Fully automated therapeutic drug monitoring of anti-epileptic drugs making use of dried blood spots

Abstract

Fully automated dried blood spot (DBS) extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, decrease the hands-on time associated with conventional DBS analysis, resulting in a higher sample throughput, making the technique more compatible with a high-capacity bioanalytical workflow. The aim of this study was to develop and validate an LC-MS/MS method, using a DBS-MS 500 autosampler, for the determination and quantification of four anti-epileptic drugs (carbamazepine, valproic acid, phenobarbital and phenytoin) and one active metabolite (carbamazepine-10,11-epoxide) in DBS samples. Method development included thorough optimization of the fully automated extraction procedure (i.e. extraction solvent, extraction (loop) volume, internal standard application, internal standard drying time, etc.). The method was fully validated based on international guidelines. Accuracy (%bias), as well as precision (%RSD) (with a single exception) were below 13%. Neither carry-over nor unacceptable interferences were observed. All compounds were stable in DBS for at least 1 month when stored at room temperature, 4 °C and -20 °C and for at least 4 days when stored at 60 °C. Internal standard-corrected matrix effects were below 8%, with %RSDs below 9.1%. Reproducible relative recovery values (around 60% for all analytes) were obtained and the effect of the hematocrit on the relative recovery was overall limited. Successful application on capillary patient samples originating from developing countries demonstrated the applicability of the developed procedure in a remote setting.

Keywords: Anti-epileptic drugs; Automated extraction; Dried blood spots; Liquid chromatography-tandem mass spectrometry; Therapeutic drug monitoring.