Reversed-phase Liquid Chromatography Column Research Articles

Characteristics of organic acid profiles in 16 species of wild growing edible mushrooms

ABSTRACTThe aim of study was to determine 9 organic acids in nine aboveground and seven wood-growing wild edible mushroom species originated from an area under the direct influence of a busy trunk road in Poland. The organic acids in the extracts of samples were identified by reversed-phase column liquid chromatography (RPLC). The presented results show that all the mushroom species were characterized by high variation in the profile and content of the analyzed acids. Each of mushroom species contained oxalic acid, the profiles of the other acids strictly depended on mushroom species. Among aboveground species, the highest total content of organic acids was found in Lepista gilva (267.5 ± 26.6 mg g−1 dry weight (DW)), while Laccaria amethystina was characterized by the lowest content (37.7 ± 6.5 mg g−1 DW). Within wood-growing species, the highest content of organic acids was determined in Flammulina velutipes (171.9 ± 26.7 mg g−1 DW), whereas the lowest content (34.2 ± 2.9 mg g−1 DW) of the studied acids was observed in Grifola frondosa.

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).

Analysis of amino acids using serially coupled columns.

Single conventional columns in reversed-phase liquid chromatography are insufficient for analysing the isoindoles of primary amino acids due to their limited functionality. An interesting possibility for increasing the separation power is the combination of several columns of different nature, where the length is modified by coupling small segments. This approach may require a considerable investment to have multiple lengths for each stationary phase. However, the combination of only two columns of fixed length can be enough to resolve satisfactorily relatively complex mixtures, provided that an optimised gradient program is applied. In this work, a mixture of 19 primary amino acid isoindoles found in proteins was analysed. Four stationary phases were assayed: C18, pentafluorophenyl-C18, C4 and cyano. The mixture of isoindoles was successfully resolved in practical times using a pentafluorophenyl-C18 column coupled to a C4 column, in spite of the extremely poor performance obtained when each column is used isolatedly, independently of the length. The extreme diversity in the polarities of the isoindoles and the need of extrapolating the retention behaviour in certain regions of the solvent content domain makes the modelling of the retention behaviour of the isoindoles particularly difficult. Nevertheless, the predicted optimal separations were very satisfactory.

Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50–110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200–400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC–MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms.

Determination of tristyrylphenol ethoxylates by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry method for the analysis of tristyrylphenol ethoxylates (TSPnEO) was established. The separation effects of TSPnEO in the four liquid chromatography modes, including reversed-phase liquid chromatography column (XBridge C18, 150 mm×2.1 mm, 3.5 μ m), hydrophilic interaction liquid chromatography column (XBridge HILIC, 150 mm×2.1 mm, 3.5 μ m), amide column (XBridge Amide, 150 mm×2.1 mm, 3.5 μ m) and C18 column (XBridge C18, 50 mm×2.1 mm, 5 μ m) combined with silica column (Nova-Pak Silica, 150 mm×2.1 mm, 4 μ m) were investigated. And the separation effects and sensitivity of TSPnEO in four mobile phases, such as 5 mmol/L ammonium acetate aqueous solution-acetonitrile, 0.1% (v/v) formic acid aqueous solution-acetonitrile, water-acetonitrile and water-methanol, and three eluent gradient conditions were optimized too. The ionization behavior of TSPnEO in electrospray ionization (ESI) was also studied. The results showed that in the ESI positive ion mode, the identified adducts were[M+NH4]+, and the distribution characterization was in Poisson-like distribution. The separation of TSPnEO (n=5-18) was achieved via C18 column combined with the silica column and water-acetonitrile as mobile phases according to the degree of ethoxylation.

Hydrophilic interaction and reversed phase mixed-mode liquid chromatography coupled to high resolution tandem mass spectrometry for polar lipids analysis

A hydrophilic interaction liquid chromatography (HILIC) fused-core column (150×2.1mm ID, 2.7μm particle size) and a short reversed-phase liquid chromatography (RPLC) column (20mm×2.1mm ID, 1.9μm) were serially coupled to perform mixed-mode chromatography (MMC) on complex mixtures of phospholipids (PL). Mobile phase composition and gradient elution program were, preliminarily, optimized using a mixture of phosphatidylcholines (PC), phosphatidylethanolamines (PE), their corresponding lyso-forms (LPC and LPE), and sphingomyelins (SM). Thus a mixture of PC extracted from soybean was characterized by MMC coupled to electrospray ionization (ESI) high-resolution Fourier-transform mass spectrometry (FTMS) using an orbital trap analyzer. Several previously undiscovered PC, including positional isomers (i.e. 16:0/19:1 and 19:1/16:0) of PC 35:1 and skeletal isomers (i.e. 18:1/18:2 and 18:0/18:3) of PC 36:3 were identified. Therefore, high-resolution MS/MS spectra unveiled the occurrence of isomers for several overall side chain compositions. The proposed MMC-ESI-FTMS/MS approach revealed an unprecedented capability in disclosing complexity of an actual lipid extract, thus representing a very promising approach to lipidomics.

Ameliorating effect of Erxian decoction combined with Fructus Schisandrae chinensis (Wu Wei Zi) on menopausal sweating and serum hormone profiles in a rat model.

BackgroundModified Erxian decoction (MEXD), i.e., Erxian decoction (EXD) with Fructus Schisandrae chinensis (Wu Wei Zi) added, has been used to alleviate menopausal symptoms. This study aimed to investigate the effects of MEXD on menopausal sweating and serum hormone levels in a rat model of menopause after oral administration of MEXD.MethodsQuality control of MEXD was conducted by employing a reversed-phase high performance liquid chromatography column. The three treatment groups received oral administration of MEXD in 0.5% sodium carboxylmethyl cellulose (CMC-Na) at three different doses (5.5, 11, and 22 g/kg body weight) once-daily for 6 consecutive weeks, with 10 animals per group. Huangqijing oral liquor (5 mL/kg) prepared from the roots of Huang qi (Astragalus membranaceus) with an antiperspirant effect was used as a positive control. The negative control group received the same volume of vehicle (0.5% CMC-Na). Ten 3-month-old Sprague–Dawley rats were used as a young group for comparison with the treatment groups (12–14 months old rats). Blood was collected from all animals after 3–6 weeks of treatment. At the end of the treatment, the uterine weight, ovarian weight, and body weight were recorded. Serum malondialdehyde contents and superoxide dismutase activities were determined by thiobarbituric acid colorimetric assays and chemoluminescence assays, respectively. Serum levels of estradiol, follicle-stimulating hormone, and luteinizing hormone were measured by radioimmunoassays. Rat foot pad assays were used to determine the antiperspirant activity of MEXD and histological examinations were conducted on plantar sweat glands.ResultsTreatment with MEXD (11 g/kg) significantly inhibited sweat excretion in the menopause model rats after treatment for 3 (P = 0.0026) and 6 (P < 0.0001) weeks. The decoction markedly decreased the number of secretory cells in plantar sweat glands. In addition, MEXD (11 g/kg) significantly increased the serum estradiol levels (P < 0.001) and superoxide dismutase activities (P = 0.0405). Furthermore, MEXD (11 g/kg) markedly decreased the serum levels of follicle-stimulating hormone (P = 0.001), luteinizing hormone (P = 0.0213), and malondialdehyde (P = 0.01).ConclusionModified Erxian decoction significantly inhibited sweat excretion, regulated serum levels of pituitary gonadotropins and estradiol, and exhibited antioxidative effects in a rat model of menopause.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0117-6) contains supplementary material, which is available to authorized users.

Lewisite Metabolites in Urine by Solid Phase Extraction-Dual Column Reversed-Phase Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry.

Lewisite (2-chlorovinyldichloroarsine) is a chemical warfare agent developed during World War I. A quantitative method using solid phase extraction (SPE) followed by dual column liquid chromatography (LC)-isotope dilution tandem mass spectrometry(MS-MS) was developed for the determination of (2-chlorovinyl)arsonic acid (CVAOA), a metabolite of Lewisite, in human urine. The sample was treated with hydrogen peroxide to oxidize any (2-chlorovinyl)arsonous acid (CVAA) that remained in the trivalent arsenic oxidation state. There was 1.19% (arsenic purity) of bis-(2-chlorovinyl)arsinic acid (BCVAOA), a minor Lewisite metabolite, in the stock CVAA material. The high-throughput method qualitatively assessed BCVAOA simultaneously utilizing normal-phase silica SPE followed by reversed-phase C18 LC for an orthogonal separation. The chromatographic method results in a5.8-min cycle time with adequate retention (k'=2.4) of CVAOA. The mass spectrometer was operated in positive electrospray ionization mode with quantitativem/z 186.9→61.0 and confirmation 186.9→91.0 mass transitions. This selective method demonstrated linearity, accuracy and reproducibility for the clinically relevant calibration range (25-3,200 µg/L as CVAA). The method detection limit was 3.3 µg/L as CVAA from a 10µL injection. This LC-MS-MS emergency response method has a throughput of >240 samples(2.5 extracted 96-well plates) per day.

Separation analysis of macrolide antibiotics with good performance on a positively charged C18HCE column.

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica-based reversed-phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients (r(2) > 0.9938) for all macrolides were obtained. The intra-day and inter-day recoveries were 73.7-134.7% and 80.7-119.7% with relative standard deviations of 2.5-8.0% and 3.9-16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02-1 μg/kg and limits of detection (S/N ≥ 3) of 0.01-0.5 μg/kg were achieved.

Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma.

In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.

Direct Determination of Glyphosate, Glufosinate, and AMPA in milk by Liquid chromatography/tandem mass spectrometry

A simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate in milk using a reversed-phase liquid chromatography column with weak anion/cation exchange stationary phase. One milliliter of milk was shaken with three milliliters of water containing Na2EDTA and acetic acid for 10 minutes, to precipitate the protein content in the sample. After centrifugation, the supernatant was passed through an Oasis HLB SPE to retain suspended particulates and phospholipids. The sample was directly injected and analyzed in 6 min, with no sample concentration or derivatization steps. Two multiple reaction monitoring (MRM) channels were monitored in the method for each target compound to achieve true positive identification. Three internal standards corresponding to each analyte were used to counter matrix suppression effects. The linearity of the detector response was demonstrated in the range of 4 to 1000 ng/mL for each analyte, with a minimum coefficient of determination (R2) value of more than 0.995. Through the use of this internal standard calibration method, the average recovery for all analytes at 0.025, 0.1, 0.5, and 2 µg/g (n = 7) are between 84-111% with a relative standard deviation of less than 8%.https://doi.org/10.21423/jrs-v03n02p020 (DOI assigned 8/7/2019)

Evaluation of the Kinetic Performance Differences between Hydrophilic-Interaction Liquid Chromatography and Reversed-Phase Liquid Chromatography under Conditions of Identical Packing Structure.

A protocol using trifluoroacetic acid at a temperature of 60 °C is developed for the adequate removal of the stationary phase of reversed-phase liquid chromatography (RPLC) columns. This procedure allows for studying the same column first under RPLC and subsequently under hydrophilic-interaction liquid chromatography (HILIC) conditions to isolate intrinsic differences between mass transfer properties in HILIC and RPLC from differences in packing quality. The established procedure allows for a complete removal of the stationary phase (confirmed by retention studies and thermogravimetry analyses) while leaving the structure of the packing unaffected (witnessed by an unchanged external porosity and pressure drop). Accurate plate height analysis comparing compounds at the same zone retention factor indicates a significant difference in reduced c-term (typically 40-80% larger under HILIC conditions), despite the columns otherwise being identical. Correcting for the known contributions of longitudinal diffusion (b-term) and mass transfer (cm- and cs-term) to focus on band broadening originating from eddy dispersion, similar strong differences are observed (differences of some h = 0.3 up to 1.2). These findings show that the interior structure and retention mechanism of the particles have a very strong effect on the observed eddy dispersion, a factor typically ascribed to phenomena occurring outside the particles. This also implies that comparing the quality of packings of different particle types is virtually impossible without the availability of a sound model to correct for the intraparticle effect on the observed eddy dispersion.

"Dilute-and-shoot" rapid-separation liquid chromatography tandem mass spectrometry method for fast detection of thiodiglycolic acid in urine.

A new sensitive rapid-separation liquid chromatography tandem mass spectrometry approach for the determination of thiodiglycolic acid (TDGA) in urine has been developed. The use of the "dilute-and-shoot" method helps to shorten the sample preparation stage and provides a sensitive and direct approach for TDGA determination in urine. Chromatographic separation of the analyte and other urine compounds was achieved using a reverse-phase liquid chromatography column with mobile phases consisting of 0.1% formic acid in water and acetonitrile in a gradient elution mode. For the identification and quantification of TDGA electrospray ionization-tandem mass spectrometry monitoring, two precursor-to-product ion transitions were used. The method demonstrates good linearity and has a detection limit of 50 ng mL⁻¹ in urine.

Evaluation of a new wide-pore superficially porous material with carbon core and nanodiamond-polymer shell for the separation of proteins

In this study, reversed phase liquid chromatographic columns packed with superficially porous material made of a carbon core and nanodiamond-polymer shell were evaluated for the analytical characterization of proteins. The emphasis was put on the impact of pore size on the kinetic performance when analyzing large molecules. Three different types of columns possessing an average pore size of 120, 180, and 250Å were thus evaluated. As expected, the peak capacities were improved with the 180 and above all the 250Å pore size, while the kinetic performance achieved with the 120Å were systematically lower.It was also shown that a trifluoroacetic acid (TFA) concentration of 0.3–0.5% was required when analyzing proteins, to achieve suitable peak shapes (limited broadening and tailing) with this material. Elevated temperature (>60°C) is mandatory when analyzing proteins with silica-based stationary phases, but this was not the case with this particular column made with a carbon core and nanodiamond-polymer shell, since the peak capacities were not improved at high temperature. However, there was a need to increase mobile phase temperature in the range 70–90°C when analyzing monoclonal antibodies (mAbs), to limit adsorption that often occur in RPLC with this specific class of biomolecules.Finally, the FLARE® wide-pore column was applied to real life samples of native, oxidative stressed and reduced therapeutic proteins as well as reduced, digested mAbs and antibody drug conjugates (ADCs), to highlight the possibilities offered by this column technology.

Interpretive Approaches to Optimize Serially-Coupled Columns in Reversed-Phase Liquid Chromatography

Interpretive Approaches to Optimize Serially-Coupled Columns in Reversed-Phase Liquid Chromatography

Comparative Study on Selectivity and Kinetic Performance of Recent Reversed Phase Liquid Chromatography Columns in the Separation of Protein Variants

In this study, the impact of stationary phase chemistry was investigated for the analysis of large biomolecules. For this purpose, widepore state-of the-art C18, C3, diphenyl stationary phases as well as a new superficially porous material possessing a carbon core and nanodiamond-polymer shell (composite material) were tested. To evaluate the similarities and differences between these phases, two samples were considered, including i) oxidatively stressed and reduced recombinant interferon alfa-2A variants as well as ii) light chain and heavy chain fragments of a human IgG1 monoclonal antibody Belimumab. For both samples, the overall selectivity was found to be similar on all the silica-based stationary phases (e.g. α = 1.20 - 1.25 between the two oxidized forms of interferon), while it was somewhat different on the composite material (α = 1.12 between the two oxidized forms of interferon). On the other hand, the kinetic performance of the composite phase was somewhat lower (peak capacity between 70 and 80) than that of silica-based materials (peak capacity higher than 100), except in the presence of a relatively high concentration TFA (0.5% TFA) due to the reduction of ionic interactions. At the end, when separating closely eluting proteins variants in RPLC mode, minor differences in selectivity may be more important than kinetic efficiency and therefore, the composite material might also be of interest for biomolecules characterization. Keywords: Carbon/nanodiamond/polymer shell particles, kinetic performance, protein separation, reversed phase chromatography, selectivity.

Tuning preparation conditions towards optimized separation performance of thermally polymerized organo-silica monolithic columns in capillary liquid chromatography

Tuning of preparation conditions, such as variations in the amount of a porogen, concentration of an aqueous acid catalyst, and adjustment in polymerization temperature and time, towards optimized chromatographic performance of thermally polymerized monolithic capillaries prepared from 3-(methacryloyloxy)propyltrimethoxysilane has been carried out. Performance of capillary columns in reversed-phase liquid chromatography was assessed utilizing various sets of solutes. Results describing hydrophobicity, steric selectivity, and extent of hydrogen bonding enabled comparison of performance of hybrid monolithic columns prepared under thermal (TSG) and photopolymerized (PSG) conditions. Reduced amounts of porogen in the polymerization mixture, and prolonged reaction times were necessary for the preparation of monolithic columns with enhanced retention and column efficiency that reached to 111,000 plates/m for alkylbenzenes with shorter alkyl chains. Both increased concentration of catalyst and higher temperature resulted in faster polymerization but inevitably in insufficient time for pore formation. Thermally polymerized monoliths produced surfaces, which were slightly more hydrophobic (a methylene selectivity of 1.28±0.002 TSG vs 1.20±0.002 PSG), with reduced number of residual silanols (a caffeine/phenol selectivity of 0.13±0.001 TSG vs 0.17±0.003 PSG). However, steric selectivity of 1.70±0.01 was the same for both types of columns. The batch-to-batch repeatability was better using thermal initiation compared to monolithic columns prepared under photopolymerized conditions. RSD for retention factor of benzene was 3.7% for TSG capillaries (n=42) vs. 6.6% for PSG capillaries (n=18). A similar trend was observed for columns prepared within the same batch.

Optimized Analytical Procedures for the Untargeted Metabolomic Profiling of Human Urine and Plasma by Combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)–Mass Spectrometry*[S

Profiling of body fluids is crucial for monitoring and discovering metabolic markers of health and disease and for providing insights into human physiology. Since human urine and plasma each contain an extreme diversity of metabolites, a single liquid chromatographic system when coupled to mass spectrometry (MS) is not sufficient to achieve reasonable metabolome coverage. Hydrophilic interaction liquid chromatography (HILIC) offers complementary information to reverse-phase liquid chromatography (RPLC) by retaining polar metabolites. With the objective of finding the optimal combined chromatographic solution to profile urine and plasma, we systematically investigated the performance of five HILIC columns with different chemistries operated at three different pH (acidic, neutral, basic) and five C18-silica RPLC columns. The zwitterionic column ZIC-HILIC operated at neutral pH provided optimal performance on a large set of hydrophilic metabolites. The RPLC columns Hypersil GOLD and Zorbax SB aq were proven to be best suited for the metabolic profiling of urine and plasma, respectively. Importantly, the optimized HILIC-MS method showed excellent intrabatch peak area reproducibility (CV < 12%) and good long-term interbatch (40 days) peak area reproducibility (CV < 22%) that were similar to those of RPLC-MS procedures. Finally, combining the optimal HILIC- and RPLC-MS approaches greatly expanded metabolome coverage with 44% and 108% new metabolic features detected compared with RPLC-MS alone for urine and plasma, respectively. The proposed combined LC-MS approaches improve the comprehensiveness of global metabolic profiling of body fluids and thus are valuable for monitoring and discovering metabolic changes associated with health and disease in clinical research studies.

Hydrophilic interaction chromatography: A promising alternative to reversed-phase liquid chromatography systems for the purification of small protonated bases.

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed-phase liquid chromatography mode (BEH-C18), an electrostatic repulsion reversed-phase liquid chromatography C18 column (BEH-C18+), a poly(styrene-divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm(3)). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed-phase liquid chromatography like conditions (with BEH-C18, BEH-C18+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed-phase liquid chromatography columns.