A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried plasma spots (DPS) on paper substrates, rather than conventional plasma samples. A quantitative bioanalytical high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay has been validated using paroxetine as a tool compound (range 0.2-200 ng/mL human plasma). The assay employed simple solvent extraction of a punched disk taken from the DPS sample, followed by reversed phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (precision, accuracy, linearity, sensitivity, selectivity), experiments are included to assess the effect of the volume of plasma spotted and the use of an indicating paper. The validated DPS approach was successfully applied to a clinical study utilizing pooled samples and a direct comparison of DPS and plasma was made (single oral dose of 37.5 mg of paroxetine).

IntroductionHyperglucagonemia is a key pathophysiological driver of type 2 diabetes. Although Roux-en-Y gastric bypass (RYGB) is a highly effective treatment for diabetes, it is presently unclear how surgery alters glucagon physiology. The aim of this study was to characterize the behavior of proglucagon-derived peptide (glucagon, glucagon-like peptide-1 (GLP-1), oxyntomodulin, glicentin) secretion after RYGB surgery.Research design and methodsProspective study of 19 patients with obesity and pre-diabetes/diabetes undergoing RYGB. We assessed the glucose, insulin, GLP-1, glucose-dependent insulinotropic peptide (GIP), oxyntomodulin, glicentin and glucagon responses to a mixed-meal test (MMT) before and 1, 3 and 12 months after surgery. Glucagon was measured using a Mercodia glucagon ELISA using the ‘Alternative’ improved specificity protocol, which was validated against a reference liquid chromatography combined with mass spectrometry method.ResultsAfter RYGB, there were early improvements in fasting glucose and glucose tolerance and the insulin response to MMT was accelerated and amplified, in parallel to significant increases in postprandial GLP-1, oxyntomodulin and glicentin secretion. There was a significant decrease in fasting glucagon levels at the later time points of 3 and 12 months after surgery. Glucagon was secreted in response to the MMT preoperatively and postoperatively in all patients and there was no significant change in this postprandial secretion. There was no significant change in GIP secretion.ConclusionsThere is a clear difference in the dynamics of secretion of proglucagon peptides after RYGB. The reduction in fasting glucagon secretion may be one of the mechanisms driving later improvements in glycemia after RYGB.Trial registration numberNCT01945840.

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It is common practice to utilize an internal standard (IS) to minimize variance in bioanalytical assays employing liquid chromatography coupled to mass spectrometry. For assays to be deployed in regulated drug development studies, ensuring the IS will compensate for differences in recovery, liquid handling and ionization efficiency should be determined early in the method development process. In this perspective article, we outline key considerations when selecting an IS and propose experiments to perform within the method development phase to demonstrate suitability of the IS within the assay prior to validation. Finally, a series of case studies will be presented, which illustrate analytical challenges related to internal standardization that we have observed in our laboratory.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.