Studying peptides of antibacterial fractions methods of the liquid chromatography and mass spectrometry

The fractions containing antimicrobic peptides have been purified from a haemolymph of caterpillars Galleria mellonella by chromatographic methods and studied by mass spectrometry.

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Fluconazole Pharmacokinetics in Galleria mellonella Larvae and Performance Evaluation of a Bioassay Compared to Liquid Chromatography-Tandem Mass Spectrometry for Hemolymph Specimens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00895-17 ◽

2017 ◽

Vol 61 (10) ◽

Cited By ~ 12

Author(s):

Karen Marie Thyssen Astvad ◽

Joseph Meletiadis ◽

Sarah Whalley ◽

Maiken Cavling Arendrup

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Human Exposure ◽

Galleria Mellonella ◽

Tandem Mass ◽

Time Curve ◽

Content Type ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

ABSTRACT The invertebrate model organism Galleria mellonella can be used to assess the efficacy of treatment of fungal infection. The fluconazole dose best mimicking human exposure during licensed dosing is unknown. We validated a bioassay for fluconazole detection in hemolymph and determined the fluconazole pharmacokinetics and pharmacodynamics in larval hemolymph in order to estimate a humanized dose for future experiments. A bioassay using 4-mm agar wells, 20 μl hemolymph, and the hypersusceptible Candida albicans DSY2621 was established and compared to a validated liquid chromatography-tandem mass spectrometry (LC–MS-MS) method. G. mellonella larvae were injected with fluconazole (5, 10, and 20 mg/kg of larval weight), and hemolymph was harvested for 24 h for pharmacokinetics calculations. The exposure was compared to the human exposure during standard licensed dosing. The bioassay had a linear standard curve between 1 and 20 mg/liter. Accuracy and coefficients of variation (percent) values were below 10%. The Spearman coefficient between assays was 0.94. Fluconazole larval pharmacokinetics followed one-compartment linear kinetics, with the 24-h area under the hemolymph concentration-time curve (AUC24 h) being 93, 173, and 406 mg · h/liter for the three doses compared to 400 mg · h/liter in humans under licensed treatment. In conclusion, a bioassay was validated for fluconazole determination in hemolymph. The pharmacokinetics was linear. An exposure comparable to the human exposure during standard licensed dosing was obtained with 20 mg/kg.

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Comparative study of chromatographic methods for the analysis of exopolysaccharides from archaeal biofilms

10.5194/biofilms9-58 ◽

2020 ◽

Author(s):

Martin Meyer ◽

Laura Kuschmierz ◽

Bettina Siebers ◽

Jost Wingender ◽

Oliver J. Schmitz

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Comparative Study ◽

Chromatographic Separation ◽

Extracellular Polymeric Substances ◽

Extracellular Dna ◽

Sulfolobus Acidocaldarius ◽

Separation Performance ◽

Chromatographic Methods ◽

Monomeric Composition

Microorganisms, such as archaea, favour life in a biofilm rather than the planktonic form of life. A biofilm is defined as a community of microorganisms embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS), mainly polysaccharides (PS), proteins and extracellular DNA. The polysaccharides form a three-dimensional network, which provides stability of the biofilm and mediates the adhesion to surfaces. [1] Analysis of the monomeric composition of PS requires chromatographic separation and identification by mass spectrometry (MS). A comparative study of different chromatographic methods for the analysis of the monomeric composition of exopolysaccharides from archaeal biofilms from Sulfolobus acidocaldarius has been carried out. For this study, different chromatographic separation methods, such as supercritical fluid chromatography (SFC), hydrophilic interaction liquid chromatography (HILIC) reversed-phase liquid chromatography (RP-LC) and gas chromatography (GC), each coupled to mass spectrometry, were developed and compared by means of separation performance and sensitivity, using authentic standards. The study revealed, that each method features distinct advantages and disadvantages over the other methods. For example, when using SFC-MS, no derivatization is necessary and soft ionization conditions can be used. [2] However, the HILIC-MS and RP-LC-MS methods show significantly greater separation performances for the analysis of the monosaccharide composition. [3] All investigated methods show similar quantification limits in the sub-mg/L range. Finally, the developed chromatographic methods were applied to real biofilm samples of the thermoacidophilic archaeon Sulfolobus acidocaldarius. To determine the monomeric composition of the exopolysaccharides from these archaeal biofilms, the extracellular polymeric substances were extracted from the biofilm and then the PS were hydrolyzed. &#160; Literature: [1] H.-C. Flemming, Nat. Microbiol. Rev. 2010, 8, 623 - 633. [2] M. Lafosse, J. Chromatogr. A, 1996, 720, 61-73. [3] V. Sieber, J. Chromatogr. A, 2014, 1350, 44&#8211;50.

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High-performance liquid chromatography methods for the analysis of endogenous cortisol and cortisone in human urine: comparison of mass spectrometry and fluorescence detection

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/0004563218783789 ◽

2018 ◽

Vol 56 (1) ◽

pp. 82-89

Author(s):

Katarzyna Kosicka ◽

Anna Siemiątkowska ◽

Agata Szpera-Goździewicz ◽

Mariola Krzyścin ◽

Grzegorz Bręborowicz ◽

...

Keyword(s):

Mass Spectrometry ◽

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Fluorescence Detection ◽

Human Urine ◽

High Performance ◽

Wilcoxon Test ◽

Systematic Bias ◽

Chromatographic Methods ◽

Deming Regression

Background The analysis of steroids in biological matrices is challenging. One can apply immunoassay as well as gas and liquid chromatography with various types of detection, depending on the available equipment and the experience of the analyst. The question is how the methods are interchangeable between themselves. Doubts were reported having compared immunoassays and chromatography-mass spectrometry, but there are scarce data on chromatographic methods with detection types other than mass spectrometry. Methods Here, we present the detailed comparison of two liquid chromatographic methods for the determination of free urinary cortisol and cortisone: one with fluorescence detection (high-performance liquid chromatography [HPLC-FLD]) and the other with tandem mass spectrometry (HPLC-MS/MS). The comparison was made with 199 human urine samples. The data analysis included Passing–Bablok and Deming regression, Bland–Altman test, Wilcoxon test, mountain plot and Lin’s concordance correlation coefficient. Results The validation data indicated that both methods met the requirements of the European Medicines Agency. However, the statistical analysis revealed the systematic bias between the two assays. The Passing–Bablok and the Deming tests showed that the HPLC-FLD method overestimated results for cortisol and underestimated measurements for cortisone. The Bland–Altman analysis estimated the mean differences between the methods: 18.8 nmol/L for cortisol and −16.9 nmol/L for cortisone measurement. Conclusions Both methods’ results led to the same conclusion in observational studies, but the techniques are not interchangeable. The literature data, the observations from the clinical setting and our experience clearly indicate that the future of steroid measurements will belong to chromatography coupled with mass spectrometry.

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