Hydrophilic Interaction Liquid Chromatography System Research Articles

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.

Enrichment and separation of high-polar compounds from Saussurea obvallata using solid-phase extraction combining with offline two-dimensional liquid chromatography.

The high-polar compounds from natural products are often used as medicines due to their good bioactivities. However, owing to the complexity and diversity of their structure, the separation of high-polar compounds is still a challenging work. For this, an efficient method for enrichment and separation of the high-polar compounds from Saussurea obvallata was developed. First, the target compounds were enriched from the total extract using a solid-phase extraction method. An offline two-dimensional liquid chromatography method was used for the separation of pure compounds from the enriched sample. After optimization of chromatographic conditions, high separation selectivity of target compounds was obtained on a polar-modified C18 column and a HILIC XAmide column. Hence, a two-dimensional reversed-phase × hydrophilic interactionliquid chromatography system was constructed and enlarged from the analytical level to the preparative level. In the first dimension, four fractions were obtained on the XCharge C18 column with a recovery rate of 71.2%. In the second-dimension preparation on the XAmide column, eight high-polar compounds with more than 96% purity were isolated. All compounds were isolated from Saussurea obvallata for the first time. The results demonstrated that this developed strategy is effective for preparative-scale isolation of high-polar compounds from natural products.

Development of a new LC-MS method for accurate and sensitive determination of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices.

Setting of maximum limits for a number of plant alkaloids is under discussion in the EU. The novel method developed and optimized in this study enables simultaneous determination of 21 tropane alkaloids (TAs) and 33 pyrrolizidine (PAs) together with their N-oxides (PANOs). For analysis of aqueous-methanolic extract, reversed phase ultra-high-performance liquid chromatography and tandem mass spectrometry (RP-U-HPLC-MS/MS) was employed. The method was validated for frequently contaminated matrices (i) sorghum, (ii) oregano, and (iii) mixed herbal tea. The recoveries at two spiking levels were in the range of 82-115%, 80-106%, and 78-117%, respectively, and repeatabilities were less than 19% for all analyte/matrix combinations. As regards the achieved limits of quantification (LOQ), their values were in the range of 0.5-10μgkg-1. The crucial problem encountered during method development, co-elution of multiple groups of isomeric alkaloids, was overcome by subsequent sample separation in the second chromatographic system, hydrophilic interaction liquid chromatography (HILIC), providing different separation selectivity. Lycopsamine, echinatine, and indicine (co-elution group 1) and N-oxides of indicine and intermedine (co-elution group 2), which could not be resolved on the commonly used RP column, were possible to separate fully by using the HILIC system.

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.

Development and validation of a HILIC-MS/MS multitargeted method for metabolomics applications.

The paper reports the development of a multianalyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites existing in cell culture medium used in in-vitro fertilization (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography system was selected. The analytical system comprised Ultra-HPLC with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, aminoacids, nucleotides, organic acids, and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end-point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy.

An efficient hydrophilic interaction liquid chromatographic method for the simultaneous determination of metformin and pioglitazone using high-purity silica column.

Hydrophilic interaction liquid chromatography (HILIC) provides a feasible approach to effectively separate polar compounds in complex matrices. Herein, a simple, reproducible and efficient HILIC method was developed for the simultaneous determination of pioglitazone. HCl (PIO) and metformin HCl (MET) in rabbit plasma. High-purity silica column was used for rapid and efficient separation of these co-administered drugs. The chromatographic parameters were optimized for best separation. The proposed HILIC system provides high separation efficiency with good peak shape compared to reversed phase (RP) chromatography. Additionally, a simple isocratic elution mode with a mobile phase composed of a mixture of methanol and 10mM phosphate buffer (pH 3.0) (94:6, v/v) was used and the effluent was monitored at 230nm. The method was validated in accordance with the requirements of US-FDA guidelines and was found to behave efficiently for the intended purpose. The correlation coefficient of 0.9992 was obtained in the concentration ranges of 0.5-100μgmL(-1). The limits of detection (S/N=3) and quantification (S/N=10) were 0.16 and 0.5ngmL(-1), respectively. The retention times were 3.4 and 5.0min for PIO and MET, respectively. Plasma levels were successfully determined in rabbit with satisfactory precision and accuracy. In addition, the stability tests in rabbit plasma proved reliable stability under the experimental conditions. The developed HILIC method was applied successfully to study the pharmacokinetic behaviors of the studied analytes in rabbit plasma after a single oral dose containing PIO and MET.

Evaluation of Seven Chromatographic Response Functions on Simulated and Experimentally Obtained Chromatograms in Hydrophilic Interaction Liquid Chromatography System

This paper investigates the ability of seven chromatographic response functions to measure the quality of chromatograms obtained in hydrophilic interaction liquid chromatography (HILIC). First, the functions were tested on a set of simulated chromatograms and differences in their mathematical design were discussed. Second, the functions were evaluated on the experimentally obtained chromatograms in HILIC analysis of model mixture consisted of beta agonists and antagonists. The ranking of chromatograms obtained by different functions was significantly different, implying that the accuracy of the optimization procedure is strongly dependent on the function that was selected as an output. Investigation of potential drawbacks of each function was conducted and general recommendations concerning the use of chromatographic response functions in optimization strategies are proposed.

Confident identification of isomeric N‐glycan structures by combined ion mobility mass spectrometry and hydrophilic interaction liquid chromatography

A central issue in glycan mass analysis is the ambiguity of structural assignments due to the heterogeneity and complexity of glycan structures. Ion mobility mass spectrometry (IM-MS) has the potential to separate isomeric glycans depending on their unique collisional cross section especially when coupled with hydrophilic interaction liquid chromatography (HILIC). Ten pyridylaminated biantennary N-glycans including isomeric structures were measured by electrospray ionization quadrupole-time-of-flight mass spectrometry with an ion mobility phase. We investigated which adduct ions would be suitable for good separation in the ion mobility phase. The differences in observed drift time of isomeric glycans were assessed by molecular dynamics (MD) simulations in vacuum. Connecting an HILIC system with IM-MS provided another, augmented separation mode. By selecting doubly protonated precursor ion species, we succeeded in separating a pair of isomeric glycans in the ion mobility phase with reasonable resolution. MD simulations of monogalactosylated glycan isomers indicate that the galactosylated Man α1-3 branch preferentially folds back to the core chitobiose portion to form a compact structure. IM-MS combined with HILIC resulted in even clearer separation of isomeric glycans within 15 min. A combination of IM-MS with an HILIC system is eminently suitable for the confident and rapid distinction of glycan structures within a defined mixture.

Analysis of amprolium by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We present a fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core™ column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an “extract and shoot” strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 μg L −1) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC–MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 μg kg −1).

Analysis of chlormequat and mepiquat by hydrophilic interaction chromatography coupled to tandem mass spectrometry in food samples

In this work a LC–MS/MS method for the determination of two quaternary ammonium growth regulators (chlormequat and mepiquat) in food is reported. The separation was based on hydrophilic interaction liquid chromatography (HILIC) without the use of ion-pair reagents. A gradient elution of acetonitrile and formic acid/ammonium formate buffer from 60 to 40% acetonitrile was enough to achieve a resolution >1.5 in less than 4.0 min. The HILIC system was coupled to a triple quadrupole mass spectrometer equipped with a heated electrospray probe (H-ESI) providing sub-pg LODs in SRM mode. A straightforward sample treatment (SPE C18 clean-up) was enough to provide MLODs at low ppb levels when analysing a range of food samples that covered different kinds of matrices such as fresh fruit, vegetables, fruit juices, baby food, bread, coffee and beer. Chlormequat was found in seven samples (0.8–126 ng/g) but mepiquat was only detected in bread and coffee samples (0.9–166 ng/g).

Improved hydrophilic interaction chromatography method for the identification and quantification of glucosinolates

An improved hydrophilic interaction liquid chromatography (HILIC) method has been developed to separate members of a closely related family of chemoprotective phytochemicals called glucosinolates. This method exploits the emergence of a second generation of HILIC chemistry, using a silica-based permanently zwitterionic stationary phase. These columns are more robust, durable, and glucosinolates separations are more reproducible than with the original polyhydroxyethyl aspartamide columns. Furthermore, the HILIC system that we report herein permits much greater alteration of the mobile phase composition for customized separation of glucosinolates from plant extracts, across a wide spectrum of polarity.

Hydrophilic interaction liquid chromatography–APCI–mass spectrometry determination of 5-fluorouracil in plasma and tissues

A simple and fast analytical method using hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry was developed to analyse 5-fluorouracil (5-FU) in plasma and tissues. The HILIC system overcomes problems reported in obtaining satisfactory retention of 5-FU with other types of HPLC systems. After addition of internal standard (IS) (5-Chlorouracil (5-CU)), plasma proteins were precipitated with acetonitrile, and tissue samples homogenised with a micro-dismembrator. The analysis was performed using a polymer-based column (Ashaipak NH 2) and the compounds were eluted under gradient conditions at 1 ml/min using a mobile phase containing a mixture of ammonium formate and acetonitrile. MS detection used a API 4000 mass spectrometry with heated nebulizer source and multiple reaction monitoring operated in the negative ion mode. The mass transitions of 5-FU and its internal standard were 129 m/ z → 42 m/ z and 145 m/ z → 42 m/ z, respectively. The lower limits of quantitation in plasma and tissues were about 5 ng/ml and 10 ng/g, respectively, using 25 μl of plasma and 50 mg of tissue. Good linearity, accuracy and precision were obtained in all matrices tested. The suitability and robustness of the method for in vivo samples were confirmed by analysis of mouse plasma, muscle and tumour from animals dosed with 5-FU.

Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones

A new two-step high-performance liquid chromatography (HPLC) procedure has been developed to separate modified histone H1 subtypes. Reversed-phase (RP) HPLC followed by hydrophilic-interaction liquid chromatography (HILIC) was used for analytical and semi-preparative scale fractionation of multi-phosphorylated H1 histone subtypes into their non-phosphorylated and distinct phosphorylated forms. The HILIC system utilizes the weak cation-exchange column PolyCAT A and an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine buffer (pH 3.0) in the presence of 70% (v/v) acetonitrile. The identity and purity of the individual histone subfractions obtained was assayed by capillary electrophoretic analysis. The results demonstrate that application of the combined RP-HPLC-HILIC procedure to the analysis and isolation of modified H1 histone subtypes provides an innovative and important alternative to traditional separation techniques that will be extremely useful in studying the biological function of histone phosphorylation.