analysis a diastolic pressure over 124 mmHg resulted predictive of total inadherence. Conclusions: TDM can be a gold-standard for evaluating therapeutic adherence and this method results eligible for a clinical routine use. We managed to discern cases of inadherence, preserving some patients from an invasive and expensive surgery. Figure. No caption available.

[PP.01.15] ACCURACY OF HOME BLOOD PRESSURE MONITORING IN ARTERIAL HYPERTENSION DIAGNOSIS

Objective: The arterial hypertension diagnosis is based on office blood pressure measurement, and current guidelines suggest the use of out-of-office blood pressure measurement techniques in specific cases, as suspected white-coat or masked hypertension. Home Blood Pressure Monitoring (HBPM) is recommended as a complementary method to Ambulatory Blood Pressure Monitoring (ABPM). However usually HBPM is only used for implementing blood pressure control in treated patients. We tried to identify the accuracy between HBPM and ABPM in untreated patients. (We tried to identify HBPM accuracy between to ABPM in untreated patients.) Design and method: We enrolled 83 consecutive untreated patients who performed ABPM in our Hypertension Unit and completed a short HBPM schedule (two measurements, twice daily, for four days) between November 2011 and June 2015. Patients were instructed about HBPM in accord to current hypertension guidelines and they used validated automated arm devices. We compared the accuracy between the two techniques and the HBPM ability to identify arterial hypertension in comparison with ABPM. Results: Pearsons correlation coefficient between HBPM 4-day average and day-time ABPM values was 0.59 for systolic blood pressure (SBP) and 0.77 for diastolic blood pressure (DBP). Bland-Altman analysis revealed a mean difference of -5.68 mmHg, SD 8.82 mmHg for SBP, and -4.64, SD 6.33 mmHg for DBP. ROC curves described AUC for SBP of 0.75 and for DBP of 0.877. The ABPM identify as hypertensive 54 subjects on 83 (65.1%), the HBPM 29 subjects (34.9%), p-value 0.01609. Conclusions: HBPM has a moderate correlation and a moderate accuracy in the identification of arterial hypertension compared with ABPM. Although HBPM is recommended as alternative method respect to ABPM, in untreated patients it is not reliable for arterial hypertension diagnosis and probably it is not able to identify specific hypertension patterns, in contrast with current guidelines.

[PP.23.13] ANALYSIS OF ELIGIBILITY CRITERIA FOR RENAL SYMPATHETIC DENERVATION

Objective: Resistant hypertension (RH) is a rare condition that affects approximately 10% of hypertensive population, its defined as blood pressure (BP) > 140/90 mmHg despite three full doses antihypertensive drugs including a diuretic. True RH is confirmed when pseudo-hypertension, secondary hypertension or poor adherence are excluded. Percutaneous radio-frequency catheter-based renal sympathetic denervation (DRN) is one of the most used invasive treatments for these patients. The goal of the study is to assess the percentage of eligibility to DRN and analyze the exclusion criteria in a group of resistant hypertensive patients. Design and method: We retrospectively analyzed data of 35 patients (63% female) referred to our Hypertension Unit between June 2011 and June 2014. We considered eligible for DRN subjects with office systolic blood pressure > = 160 mmHg and patients with severe hypertension treated with fewer drugs for poly-intolerances/allergies. Secondary hypertension form and white coat hypertension were excluded. Patients with confirmed true resistant hypertension underwent CT angiography in order to check the renal anatomic criteria of eligibility to the DRN. Results: 35 caucasian patients (63% female) referred to our Hypertension Unit between June 2011 and June 2014 for DRN assessment. At the first evaluation median systolic and diastolic office BP were 179 ± 25 mmHg and 105 ± 20 mmHg; six month later, after appropriate changes in lifestyle and drug therapy, systolic/diastolic office BP was reduced of 18/8 mmHg (p < 0.05). In the most of cases, patients had to BP control with introduction of antialdosteronic (35%). In our sample, 27 patients were considered unsuitable for the DRN for many reason: blood pressure control with optimization of drug therapy (52%), evidence of white coat effect (22%), secondary hypertension (22%), lack of true resistant hypertension (18%), absence of consent to the procedure (29%). Conclusions: A carreful patients selection in Specialistic Center is necessary before DRN; indeed frequently a good BP control is obtained with appropriate drug therapy changes and exclusion of secondary forms.

[PP.08.16] AUTOMATICALLY CLASSIFYING ESSENTIAL ARTERIAL HYPERTENSION FROM PHYSIOLOGICAL AND DAILY LIFE STRESS RESPONSES

Objective: Essential arterial hypertension (EAH) might be correlated with an abnormal response to stressful life events. For understanding the psychological dimensions associated with EAH, we investigate individual reactions to stressful events in everyday life. We aim to assess the combined influence of individual perception of stress and physiological signals by continuously monitoring patients with EAH. Design and method: Two groups of subjects, balanced by gender, were compared: twelve normotensive, and twelve diagnosed with EAH grade I or II controlled by therapy, without organ damage (average age 49,2, average BMI 25,43 Kg/m2, mean office arterial pressure 127,57/83,57, average heart rate 73,5, average number of drugs 1,5). Patients performed ambulatory blood pressure monitoring 24 hours (ABMP) to rule out white coat effect. Controls were checked to rule out hidden hypertension. Blood Volume Pulse (BVP), Heart Rate Value (HRV), Galvanic Skin Response (GSR) and skin temperature were continuously recorded by Empatica E3 device. An iPhone application capable of storing and streaming physiological signals was provided; it elicited annotations from subjects to report their psychological state, perceived and reported stress and workload, and daily activities. We used behavioural analytics algorithms to analyse and classify the recorded data. Results: Two dimensions of emotional response were identified - emotional suppression, and anxious anticipation of stressful events. Subjects with high and low scores in emotional inhibition are equally represented in the two groups, but subjects with high scores on anticipatory anxiety were more represented in the EAH category. The EAH subjects recorded higher percentage of both anticipated and reported stress. Physiological signal streams showed that EAH subjects significantly differ from controls. HRV features showed significant differences between normotensive and hypertensive patient groups. Combining HRV features with other physiological signals such as GSR and BVP provided a high accuracy in distinguishing between the two groups. Conclusions: Hypertension category tends to overestimate stressful events, and are incline towards anticipatory anxiety. They also exhibit different physiological responses to stress when compared to normotensive subjects. This pattern is relevant to the postulated links between hypertension and patterns of emotional response.