Dataset Information

Optimized Extraction of Amikacin from Murine Whole Blood.

ABSTRACT: Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.

SUBMITTER: Sardella R

PROVIDER: S-EPMC7865403 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Optimized Extraction of Amikacin from Murine Whole Blood.

Sardella Roccaldo R Xiroudaki Styliani S Mercolini Laura L Sabbatini Samuele S Monari Claudia C Perito Stefano S Ianni Federica F Vecchiarelli Anna A Giovagnoli Stefano S

Molecules (Basel, Switzerland) 20210127 3

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction met ...[more]

PMID: 33513993

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Project description:The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some β-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

Dataset Information

Optimized Extraction of Amikacin from Murine Whole Blood.

Publications

Optimized Extraction of Amikacin from Murine Whole Blood.

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