Hematocrit-independent recovery of immunosuppressants from DBS using heated flow-through desorption.

BACKGROUND: We applied our concept for automated flow-through desorption of DBS to investigate the effect of desorption temperature on recovery. For this purpose, a method has been developed for the determination of four immunosuppressants in DBS. RESULTS: We compared recoveries of four immunosuppressants for measurements with and without temperature-enhanced desorption at different hematocrit (Ht) levels. Temperature-enhanced desorption increased recovery substantially for tacrolimus, sirolimus and everolimus at all Ht values and for cyclosporine At high Ht. In addition, recovery became largely independent from Ht variations. Under the optimized conditions, a brief validation using spiked blood samples showed that the method complies with acceptance criteria for quantitative bioanalysis. CONCLUSION: This method enables a quantitative analysis of immunosuppressants in DBS independent from the Ht.

Exploration of a new concept for automated dried blood spot analysis using flow-through desorption and online SPE-MS/MS.

BACKGROUND: A new concept for simple and generic automation of dried blood spot (DBS) analysis is evaluated. Flow-through desorption of the blood spot is coupled online to SPE and MS/MS without using a LC column. Blood, spiked with a mixture of test compounds is spotted on paper, dried and then desorbed by means of a prototype clamping device. RESULTS: Conditions for extraction and chromatography on a single SPE cartridge were optimized with respect to clean-up, separation and ionization suppression. Addition of internal standard using loop-injection upstream of the clamped blood spot was examined and proved to be simple and reliable. Validation results for the 1-1000 ng/ml range are well within acceptance criteria for bioanalysis. CONCLUSION: Results demonstrate feasibility of the DBS SPE-MS/MS concept for efficient automation of the entire DBS analysis workflow.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma.

BACKGROUND: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. METHODS: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. RESULTS: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. CONCLUSION: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.