Low-dose acetylsalicylic acid therapy monitored with ultra high performance liquid chromatography

Assessment of compliance in patients on low-dose acetylsalicylic acid (ASA) therapy is limited to interview, pill counting, witnessed ASA intake, and measurement of thromboxane B2 levels. We validated a new, sensitive assay for analyzing blood levels of ASA and the metabolite salicylic acid (SA).

Blood samples were withdrawn from 10 healthy volunteers and 50 patients with stable coronary artery disease, before and after intake of 75 mg ASA. Plasma was mixed with acetonitrile, and 2-methylbenzoic acid was added as internal standard. After filtration, ASA and SA were quantified with ultra high performance liquid chromatography (UHPLC). Separation was accomplished with an isocratic flow of acetonitrile in phosphate buffer pH2.5, and a Zorbax RRHD C18 analytical column. Detection was achieved with wavelength photodiode array detection set at 237 nm.

The ASA- and SA-assay showed linearity (r(2)>0.999) within the range of 0.2-200 μg/mL, and with detection limits of 170 ng/mL and 53 ng/mL. The intra- and inter-day imprecision for ASA and SA were 2.2-5.9%, 3.4-11.7% and 1.8-8.0%, 3.8-9.4%, respectively. Recovery was between 89 and 103% for both assays. More than 60 coadministered drugs were investigated for interference, and only one drug interfered with the ASA-assay and none with the SA-assay. ASA was measurable 1h after intake of 75 mg ASA, and SA was measurable 1 and 6h after ASA intake.

We developed a fast and reliable UHPLC assay with a high sensitivity and selectivity, suitable for monitoring of compliance in patients treated with low-dose ASA.