Internal standard addition in bioanalysis of dried blood spots: results from the EBF DBS-microsampling consortium

In the framework of wider exploration of the application of Dried Blood Spots (DBS) in bioanalysis, by the DBS consortium of the European Bioanalytical Forum (EBF), one team of five laboratories investigated the merits of the various ways of internal standard addition prior to LC-MS/MS analysis. A set of 22 pharmaceutical compounds with log P in the range of 0 to 10 was selected for this purpose. Assessments were made of precision, recovery, and of the effects of prolonged storage. Assay precision was not significantly different for 3 months aged samples as compared to ‘fresh’ samples stored for 7 – 22 days. Extraction recovery from 3 months aged spots decreased for some of the analytes. In these cases, the most widely employed addition of IS in the extraction solvent does not adequately compensate for recovery. From the overall results, it is clear that there is no ‘one size fits all’ approach to IS addition in DBS bioanalysis. For some assays the procedure of spiking the IS to the extraction solvent looks perfectly acceptable, but for other assays this way of adding the IS will not compensate for changes in extraction recovery from aged dried blood spots

Mephenytoin as a probe for CYP2C19 phenotyping: effect of sample storage, intra-individual reproducibility and occurrence of adverse events

Aims To further evaluate mephenytoin as a probe for CYP2C19 phenotyping. Methods Healthy subjects (n=2638) were phenotyped using the urinary (S)-mephenytoin to (R)-mephenytoin ratio. This method was evaluated for (a) the stability of the S/R-ratio following sample storage, (b) the intraindividual reproducibility of the ratio, and (c) the occurrence of adverse events. Results After prolonged storage, the S/R-ratio of samples from extensive metabolisers (EM) increased up to 85%. In 1.5% of the cases (1 out 66), this led to incorrect classification of phenotype. In EMs, but not in poor metabolisers (PMs), the S/R-ratio increased after acid treatment. The intraindividual reproducibility of the mephenytoin phenotyping procedure was 28%. No major side-effects were observed and there was no relationship between the incidence of side-effects and the phenotype of the subject. Conclusions After prolonged storage the S/R-ratio significantly increased in EMs and, although low, the risk of incorrect classification should not be ignored. Our data support the use of mephenytoin as a safe drug for CYP2C19 phenotyping