Performance comparison between solid phase extraction and magnetic carbon nanotubes facilitated dispersive-micro solid phase extractions (Mag-CNTs/d-µSPE) of a cyanide metabolite in biological samples using GC–MS

Dispersive-micro solid phase extraction (d-µSPE) has gained increasing attention due to its convenience, effectiveness, and flexibility for sorbent selection. Among a various selection of materials, magnetic carbon nanotubes (Mag-CNTs) is a promising d-µSPE sorbent with excellent separation efficiency in addition to its high surface area and adsorption capability. In this work, two different surface-modified Mag-CNTs, Mag-CNTs-COOH and Mag-CNTs-SO3H, were developed to facilitate d-µSPE (Mag-CNTs/d-µSPE). The cyanide metabolite, 2-aminothiazoline-4-carboxylic acid (ATCA), was selected to evaluate their extraction performance using gas chromatography–mass spectrometry (GC–MS) analysis. The Mag-CNTs-COOH enabled a one-step derivatization/desorption approach in the workflow; therefore, a better overall performance was achieved. Compared to the Mag-CNTs-SO3H/d-µSPE and SPE workflow, the one-step desorption/derivatization approach improved the overall extraction efficiency and reduced solvent consumption and waste production. Both Mag-CNTs/d-µSPE workflows were validated according to ANSI/ASB 036 guidelines and showed excellent analytical performances. The limit of detection (LOD) and limit of quantitation (LOQ) of ATCA in synthetic urine were 5 and 10 ng/mL, respectively, and that in bovine blood were achieved at 10 and 60 ng/mL. The SPE method’s LOD and LOQ were also determined at 1 and 25 ng/mL in bovine blood samples. The Mag-CNTs/d-µSPE methods demonstrated great potential to extract polar and ionic metabolites from biological matrices. The extraction processes of ATCA described in this work can provide an easier-to-adopt procedure for potential routine forensic testing of the stable biomarker in cyanide poisoning cases, particularly for those cases where the cyanide detection window has passed.

How long does the mRNA remains stable in untreated whole bovine blood?

Background: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. Methods and Results: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4°C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage.Conclusion: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

In vivo / in vitro Correlation of Pharmacokinetics of Gentamicin, Vancomycin, Teicoplanin and Doripenem in a Bovine Blood Hemodialysis Model

Background: Elimination of a drug during renal replacement therapy is not only dependent on flow rates, molecular size and protein binding, but is often influenced by difficult to predict drug membrane interactions. In vitro models allow for extensive profiling of drug clearance using a wide array of hemofilters and flow rates. We present a bovine blood based in vitro pharmacokinetic model for intermittent renal replacement therapy.Methods: Four different drugs were analyzed: gentamicin, doripenem, vancomicin and teicoplanin. The investigated drug was added to a bovine blood reservoir connected to a hemodialysis circuit. In total seven hemofilter models were analyzed using commonly employed flow rates. Pre-filter, post-filter and dialysate samples were drawn, plasmaseparated and analyzed using turbidimetric assays or HPLC. Protein binding of doripenem and vancomycin was measured in bovine plasma and compared to previously published values for human plasma.Results: Clearance values were heavily impacted by choice of membrane material and surface as well as by dialysis parameters such as blood flow rate. Gentamicin clearance ranged from a minimum of 90.12 ml/min in a Baxter CAHP-170 diacetate hemofilter up to a maximum of 187.90 ml/min in a Fresenius medical company Fx80 polysulfone model (blood flow rate 400 ml/min, dialysate flow rate 800 ml/min). Clearance of Gentamicin vs Vancomicin over the F80s hemofilter model using the same flow rates was 137.62 mL vs 103.25 ml/min. Doripenem clearance with the Fx80 was 141.25 ml/min.Conclusion: Clearance values corresponded very well to previously published data from clinical pharmacokinetic trials. In conjunction with in silico pharmacometric models. This model will allow precise dosing recommendations without the need of large scale clinical trials.

Physicochemical and microbiological changes of bovine blood due to high hydrostatic pressure treatment

Bovine blood samples were treated with high hydrostatic pressure (HHP) to examine the changes that may occur in the blood related to its colour, microbiological characteristics, protein denaturation, and dynamic viscosity. Pressure treatments were carried out from 100 to 600 MPa in 100 MPa scale up, with 5 min holding time. The blood samples were treated with anticoagulant (EDTA) to eliminate the possible measurement distorting effects. We found that 2 log reduction in the microbial load could be achieved with a pressure treatment above 400 MPa. According to the protein denaturation measurements (DSC), blood proteins were resistant to pressure treatment, even at 300–400 MPa a substantial part of proteins remained in native state. The colour of the samples got darker with the rising pressure, however, visible colour change was observed only above 400 MPa. It can be established, that the HHP treatment was suitable to increase the microbiological stability of blood, without significantly changing its techno-functional properties.