Development and validation of a fully automated online human dried blood spot analysis of bosentan and its metabolites using the Sample Card And Prep DBS System.

This paper describes the development and validation of a liquid chromatography (LC)-electrospray ionization tandem mass spectrometry assay for the fully automated simultaneous determination of bosentan, a dual endothelin receptor antagonist used in the treatment of pulmonary arterial hypertension, and its three primary metabolites hydroxy bosentan (Ro 48-5033), desmethyl bosentan (Ro 47-8634), and hydroxy desmethyl bosentan (Ro 64-1056) in human dried blood spots (DBS) by use of the Sample Card And Prep (SCAP) DBS System. The system enabled the online extraction of compounds from filter paper cards without the need for punching and sample pretreatment. This was realized by automatic introduction of DBS sample cards into the LC flow via a pneumatically controlled clamp module. Using a three-column setup comprised of two pre columns for successive online DBS sample cleanup and a Synergi™ POLAR-RP C(18) analytical column for chromatographic separation under gradient conditions with a mobile phase A consisting of 1% acetic acid and a mobile phase B consisting of 1% acetic acid in methanol/2-propanol (80/20, v/v). MS/MS detection was performed in the positive multiple reaction monitoring mode using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray™ source. The total run time was 9.0min. The individual phases of online human DBS analysis were synchronized by automated valve switching. The analytical method was shown to be sensitive and selective with inter-day accuracy and precision of 91.6-108.0% and 3.4-14.6%, respectively, and it exhibited good linearity (r(2)≥0.9951 for all analytes) over the concentration range of 2ng/mL (5ng/mL for Ro 47-8634)-1500ng/mL. The analytes were stable in human DBS over 3.5 months at ambient temperature and accurate and precise results were obtained when using a blood spot volume between 20 and 30μL. Furthermore, no apparent (-8.9 to 12.6%) impact of hematocrit values ranging from 0.35 to 0.65 was observed on the quantification of the analytes. The system allowed very good recoveries of all analytes, between 83.0% and 92.3% for bosentan, between 94.4% and 100% for Ro 48-5033, between 98.0% and 100% for Ro 47-8634, and between 94.3% and 100% for Ro 64-1056. The validation demonstrated that the SCAP DBS System provides a robust automated platform for DBS analysis.