Automated high-capacity on-line extraction and bioanalysis of dried blood spot samples using liquid chromatography/high-resolution accurate mass spectrometry Regina V. Oliveira 1† , Jack Henion 1 * and Enaksha R. Wickremsinhe 2 1 Quintiles Bioanalytical and ADME Laboratories, 19 Brown Rd., Ithaca, NY 14850, USA 2 Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA RATIONALE: Pharmacokinetic data to support clinical development of pharmaceuticals are routinely obtained from liquid plasma samples. The plasma samples require frozen shipment and storage and are extracted off-line from the liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems. In contrast, the use of dried blood spot (DBS) sampling is an attractive alternative in part due to its benefits in microsampling as well as simpler sample storage and transport. However, from a practical aspect, sample extraction from DBS cards can be challenging as currently performed. The goal of this report was to integrate automated serial extraction of large numbers of DBS cards with on-line liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAMS) bioanalysis. METHODS: An automated system for direct DBS extraction coupled to a LC/HRAMS was employed for the quantification of midazolam (MDZ) and α-hydroxymidazolam (α-OHMDZ) in human blood. The target analytes were directly extracted from the DBS cards onto an on-line chromatographic guard column followed by HRAMS detection. No additional sample treatment was required. The automated DBS LC/HRAMS method was developed and validated, based on the measurement at the accurate mass-to-charge ratio of the target analytes to ensure specificity for the assay. RESULTS: The automated DBS LC/HRAMS method analyzed a DBS sample within 2 min without the need for punching or additional off-line sample treatment. The fully automated analytical method was shown to be sensitive and selective over the concentration range of 5 to 2000 ng/mL. Intra- and inter-day precision and accuracy was less than 15% (less than 20% at the LLOQ). The validated method was successfully applied to measure MDZ and α-OHMDZ in an incurred human sample after a single 7.5 mg dose of MDZ. CONCLUSIONS: The direct DBS LC/HRAMS method demonstrated successful implementation of automated DBS extraction and bioanalysis for MDZ and α-OHMDZ. This approach has the potential to promote workload reduction and sample throughput increase. Copyright © 2014 John Wiley & Sons, Ltd. Dried blood spot (DBS) sampling is well established globally as a method of collecting and storing blood samples on cellulose paper for screening of inborn errors of metabolism, [1,2] HIV detection/monitoring [3] and for therapeutic drug monitoring. [4,5] More recently, the determination of drug concentrations in whole blood by analysis of DBS samples has been adopted within the pharmaceutical industry for pharmacokinetic (PK) and toxicokinetic (TK) studies to support preclinical and clinical drug development. [6–8] The key benefits of DBS sampling and analysis include minimally invasive sample collection, reduced blood volume requirements leading to reduced animal use and cost effectiveness in terms of sample handling, as well as storage and transport, especially when compared to conventional wet samples. These advantages and new opportunities for DBS analysis are discussed in recent publications. [9–13] Despite the potential benefits of DBS for bioanalysis, its implementation into bioanalytical workflow demands the same rigor of method development and validation that is generally expected from current regulatory bioanalysis. [14] Considerations when developing a DBS assay include, but are not limited to, the type of DBS cards employed, the extraction solvent used to recover the analyte from the paper substrate, the application of the internal standard, on-card stability of the target analytes, influence of hematocrit, and the spot diameter to be sampled. In recent years, bioanalytical methods for the determination of compounds in DBS samples have shown that DBS techniques can provide a viable approach for quantitative measurements of drugs and their metabolites. [15–19] There is an imminent need for efficient workflow in bioanalytical laboratories processing large numbers of DBS samples, allowing for reduced sample handling and improved sample throughput. One possible way to improve established DBS workflow is by employing either * Correspondence to: J. Henion, Quintiles Bioanalytical and ADME Laboratories, 19 Brown Rd., Ithaca, NY 14850, USA. E-mail: henionj@advion.com † Present address: Chemistry Department, Federal University of São Carlos, São Carlos, SP, 13565-905, Brazil Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 2415–2426 Research Article Received: 1 July 2014 Revised: 25 August 2014 Accepted: 26 August 2014 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2014, 28, 2415–2426 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7033 2415