GC-MS Metabolomic Profiling of Protic Metabolites Following Heptafluorobutyl Chloroformate Mediated Dispersive Liquid Microextraction Sample Preparation Protocol.

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards.

Profiling of urinary amino-carboxylic metabolites by in-situ heptafluorobutyl chloroformate mediated sample preparation and gas chromatography-mass spectrometry.

A novel 1,1,1,2,2,3,3-heptafluorobutyl chloroformate reagent (HFBCF) was examined for in-situ derivatization of amino-carboxylic metabolites in human urine. The arising reaction products exhibit greatly reduced polarity which facilitates combining the derivatization and liquid-liquid microextraction (LLME) from an aqueous urine into an isooctane phase and immediate gas chromatographic-mas spectrometric analysis (GC-MS). The sample preparation protocol is simple, proceeds without an alcohol excess and provides cleaner extracts than other urinary GC-MS based methods.

Survey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography.

Trichloroacetic acid, perchloric acid, phosphotungstic acid, acetonitrile, methanol and some other organic solvents were evaluated for their ability to provide protein and lipid-free plasma supernatants. The residual proteins, total cholesterol and triacylglycerols were assayed in the supernatant on a Beckman Analyzer instrument. The free cholesterol and the neutral lipids were further analyzed by means of high-temperature GC analysis.

Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography-mass spectrometry.

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols.

Prevalence and age-related infection of Cryptosporidium suis, C. muris and Cryptosporidium pig genotype II in pigs on a farm complex in the Czech Republic.

A total of 413 pig faecal samples were collected from pre-weaners (119), starters (131), pre-growers (123) and sows (40) from a farm with a closed breeding system segmented into two breeding complexes and a growing complex in the region of Vysocina, Czech Republic and screened for the presence of Cryptosporidium using staining methods and genotyping (SSU rRNA). Cryptosporidium oocysts were detected by microscopy in the faeces of 21.1% of the samples (87/413).

Molecular characterization of Cryptosporidium isolates from pigs at slaughterhouses in South Bohemia, Czech Republic.

A total of 123 fecal samples of slaughtered finisher pigs and 21 sows from 14 farms were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method. Positive samples were molecularly characterized by direct sequencing of partial small subunit ribosomal RNA (SSU rRNA) and GP60 partial genes and polymerase chain reaction restriction fragment length polymorphism of SSU rRNA.

First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic.

Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people. The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E.