Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.     

Praziquantel analogs with activity against juvenile Schistosoma mansoni

Six amide and four urea derivatives of praziquantel were synthesized and tested for antischistosomal activity against juvenile and adults stages of Schistosoma mansoni in infected mice. Only one of these had significant activity against adult worms, but, unlike praziquantel, six of these had low to modest activity against juvenile worms. A praziquantel ketone derivative had the best combination of activity against juveniles and adults, but it had no effect on the motility of adult S. mansoni in ex vivo culture. Cytochrome P450 metabolic stability data support the hypothesis that the major trans-cyclohexanol metabolite of praziquantel plays an important role in the antischistosomal activity of this drug.     

Discovery,synthesis and characterization of a series of (1-alkyl-3-methyl-1H-pyrazol-5-yl)-2-(5-aryl-2H-tetrazol-2-yl)acetamides as novel GIRK1/2 potassium channel activators

The present study describes the discovery and characterization of a series of 5-aryl-2H-tetrazol-3-ylacetamides as G protein-gated inwardly-rectifying potassium (GIRK) channels activators. Working from an initial hit discovered during a high-throughput screening campaign, we identified a tetrazole scaffold that shifts away from the previously reported urea-based scaffolds while remaining effective GIRK1/2 channel activators. In addition, we evaluated the compounds in Tier 1 DMPK assays and have identified a (3-methyl-1H-pyrazol-1-yl)tetrahydrothiophene-1,1-dioxide head group that imparts interesting and unexpected microsomal stability compared to previously-reported pyrazole head groups.     

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(?)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.     

Tandem affinity monolithic microcolumns with immobilized protein A, protein G', and antibodies for depletion of high abundance proteins from serum samples: integrated microcolumn-based fluidic system for simultaneous depletion and tryptic digestion

Affinity monolithic microcolumns with immobilized affinity ligands including protein A, protein G' and polyclonal antibodies were developed for the microscale depletion of the top eight most abundant proteins in human serum. These various affinity microcolumns were evaluated for their sample loading capacities with the standard protein substrates. In general, the sample loading capacity of protein A and protein G' was about 7-25 fold higher than that of the antibody-based affinity columns. The macroporous nature of the monolithic columns, which offers high permeability in pressure-driven flow, allowed the design of long tandem affinity columns for the simultaneous depletion of the top eight most abundant proteins in a single run. The tandem format could be extended to include additional affinity monolithic columns to deplete other proteins for which specific antibodies are available without running into high inlet pressure. Furthermore, the tandem affinity columns were integrated with immobilized trypsin monolithic columns to achieve the simultaneous depletion and digestion of proteins. The various formats investigated in this study could be down scaled to achieve nanoLC or up scaled to perform conventional HPLC depending on the size of the proteomic samples.     

Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography

An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 microm, 150 mm x 2 mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 microg/ml for plasma and amniotic fluid and 0.2-50 microg/ml for the placental and fetal tissues.