Workshop report and follow-up--AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples--implications of Crystal City recommendations.
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Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR.
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Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses. (+info)
The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MS.
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Development of spray deposition/MALDI-TOFMS and its application to the rapid screening of hydrolysis products derived from nitrogen mustards.
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A novel method for preparing samples for use in MALDI-TOFMS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) is described. Seven hydrolysis products derived from nitrogen mustards and CHCA (alpha-cyano-4-hydroxycinnamic acid) were selected as model compounds and the matrix, respectively. A capillary atomizer was used for evaporative and spray deposition of the sample/matrix solution, leading to the formation of a freestanding film that coated and accumulated on the MALDI substrate (i.e., sample plate). Compared to the traditional method for MALDI, which involves the production of dried droplets, the surface roughness was reduced, resulting in the accumulation of the sample-doped matrix on the sample plate. This resulted in an increase in the limit of detection of 1 - 2 orders of magnitude. In order to compare the structures of the sample-doped matrices obtained by the traditional dried droplet method versus the spray deposition method (developed in this study), the matrices were examined by SEM (scanning electron microscopy). The design of the capillary atomizer and details of the experimental conditions are reported. The application of this method to the above seven degradation products was successful, suggesting that it has great potential for use as a routine monitoring tool. (+info)
Optimization of a solid-phase extraction protocol for fractionation of selected steroids using retention data from micro thin-layer chromatography.
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In this paper a simple protocol is described for estimating of solid-phase extraction (SPE) elution volumes of steroids based on retention data generated from micro-planar chromatography. Particularly, the retention of selected steroids, including estrogens and progestagens, was studied on wettable with water octadecylsilica HPTLC plates and mobile phases composed of methanol:water mixtures ranging from 20 to 100% (v/v). It was found that TLC retention data can be linearized by plotting R(M) values of steroids against a reciprocal form of the organic modifier molar fraction (1/X(s)). Using such a mathematical approach, the retention parameter of steroids investigated could be easily back-calculated for a wide range of mobile-phase compositions, using few initial experimental data points. The hold-up time of SPE cartridges filled with 0.5 g of C-18 adsorbent was determined experimentally, and appropriate retention factor values (k(SPE)) for components of interest studied were calculated. Using an appropriate slope and intercept coefficients of the linear-regression equation formed as log k(SPE) = aR(M) + b, the steroids SPE elution volumes were predicted beyond the experimental data range that was available for a solid-phase extraction experiment, particularly for mobile phases that contained a high level of water. (+info)