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

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 100μL of each sample were added with 40μL of internal standard and 860μL of water:acetonitrile 90:10, acidified with 0.05% formic acid. 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 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.