A Low-Molecular-Weight Compound Derived from Human Leukocytes Determines a Bactericidal Activity of the Interferon Preparation.

The aim of this study was to characterize the structure and mode of action of antimicrobials derived from a commercial preparation of alfa-interferon. By combination of semi-preparative/analytical reversed-phase high-performance liquid chromatography, we isolated and purified a novel active substance based on carbohydrate with a complex of amino acids, which determines antimicrobial property of commercial preparation of interferon. A size-exclusion chromatography was performed and LC/ESI-MS revealed molecular masses of active substance were in the range of 180-249 Da. Edman sequencing identified phenylthiohydantoin (PTH) derivatives which consisted a set of preliminary (Asp, Glu, Gly, and Ala) and minor amino acids (Leu and Thr) at equimolar ratio. Thus, the purified active substance is a compound containing the complex of amino acids connected with carbohydrate background and called leucidin. Leucidin demonstrated antimicrobial activity against the model Escherichia coli (E. coli) K12 strain at a minimal inhibitory concentration of 20 µg mL-1. The revealed antimicrobial mechanism of action is associated with violation of the bacterial cell wall leading to a SOS response and bacterial autolysis. Despite the preliminary nature of the results, obtained data allowed us to discover the previously unknown leukocyte-derived antimicrobial molecules.

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Thermodynamic enantioseparation behavior of phenylthiohydantoin-amino acid derivatives in supercritical fluid chromatography on polysaccharide chiral stationary phases.

Thirteen pairs of enantiomers belonging to the same structural family (phenylthiohydantoin-amino acids) were analyzed on two polysaccharide chiral stationary phases, namely, tris-(3,5-dimethylphenylcarbamate) of amylose (Chiralpak AD-H) or cellulose (Chiralcel OD-H) in supercritical fluid chromatography with a carbon dioxide/methanol mobile phase (90:10 v/v). Five different temperatures (5, 10, 20, 30, 40°C) were applied to evaluate the thermodynamic behavior of these enantioseparations. On the cellulose stationary phase, the retention, and separation trends were most similar among the set of probe analytes, suggesting that the chiral cavities in this stationary phase have little diversity, or that all analytes accessed the same cavities. Conversely, the retention and separation trends on the amylose phase were much more diverse, and could be related to structural differences among the set of probe analytes (carbon chain length in the amino acid residue, secondary amine in proline, existence of covalent rings, or formation of pseudo-rings via intramolecular hydrogen bonds). The large variability of behaviors on the amylose phase suggests that the chiral-binding sites in this chiral stationary phase have more variety than on the cellulose phase, and that the analytes did access different cavities.

Polymeric alkenoxy amino acid surfactants: V. Comparison of carboxylate and sulfate head group polymeric surfactants for enantioseparation in MEKC.

In this work, six amino acid derived (L-leucinol, L-leucine, L-isoleucinol, L-isoleucine, L-valinol, and L-valine) polymeric chiral surfactants with carboxylate and sulfate head groups that were recently synthesized in our laboratory [30, 33, 35] are compared for the simultaneous enantioseparation of several groups of structurally similar analytes under neutral and basic pH conditions. The physicochemical properties of the monomers and polymers of both classes of sulfated and carboxylated surfactants are compared. In addition, cryogenic high-resolution electron microscopy showed tubular structures with distinct order of the tubes of 50-100 nm width. A Plackett-Burmann experimental design is used to study the factors that influence the chiral resolution and analysis time of ten structurally related phenylethylamines (PEAs). It is observed that increasing the number of hydroxy groups on the benzene ring of the PEAs resulted in deterioration of enantioseparation using any of the six polymeric surfactants. For all three classes of PEAs, polysodium N-undecenoxycarbonyl-L-amino acidate (poly-L-SUCAA)-type surfactants provided enhanced resolution compared to that of polysodium N-undecenoxycarbonyl-L-amino acid sulfates (poly-L-SUCAASS). Several classes of basic and neutral chiral compounds (e.g., beta-blockers benzoin derivatives, PTH-amino acids, and benzodiazepines) also provided improved chiral separations with poly-L-SUCAA. Among the poly-L-SUCAAs, polysodium N-undecenoxycarbonyl-L-isoleucine sulfate (poly--SUCL) exhibited overall the best enantioseparation capability for the investigated basic and neutral compounds, while among the poly-L-SUCAASs, polysodium N-undecenoxycarbonyl-L-isoleucine sulfate (poly-L-SUCILS), and polysodium N-undecenoxycarbonyl-L-valine sulfate (poly-L-SUCVS) proved to be equally effective for enantioseparation. This work clearly demonstrates that variation in the head group of polymeric alkenoxy amino acid surfactants has a significant effect on chiral separations.

Simultaneous optimization of the resolution and analysis time in micellar liquid chromatography of phenyl thiohydantoin amino acids using Derringer's desirability function.

The chemometrics approach was applied for simultaneous optimization of resolution and analysis time of nine phenyl thiohydantoin amino acids in micellar liquid chromatography. Derringer's desirability function, a multi-criteria decision making method, was tested for the evaluation of the two different chromatographic performance goals. The effect of five experimental parameters on a chromatographic response function formed using two sigmoidal desirability functions was investigated. The sigmoidal functions were used to transform the optimization criteria, resolution and analysis time, into the desirability values. The factors studied were the concentration of sodium dodecyl sulfate, alkyl chain length of the alcohol used as the organic modifier, organic modifier content, mobile phase pH and temperature. The experiments were performed according to a face-centred cube response surface experimental design to map the chromatographic response surface. Then, calculated chromatographic response functions were fitted to a polynomial model. The obtained regression model was characterized by both descriptive and predictive ability (R2 = 0.988 and R(2)cv = 0.973). The model was verified, as good agreement was observed between the predicted and experimental values of the chromatographic response function in the optimal condition. Based on the results of the study, combination of response surface mapping with Derringer's desirability function allows to predict the best operating condition in micellar liquid chromatography of phenyl thiohydantoin amino acids with respect to resolution and analysis time.

Enantioselectivity of structurally modified poly(sodium undecenoyl-L-leucinate) by insertion of Triton X-102 surfactant molecules.

The effectiveness of Triton X-102 (TX-102), as a structural modifier of the polymeric surfactant sodium undecanoyl-L-leucinate (L-SUL) was investigated for enhanced enantiomeric recognition of various chiral compounds in micellar electrokinetic chromatography (MEKC). Increasing concentrations of TX-102 were separately added into the micellar solutions of L-SUL and then polymerized to form poly-L-SUL. The resulting polymers were purified by use of 3500 molecular-weight-cutoff (MWCO) dialysis membranes. Fluorescence and pulsed field gradient-nuclear magnetic resonance (PFG-NMR) techniques were used to elucidate the structural effects of TX-102 on poly-L-SUL. Evaluation of data from fluorescence measurements suggested an increase in polarity with increasing concentration of TX-102. However, the polarity decreased at higher concentrations of TX-102. Evaluation of data from PFG-NMR suggested an increase in hydrodynamic radius upon increasing the concentration of TX-102. The racemates of coumarinic and phenythiohydantoin amino acid derivatives, and pindolol were used as test analytes in MEKC. A notable increase in resolution and capacity factors of the test analytes was observed when the modified poly-L-SUL was used in MEKC measurements. Examination of the data obtained from fluorescence, PFG-NMR, and MEKC suggests a strong correlation between the polarity and the hydrodynamic radii of TX-102 modified micelles and the enantiomeric resolution of the test analytes.

Separation of phenylthiohydantoin amino acids by capillary electrochromatography.

Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.

Rapid separation of phenylthiohydantoin amino acids: ambient pressure ion-mobility mass spectrometry (IMMS).

An electrospray ionization (ESI) ambient pressure ion-mobility spectrometer (APIMS) interfaced to an orthogonal reflector time-of-flight mass spectrometer (TOFMS) was evaluated for the first time as a detector for the identification of phenylthiohydantoin (PTH)-derivatized amino acids, the final products in the Edman sequencing process of peptides and proteins. The drift and flight times of the twenty common PTH amino acids were characterized by a well-defined 2-D mobility/mass spectral pattern. The combination of mobility/mass modes of analysis gave rise to a unique trend-line formation for the series of PTH amino acids. In addition, each PTH amino acid had a unique reduced mobility constant K(o), thus enabling the differentiation of all the amino acid derivatives including the PTH-leucine and PTH-isoleucine isomers. More importantly it was shown that it was possible to resolve a complete reference mixture of PTH amino acids in a single experimental run in less than 1 min. Detection limits for the PTH amino acids were found to range from 1.04 to 3.52 ng; indicating that the limits of detection were less than 17.0 pmol for all of the PTH amino acids.

High-sensitivity detection of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by capillary liquid chromatography-microelectrospray ion trap mass spectrometry.

We describe the separation and detection at the low-femtomole level of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins (311-PTHs) by capillary liquid chromatography-microelectrospray ion trap mass spectrometry. Highest sensitivity was obtained in the multiple-ion monitoring operating mode in which we detected 311-PTHs at the 5-fmol level with a signal-to-noise ratio of approximately 10. We investigated the fragmentation patterns of the isobaric 311-PTH isoleucine and 311-PTH leucine by electrospray ionization ion trap tandem mass spectrometry. The compounds could be differentiated by a fragment ion of mass m/z = 366.1 which was specific for the breakdown of 311-PTH leucine, thus allowing for the unambiguous identification of the 311-PTH derivatives of all 20 naturally occurring amino acids by their masses and fragmentation patterns.

Micellar electrokinetic chromatography with electroosmotic step gradient elution.

The peak capacity, defined as the maximum number of resolvable peaks, is dependent on the range of the migration-time window in micellar electrokinetic chromatography (MEKC). By applying various radial electric potential gradients across the capillary wall, the direct control of the zeta potential and the electroosmotic flow in MEKC results in a significant increase in the migration-time window and a great enhancement of the separation resolution. The enhancement in the separation resolution is obtained at a large expense in analysis time. To optimize both the separation efficiency and the resolution in MEKC, the dynamic control of electroosmosis and the migration-time window during the separations is demonstrated with the use of neutral and charged phenylthiohydantoin-amino acids (PTH-amino acids) as a model system. A simple mathematic model which is capable of predicting migration times of analytes in electroosmotic step gradient elution MEKC is presented.

The use of mixed surfactants in micellar electrokinetic chromatography.

The use of mixed surfactants for the separation of nine phenylthiohydantoin amino acids (PTH amino acids) by micellar electrokinetic chromatography (MEKC) was examined. The potential of a mixed surfactant system, consisting of cationic and anionic surfactants, for enhancing the selectivity of MEKC separations was explored. Both sodium dodecyl sulfate (SDS) and cetyltrime-thylammonium bromide (CTAB) in the electrophoretic medium were found to improve the separation of the PTH-amino acids compared to the use of CTAB or SDS alone. Complete separation of the PTH amino acids was achieved using a buffer containing 2.5 mM CTAB and 40 mM SDS. The analysis was performed without the need to reverse the polarity of the electrodes, typically required if oppositely charged micelles are used. The migration behavior of the nine PTH amino acids using the mixed surfactant system was compared with MEKC in presence of SDS alone.

Micellar capillary electrophoresis separation and thermo-optical absorbance detection of products from manual peptide sequencing.

Micellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side-products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side-reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.

An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed-phase HPLC.

An optimization procedure for the separation of 24 PTH-amino acids by high-performance liquid chromatography on an inexpensive Merck Superspher Si 60 RP-8, (4.0 x 250 mm) column with PTH-Nle as an internal standard is described. The effects of pH, ionic strength, temperature and gradient were investigated. Using conventional HPLC equipment, the practical detection limit is about 5 pmol.

A method for the unambiguous identification of tryptophan in automated protein sequence analysis.

Some widely used standard protocols for the separation of phenylthiohydantoin amino acid derivatives by reverse-phase gradient HPLC do not provide separation of the phenylthiohydantoin derivative of tryptophan (PTH-Trp) from diphenylurea (DPU), a by-product generated during Edman degradation of proteins in variable amounts. Furthermore, PTH-Trp is usually recovered in low yield under typical experimental conditions used with automated sequencing equipment. These factors may compromise the unambiguous assignment of tryptophan residues in automated protein sequence analysis, especially when sequencing is performed at high sensitivity. We devised a reverse-phase HPLC method which allows the separation of DPU and PTH-Trp and therefore the correct assignment of PTH-Trp. The method is based on a modification of the HPLC gradient used to elute and separate all PTH amino acids of interest. With Applied Biosystems Model 477A protein sequencers with on-line PTH amino acid identification, the correct assignment of tryptophan was consistent and reproducible even when sequencing at very high sensitivity (5 pmol).

Preparation and characterization of 5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantoin, the phenylthiohydantoin derivative of 3-nitrotyrosine.

The phenylthiocarbamoyl derivative of 3-nitrotyrosine was synthesized according to the known Edman method and then converted to its phenylthiohydantoin derivative [5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantion] by incubation in 0.5M HCl for 24 h at room temperature. After drying over P2O5 the chromatographically pure substance could be obtained by double recrystallization from hot acetic acid. It could be established that a shorter incubation time leads to an incomplete conversion and higher temperatures cause polymerization of the product. The compounds could be characterized by thin-layer and high-performance liquid chromatography, melting point, elemental analysis as well as NMR- and absorption spectroscopy.

Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography.

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate.

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.