Mixed-mode High-performance Liquid Chromatography Research Articles

Use of Commercial Mixed-Mode Stationary Phases and Sorbents in the High-Performance Liquid Chromatography Analysis and Solid-Phase Extraction of Ionized and Hydrophilic Bioactive Compounds.

Mixed-mode high-performance liquid chromatography (HPLC) is increasingly used for the analysis of ionic and highly hydrophilic drugs, which are difficult to separate by conventional single-mode HPLC. In the former case, chromatographic separation is achieved using one of the several commercially available mixed-mode stationary phases, typically combinations of reversed and ion-exchange phases. Moreover, mixed-mode stationary phases can be used as solid-phase extraction (SPE) sorbents. This review focuses on the recent applications of mixed-mode stationary phases in the chromatographic analysis of bioactive compounds, such as drugs, herbicides, and pesticides. Specifically, we briefly summarize HPLC methods utilizing mixed-mode stationary phases and SPE pretreatment procedures utilizing mixed-mode sorbents developed in the last decade, thus providing a reference work for overcoming the difficulties in analyzing ionized or hydrophilic drugs by conventional reversed-phase chromatography.

A chiral porous organic polymer COP-1 used as stationary phase for HPLC enantioseparation under normal-phase and reversed-phase conditions.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel-Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4'-bis(chloromethyl)-1,1'-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) (n = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m-1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation.

Monomer-mediated fabrication of microporous organic network@silica microsphere for reversed-phase/hydrophilic interaction mixed-mode chromatography

Microporous organic networks (MONs) are promising in high performance liquid chromatography (HPLC) with large specific surface area, good hydrophobicity and stability. However, their superhydrophobic structures restrict MONs-based HPLC only in reversed-phase mode. To decrease the hydrophobicity of pristine MONs and to expand their broad application in HPLC, here we described the monomer-mediated fabrication of core-shell MON-2COOH@SiO2 microspheres for reversed-phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode chromatography for the first time. The –COOH groups were introduced into MONs’ skeleton to improve their hydrophilicity and to provide hydrophilic interaction sites. The MON-2COOH was grafted onto silica via a monomer mediated method to produce monodispersed core-shell microspheres. By adjusting the concentration of reactants, the thickness of MON-2COOH shell was easily manipulated. The packed MON-2COOH@SiO2 column showed high resolution and selectivity for separating both hydrophobic (alkylbenzenes, polycyclic aromatic hydrocarbons, anilines and phenols) and hydrophilic (nucleoside and nucleic bases) probes, highlighting the promise of MONs in mixed-mode HPLC. The MON-2COOH@SiO2 column also achieved good separation to sulfonamides, nonsteroidal anti-inflammatory drugs, flavonoids and phenylurea herbicides, and offered better resolution than commercial C18 and pristine SiO2 column. Multiple retention mechanisms were also found on MON-2COOH@SiO2 packed column, underlining the great potential of MONs in mixed-mode HPLC.

Separation of fosetyl and phosphonic acid in food matrices with mixed-mode HPLC column coupled with tandem mass spectrometric detection and method application to other highly polar pesticides

The aluminum salt of fosetyl (tris(ethyl phosphonate)) is an antifungal agrochemical. This paper presents a novel high performance liquid chromatography (HPLC) method for the simultaneous determination of fosetyl and the phosphonic acid, its main metabolite, in food samples. The method is based on an ion-displacement separation performed on the recently released Luna Omega PS C18 mixed-mode HPLC column. Baseline separation of fosetyl and phosphonic acid was feasible. This was achieved by optimizing the mobile phase composition and by introducing ethylenediaminetetraacetate for all matrices in the generally used extraction medium for polar pesticides and the injection solution. The binary mobile phase consisted of 10% (v/v) methanol in water and aqueous formate buffer (pH = 3.5) in gradient elution mode. The main advantages of the method over previous method include the stable retention time and peak resolution without the need for long column priming, conditioning or regeneration. Moreover, the approach was tested with other polar pesticides including glyphosate, glufosinate, and perchlorate and showed fit-for-purpose separation. The method was validated for spinach, cherry, and wheat flour samples, and was successfully applied on oat flour and arugula quality control samples. The results obtained for the five analytes met the requirements set by EU. The limit of quantifications was much lower than the maximum residue limits and ranged from 0.02 to 0.20 mg/kg.

Comprehensive ethoxymer characterization of complex alcohol ethoxy sulphate products by mixed-mode high-performance liquid chromatography coupled to charged aerosol detection

The present work describes a simultaneous mixed-mode high performance liquid chromatography (HPLC) method combined with a universal and non-selective-response detector for the complete ethoxymer profiling of alcohol ethoxy sulphate mixtures. The optimized HPLC methodology combines the dual hydrophilic (HILIC) and reversed-phase selectivity of a surfactant-type column in order to render a comprehensive and simultaneous separation of more than 50 endogenous ethoxymers in a single analysis. Furthermore, an accurate quantitation of every single analyte was achieved using a final universal charged aerosol detector (CAD) including specific mathematical processing tools. Results obtained helped describing a complete alkyl chain and ethoxymer distribution of the investigated AES samples. Method validation evidences provided reliability of the individual ethoxymer contributions determined with the proposed HPLC−CAD methodology. Regarding accuracy including independent nuclear magnetic resonance (NMR) experiments, an excellent correlation was found between the structural information provided by a COSY NMR spectrum and the CAD results regarding the mono/polyethoxylated and the non-ethoxylated/ethoxylated distribution. Additional calculations including the average molecular weight and the degree of ethoxylation for the reference AES sample showed minimum differences (relative error < 1 %) between the two considered techniques. An outstanding precision and linearity along the working concentration range (r2>0.999) was also observed. The individual limit of detection for the target sulphate ethoxymers was determined to be in the low ppm range. Further validated distribution profiles for a large number of AES samples demonstrated the applicability of the optimized HPLC−CAD methodology to routine surfactant screenings. Therefore, the hereby developed methodology provided extensive information regarding the detailed individual ethoxymer profile of AES formulations, which can be extremely useful for the surfactant industry in order to gain information on specific synthesis routes and/or detergency properties.

Preparation and evaluation of a reversed-phase/hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase functionalized with dopamine-based dendrimers

In this work, a novel dendritic stationary phase was synthesized by the repeated grafting of 1,4-butanediol diglycidyl ether (BDDE) and dopamine (DA) on the surface of silica for performing mixed-mode high-performance liquid chromatography (MHPLC). Elemental analysis (EA), thermogravimetric analysis (TGA) and Fourier transform infrared spectrometry (FT-IR) showed the successful preparation of the dendritic stationary phase. The prepared stationary phase showed the retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and ion-exchange chromatography (IEC) under different mobile phase conditions. In detail, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and hydrophobic positional isomers were separated successfully in the RPLC mode. The baseline separation of nucleobases, nucleosides and flavonoids was achieved under HILIC mode, respectively. Meanwhile, some acidic and basic analytes were used to evaluate the IEC mode. The effects of different chromatographic conditions, such as acetonitrile content, salt concentration and pH in the mobile phase, on the different chromatographic modes were also investigated. In addition, the application of the mixed-mode dendritic stationary phase was demonstrated by the analysis of traditional Chinese medicine (TCM), including Carthamus tinctorius L. and Abelmoschus manihot (Linn.) Medicus. Interestingly, the stationary phase also has the ability for the capture and separation of boric acids. These meaningful applications confirmed that the mixed-mode dendritic stationary phase can be potentially applied in the analysis of complex samples.

Impurity profiling of drug candidates: Analytical strategies using reversed-phase and mixed-mode high-performance liquid chromatography methods

The development of new active pharmaceutical ingredients (API) requires accurate impurity profiling. Nowadays, reversed-phase HPLC (RPLC) on C18 stationary phase is the method of first choice for this task and usually employed in generic screening methods. However, this method sometimes fails, especially when the target analyte is not sufficiently retained, making impurity analysis difficult or even impossible. In such cases, a second method must be available.In the present paper, we compare the merits of RPLC on C18 phase to those of previously optimized alternative methods, based on the analysis of a large and diverse set of small-molecule drug candidates. Various strategies are considered: RPLC on C18 phase but with different mobile phase composition (acidic or basic), RPLC with a pentafluorophenyl stationary phase, or mixed-mode HPLC with both bimodal and trimodal stationary phases. First, method performances were compared in terms of response rate (proportion of compounds eluted) and peak shapes for a large set of synthetic drugs (140) with structural diversity and their orthogonality was evaluated. Then a subset of compounds (25) containing varied impurity profiles was used to compare the methods based on the capability to detect impurities and evaluate the relative purity of the API.

Study on the Mixed-Mode HPLC Separation of Shuang-Huang-Lian and Xue‑Bi‑Jing Injections Based on an Ionic Liquid Column

Both Shuang-Huang-Lian and Xue-Bi-Jing injections are clinically proven efficient and low cost medicines. However, the studies of their chromatographic separation are limited. In this study, a column based on ionic liquid stationary phase was applied for the first time for the analysis of these two injections. For Shuang-Huang-Lian injection, U-shaped plots of retention factors vs. mobile phase constitutions were obtained. This unique kind of separation behavior allowed a flexible adjustment of retention by tuning the mobile phase constituents. Then acids group was analyzed with hydrophilic chromatography mode, while glycosides group was analyzed with reversed phase chromatography mode. Similarly, constituents in Xue-Bi-Jing injection were analyzed with mixed mode high performance liquid chromatography (HPLC). Higher, medium and lower polarity groups were analyzed with hydrophilic interaction, reversed phase and normal phase liquid chromatography modes, respectively. Results from this work provided valuable information for the separation of complicated systems such as traditional Chinese medicine injections.

Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry.

We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed-mode high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40min. Chromatographic separation was governed by a silica-based mixed-mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1μL, we obtained detection limits <3μM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization-based methods. This method represents an alternative to derivatization-based methods and can be applied in areas where sample matrices are highly complex.

Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assay

Determination of excipient content in drug formulation is an important aspect of pharmaceutical formulation development and for analytical testing of the formulation. In this study, the influence of polysorbate subspecies, in particular mono- and poly-esters, for determining polysorbate (PS) content were investigated by comparing three of the most widely used PS quantitation approaches, the Fluorescence Micelle Assay (FMA) and Mixed-Mode High Performance Liquid Chromatography coupled with Charged Aerosol Detection (MM-CAD) or Evaporative Light Scattering Detection (MM-ELSD). FMA and MM-CAD were employed to investigate the quantitation behavior of PS20 and PS80 subspecies and corresponding degradation products in placebo formulations using forced degradation conditions at 40°C for up to 12 weeks. While both methods allowed accurate and comparable quantification of neat PS at the beginning of stress studies, pronounced differences in content determination between the methods were observed at later time points, which were attributable to substantial differences in the contribution of individual mono- and poly-esters to the overall quantitation results. It was particularly surprising to find that the main component of PS20, polyoxyethylene sorbitan monolaurate, did not show a signal at the studied concentration using FMA. Moreover, the degradation of polysorbate poly-esters, was reflected much stronger in FMA than MM-CAD results. Additional experiments employing chemical oxidation and base hydrolysis to degrade PS20, quantified by FMA and MM-ELSD, also show preferential reduction in certain subspecies depending on the degradation pathway involved. For PS20 degraded by chemical oxidation, quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with base hydrolysis.

Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity

Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca2+-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1μM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300μM Trp-Val or Trp-Ile. On the basis of Ca2+-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca2+-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6μM, respectively.

Separation of cannabinoids on three different mixed-mode columns containing carbon/nanodiamond/amine-polymer superficially porous particles.

Three mixed-mode high-performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine-polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C18 ), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed-mode column (C18 ) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed-mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C18 ) mixed-mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.

Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

β-Diketones: Peak shape challenges and the use of mixed-mode high-performance liquid chromatography methodology to obtain fast, high resolution chromatography

Historically, indirect methods have been used for the HPLC analysis of β-diketone compounds because of the very poor peak shapes and resolution obtained on conventional HPLC stationary phases. In this paper we demonstrate that it is possible to obtain good peak shapes for underivatised β-diketone compounds, in a simulated reaction mixture, using only conventional mobile phases with mixed-mode stationary phase HPLC columns. Optimum conditions were obtained using the mixed-mode reversed-phase strong anion exchange column Primesep B, supplied by SIELC Technologies, with a 0.1% aq. TFA/MeOH gradient method and a column temperature of 55 °C.

Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H + form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.

Analysis of nonylphenol and nonylphenol ethoxylates in environmental samples by mixed-mode high-performance liquid chromatography–electrospray mass spectrometry

A new method is described based on mixed-mode high-performance liquid chromatography with electrospray mass spectrometry detection for comprehensive quantitative analysis of nonylphenol (NP) and nonylphenol ethoxylates (NPEOs) in wastewater and sediment. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing a methanol–water gradient on a mixed-solvent gel filtration column designed for MS interfacing. Quantitative accuracy and precision of the method were improved by the use of custom-synthesized [ 13C 6]NPEO analogs as isotope-dilution surrogate standards. Method detection limits for NP and individual NPEOs ranged from 1 to 55 pg injected on column.

Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography

A mixed-mode high-performance liquid chromatography (HPLC) method that resolves the six known non-allelic variants of chicken erythrocyte histone H1 is described. Common, but previously unknown, allelic variants of H1 that comigrate in polyacrylamide gel electrophoresis are also resolved. The resolution of H1 variants achieved by this method should be useful in determining the functional significance of H1 sequence heterogeneity and in analyses of post-translational modification of H1. Furthermore, the principles behind the separation should be applicable to analyses of polymorphism in other proteins.

19] Analysis of synthetic peptides by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) has proved extremely versatile for the isolation/purification of peptides varying widely in their sources, quantity, and complexity. High-performance chromatographic techniques are suited to the purification of a single peptide from the kind of complex peptide mixtures encountered following solid-phase peptide synthesis, where impurities are closely related to the peptide of interest (deletion, terminated, or chemically modified peptides), perhaps missing only one amino acid residue, and, hence, may be difficult to separate. This chapter focuses on HPLC applications of particular interest to researchers utilizing solid-phase synthesis methods: (1) problem-solving approaches to difficult peptide separations, (2) HPLC monitoring of formation of desired product, (3) and HPLC as a diagnostic tool to detect unwanted side-chain modification. The use of reversed-phase chromatography (RP-HPLC), the most important mode of HPLC for synthetic peptidepurification, is focussed; in addition, the exciting potential of a novel mixed-mode HPLC technique, hydrophilic interaction/cation-exchange chromatography (HILIC/CEC), is demonstrated.

Lophozozymus pictor toxin: A fluorescent structural isomer of palytoxin

Purified Lophozozymus pictor toxin (LPTX) shares many properties similar to palytoxin (PTX). LPTX and palytoxin isolated from Palythoa caribaeorum (C-PTX) have similar mol. wts of approx. 2680 on ionspray mass spectrometry (MS). In addition, antibodies against PTX could recognize and bind LPTX. Mixed mode high-performance liquid chromatography (HPLC) of LPTX, C-PTX and H-PTX (isolated from Palythoa tuberculosa) showed a major PTX component common to all three with the characteristic PTX-like UV spectrum at a retention time ( R t) of 17 min. However, LPTX exhibits fluorescence but PTX of equivalent toxicity does not. LPTX showed a unique peak at R t of approx. 22 min on mixed mode HPLC. In addition, LPTX and C-PTX showed different ion fragmentation patterns on MS/MS. These results suggest that LPTX and the palytoxins are structural isomers, containing at least one difference which gives rise to fluorescence in LPTX.

Speciation of toxicologically important transition metals using ion chromatography with inductively coupled plasma mass spectrometric detection

Speciation of vanadium(IV) and (V) using a mixed mode high-performance liquid chromatography column with inductively coupled plasma mass spectrometry detection was investigated. Nickel(II), chromium(III) and (VI) were also separated using the same column. The effect on the separation by varying the pH of the mobile phase and the ionic strength were studied. The absolute detection limits in aqueous media were 0.024 ng for vanadium(v) and 0.114 ng for vanadium(IV). The speciation of vanadium in National Institute of Standards and Technology Standard Reference Material (SRM) 2670 (Toxic metals in Freeze-Dried Urine, Elevated Level) was also accomplished. Using a slight modification of the mobile phase, the speciation of chromium(III) and (VI), as well as nickel(II) was accomplished. Absolute detection limits for chromium(III) at m/z 52 and 53 were 0.042 and 0.017 ng, respectively and for chromium(VI) at the same masses were 0.055 and 0.022 ng, respectively. Absolute limits of detection for nickel obtained were 0.031 ng and the speciation of nickel(II) in SRM 2670 was also demonstrated.