Hydrophilic Interaction Liquid Chromatography Column Research Articles

Metabolomic Investigation of Staphylococcus aureus Antibiotic Susceptibility by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcus aureus is a major human pathogen that can readily acquire antibiotic resistance. For instance, methicillin-resistant S. aureus represents a major cause of hospital- and community-acquired bacterial infections. In this chapter, we first provide a detailed protocol for obtaining unbiased and reproducible S. aureus metabolic profiles. The resulting intracellular metabolome is then analyzed in an untargeted manner by using both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry. Such analyses are done in conjunction with our in-house spectral database to identify with high confidence as many meaningful S. aureus metabolites as possible. Under these conditions, we can routinely monitor more than 200 annotated S. aureus metabolites. We also indicate how this protocol can be used to investigate the metabolic differences between methicillin-resistant and susceptible strains.

Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20–500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.

A rapid UHPLC-HILIC method for algal guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and the potential separation mechanism

A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmol g−1 cells within 12 min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.

Investigating hydrophilic and electrostatic properties of surfactants using retention on two mixed-mode liquid chromatographic columns

In environmental risk assessment, it is essential to understand the relationship between molecular structure and fate and toxicity of organic contaminants. For surfactants, physico-chemical parameters which can reflect the interactions that determine surfactant behavior are not well defined and are therefore needed for the development of robust quantitative structure-activity relationships (QSAR). For the present study, we have measured HPLC retention times of several hydrocarbon and perfluorocarbon surfactant groups on a mixed-mode weak anion-exchange (WAX) and mixed-mode hydrophilic interaction liquid chromatography (HILIC) stationary phase. The nonionic alcohol ethoxylates are well retained on the HILIC column. Retention of anionic surfactants on the HILIC column is likely influenced by the degree of hydration of the surfactants and electrostatic repulsion from silanol groups. Less hydrated anionic surfactants (perfluoroalkyl carboxylates, perfluoroalkyl sulfonates and alkyl sulfates) show minimal hydrophilic interaction while other better hydrated anionic surfactants (alkyl carboxylates and alkyl sulfonates) are well retained. The retention mechanism of surfactants on both columns seems to be related to their degree of hydration, albeit expressed in different retention behavior: generally, retention on the WAX phase increases when retention on the HILIC phase decreases, and vice versa. The retention times from both columns were used to calculate retention factors (k’) and these were subsequently used in calculating parameters that reflect the electrostatic property (kAX) and hydrophilic property (kHILIC) that determine the interaction between the hydrophilic part of the surfactant and the stationary phase. In further development of predictive models, we suggest the use of kAX for anionic surfactants and kHILIC for nonionic surfactants.

Fast and sensitive method for the determination of trace 1,2,4-triazole and 4-amino-1,2,4-triazole by hydrophilic interaction liquid chromatography (HILIC) in antifungal drug substance

An HILIC method was developed to quantitatively determine trace amounts of 1,2,4-triazole and 4-amino-1,2,4-triazole in fluconazole drug substance. Separation was achieved on HILIC column using isocratic elution with mixture of water, acetonitrile and phosphoric acid mobile phase and UV detection at 205 nm. The method was validated according to ICH guideline requirements and was determined to be linear in the range of 0.6–22.0 μg/mL. Method accuracy and precision were assessed by spiking 1,2,4-triazole and 4-amino-1,2,4-triazole at three levels over the range of 2.5–15 μg/mL, the recoveries ranged from 78.6% to 95.7% for 1,2,4-triazole and 94.6% to 103.4% for 4-amino-1,2,4-triazole. The intermediate precision was represented by the overall %RSD of 6.9% (n = 9) for 1,2,4-triazole and 3.6% (n = 9) for 4-amino-1,2,4-triazole. The method was also shown to be repeatable at concentration levels of 2.5, 12.5 and 15 μg/mL with n = 3 %RSD values of 3.7%, 1.0% and 1.3% for 1,2,4-triazole, and 0.3%, 1.2% and 0.6% for 4-amino-1,2,4-triazole. The method was found to be specific, stability-indicating and has a detection limit of 0.8 μg/mL and a quantitation limit of 2.5 μg/mL. Finally, the method was demonstrated to be robust to small variations of key chromatographic parameters.

Poly(2-carboxyethyl acrylate-co-ethylene glycol dimethacrylate) monolithic precursor. Part II. Carbodiimide assisted post-polymerization modification with tris and d-Glucamine for use in hydrophilic interaction capillary liquid chromatography

A carboxylated precursor monolith (carboxy monolith), namely poly(2-carboxyethyl acrylate-co-ethylene glycol dimethacrylate), was prepared by the in situ co-polymerization of 2-carboxyethyl acrylate (CEA) with ethylene glycol dimethacrylate (EDMA), see the preceding article in this issue. This carboxy monolith was subjected to post-polymerization modification (PPM) with Tris and d-Glucamine in the presence of N-(3-dimethylamineopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), a water soluble carbodiimide. EDAC with its zero-length carboxyl-to-amine coupling ability works by activating the surface carboxyl groups of the monolith for direct reaction with the primary amines of Tris and d-Glucamine via amide bond formation. The Tris-amide (TA) and d-Glucamide ligands imparted the poly(CEA-co-EDMA) monolith a polar character for use in hydrophilic interaction liquid chromatography (HILIC). The TA- and d-Glucamide-poly(CEA-co-EDMA) monolithic capillary columns were evaluated using neutral and charged polar analytes, such as phenolic acids, nucleic acid bases and nucleosides. The difference in polarity between these monolithic capillary columns is noticeable in terms of the numbers of hydroxyl group per ligands, and therefore, the d-Glucamide-poly(CEA-co-EDMA) monolith bearing five hydroxyl groups per ligand could separate more polar species compared to the TA-poly(CEA-co-EDMA) monolith which has three hydroxyl groups per ligand. Solute’ retention on both types of HILIC columns were reproducible from run-to-run, day-to-day and column-to-column.

Simultaneous determination of 14 oral antihyperglycaemic drugs in human urine by liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid assay based on hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry was developed and validated for the simultaneous determination of metformin and 13 other oral antihyperglycaemic drugs in human urine using metoprolol as an internal standard. A simple sample clean-up procedure using the "dilute and shoot" approach enabled fast and reliable analysis. Chromatographic separation was performed on a HILIC column using an elution gradient of mobile phase A, composed of 1mM ammonium formate (pH 5), and mobile phase B, composed of acetonitrile, at a flow rate of 0.35mL/min. Quantitation was performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode by using electrospray ionization in positive ion mode. The total chromatographic run time was 20min. Calibration curves for each analyte were linear over concentration ranges of 2-300, 5-400, or 20-500ng/mL, with a coefficient of determination above 0.99. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision, system suitability, robustness, and stability. Inter-batch and intra-batch coefficients of variation across four validation runs were≤13.62%. The present method was successfully applied for the analysis of metformin and nateglinide in urine samples after their oral administration to healthy human subjects under fasted conditions.

Fluorous-assisted metal chelate affinity extraction for nucleotides followed by HILIC-MS/MS analysis

We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 43.2 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides.

Discriminant and Simultaneous HPLC Analysis of Reducing and Non-reducing Monosaccharides on a Polyethyleneimine-attached Hydrophilic Interaction Liquid Chromatography Column

Discriminant and Simultaneous HPLC Analysis of Reducing and Non-reducing Monosaccharides on a Polyethyleneimine-attached Hydrophilic Interaction Liquid Chromatography Column

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy.

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1mm, 1.7μm). Serum samples (250μL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000ng/mL for HGA and from 10 to 1000ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000ng/mL; ~8.5-150ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

Characterization of the Retention of Artificial Sweeteners by Hydrophilic Interaction Liquid Chromatography

ABSTRACTThe aim of this work was to study the chromatographic behavior of polar artificial sweeteners on three hydrophilic interaction liquid chromatography (HILIC) columns: Kinetex HILIC (unbounded silica, core shell), ZIC HILIC (sulfoalkylbetain) and TSKgel Amide-80 (amide). The studied analytes were dipeptides (aspartame, neotame, advantame, alitame), sulfamates (acesulfame, saccharin, cyclamate), and sugars (sucralose, neohesperidin dihydrochalcone). Critical parameters such as the mobile phase pH, buffer concentration, column temperature, and organic modifier were studied for the investigation of the retention mechanism. Partition and adsorption models were also used. The retention mechanism of dipeptides seems to be partition for the aqueous layer and weak electrostatic interactions or hydrogen bonds. For the sulfamates, on Kinetex and ZIC HILIC columns, it is likely that partitioning to the aqueous layer is combined with the ERLIC phenomena at pH 5.5. For the TSKgel Amide, the partition and the hydrogen bonds formed between the analytes and carbamoyl groups of the stationary phase play an important role in the retention mechanism. For sucralose, partition seems to be the predominant mechanism in every column, while, for neohesperidin dihydrochalcone, both partition to aqueous layer and hydrogen bonding seems to contribute to the retention. This is also the first report of a method for the chromatographic determination of advantame, a new sweetener.

Association between B vitamins and schizophrenia: A population-based case-control study

To explore the association between schizophrenia and six types of B vitamins, including choline, biotin, riboflavin, pyridoxamine, pyridoxine and nicotinamide, based on the hydrophilic interaction liquid chromatography column (HILIC) Liquid Chromatography-Mass Spectrometry (LC-MS) platform. We conducted the case-control study between November 2015 and September 2016 in Weifang, Shandong Province, China. Blood samples from 128 cases of schizophrenia and 101 controls were collected, and B vitamin were measured by LC-MS coupled with HILIC. The HILIC UPLC-MS based analysis of serum B vitamins levels from 128 cases (30 cases with first-episode, 98 cases with relapse) and 101 controls were performed. The results indicated that lower pyridoxine level and schizophrenia was related. (total cases versus controls: β= −0.215, 95% CI: −0.271, −0.125, p < 0.001; first-episode cases versus controls: β = −0.190, 95% CI: −0.277, −0.103, p < 0.001). Higher nicotinamide level was also associated with schizophrenia after adjusting confounders (β = 0.343, 95% CI: 0.022, 0.664, p = 0.036). Other four B vitamins, including biotin, riboflavin, pridoxamine and choline, were showed no statistically difference in cases versus controls, first episode cases versus relapse cases. Two types of B Vitamins, pyridoxine and nicotinamide, show significant association with the schizophrenia.

Simultaneous determination of three pesticide adjuvant residues in plant-derived agro-products using liquid chromatography-tandem mass spectrometry

Herein, an accurate and reliable isotope-labelled internal standard method was developed and validated for simultaneous determination of three polar pesticide adjuvants, namely 2-pyrrolidone, N-methyl-2-pyrrolidone, and N-ethyl-2-pyrrolidone in plant-derived agro-products. Matrices, including apple, cabbage, tomato, cucumber, rice, and wheat were extracted with a modified quick, easy, cheap, effective, rugged, and safe “QuEChERS” method and purified with a new clean-up sorbent (Z-Sep). A hydrophilic interaction liquid chromatography column (HILIC), exhibiting a lipophilic-hydrophilic character, was used to separate the three analytes over 10min using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Matrix effects in various matrices were evaluated and an isotope-labelled internal standard method was employed to compensate for ion enhancement/suppression effects. At three fortification levels (2.0, 5.0, and 20.0μg/kg), the mean recoveries ranged from 78.5 to 112.1% with relative standard deviations (RSDs)<11.0% for all tested analytes. The limits of detection (LODs) and quantification (LOQs) were 0.04–0.45 and 0.12–1.58μg/kg in various matrices, respectively. The developed experimental protocol was successfully applied to monitor different samples purchased from local markets in Beijing, China. In conclusion, the developed method exhibited both high sensitivity and satisfactory accuracy and is suitable for the simultaneous determination of the three tested pesticide adjuvant residues in agro-products of plant origin.

Determination of chlormequat and mepiquat residues and their dissipation rates in tomato cultivation matrices by ultra-performance liquid chromatography-tandem mass spectrometry.

This study described the development and validation of a simple, rapid, specific and sensitive method for detecting chlormequat chloride (CQ) and mepiquat chloride (MQ) residues in tomato cultivation matrices covering soil, water, seedling samples. The dissipation rates of CQ and MQ in tomato cultivation matrices were also determined in this study. A Hydrophilic Interaction Liquid Chromatography (HILIC) column was used for chromatographic separation. A triple quadrupole mass spectrometer equipped with an electrospray ionisation source in positive ion mode by multiple reaction monitoring was used for detection. Soil samples were extracted with accelerated solvent extraction (ASE) and cleaned up with WCX phase extraction column; water samples were extracted with WCX phase extraction column; seedling samples were extracted with methanol-ammonium acetate solution. LODs and LOQs of CQ and MQ were 0.02μg/kg and 0.1μg/kg in soil samples, 0.005ng/mL and 0.02ng/mL in water samples, and 0.05μg/kg and 1.0μg/kg in seedling samples, respectively. The mean recovery rate of CQ in soil, water and seedling samples ranged from 76.98% to 111.60%. While the mean recovery rate of MQ in soil, water and seedling samples ranged from 96.90% to 105.40%. The fastest to the slowest metabolising rates of CQ and MQ were as follows: soil samples>seedling samples>water samples. In conclusion, this study provided a new potential method for detecting CQ and MQ in tomato cultivation matrices using ultra-performance liquid chromatography-tandem mass spectrometry.

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography-MS/MS analysis: Application to a pharmacokinetic study.

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI-MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile-ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.

Isolation of Three Triterpene Saponins, Including Two New Oleanane Derivatives, from Soldanella alpina and Hydrophilic Interaction Liquid Chromatography-Evaporative Light Scattering Detection of these Three Saponins in Four Soldenella Species.

The genus Soldanella is one of the few endemic to Europe. Some of its species have relevance in local traditional medicine. Earlier work has indicated the possible presence of saponins in S. alpina. To investigate S. alpina and other related species for the occurrence of saponins. Following sequential extraction with n-hexane, dichloromethane and ethyl acetate the subsequent methanolic extract of S. alpina roots was fractionated after solvent precipitation using fast centrifugal partition chromatography and column chromatography. Structures were elucidated by LC-MSn , high-resolution MS, hydrolysis experiments and one-dimensional (1D)- and two-dimensional (2D)-NMR. A hydrophilic interaction liquid chromatography method was developed to quantitate saponins in the leaves and roots of four Soldanella species. Three triterpene saponins, two of them new natural products, were isolated from S. alpina. Based on an epoxyoleanal aglycone substituted with four sugar units, they were analytically quantitated using a Kinetex 2.6 μm hydrophilic interaction liquid chromatography (HILIC) column together with a mobile phase comprising of ammonium acetate, water and acetonitrile. Method validation confirmed that the assay meets all requirements in respect to linearity, accuracy, sensitivity and precision. All four Soldanella species investigated contained the three saponins. The lowest total level of the three saponins (1.09%) was observed in S. montana leaves while the highest saponin content (5.14%) was determined in S. alpina roots. The detection of saponins within the genus Soldanella is an indication that further phytochemical examination of this genus may reveal more secondary metabolites of interest. Copyright © 2017 John Wiley & Sons, Ltd.

Synthesis, characterization, and application of a novel multifunctional stationary phase for hydrophilic interaction/reversed phase mixed-mode chromatography

A novel multifunctional stationary phase based on silica gel was synthesised starting from L- isoleucine and 4-phenylbutylamine and evaluated as a hydrophilic interaction/reversed-phase mixed-mode stationary phase for high-performance liquid chromatography (HPLC). The prepared stationary phase was characterized by elemental analysis, infrared spectroscopy (IR), scanning electron microscopy (SEM), Brunauer, Emmett and Teller (BET) and solid-state 13C nuclear magnetic resonance (NMR). The mechanisms involved in the chromatographic separation are multi-interaction, including hydrophobic, π-π, hydrogen-bonding, dipole-dipole and ion-dipole interactions. Based on these interactions, successful separation could be achieved among several aromatic compounds having different polarities under both hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) condition. Nucleotides/nucleosides were separated in the HILIC mode. The effects of different separation conditions, such as pH value, mobile-phase content, column temperature, buffer concentration and flow rate, on the separation of nucleotides/nucleosides in HILIC mode were investigated. The seven nucleotides/nucleosides were separated within 22min, while six of them were separated within 10min by isocratic elution. To determine the influence of the new multifunctional stationary phase under the RP condition, a number of moderately and weakly polar and nonpolar compounds, such as 10 substituted anilines and eight substituted phenols were separated successfully under the RP condition within 14 and 15min, respectively. Additionally, nine mixtures of polar/nonpolar test compounds were simultaneously separated within 19min, while seven of them were separated within 12min, under HILIC/RP mixed-mode conditions. Chromatographic parameters, such as the retention factor and peak asymmetry factor, were calculated for all of the analytes, while the theoretical plate number was calculated for analytes separated by isocratic elution. Compared to traditional C18 and commercial HILIC columns, the new stationary phase exhibited both HILIC and RPLC performance, and the scope of analyte separation was thus enlarged.

Assessment of intra-particle diffusion in hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography under conditions of identical packing structure

A recently developed stripping protocol to completely remove the stationary phase of reversed-phase liquid chromatography (RPLC) columns and turn them into hydrophilic interaction liquid chromatography (HILIC) columns with identical packing characteristics is used to study the underlying mechanisms of intra-particle diffusion in RPLC and HILIC. The protocol is applied to a column with a large geometrical volume (250×4.6mm, 5μm) to avoid extra-column effects and for compounds with a broad range in retention factors (k” from ∼0.6 to 8). Three types of behavior for the intra-particle diffusion (Dpart/Dm) in RPLC versus HILIC can be distinguished: for nearly unretained compounds (k”<0.6), intra-particle diffusion in HILIC is larger than in RPLC; for compounds with intermediate retention behavior (k”∼0.9–1.2), intra-particle diffusion in HILIC and RPLC are similar; and for well retained compounds (k”>1.8), intra-particle diffusion in RPLC is larger than in HILIC. To explain these observations, diffusion in the stationary phase (γsDs) and in the stagnant mobile phase in the mesopore zone (γmpDm) are deduced from experimentally determined values of the intra-particle diffusion, using models derived from the Effective Medium Theory. It is demonstrated that the larger intra-particle diffusion obtained for slightly retained compounds under HILIC conditions is caused by the higher mesopore diffusion in HILIC (γmp=0.474 for HILIC versus 0.435 for RPLC), while the larger intra-particle diffusion obtained for strongly retained compounds under RPLC conditions can be related to the much higher stationary phase diffusion in RPLC (γsDs/Dm=0.200 for RPLC versus 0.113 for HILIC).

Modeling of HILIC retention behavior with theoretical models and new spline interpolation technique

When it is taken into account that hydrophilic interaction liquid chromatography (HILIC) as an analytical method is relatively young compared with the other techniques, retention modeling could still bring scientifically valuable data to the field. Therefore, in this paper, olanzapine and its 8 impurities were selected as a test mixture, considering that they have never been analyzed in HILIC before. Their investigation on 4 different HILIC columns (bare silica, cyanopropyl, diol and zwitterionic) has been performed. The mixture of 9 structurally similar substances allows the examination of complex HILIC retention behavior depending on the chemical properties of the analytes, as well as of the stationary phase. To describe the nature of the relationship between the retention and the stronger eluent content in the mobile phase, we fitted experimentally obtained data to several theoretical (localized adsorption, nonlocalized partition, quadratic, and mixed) models. Results show that the best fit is the quadratic model with the highest R2 and cross‐validated coefficient of determination (Q2) values, but its usage has some drawbacks. With the aim to improve the possibility to predict retention behavior in HILIC, a new empirical model was proposed. For that purpose, a spline interpolation technique was performed, by dividing the experimental range into several subdivisions. This type of interpolation was performed for the first time in the chromatographic field. The estimation of the polynomial equations was performed using Q2 values. Obtained Q2 values pointed out the goodness of fit of the model, as well as its good predictive capabilities. In the end, the prediction capabilities were experimentally verified, under randomly chosen conditions from the experimental range. The errors in prediction were all under 10%, which is satisfying for HILIC.

Analysis of illegal peptide drugs via HILIC-DAD-MS

Biopharmaceuticals have established themselves as highly efficient medicines, and are still one of the fastest growing parts of the health-product industry. Unfortunately, the introduction of these promising new drugs went hand in hand with the creation of a black market for illegal and counterfeit biotechnology drugs. Particularly popular are the lyophilised peptides with a molecular weight of less than 5kDa. Most of them are meant for subcutaneous injection and are easily accessible via the internet. In recent years, different methods based on reversed phase liquid chromatography have been developed to detect and quantify these peptides. The emerging of more polar peptides however requires the introduction of other separation techniques. Therefore, we set out to develop and validate an analytical method based on hydrophilic interaction liquid chromatography (HILIC) to identify and quantify the most frequently encountered illegal peptides on the European market. For this objective, five different HILIC columns were selected and screened for their chromatographic performance. Among those columns, the ZIC HILIC column showed the best performance under the tested screening conditions in terms of resolution and symmetry factor for the targeted peptide set. Hence, the operational conditions were further optimised for the identification of illegal preparations via mass spectrometry (MS) and quantification via UV. Validation was performed via accuracy profiles based on the ISO 17025 guideline. The obtained validated HILIC-method allows for the detection and quantification of the most frequently encountered illegal peptides on the internet in a total run time of 35min including post gradient equilibration and online cleaning step. Combined with a previously developed RPLC-method, the ZIC HILIC system allows for the detection and quantification of a wide spectrum of illicit peptide drugs available on the internet. Furthermore, the developed method could also be envisaged for the detection of new emerging polar peptide drugs.