Optimisation of an Automated DNA Extraction Method for Bone and Teeth Samples and Applicability to Two Forensic Cases

Bones and teeth are highly challenging sources of DNA in forensic science and human remains identification, requiring multiple laborious processing steps. In this study, we compared an organic phenol–chloroform method to the QIAamp® DNA Investigator and PrepFiler Express BTA™ methods in order to identify the most efficient automated DNA extraction method for bones and teeth. Results from individual tooth powder replicates showed that the PrepFiler Express BTA™ method extracted the highest yields of DNA per mg of tooth powder, returning a minimum of 20/21 PowerPlex® 21 loci. Samples extracted using the organic extraction or QIAamp® DNA Investigator methods produced PowerPlex® 21 profiles displaying a ski-slope morphology. The improved DNA quality and yield from the PrepFiler Express BTA™ method was verified using aged samples, where higher DNA yields per mg of powder and more informative profiles were obtained. Furthermore, the PrepFiler Express BTA™ method subsequently provided useful DNA profiles for two forensic cases involving degraded bone samples. Overall, this study showed that the PrepFiler Express BTA™ chemistry is a reliable and robust method for DNA extraction from bone and teeth samples, and will allow larger numbers of samples to be efficiently extracted in the event of a Disaster Victim Identification event.

A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

Method Validation of Drug Quantification Poster

A lowest detection limit with straight linearity was obtained by developing a method to analyze Phenacetin (Phe) in both aqueous and organic extraction by using Liquid chromatography Triple Quoadrpole mass with electrospray ionization (LCMSMS/ESI). The validation of the developed method was carried out according to ICH Harmonized Tripartite guideline. Validation criteria obtained were; the method detection limit MDL is 0.089 ng/ml, method quantification limit MQL is 0.19 ng/ml while the calibration curve linear from 0.1 to 1000 ng/mL with correlation coefficient R2 is 0.9994, Accuracy and precision up to 97% and the repeatability inter and intraday for six replicates of three concentration with RSD 2.1%. Separation occurred using Nova Pack C18 4 um, 150 x 3.9 mm column, using acetonitrile: 0.1% Formic acid 60:40% (v:v) at flow rate 1ml/min. the detector was triple quad mass spectrometry at multi-reaction mode MRM to detect parent mass 180.1 at frag 97 to transition fragments 110 and 138 at collision voltages 16 and 12 respectively.

Extraction Procedure Optimization of Atenolol from Dried Plasma Spots

Aims: Atenolol is one of the β-blockers widely used for the treatment of hypertension and other cardiovascular diseases. To simplify the methods for determining of drugs concentrations in blood and plasma the dried spots assays (dried blood spots or dried plasma spots) could be used. In this case high sensitive detector like mass-spectrometer is required as well as high level of drug recovery from dried spot. In this study the extraction of atenolol from dried plasma spots (DPS) was studied to offer the optimum parameters of extraction method. Study Design: Short research articles. Place and Duration of Study: Core Facility of Mass Spectrometric Analysis, Institute of Chemical Biology and Fundamental Medicine SB RAS, between January and October 2019. Methodology: The organic extraction method was chosen for evaluation as the most suitable for LC-MS assay. Several parameters: % of organic solvent, presence or absence of 0.1% formic acid, time, volume and temperature of extraction were investigated to find the best combination for atenolol recovery from DPS for further LC-MS analysis. Results: Results showed that the solvent composition and temperature has main influence on the extraction. The effect of extraction time and volume of solvent have no significant influence on atenolol recovery. Pure acetonitrile is the worst solvent for atenolol extraction from DPS. The solvents: MeOH:H2O (60:40, v:v), MeOH:0.1% FA in H2O (60:40, v:v), ACN:0.1% FA in H2O (50:50, v:v) or ACN:MeOH (60:40, v:v) provide the best recovery of atenolol . The optimum extraction temperature is 40°C, time of extraction is 15-30 min and volume of solvent - 200-300 μL. Conclusion: Several solvents acceptable for LC-MS analysis with optimized recovery parameter from DPS can be used for routine extraction of atenolol.