原子转移自由基聚合制备新型接枝聚α-D-吡喃半乳糖苷色谱固定相及其性能

以2-溴异丁酰溴为引发剂,CuCl/CuC12/Me6TREN为催化体系,在室温条件下采用原子转移自由基聚合(ATRP)法将单体6-O-甲基丙烯酰基-1,2,3,4-双-O-亚异丙基-α-D-吡喃半乳糖苷(6-O-methacryloyl-1,2,3,4-di-O-isopropylidene-α-D-galactop...     

Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence

Hair analysis is a powerful tool for retrospective drug analysis. By determining the minor ethanol metabolites ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair, even a previous consumption of alcohol is detectable. However, previous studies showed a lack of correlation if both parameters are determined simultaneously. A further study was conducted to confirm or refute these results. One hundred and sixty hair samples were analyzed for EtG and FAEE in the context of driving ability. In 109 cases, alcohol abstinence was clearly proven and was excluded in 15 cases. In 36 cases, ambiguous results were found. Possible reasons for the deviating results are discussed. It is recommended, that in context of driving ability diagnostics the EtG result is determinant. In critical cases FAEE concentrations can be determined for checking purposes, but a negative FAEE result cannot refute a determined EtG concentration >7 pg/mg.     

Investigations on the influence of different grinding procedures on measured ethyl glucuronide concentrations in hair determined with an optimized and validated LC-MS/MS method

Ethyl glucuronide (EtG) analysis in hair is a suitable method for the retrospective determination of previous alcohol consumption. According to the German guidelines, EtG abstinence is improbable at c _EtG > 7 pg/mg in the proximal 3 cm of scalp hair. The chromatography of the routinely used liquid chromatography-tandem mass spectrometry procedure was optimized by replacing the stationary phase. To simplify sample preparation, two different mills were tested, and an optimized grinding process was developed. The new method was successfully validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry. Despite a simple extraction procedure without any cleaning steps, a very high sensitivity (limit of detection, 1.7 pg/mg; limit of quantitation, 2.3 pg/mg) could be achieved. Competitive analysis showed significantly higher EtG concentrations in pulverized versus cut hair samples. The strong impact of sample preparation on the determined EtG concentrations suggests the introduction of a standardized sample preparation method to produce comparable results.     

Determination of ethyl glucuronide and fatty acid ethyl esters in hair samples

Hair testing for alcohol biomarkers is an important tool for monitoring alcohol consumption. We propose two methods for assessing alcohol exposure through combined analysis of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) species (ethyl myristate, palmitate, stearate and oleate) in hair (30 mg). EtG was analysed by liquid chromatography–tandem mass spectrometry, while FAEEs were analysed by gas chromatography–tandem mass spectrometry using electron impact ionization. Both methods were validated according to internationally accepted guidelines. Linearity was proven between 3 and 500 pg/mg for EtG and 30–5000 pg/mg for FAEEs, and the limits of quantification were 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Precision and accuracy were considered adequate, processed EtG samples were found to be stable for up to 96 h left in the injector and processed FAEEs samples for up to 24 h. Matrix effects were not significant. Both methods were applied to the analysis of 15 authentic samples, using the cut‐off values proposed by the Society of Hair Testing for interpretation. The results agreed well with the self‐reported alcohol consumption in most cases, and demonstrated the suitability of the methods to be applied in routine analysis of alcohol biomarkers, allowing monitoring consumption using low sample amounts.     

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F13C9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C8) and octadecyl (C18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C18 phase, F13C9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C8, C18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C18 or C8 phases and was even disadvantageous on F13C9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.     

Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material

The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed‐phase and normal‐phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride‐based stationary phases.     

Preparation and Evaluation of Poly-l-Lysine Stationary Phase for Hydrophilic Interaction/Reversed-Phase Mixed-Mode Chromatography

In this work, a poly-l-lysine-grafted stationary phase was synthesized by polymerization of N-carboxyanhydride of l-lysine initiated by 3-aminopropylated silica. The resulting material was characterized by FT-IR spectra, elemental analysis and thermogravimetric analysis, which clearly indicated that the new phase had been prepared successfully. The retention of polar solutes depending on acetonitrile content in mobile phase exhibited ??U-shaped?? curves, which was an indication of hydrophilic interaction liquid chromatography (HILIC)/reversed-phase liquid chromatography (RPLC) mixed-mode retention behavior. The retention mechanisms in HILIC and RPLC modes also were investigated. Phenol compounds, aniline compounds and hydrophilic compounds were separated in RPLC or HILIC mode on the new stationary phase, respectively. This result shows that the new phase could be used for both RPLC and HILIC applications, providing greater flexibility for real sample analysis.     

Structural organization of ethyl esters based on hexafluoropropene oxide oligomers: Inverse gas chromatography study

Samples of ethyl esters of perfluorinated acids based on hexafluoropropene oxide oligomers with molecular masses of 2000, 3100, and 4500 are used as stationary phases, and their interactions with low-molecular-mass compounds (sorbates) of different natures are studied by inverse gas chromatography. The linear dependence of the specific retention volumes for alkanes, cyclohexane, aromatic hydrocarbons, tetrachloromethane, and trifluorotrichloroethane (CFC-113) on inverse molecular masses of the oligomer samples is ascribed to the quasi-biphasic structure of the liquid oligomers. The experimental dependences of specific retention volumes on inverse molecular masses of oligomer samples make it possible to estimate the main thermodynamic characteristics of sorbate interaction with individual quasi-phases and their solubility parameters directly related to the cohesion-energy density.     

Evaluation of the separation characteristics of application-specific (volatile organic compounds) open-tubular columns for gas chromatography

The solvation parameter model is used to characterize the separation characteristics of two application-specific open-tubular columns (Rtx-Volatiles and Rtx-VGC) and a general purpose column for the separation of volatile organic compounds (DB-WAXetr) at five equally spaced temperatures over the range 60-140 degrees C. System constant differences and retention factor correlation plots are then used to determine selectivity differences between the above columns and their closest neighbors in a large database of system constants and retention factors for forty-four open-tubular columns. The Rtx-Volatiles column is shown to have separation characteristics predicted for a poly(dimethyldiphenylsiloxane) stationary phase containing about 16% diphenylsiloxane monomer. The Rtx-VGC column has separation properties similar to the poly(cyanopropylphenyldimethylsiloxane) stationary phase containing 14% cyanopropylphenylsiloxane monomer DB-1701 for non-polar and dipolar/polarizable compounds but significantly different characteristics for the separation of hydrogen-bond acids. For all practical purposes the DB-WAXetr column is shown to be selectivity equivalent to poly(ethylene glycol) columns prepared using different chemistries for bonding and immobilizing the stationary phase. Principal component analysis and cluster analysis are then used to classify the system constants for the above columns and a sub-database of eleven open-tubular columns (DB-1, HP-5, DB-VRX, Rtx-20, DB-35, Rtx-50, Rtx-65, DB-1301, DB-1701, DB-200, and DB-624) commonly used for the separation of volatile organic compounds. A rationale basis for column selection based on differences in intermolecular interactions is presented as an aid to method development for the separation of volatile organic compounds.     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

磷酰胆碱色谱固定相的制备及其在分离生物大分子中的应用

以三氯氧磷、氯化胆碱和甲基丙烯酸-2-羟乙酯(HEMA)为原料合成了含磷酰胆碱的单体2-甲基丙烯酰氧乙基磷酰胆碱(MPC). 在硅胶表面嫁接聚合MPC, 得到磷酰胆碱两性离子交换色谱填料. 研究了该填料对标准蛋白的分离性能及pH对蛋白质保留的影响. 结果表明, 该填料对溶菌酶和牛血清白蛋白的动态吸附容量分别为13.8和18.7 mg/g, 其基质磷酰胆碱色谱固定相可同时基线分离两种酸性和两种碱性蛋白.     

磺酸甜菜碱型两性离子亲水作用色谱固定相的制备及色谱性能评价

以丙烯酸二甲氨基乙酯(DMAEA)和1,3-丙磺酸内酯为原料,合成了含磺酸甜菜碱型两性离子的N,N-二甲基-N-丙烯酰氧乙基-N-丙基磺酸铵(DMAEAPS)功能单体,通过原子转移自由基聚合(ATRP)技术将其接枝到硅胶表面,制备了磺酸甜菜碱型两性离子色谱固定相(Sil-DMAEAPS)。研究了该固定相对安息香、维生素B6、芸香叶苷、对香豆酸和咖啡酸5种极性溶质的亲水作用色谱分离性能。结果表明,在典型的亲水作用色谱条件下,极性溶质的保留主要由静电作用和亲水作用控制;而在典型的反相色谱条件下,极性溶质则表现出反相柱的分离特征。与ZIC-HILIC商品柱进行对比,自制色谱柱对5种极性溶质表现出不同的分离选择性。将自制色谱柱用于芦丁片中芸香叶苷含量的测定,操作方法简单,为极性样品的分离提供了新方法。     

Temperature effects on solute retention for hydride-based stationary phases

The effect of solute retention is investigated for two hydride-based stationary phases: bidentate C18 and cholesterol. Several small molecules and peptides are used as solutes. In the reversed-phase mode, most compounds have the expected temperature behavior, i.e. decreasing retention with increasing temperature. Two analogs of the drug lisinopril do exhibit the opposite behavior on the cholesterol column; increasing retention with increasing temperature. In the aqueous normal phase (ANP) mode, more compounds, particularly certain peptides, have this unusual retention behavior with respect to temperature. These preliminary studies indicate that as ANP retention becomes stronger, there is a greater possibility of observing increasing retention with increasing temperature.     

Heterogeneity of adsorption mechanisms in chiral normal-phase liquid chromatography. 2-Propanol and ethyl acetate adsorption equilibria

The adsorption equilibria of two commonly employed strong mobile phase modifiers, ethyl acetate and 2-propanol, on a polysaccharide-based chiral stationary phase have been studied by modeling nonlinear perturbation peaks measured after equilibration of the column with hexane (the weak component of the binary mixture). The investigation of adsorption processes from dilute solutions for species that are strongly retained on the stationary phase could be performed by this approach. On the opposite, limits of traditional linear perturbation technique for isotherm determination, in presence of strong interactions, have been evidenced. Alcohol adsorption has been modeled by a single Langmuir isotherm, while the ester has required a BiLangmuir model. These findings have found to be in a semi-quantitative agreement with available spectroscopic data about 2-propanol and ethyl acetate adsorption on thin silica sol-gel films in contact with a weak solvent. Experimental features observed for racemic separation on polysaccharide-based chiral stationary phases, such as the dependence of the separation factor on the amount and type of the employed additive, have been explained in light of these measurements.     

Insights into the Retention Mechanism on a Pentafluorophenylpropylsiloxane-Bonded Silica Stationary Phase (Discovery HS F5) in RP-LC

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.     

Effect of stationary phase polarity on the retention of ionic liquid cations in reversed phase liquid chromatography

Chromatographic analysis of ionic liquids on different types of packings offers interesting possibility to determine their retention mechanism. As a consequence, the major interactions between stationary phase ligands and analyzed chemical entities can be defined. The main aim of this work was to analyze cations of ionic liquids on chemically bonded stationary phases with specific structural properties. The attempt to predict the main interactions between positive ions of ionic liquids and stationary phase ligands was undertaken. For that purpose, butyl, octyl, octadecyl, phenyl, aryl, mixed, alkylamide, and cholesterolic packings were chosen and applied to the analysis of six most commonly used ionic liquids' cations. Obtained results indicate mainly dispersive and pi-pi type of interaction part in the retention mechanism of analyzed compounds.     

Evaluation of polymeric methacrylate-based monoliths in capillary electrochromatography for their potential to separate pharmaceutical compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.     

High-performance liquid chromatographic separation of dihydropyrimidine racemates on polysaccharide-derived chiral stationary phases

The chromatographic behavior of a set of racemic dihydropyrimidines (DHPMs) has been evaluated on two polysaccharide-derived chiral stationary phases under normal phase conditions. One of these is coated, the other chemically immobilized. The outstanding solvent compatibility of the immobilized chiral stationary phase (CSP) permits the use of solvents such as ethyl acetate (EtOAc) that are unsuitable for coated supports, for which traditional 2-propanol:hexanes mixtures have been employed. Drastic changes in the chromatographic retention and resolution of DHPMs and, in general, in the separation performances have been observed for the two systems. From a practical point of view, EtOAc has been proven to be a better choice for the separation of this important class of compounds. By comparing molecules different in specific positions of their scaffolds, hypotheses concerning the role of individual chemical groups on retention and selectivity have been done. These effects have been quantified, in some cases, in terms of standard Gibbs energy variations. Even if no chromatographic measurements have been made under nonlinear conditions, clear indications of the potential use of immobilized chiral adsorptive media operated with EtOAc:hexanes mixtures for preparative separations of DHPMs have been evidenced.