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The CAMAG thin-layer chromatography mass spectrometer (TLC--MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide 'chemical space' for pharmaceutical-type molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC--MS analysis). Investigations were performed to optimize extraction time, minimize sample-to-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity. 10.1021/ac901985e

A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried blood spots (DBS) on paper, rather than conventional plasma samples. A quantitative bioanalytical HPLC-MS/MS assay requiring small blood volumes (15/[micro]L) has been validated using acetaminophen as a tool compound (range 25 to 5000 ng/mL human blood). The assay employed simple solvent extraction of a punch taken from the DBS sample, followed by reversed phase HPLC separation, combined with selected 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), a number of experiments were performed to specifically demonstrate the quality of the quantitative data generated using this novel sample format, namely, stability of the analyte and metabolites in whole human blood and in DBS samples; effect of the volume of blood spotted, the device used to spot the blood, or the temperature of blood spotted. The validated DBS approach was successfully applied to a clinical study (single oral dose of 500 mg or 1 g acetaminophen).

Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A postcolumn concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 [micro]L/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, postcolumn addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 [micro]L/min flows is referred to as a nanosplitter because it achieved high split ratios (2000:1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 [micro]L/main). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 [micro]L/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 (mu)L/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic 'A' ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.

Volumetric absorptive microsampling (VAMS) is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and homogeneity issues associated with conventional dried blood spot (DBS) sample when a subpunch is taken. The VAMS sampler absorbs a fixed volume of blood ( ~10 μL) in 2-4 s with less than 5% volume variation across the hematocrit range of 20-70% with low tipto- tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios. [ABSTRACT FROM AUTHOR]