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. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.

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. Moreover, thiol metabolites bound in disulfide bonds can be released by reduction with tris(3-hydroxypropyl)phosphine (THP) prior to the developed derivatization and LLME step. In order to evaluate potential of the novel method for GC-MS metabolomics, reaction products of 153 urinary metabolites with HFBCF, particularly those possessing amino and carboxyl groups (56 amino acids and their conjugates, 84 organic acids, 9 biogenic amines, 4 other polar analytes) and two internal standards were investigated in detail by GC-MS and liquid chromatography-mass spectrometry (LC-MS). One hundred and twenty metabolites (78%) yielded a single product, 25 (16%) and 2 metabolites (2-methylcitrate, citrate) generated two and more derivatives. From the examined set, analytically applicable products of 5 metabolites were not detected; the derivatives of 3 metabolites were only suitable for LC-MS analysis. Electron ionization (EI) of the examined analytes contained characteristic, diagnostic ions enabling to distinguish related and isomeric structures. The new method was validated for 132 metabolites using two internal standards in artificial urine and with special attention to potential disease biomarker candidates. The developed sample preparation protocol was finally evaluated by means of a certified organic acid standard mixture in urine and by GC-MS analysis of 100 morning urines obtained from healthy patients (50 males and 50 females), where 112 physiological metabolites were quantified in a 25 µL sample aliquot. The quantification data for the set were satisfactory, most metabolites were found within the range reported in the reference human metabolome (HMDB) database and literature. The reported results suggest that the described method has been a novel promising tool for targeted GC-MS based metabolomic analysis in urine.

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. The conditions for the deproteinizing step were optimized for minimal lipoprotein disruption. A substantial difference regarding contamination by the lipids was found if the plasma supernatant or the whole serum were treated with an alkyl chloroformate reagent. Three plasma sulfur amino acids and the aromatic ones were chosen as model compounds to evaluate compatibility of the precipitation methods with a subsequent methyl chloroformate-mediated derivatization and GC-MS analysis. The results of the total homocysteine assay matched well with that obtained using a commercial immunoassay. Precipitation with trichloroacetic acid has proven to be a method of choice for the analysis of the acido-basic analytes by GC-MS via chloroformates.

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. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 µmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

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). Sequence analyses and RFLP identified C. suis in 44, Cryptosporidium pig genotype II in 23 and C. muris in 2 samples. No mixed infections were found. Pigs under 7 weeks of age were infected with C. suis only. Cryptosporidium pig genotype II was found in animals from 7 weeks of age. No relationship was found between diarrhoea and any Cryptosporidium infection in any of the different age groups (P<0.05). The pre-weaned pigs shed significantly more Cryptosporidium oocysts than older pigs and it was associated with C. suis infection.

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. Cryptosporidium oocysts were microscopically identified in 36 finishers (29%) and two sows (10%). Twenty-one mono-infections of Cryptosporidium pig genotype II and 15 mixed-infection of Cryptosporidium pig genotype II and Cryptosporidium suis in finishers were found. Both sows were infected with the Cryptosporidium parvum subgenotype IIaA16G1R1, which is reported from pigs for the first time.

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. bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity. Spores were detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR) identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.