3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole: a fascinating molecular framework to study the enantioseparation ability of the amylose (3,5-dimethylphenylcarbamate) chiral stationary phase. Part I. Structure-enantioselectivity relationships

Chiral stationary phases (CSPs) based on amylose (3,5-dimethylphenylcarbamate) (ADMPC) exhibit awide-range of enantioselectivity in high-performance liquid chromatography (HPLC) and supercriticalfluid chromatography (SFC). Although this class of CSPs has been extensively used, chiral discriminationsat receptorial level, which are useful to develop predictive molecular models, have been rarely reportedin the literature.Herein, we describe the results obtained in the enantioselective HPLC of a set of six C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives on the ADMPC-based Chiralpak AD-3 CSP (CSP) under normal-phaseand polar organic conditions. Using pure methanol as a mobile phase the exceptional enantioseparationfactor value of 50 at 25◦C was found for one of the investigated analytes. To the best of our knowledge, theenantiomeric bias represents the most outstanding enantioseparation ever recorded on ADMPC-basedCSPs.Systematic variations in chemical groups in specific positions of the 3-(phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole molecular framework resulted in peculiar changes in retention andenantioselectivity. A careful analysis of the chromatographic data permitted to advance some hypothesesconcerning the role played by the individual chemical groups in determining the exceptional enantiosep-aration.In particular, under methanol-rich mode, the prenyl moiety of the second eluted enantiomer of thebetter resolved analyte was recognized as a critical structural element to establish direct and favorablesolvophobic interactions with apolar portions of selector

Mixed-Mode Chromatography of Mixed Functionalized Analytes as the Homologues of Benzalkonium Chloride. Application to Pharmaceutical Formulations

[Abstract] In this work, a retention behavior based on mixed-mode reversed-phase (RP)/hydrophilic interaction liquid chromatography (HILIC) was observed for benzalkonium chloride (BAK) using a core-shell column functionalized with biphenyl groups. Although in the literature, the U-shaped retention was reported for polar compounds in mixed functionalized phases, in the present work, the behavior was dependent upon the chemical structure of the analyte with mixed functionality (ammonium group, a benzyl group and an alkyl chain) and on the high selectivity of the chromatographic column. The bimodal retention was observed for the four BAK homologues using a content of acetonitrile from 65 to 95% in the mobile phase. The data were adjusted to polynomial equations which allow for modeling and predicting the U-shaped retention. The salt concentration (50 and 100 mM), anion (formate and acetate) and cation (ammonium and triethylammonium) of the salt, pH (4 and 5) in the mobile phase were studied in order to understand their influence on the two retention modes. Significant electrostatic interactions were involved in the two retention modes, especially with a content of acetonitrile higher that 90%. Linear relationships between the retention factors of the four homologues were found in a wide range of %acetonitrile when the salt and triethylamine concentration, pH and nature of salt were changed. The differences found on the retention of the homologues, when increasing the alkyl chain length, were more significant in the RP mode due to predominant hydrophobic interactions. A pH decrease and a salt concentration increase caused a retention decrease for both modes. A decrease on of the retention was observed when acetate anion was replaced by formate anion. The different order of the polynomial equations according to the used mobile phase confirmed its relevant role in the interactions with the analytes and stationary phase. A mobile phase was selected (85% acetonitrile, pH 4 and 100 mM ammonium formate) for the BAK determination in cutaneous, otic and ophthalmic formulations with different active pharmaceutical ingredients and excipients. Low sample volume (500 μL) and short analysis time ( 0.999, % RSD <4.5% for intra-day precision and <5.8% for inter-day precision, and recoveries in the 92–105% range) was obtained.This work was supported by Xunta de Galicia, Spain (Programa de Consolidación y Estructuración de Unidades de Investigación Competitivas- Program for the Consolidation and Structuring of Competitive Research Units ED431C 2017/28-2017-2020)Xunta de Galicia; ED431C 2017/28-2017-202

Unusual separations with macrocyclic glycopeptide chiral stationary phases and chromatographic analysis and characterization of microbes

The macrocyclic glycopeptide stationary phases have been widely used in enantiomeric separations. Some unusual aspects of this class of stationary phases were investigated in this work. The reversal of elution order among members of this class of stationary phases was studied. These chiral stationary phases were not only used in enantiomeric separations, but also for separating closely related peptides. Many of the biologically important peptides were separated with mobile phases that were compatible with ESIMS detection.;A hydrophilic OH-functionalized polystyrene divinylbenzene spherical porous particle packing was used to develop a size exclusion column for microbial analysis. The theory of the close-packing of spheres is proposed to show that common bacteria should be able to perfuse in the inter particle voids of the 50 mum spherical packing. The size exclusion column coupled with polarimetric detection was used to follow the growth of three different bacteria. Coupling this separation method to an ICP magnetic sector mass spectrometer provides an effective means to study the microbial uptake of heavy metals (i.e., uranium) from their surrounding environments. Multiple metal ions can be monitored in the microorganism and in the surrounding solution. Crucial information can be provided for the remediation of radioactive waste sites by using this method. The effect of uranium on microbial growth is also discussed

Synthesis and Evaluation of Silica Hydride-Based Fluorinated Stationary Phases

Silica-based fluorinated bonded stationary phases have shown enhanced selectivity with altered elution orders for molecules differing in hydrophobicity and hydrophilicity in comparison with C8 and C18 reversed phase columns. Hence, two novel silica hydride-based fluorinated bonded phases have been synthesized using a hydrosilation procedure to exploit fluorine-based unique selectivity for polar basic metabolites. Bonded moieties have been characterized by elemental and spectral analyses. Silica hydride-based aqueous normal phase (ANP) chromatography has retention behavior similar to normal phase chromatography, except for the use of water as a part of the binary solvent (\u3e60 % acetonitrile : water). In ANP, a higher percentile of nonpolar mobile phase shows increased retention for acids and bases, and nonpolar solutes can also be retained as in reversed phase chromatography (RPC). The synergistic effects of fluorinated phases\u27 altered selectivity and aqueous normal phase retentivity have been explored for small polar metabolites using high performance liquid chromatography (HPLC) coupled with several detectors. Hydride-based fluorinated stationary phases showed good stability and remarkable reproducibility in retention time with %RSD \u3c 1

Simultaneous Determination of Escitalopram Impurities Including the R-Enantiomer on a Cellulose Tris(3,5-Dimethylphenylcarbamate)-Based Chiral Column in Reversed-Phase Mode

A high-performance liquid chromatographic method was developed for the simultaneous determination of the related substances – three potential chemical impurities from escitalopram synthesis -, and the enantiomeric purity of escitalopram. The separation capacity of seven different polysaccharide-type chiral columns, including three amylose-based (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2) and four cellulose-based (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3 and Lux Cellulose-4) were screened in polar organic, and reversed-phase mode. Lux Cellulose-1, based on cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector with acetonitrile-water mixture containing 0.1% diethylamine was identified as the most promising system. Using “one factor at a time” optimization technique the effect of column temperature, flow rate and mobile phase constituents on separation performance was checked and the critical resolution values were determined. U-shaped retention pattern was obtained when plotting the retention factors of the citalopram enantiomers versus the water content of the binary mobile phases on Lux Cellulose-1 column. Thermodynamic analysis revealed an enthalpy-driven enantioseparation in polar organic and in reversed-phase mode as well. For further method optimizations a L9 orthogonal array table was employed. Using the optimized parameters (Lux Cellulose-1 column with 0.1% (v/v) diethylamine in water/acetonitrile 55/45 (v/v), 0.8 mL/min flow rate at 25 °C) baseline separation were achieved between all compounds. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation and proved to be suitable for routine quality control of related substances and the enantiomeric purity of escitalopram

Uudsed eluendilisandid LC-MS baasil bioanalüütilistele metoodikatele

Väitekirja elektrooniline versioon ei sisalda publikatsiooneKäesoleva töö eesmärk oli laiendada teadmisi uudsete eluendilisandite kasutamiseks bioanalüütilises keemias kasutades vedelikkromatograafia-massispektromeetria (LC-MS) analüüsimeetodit. Nende kahe instrumendi: LC ja MS ühendamine laiendab määratavate analüütide hulka, kuigi seab ka piiranguid mobiilfaasi lenduvuse osas. Seejuures mitmed mittelenduvad eluendilisandid, mis aitavad suurendada LC analüüsi efektiivsust ja on küll laialdaselt kasutatud traditsiooniliste UV/Vis detektori puhul, kuid pole kasutatavad MS detektoritele. Uudsete fluoroalkoholide kasutamine eluendilisanditena võimaldab LC-MS analüüsile paljulubavat tulevikku. Fluoroalkoholid kui nõrgad happelised ühendid, millel on ioonpaaride moodustamise võimekus ja mis soodustavad analüütide ioniseerumist MS detekteerimisel, võimaldavad nii keerulistest segudest paremini komponente eraldada kui ka neid paremini detekteetida. Selleks, et uurida mõju ravimite retentsioonile pöördfaas-vedelikkromatograafias, testiti mitmeid paljulubavaid ühendeid aluselise pH juures. Lisandite uurimiseks kasutati erinevaid analüütilisi kolonne - C18, bifenüül ja pentafluorofenüül (PFP) ning uuriti mudel-analüütide ja üldlevinud ravimite ning narkootikumide käitumist. Töö raames leiti, et kasutatud fluoroalkoholid mõjutavad analüüsi vähendades happeliste analüütide retentsiooni ja suurendades aluseliste analüütide retentsiooni. Uudsed eluendilisandid rakendati ka kahes praktilises rakendusnäites. Esiteks kasutati neid uinutite morfiin, klonidiin ja midasolaam (ning nende metaboliitide) ülitundlikuks mõõtmiseks. Teiseks kasutati uudseid lisandeid südame- ja veresoonkonna ravimite milrinooni ja dobutamiini tundlikkuks mõõtmiseks. Neid kahte bioanalüütilist metoodikat kasutati ravimite ja metaboliitide mõõtmiseks laste vereplasmast farmakokineetiliste ja farmakodünaamiliste uuringute tarbeks. Uudsete eluendilisandite mõju analüütide ioniseerumisele võimaldas mõlema metoodikaga saavutada erakordselt madalaid määramispiire, vajades seejuures minimaalset proovikogust, mis oli sobilik just antud patsientide grupile.The aim of this thesis was to expand knowledge about novel eluent additives used in bioanalytical chemistry using liquid chromatography-mass spectrometry (LC-MS) analysis technique. The combination of the two instruments, an LC and MS expands the limits of analytes that can be detected and quantified. However, it also introduced considerable limitations in terms of mobile phase volatility. Thus, many eluent additives which helped to increase the efficiency of LC analysis and were widely used when UV/Vis was a detector did not pass the criteria anymore. However, a promising perspective in LC-MS analysis is offered by novel fluoroalcoholic eluent additives. They are weakly acidic compounds with ion-pairing capabilities and with a positive influence on analyte ionisation in the MS source. To research the influence on retention patterns of pharmaceuticals, several novel eluent additives were tested in the basic mobile phase pH range. Different analytical column stationary phases - C18, biphenyl and pentafluorphenyl (PFP) - were investigated, both with model analytes and a large set of common toxicology screening compounds. It was observed that the use of these fluoroalcohols influenced the analysis by decreasing retention of acidic analytes and increasing retention for analytes with basic properties. Two practical applications employing novel eluent additives were developed and validated for the pharmaceuticals morphine, clonidine, midazolam, and their metabolites, as well as, for milrinone and dobutamine. The two bioanalytical methods were used to obtain data for pharmacokinetic and pharmacodynamic studies in paediatric patients. Both methods reached exceptionally low limits of quantification, with minimal sample amount used due to the positive influence of novel eluent additives on analyte ionisatio

LC-MS analysis of related peptides and anions in the positive mode

This dissertation focuses on the use of LC-MS for the analysis of related peptides and anions in the positive mode. Separating closely related peptides (those differing by one or two amino acids or the chirality of a single amino acid) can be challenging using reversed-phase liquid chromatography (LC), ion exchange LC, or using ion-pairing agents. Also, the mobile phases that give the best separations in these modes may not be electrospray ionization mass spectrometry (ESI-MS) compatible. Macrocyclic glycopeptide stationary phases were investigated as an alternative to the standard C18 stationary phase for the separation of related peptides. On the macrocyclic glycopeptide stationary phases, high selectivity was observed for single amino acid substitutions (achiral and chiral) regardless of the position of the substitution in the sequence for peptides of thirteen amino acids or less. Selectivity of the macrocyclic glycopeptide stationary phases for a series of diastereomers and larger peptides was also explored and compared to separations achieved on a standard C18 stationary phase. MS compatible mobile phases were used whenever possible.;Negative ion mode is often used for the detection of anions in LC-ESI-MS applications. However, operating in negative ion mode tends to be more problematic than positive ion mode. Singly charged anions can be detected in the positive ion mode if the anions are paired with a dicationic reagent to form a complex with the anion that retains an over all positive charge. This method was also expanded to divalent anions through the use of tricationic reagents. When the anion pairs with the tricationic reagent, an overall positive charge is retained and enables detection by ESI-MS in the positive mode. Different cationic reagents were found to vary tremendously in their ability to pair with anions and produce sensitive ESI-MS signals. The effect of these structural elements on the detection sensitivity of the complex is examined empirically. A comparision of signal to noise ratios achieved in positive and negative modes also is presented