Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. I. Injection procedure and column stability

We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.     

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.     

On-line liquid chromatography/thermospray mass spectrometry in the analysis of oligosaccharides.

The analysis of intact neutral oligosaccharides by on-line liquid chromatography/thermospray mass spectrometry is described. Molecular-weight information on oligomers up to a degree of polymerization of 10 is obtained using an aqueous mobile phase containing 10(-4) mol/L sodium acetate, which was found to be compatible with thermospray interfacing and ionization. Ions due to sodiated and disodiated oligosaccharides are observed under these conditions without fragmentation. The aqueous 10(-4) mol/L sodium acetate mobile phase is demonstrated to be applicable in the separation of mixtures of oligosaccharides on a reversed-phase octadecyl-modified silica column.     

Reversed‐phase separation parameters for intact proteins using liquid chromatography with triple quadrupole mass spectrometry

The separation of intact proteins is inherently more complex than that of small molecules using reversed‐phase liquid chromatography. The goal of this work was to determine a reasonable set of operational parameters (a recommended starting point for other analysts) for the separation of intact proteins and their detection by triple quadrupole mass spectrometry. Although protein separations have been studied for many years, the direct detection of intact proteins with mass spectrometry requires special considerations of mobile phase additives to achieve efficient separation and sensitive detection. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as model analytes to investigate chromatographic method development using a triple quadrupole mass spectrometer and detection by multiple reaction monitoring. Chromatographic parameters including the concentration of trifluoroacetic acid, flow rate, gradient slope, temperature, mobile phase composition, and stationary phase chemistry were evaluated. Protein charge state profiles were also monitored for temperature and modifier effects. An optimized method using 0.2 mL/min flow rate, 15% gradient slope, and 75°C with a combined trifluoroacetic acid and formic acid modified mobile phase was developed.     

Pharmaceutical analysis by supercritical fluid chromatography: Optimization of the mobile phase composition on a 2-ethylpyridine column

The separation of neutral, acidic, and basic pharmaceuticals with diverse physicochemical properties by packed column supercritical fluid chromatography (pSFC) on a 2-ethylpyridine column (25 cmx4.6 mm id, 3 mum particles) is presented. The optimization strategy involved separations at 100% methanol (MeOH) and at 50% MeOH/50% ACN while keeping the peak symmetry additives formic acid (FA) and isopropylamine (IPA) at constant levels of 0.25% v/v. By plotting the adjusted retention times as a function of the MeOH/ACN ratio, an optimal modifier ratio composition of 65% MeOH/35% ACN was found. The total set of 26 neutral, acidic, and basic pharmaceuticals was analyzed and the optimal composition experimentally verified. This mobile phase composition is currently used in pharmaceutical method development and open-access generic screening environments.     

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.     

High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol‐modified CO2 and water as the polar additive

The separation of four water soluble nucleobases (thymine, uracil, adenine, and cytosine) via supercritical fluid chromatography with a CO₂‐based mobile phase containing an alcohol modifier and additive is described. Methanol, ethanol, 1‐propanol, and 1‐butanol were examined in conjunction with water as a neutral additive. Packed column stationary phases included silica bonded diol, cyanopropyl, and 2‐ethyl pyridine. Thymine and uracil eluted with good peak shapes without additive, while adenine and cytosine yielded late eluting, severely tailing peaks. The addition of up to 5% water to each of the five alcohols gave rise to much sharper peaks that eluted under gradient conditions in less than 10 min with no baseline noise. Results with water under identical chromatographic conditions were compared with formic acid and ammonium acetate as additives. Water proved to be much superior to formic acid, and it was comparable to ammonium acetate. The role of water was speculated to not only enhance the solvating power of the binary mobile phase for water soluble analytes, but the common elution pattern exhibited by each of the three stationary phases suggested that water had altered the surface chemistry of the packed phase.     

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

Hydrophilic interaction chromatography has been applied for the separation of epirubicin and its analogues using high-purity silica column with aqueous-organic mobile phase. Parameters affecting the chromatographic behavior of the solutes such as organic modifier, buffer pH, ionic strength and sample size, have been investigated. Of utmost importance for successful separation of these analogues is the choice of organic modifier, since it impacts both the solvent selectivity and the ionization of silica silanols as well as buffer solution, and consequently the retention behavior of solutes. Acetonitrile was shown to offer superior separation of these analogues to methanol, isopropanol or tetrahydrofuran. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange. In addition, an irreversible adsorption of these compounds was found on silica in the weakly acidic or neutral mobile phases, and the effect of various factors on irreversible adsorption was also preliminarily discussed. More significantly, these basic compounds have exhibited peaks with a slanted front and a sharp tail, a typical overloading peak profile belonging to the behavior of competitive anti-Langmuir isotherm by increasing the sample size at the experimental conditions.     

Influence of solvent effects on retention for a porous polymer sorbent in reversed phase liquid chromatography

Summary The influence of acetonitrile, methanol and isopropanol as retention selectivily modifiers in reversed phase liquid chromatography on a poly(styrene-divinylbenzene) macroporous polymer sorbent (PLRP-S) is evaluated using the solvation parameter model. Retention results from a combination of adsorption and partitioning and is influenced by the equilibrium absorption of organic solvent by the polymer from the mobile phase. The sorption of solutes is dominated by the ease of cavity formation in or on the solvated sorbent, with a small contribution from lone pair-lone pair electron interactions. All polar interactions, such as dipole-type and hydrogenbond formation, are more favorable in the mobile phase and reduce retention. Changes in the uptake of organic solvent from the mobile phase affect kinetic properties of the column such as band broadening and porosity as well as retention. The PLRP-S solvated sorbent is suitable for solid-phase extraction and is more retentive than typical silica-based, bonded phase sorbents for extraction from water. As a surrogate system for estimating solute lipophilicity and biological activity through retention-property correlations it provides a poor fit for hydrogen-bond acid solutes and is too dipolar/polarizable to fit some models.     

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.     

Quantitative Analysis of Amoxicillin, Clavulanic Acid, and Ticarcillin in Multi-Wells by Liquid Chromatography/Electrospray Ionization Mass Spectrometry as a Strategy for Quality Control

Liquid chromatography combined with electrospray ionization tandem mass spectrometry in both positive and negative ion modes was used to separate and detect amoxicillin, clavulanic acid, and ticarcillin in three matrices: H₂O, Cation Adjusted Muller Hinton Broth (CAMHB), and 5% Lysed Horse Blood (LHB) in CAMHB. Direct precipitation of protein with acetonitrile (CH₃CN) was used for sample clean up. Chromatographic separation was accomplished using an octadecyl silica (C₁₈) bonded phase column. The mobile phase was 1% aqueous formic acid and 1% formic acid in CH₃CN using a gradient elution program. Ampicillin was used as an internal standard. The mass spectrometer showed excellent linearity in the calibration of all drugs. Amoxicillin and clavulanic acid in a range of 10–15 ng mL^?1 were easily quantified. The method was used successfully to quantify these drugs in a series of multi-well panels.     

Determination of lansoprazole enantiomers in dog plasma by column‐switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study

Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column‐switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)‐pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed‐phase chromatography on a C₁₈ column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ‐RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra‐ and inter‐day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)‐lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (–)‐lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silica-based C₁₈ material proved superior to the polymer materials tested.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Peculiarities of Mobile Phases Containing Formic Acid

Formic acid is often used for pH adjustment in RP–LC mobile phases. However, under certain circumstances, formic acid can also behave as an organic co-solvent. This study illustrates two scenarios when the effects of formic acid as co-solvent will be most noticeable. One scenario is for water-rich mobile phases, in which the addition of formic acid will cause small but significant decreases in retention of neutral analytes. The other scenario is in gradient chromatography. It is demonstrated that, during acetonitrile solvent gradients, desorption of formic acid from the column stationary phase occurs simultaneously, potentially causing a spike in UV detector baselines at the beginning of gradients. A pK _a value of formic acid obtained in 50% acetonitrile is also reported and the impact of organic solvent on the dissociation of formic acid is discussed.     

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.

     

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.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.