Determination of Atenolol in Human Plasma by Pseudo Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

Previous HPLC determination of atenolol on reversed-phase packings has often required a mobile phase containing three components: organic modifier, buffer and ion-pairing reagent or organic amine. In addition to the complexity of the eluents employed, alkyl sulphonates and organic amines in the mobile phase can reduce the life of silica-based bonded columns. A new simple, rapid and sensitive method—pseudo reversed-phase liquid chromatography/tandem mass spectrometry has been developed for the analysis of atenolol in human plasma using bare silica as the stationary phase coupled with a simple mobile phase consisted of 5% acetonitrile and 95% formate buffer. The optimization of separation is fast and easy. The assay was validated for the concentration range 1–100 ng mL^?1 with a detection limit of 1 ng mL^?1. Moreover, the silica column was durable with the mainly aqueous eluents. No obvious loss in performance was observed for 30,000 column volumes of eluent.     

Separation of erythromycin and related substances on base-deactivated reversed-phase silica gel columns

An official liquid chromatographic method for the analysis of erythromycin and related substances, which is based on a polymer reversed-phase, is described in the European Pharmacopoeia and in the United States Pharmacopeia. The pH of the mobile phase used in this system is 9.0. Recent advanced technology has led to the introduction of a new generation of silica-based reversed-phase column packings, which are claimed to be much more stable towards bases. They are useful for the analysis of basic compounds. Studies to verify the separation of erythromycin and related substances on Hypersil BDS C₁₈, Luna C₁₈(2), Inertsil ODS-2 and Supelcosil ABZ+ have been performed and the results are presented. It is shown that these base-deactivated phases give a better sensitivity and selectivity towards erythromycins than the polymer phase, provided that an adapted mobile phase is used. This is the first liquid chromatographic method described for the separation of erythromycin D from erythromycin A.     

溶质在动态改性氧化锆液相色谱柱上的保留行为

分别采用硬脂酸、环糊精和十二烷基磺酸钠动态改性自制的ZrO2微球,研究了流动相中甲醇和改性剂浓度对苯酚及苯甲酸的衍生物、苯胺衍生物及芳香烃类化合物的色谱保留行为的影响。中性及碱性化合物的保留时间较短,色谱峰对称;酸性化合物保留时间较长,色谱峰拖尾较严重。改性氧化锆表现出反相色谱性能。     

Purification of the hydrophilic antibiotics epidermin,gallidermin and nikkomycin Z by preparative reversed-phase HPLC

Summary Preparative reversed-phase HPLC techniques for the separation of different hydrophilic fermentation products have been developed to shorten the comples and time-consuming isolation procedure. Separations were performed with low adsorptive reversed-phase packings and, additionally with strong acidic volatile modifiers to prevent adsorption, especially of the basic polypeptide antibotics epidermin and gallidermin on the stationary phases. The complex nature of the crude products required a particle size of 10 micron to separate the substances in a purity between 94% and 100%. The loading was limited to 100mg per injection on a 16 mm i.d. column, which corresponds to 4×10^–3 g/g stationary phase.     

Reversed-phase high-performance liquid chromatography of dinucleoside polyphosphates

The reversed-phase high-performance liquid chromatographic separation of purine dinucleoside polyphosphates on octadecyl- and phenyl-bonded silica packings using phosphate-based eluents was studied. The effects of pH, ionic strength and the content of the organic modifiers methanol and acetonitrile in the mobile phase on the retention and other chromatographic parameters are reported. The data obtained were used to establish an isocratic assay for diguanosine and diadenosine polyphosphates.     

Comparative study of capillary electroendosmotic chromatography and electrically assisted gradient nano-liquid chromatography for the separation of peptides

Capillary electroendoendosmotic chromatography (CEC), being a hybrid of high-performance liquid chromatography (HPLC) and capillary electrophoresis, offers considerable changes to enhance column efficiency, speed of analysis and additional selectivity as compared to the parent methods. The analytes are driven by the electroendosmotic flow (EOF) and separated by surface-solute interactions as well as by differences in electromigration. In this paper on the separation of peptides on C18 reversed-phase and mixed-mode (sulphonic acid-n-alkyl) packings in CEC and electrically assisted reversed-phase gradient nano-LC are investigated. It is shown that mixed mode packings generate a higher EOF than reversed-phase packings that is scarcely dependent on the pH of the eluent. Applying a potential in gradient elution reversed-phase nano-LC of peptides shortens the analysis time as compared to separations without a potential. Electrically assisted reversed-phase gradient elution nano-LC is a powerful separation tool for analysis of tryptic digests. Peptides can be successfully resolved in acidic organic mobile phase at pH 2-3 with and without trifluoroacid as ion pairing reagent under isocratic conditions. It is demonstrated that CEC with mixed mode packing and an eluent of pH 2.3 with varying acetonitrile content can be applied to monitor impurities in a synthetic peptide.     

Reversed-phase separations on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase

Summary The chiral separation of eight racemic compounds has been investigated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral column under reversed-phase conditions. The discrimination mechanism under reversed-phase conditions is discussed. Addition of acid to the mobile phase is necessary for resolution of acidic racemic compounds. The presence of ion-pair reagent in the mobile phase is a key factor in the resolution of basic racemic compounds. Retention of the racemates is also affected by addition of acid or salt. The anion in buffer not only interacts with the racemete, but also with the polysaccharide derivative on the silica gel surface.     

Exclusion chromatography of polypropylenamine dendrimers

Size-exclusion chromatography of polypropylenamine (POPAM) dendrimers is investigated. The nitrile-terminated half-generations can be analysed on polystyrene-divinylbenzene-based column packings using tetrahydrofuran as a mobile phase. Several basic and acidic aqueous phase systems were compared for exclusion chromatography of POPAM-amine dendrimers. The optimum system consists of a reversed-phase silica stationary phase deactivated by tetraazacyclotetradecane and a mobile phase of 0.25 M formic acid at 60°C. Several by-products were identified by thermospray mass spectrometric detection.     

亲水作用色谱及其在碱性药物分析中的应用

亲水作用色谱是采用极性固定相、高含量极性有机溶剂-水相缓冲液为流动相的一种分离技术，它能有效地保留反相色谱中保留弱或不保留的强极性碱性药物。本文综述了以未键合硅胶为固定相的亲水作用色谱分离碱性药物的机理、特点及其应用的最新进展。     

万古霉素键合固定相的分离性能

万古霉素作为一种大环抗生素,具有复杂的分子结构。在充分考虑万古霉素分子结构特征的情况下,采用戊二醛间隔臂法制备了万古霉素键合固定相,在反相、亲水、离子交换等分离模式下研究了其色谱分离性能。结果表明,当流动相中有机调节剂含量较低时,该色谱柱表现出典型的反相色谱分离模式特征;随着有机调节剂含量的增加,逐渐转变成亲水模式,分离特性发生明显改变。由于万古霉素分子结构中含有可以解离的氨基,因此该固定相也能够用于阴离子交换模式下的分析方法的发展。分别在反相、亲水和阴离子交换模式下,将其应用于扑尔敏等多种非对映体药物和新型甜味剂甜菊糖的高效液相色谱分离;仅通过改变分离条件,即可在3种不同分离模式下完成分离。这些结果可以为新型色谱固定相的设计,以及发展采用特殊结构改性基团的色谱固定相在相应分离模式下的分析方法提供指导。     

Monitoring free silanols on reversed-phase supports with peptide standards

Summary A series of synthetic peptide HPLC standards has been designed for use in monitoring free silanols on silica-based reversed-phase supports. The four standards (+ 1 to + 4 net charge) showed great versatility in monitoring silanol activity over a pH range of 2.0 to 7.0. We have developed a silanol monitoring system based on the sensitivity of the standards to ionic interactions with reversed-phase packings at pH 7.0. This monitoring procedure involves chromatographing the peptides at pH 7.0 with a combined acetonitrile and sodium perchlorate linear AB gradient and is generally applicable to reversed-phase packings exhibiting a wide range of silanol concentrations. These standards not only demonstrate the presence and extent of free silanols, they also allow the researcher to determine what changes must be made to the mobile phase composition to minimize ionic interactions. In addition, the standards, in conjunction with the double gradient monitoring system at pH 7.0, will aid researchers and manufacturers in the improvement and development of reversed-phase matrices.     

Evaluation of Micro-HPLC and Various Stationary Phases in Reversed-Phase Mode for the Separation of Styrene Oligomers

Abstract

The potential of the micro-HPLC technique and various reversed-phase packing materials for the separation of styrene oligomers is evaluated. The results indicate that longer carbon chains and/or copolymer materials with small-diameter particles are superior to other type packings for the separation of oligomers with methanol as mobile phase. Micro-HPLC promises the possibility of separating conformational isomers of styrene oligomers.     

Enantioseparations of basic and bifunctional pharmaceuticals by capillary electrochromatography using polysaccharide stationary phases

A fast screening strategy was developed in capillary electrochromatography (CEC) for the chiral separation of basic and bifunctional compounds. The screening conditions were determined on polysaccharide chiral stationary phases using 15 pharmaceutical compounds. The content and type of organic modifier, as well as the pH of the mobile phase appeared to have the largest influence on the chiral resolution. It was seen that for acidic compounds, our approach was not suitable. A generic mobile phase for basic and bifunctional compounds was determined. The testing on 20 additional compounds showed that the proposed mobile phase performed well since enantioselectivity was observed for 86% of the investigated compounds. A comparison of CEC and reversed-phase liquid chromatography (RPLC) results was attempted to demonstrate the potential of the used technique for chiral method development.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.     

Two fluoroalcohols as components of basic buffers for liquid chromatography electrospray ionization mass spectrometric determination of antibiotic residues

Two fluoroalcohols--1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB)--were evaluated for the first time as volatile buffer acids in the basic mobile phase for reversed-phase chromatography with electrospray ionization-mass spectrometric (LC-ESI-MS) detection of five antibiotics. Chromatographic separation as well as positive and negative ion ESI-MS intensities using these novel buffer components were compared to traditional buffer systems. Overall, the highest signal intensities and best chromatographic separation for the five antibiotics (ciprofloxacin, norfloxacin, ofloxacin, sulfadimethoxine and sulfamethoxazole) were achieved using 5 mM HFIP as the buffer acid to methanol : water mobile phase (pH of the aqueous component adjusted to 9.0 with ammonium hydroxide). Comparable results were achieved using 5 mM HFTB (pH adjusted to 9.0 with ammonium hydroxide). The suitability of HFIP for analysis of antibiotic residues in lettuce is demonstrated.     

Native and modified alumina,titania and zirconia in normal and reversed-phase high-performance liquid chromatography

Summary Chromatographic properties of silica, alumina, titania and zirconia have been investigated in normal phase mode in the separation of test mixtures of basic, neutral and acidic compounds. In contrast to silica the chromatographic behaviour revealed the basic properties of the alumina, titania and zirconia surfaces. Therefore, separation of basic compounds on these packings seems very promising. Lypophilic packings have been synthesized by modification of titania, zirconia and alumina with organosilanes and polymers and tested for the separation of basic compounds and proteins. High hydrolytic stability of the modified packings was observed during separations with strong alkali and acidic eluents.     

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.     

Retention properties of internal-surface reversed-phase silica packing and recovery of drugs from human plasma

The properties of internal-surface reversed-phase silica packings developed for use in high-performance liquid chromatography, were investigated with regard to the effects of mobile phase conditions (pH, ionic strength, nature and concentration of organic modifiers, ion-pairing agent and surfactant) on the retention of some selected compounds having different polarities. The results indicated that pi-electron interactions play the main role in solute retention, although it is weaker than that on phenylsilica supports, and that weak cation-exchange properties exert a secondary effect on the retention of ionic solutes. The ion-pairing effect was found to be markedly weakened in mobile phases containing an anionic counter ion. In contrast, the addition of a surfactant (SDS) caused a marked increase in the retention of certain drugs at low pH and expanded the pH range of the mobile phase applicable to plasma samples. Based on these findings, the separation and recovery of several drugs (probenecid, lidocaine, cefpiramide and cefaclor) from human plasma were investigated, with emphasis on their protein bindings. The contour chromatogram of human plasma is also demonstrated in relation to the selection of the detection wavelength.     

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.     

Cholesterol bonded phase as a separation medium in liquid chromatography. Evaluation of properties and applications

An extensive survey of the properties and separation capabilities of a cholesterol bonded phase is reported. The intermediate hydrophobic/hydrophilic properties of the bonded cholesterol material allows this stationary phase to be used for both reversed-phase and aqueous normal-phase separations. Interesting high selectivity is reported for the structural isomers of some antibiotics. The cholesterol bonded material does not display "phase collapse" in high aqueous content mobile phases. Variable temperature studies demonstrate that substantial structural changes of the bonded moiety occur that might be used to control selectivity. Finally, separation of some enantiomers of compounds with a variety of chemical structures is reported under reversed-phase conditions indicating that the cholesterol material may be chiral stationary phase with a broad range of applicability.