Mobile Phase Components Research Articles

Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry.

We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.

Application of protein A-modified capillary-channeled polymer polypropylene fibers to the quantitation of IgG in complex matrices

Polypropylene (PP) capillary-channeled polymer (C-CP) fibers loaded with recombinant Staphyloccocus aureus protein A (rSPA) were used as an affinity chromatography stationary phase for the quantitation of immunoglobulin G (IgG) in complex biological matrices. Optimization of the chromatographic method regarding mobile phase components and load/elution conditions was performed. The six-minute analysis, including a load step with 12mM phosphate at pH 7.4, an elution step with 0.025% phosphoric acid and a re-equilibration step, was employed for quantitation of IgG1 from 0.075 to 3.00mgmL−1 in an IgG-free CHO cell supernatant matrix. Quantification of IgG1 content in a different CHO cell line was accomplished using the external calibration curve as well as using a standard addition approach. The high level of agreement between the two approaches suggests that the protein A-modified C-CP fiber phase is immune from matrix effects due to concomitant species such as host cell proteins (HCPs), host cell DNA, media components and other leachables and extractables. The inter-day and intra-day precision of the method were 3.1 and 3.5%RSD respectively for a single column. Column-to-column variability was 1.31 and 6.62%RSD for elution time and peak area, respectively, across columns prepared in different batches. The method reported here is well-suited for IgG analysis in complex harvest cell culture media in both the development and production environments.

New Insights into the Effects of Tetrahydrofuran and Dioxane on the Selectivity of Ternary Eluent Compositions in RP-HPLC Systems

The present paper discusses the tendencies in the retention changes of a diverse set of organic model compounds on HPLC C18 stationary phases in aqueous ternary eluent mixtures containing tetrahydrofuran or 1,4-dioxane, and one short chain aliphatic alcohol (ethanol, n-propanol or isopropanol). The set of compounds consisted of steroid and non-steroid molecules with different hydrogen bond donor and/or acceptor abilities. Every eluent mixture contained 75 V/V% water and 25 V/V% organic solvent(s). The composition of the mixture was changed in 5 V/V% steps, starting with the binary alcohol–water mixtures and finishing with the binary ether–water mixtures. The results show clearly the dependency of retention times on the eluent composition, the size of the molecules, and the occurence of the hydrogen bond donor/acceptor groups. The isopropanol–tetrahydrofurane–water mixtures resulted in selective changes in the retention times of the compounds with acidic groups or with other non-acidic OH or NH protons in the neighborhood of electron-withdrawing groups. Every compound has shown elevated retention times in the isopropanol–dioxane–water, n-propanol–tetrahydrofuran–water, or n-propanol–dioxane–water mixtures. Clear trends could not be observed in the eluents with ethanol. The probable reason for the retention enhancement is the adsorption of the organic components of the mobile phase on the surface of the stationary phase. The different effects of the alcohols may originate from the interaction of their varying aliphatic alkyl chains with the C18 chains. This phenomenon may result in different availability of the C18 chains for dioxane, tetrahydrofuran and the model compounds.

Development and validation of a fast SFC method for the analysis of flavonoids in plant extracts

Flavonoids from plants always show a wide range of biological activities. In the present study, a rapid and highly efficient supercritical fluid chromatography (SFC) method was developed for the separation of 12 flavonoids. After careful optimization, the 12 flavonoids were baseline separated on a ZORBAX RX-SIL column using gradient elution. A 0.1% phosphoric acid solution in methanol was found to be the most suitable polar mobile phase component for the separation of flavonoids. From the viewpoint of retention and resolution, a backpressure of 200bar and a temperature of 40°C were shown to give the best results. Compared with a previously developed reverse phase liquid chromatography method, the SFC method could provide flavonoid separations that were about three times faster, while maintaining good peak shape and comparable peak efficiency. This SFC method was validated and applied to the analysis of five flavonoids (kaempferol, luteolin, quercetin, luteoloside, buddleoside) present in Chrysanthemum morifolium Ramat. from different cultivars (Chuju, Gongju, Hangju, Boju). The results indicated a good repeatability and sensitivity for the quantification of the five analytes with RSDs for overall precision lower than 3%. The limits of detection ranged from 0.73 to 2.34μg/mL, while the limits of quantification were between 2.19 and 5.86μg/mL. The method showed that SFC could be employed as a useful tool for the quality assessment of Traditional Chinese medicines (TCMs) containing flavonoids as active components.

Identification of the structural characteristics of the asphaltenes in the tetrahydrofuran-microwave-extracted portions from two Chinese coals

This work aimed to evaluate the detailed chemical structures of the asphaltenes in the tetrahydrofuran-microwave-extracted portions from two Chinese coals, namely, Shenfu and Zhundong coal (SF, ZD) and to clarify their characteristics on chemical composition at molecular level. Based on the analyses of diffuse reflectance Fourier transform infrared spectroscopy (DRIFT) and Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), the detailed chemical structure of a series of basic and non-basic nitrogen- and oxygen-containing class species in the asphaltenes were clarified. The results revealed that condensed nitrogen-containing alkylaromatic structures with amidogens (1–3) were the main structure units in N4Oy (y=0–2) class species due to the high double bond equivalent (DBE) values for both the asphaltene from SF (ASF) and the asphaltene from ZD (AZD). Acidic O2–O7 class species with two or three aromatic rings were the main structure units, among which O4 and O2 class species had the highest relative abundances (RAs) in ASF and AZD, respectively. Acidic oxygen-containing components in AZD had narrower range of DBE and carbon number and mainly consisted of aromatic structure units with two rings. On the contrary, the average DBE values of O4–O7 class species in ASF had positive relationship with oxygen atom number which could be attributed to the introduction of phenol groups. The results provided more detailed information on the structural characteristics of polar and aromatic-rich components of mobile phase in coal.

Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography

Enhanced fluidity liquid chromatography using the hydrophilic interaction retention mechanism (EFLC-HILIC) is studied as an alternative separation mode for analyzing oligosaccharides and other sugars. These carbohydrates, which are important for the study of foods and biological systems, are difficult to comprehensively profile and either require a non-green, expensive solvent (i.e. acetonitrile) or derivatization of the analytes at the expense of time, sample loss, and loss of quantitative information. These difficulties arise from the diverse isomerism, mutarotation, and lack of a useable chromophore/fluorophore for spectroscopic detection. Enhanced fluidity liquid chromatography is an alternative separation method that involves the use of conventional polar solvents, such as methanol/water mixtures, as the primary mobile phase component and liquid carbon dioxide (CO2) as the modifier in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis and higher efficiency. This work illustrates an optimized EFLC-HILIC separation of a test mixture of oligosaccharides and simple sugars with a resolution greater than 1.3 and an analysis time decrease of over 35% compared to a conventional HPLC HILIC-mode analysis using acetonitrile/water mobile phases.

A Simple and Improved HPLC-PDA Method for Simultaneous Estimation of Fexofenadine and Pseudoephedrine in Extended Release Tablets by Response Surface Methodology

A simple RP-HPLC method has been developed for simultaneous estimation of fexofenadine and pseudoephedrine in their extended release tablet. The method was developed based on statistical design of experiments (DoE) and Response Surface Methodology. Separation was achieved on double end-capped C18 column (250 mm × 4 mm, 5 μm). In this experiment, two components of mobile phase, namely, acetonitrile (% v/v) and methanol (% v/v), were the factors whereas retention and resolution of the chromatographic peaks were the responses. The effects of different composition of factors on the corresponding responses were investigated. The optimum chromatographic condition for the current case was found as an isocratic mobile phase consisting of 20 mM phosphate buffer (pH 6.8) and acetonitrile and methanol in a ratio of 50 : 36 : 14 (% v/v) at a flow rate of 1 mL/min for 7 minutes. The retention of pseudoephedrine and fexofenadine was found to be 2.6 min and 4.7 min, respectively. The method was validated according to the ICH and FDA guidelines and various validation parameters were determined. Also, forced degradation studies in acid, base, oxidation, and reduction media and in thermal condition were performed to establish specificity and stability-indicating property of this method. Practical applicability of this method was checked in extended release tablets available in Bangladeshi market.

Lipophilicity estimation and characterization of selected steroid derivatives of biomedical importance applying RP HPLC

The present paper deals with chromatographic lipophilicity determination of twenty-nine selected steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC) combined with two mobile phase, acetonitrile-water and methanol-water. Chromatographic behavior of four groups (triazole and tetrazole, toluenesulfonylhydrazide, nitrile and dinitrile and dione) of selected steroid derivatives was studied. Investigated compounds were grouped using principal component analysis (PCA) according to their logk values for both mobile phases. Grouping was in the very good accordance with the polarity and lipophilicity of the investigated compounds. QSRR (quantitative structure-retention relationship) approach was used to model chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods. The most influential molecular descriptors were lipophilicity descriptors that are important for molecules ability to pass through biological membranes and geometrical descriptors. All established LR-QSRR and MLR-QSRR models were statistically validated by standards, cross- and external validation parameters as well as with two graphical methods. According to all these assessments, MLR models were better for chromatographic lipophilicity prediction. It was shown that chromatographic systems with methanol-water were better for modeling of logk than systems with acetonitrile-water, as well as the systems that contained lower volume fractions of organic component in mobile phase. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates of biomedical importance.

Separation and determination of estrogen in the water environment by high performance liquid chromatography-fourier transform infrared spectroscopy.

The components for connecting high-performance liquid chromatography (HPLC) with Fourier-transform infrared spectroscopy (FTIR) were investigated to determine estrogen in the water environment, including heating for atomization, solvent removal, sample deposition, drive control, spectrum collection, chip swap, cleaning and drying. Results showed that when the atomization temperature was increased to 388 K, the interference of mobile phase components (methanol, H2O, acetonitrile, and NaH2PO4) were completely removed in the IR measurement of estrogen, with 0.999 of similarity between IR spectra obtained after separation and corresponding to the standard IR spectra. In experiments with varying HPLC injection volumes, high similarity for IR spectra was obtained at 20 ul injection volume at 0.01 mg/L BPA while a useful IR spectrum for 10 ng/L BPA was obtained at 80 ul injection volume. In addition, estrogen concentrations in the natural water samples were calculated semi-quantitatively from the peak intensities of IR spectrum in the mid-infrared region.

Salting-out assisted extraction method coupled with hydrophilic interaction liquid chromatography for determination of selected β-blockers and their metabolites in human urine.

In this study, a new analytical method was developed and validated for the simultaneous analysis of β-blockers (metoprolol, propranolol, carvedilol) and their metabolites (5'-hydroxycarvedilol, O-desmethylcarvedilol, α-hydroxymetoprolol, O-desmethylmetoprolol, 5-hydroxypropranolol) in human urine. A salting-out assisted liquid-liquid extraction (SALLE) procedure was used for sample preparation. Several parameters affecting the extraction efficiency and method sensitivity including the type and volume of the extraction solvent, the type and quantity of the inorganic salt, extraction time and sample pH were investigated. Hydrophilic interaction liquid chromatography-ultraviolet detection (HILIC-UV) was used for the determination of all analytes. During method development, the effects of mobile phase components (type, pH, concentration of salt, organic modifier type and content, flow rate, column temperature) on the retention and separation of β-blockers and metabolites on the five different HILIC columns were examined. The method was linear for concentrations ranging from 0.1 to 8.0μg/mL, with determination coefficients higher than 0.993 for all analytes. The limits of quantification were in the range from 0.1 to 0.2μg/mL. Intra- and inter-day precision ranged from 0.1 to 8.9%, and accuracy was within±13% interval for all analytes. Under the optimized conditions, extraction efficiency was greater than 83.4% for determined compounds. The validated method was then applied to the measurement of β-blockers and their metabolites in human urine samples.

High-performance thin-layer chromatography and pressurized planar electrochromatography of some diastereomeric amino acid derivatives in reversed-phase system with carboxylic acid mobile phase buffers

The separation of 1-fluoro-2,4-dinitrophenyl-5-l-valine amide (FVDA) diastereomeric derivatives of aspartic acid, cysteine, and histidine by means of high-performance thin-layer chromatography (HPTLC) as well as pressurized planar electrochromatography (PPEC) techniques in systems with HPTLC RP-18W plates and the various acetonitrile—buffer mobile phases is presented. The influence of the mobile phase components, i.e., acetonitrile concentration and buffer kind on migration distance of the solute zones, was investigated. The effect of mono (formic) and various dicarboxylic acids (oxalic, malonic, maleic, malic, succinic, tartaric, and pimelic) as the mobile phase buffer components on the solute retention was studied. It is observed that an increase of acetonitrile content of the mobile phase affects the solute zone migration and retention in PPEC and HPTLC. What is more, the separation selectivity in the latter and former techniques differs. The PPEC mode presents a higher efficiency in comparison with HP...

Sorption of nitrogen-containing aromatic compounds on ultradispersed diamonds

Adsorption of a number of aniline and pyridine derivatives from water-acetonitrile solution on ultradispersed diamonds was investigated using dynamic sorption method. It was shown that the nature of functional substitutes and their position in molecules of nitrogen-containing compounds of pyridine and aniline have a pronounced effect on adsorption on the surface of ultradispersed diamonds. The dependence of chromatographic sorbate retention on the content of mobile phase could be described by a curve with a minimum. Such nonlinear relationship was explained by the change in the ratio of contribution of specific and non-specific interaction to the sorbate retention that was observed on varying the volume content of an organic component in mobile phase. The influence of temperature on sorption of pyridine and aniline derivatives on ultradisperesed diamonds was investigated. The changes in enthalpy and entropy factors of competitive sorption of sorbates were determined.

Development and Validation of a Stability Indicating HPLC Method for the Simultaneous Analysis of Esomeprazole and Itopride in Bulk and In Capsules

A rapid, simple, selective, precise, and accurate stability indicating HPLC method has been developed and validated for the simultaneous analysis of esomeprazole and itopride in bulk and in capsule form. An isocratic separation was achieved using a Hypersil C4 (250 x 4.6 mm), 5 μm particle size column with a flow rate of 1 mL/min and photodiode array detector at 272 nm. The mobile phase consisted of 0.1M dipotassium hydrogen phosphate: acetonitrile (40:60 v/v). The method was validated for selectivity, specificity, linearity, precision, accuracy and robustness. The selectivity of the method was determined by assessing interference from the placebo, components of mobile phase and common excipients in pharmaceutical formulations. Whereas, specificity was established by stress degradation studies. The method was linear over the concentration range 40 – 120 μg/mL (R 2 = 0.9999) and 150-450 μg/mL (R 2 = 0.9999) for esomeprazole and itopride, respectively. Limit of detection is 0.207 and 0.724 μg/mL & Limit of quantitation is 0.691 and 2.415 μg/mL for esomeprazole and itopride, respectively. The precision and accuracy of the method was found to be acceptable. The method was found to be robust and suitable for the simultaneous analysis of esomeprazole and itopride in a capsule formulation. Degradation products resulting from the stress studies did not interfere with the detection and quantification of esomeprazole and itopride. The proposed HPLC method is thus stability-indicating.

A systematic approach for reversed phase liquid chromatographic method development of fingolimod hydrochloride via design augmentation

A stability indicating reversed phase liquid chromatographic (RP-LC) method was developed for the analysis of fingolimod hydrochloride (FIM) and its related compounds in drug substance and its dosage form. Separation of seven related compounds and three degradation products was achieved in a C18 stationary phase with 3mM tetrabutyl ammonium hydrogen sulfate in 0.02% orthophosphoric acid and acetonitrile in gradient elution. Aqueous mobile phase components and column temperature were optimized through Design of experiments (DoE) for a fixed gradient program. Major degradation products were identified by LC-MS and degradation pathway is proposed. The validation results indicated that the method is sensitive in detection, linear, precise and accurate. The developed method was also demonstrated in UHPLC.

A reversed-phase/hydrophilic interaction mixed-mode C18-Diol stationary phase for multiple applications

A mixed-mode chromatographic packing material, C18 and diol groups modified silica (C18-Diol), was prepared with controllable hydrophobicity and hydrophilicity. It demonstrated excellent aqueous compatibility and stability in aqueous mobile phase; compared to the traditional C18 column, improved peak shape of basic analytes was also obtained. Additionally, it exhibited both reversed-phase liquid chromatographic (RPLC) and hydrophilic interaction chromatographic (HILIC) performance; the analyte separation scope was thus enlarged, demonstrated by simultaneous separation of twenty acids, bases and neutrals. More interestingly, a novel on-line two-dimensional liquid chromatography on the single column (2D-LC-1C) was established by modifying the high performance liquid chromatographic instrument only with the addition of an extra six-port two-position valve. The early co-eluted components of the extract of Lonicera japonica on the 1st-dimension (RPLC) were collected for the online re-injection to the 2nd-dimension (HILIC) by conveniently varying the mobile phase components. Six more peaks were obtained. The established system was simple, easy operation and low cost, which had advantages in analyzing complicated samples.

An interphase model for retention in liquid chromatography

An interphase model for retention is appropriate for biphasic systems in which one phase (the mobile phase) modifies the properties of the other phase through absorption of mobile phase components. This is typical of liquid chromatography, where separation occurs by the distribution of sample components between a bulk mobile phase and an interphase region in intimate contact with the mobile phase. This has profound implications for the interpretation of retention mechanisms since the properties of the stationary phase are those of the interphase region, which can be quite different to those known or perceived for the nonsolvated stationary phase. For reversed-phase chromatography, it is shown that retention properties can be adequately described by the solvation parameter model and visualized as a function of the bulk mobile phase composition by system maps. For normal-phase chromatography, a modified approach is required for inorganic oxide adsorbents to accommodate site-specific interactions (localizati...

HPLC Determination of Enantiomeric Purity of PF-04136309 Based on a Chiral Stationary Phase

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of (S)-N-[2-(3-{trans-4-hydroxy-4-[5-(pyrimidin-2-yl)pyridin-2-yl] cyclohexylamino} pyrrolidin-1-yl)-2-oxoethyl]-3-(trifluoromethyl) benzamide (PF-04136309). Baseline separation with a resolution higher than 1.8 was accomplished within 40 min using a CHIRALPAK AD (250 × 4.6 mm; particle size 5 μm) column, with n-hexane:2-propanol (70:30 v/v) as mobile phase at a flow rate of 1 mL min−1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as the resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within a concentration range between 0.01 and 1 mg mL−1 (n = 9), and recoveries between 98.17 and 101.28 % were obtained, with relative standard deviation (RSD) lower than 1.44 %. The LOD and LOQ for PF-04136309 were 3.59 and 11.54 μg mL−1 and for its enantiomer were 3.39 and 11.28 μg mL−1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of PF-04136309, especially for the analysis of bulk samples.

β-Cyclodextrin as the Mobile Phase Component for Separation of Some DNS-Amino Acid Enantiomers with HPTLC and PPEC

Separation of 5-(dimethylamino) naphthalene-1-sulphonyl (DNS-) derivatives of some amino acids (alanine, leucine, valine, phenylalanine) D- and L-enantiomers with high-performance thin layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) both with β-cyclodextrin as the component of the mobile phase is presented. The separation mechanism in the PPEC technique involves two effects (partition and electrophoresis) whereas HPTLC system employs only one (partition). The separation process in both modes was carried out with the HPTLC RP-18 W plates. Influence of variables such as: β-cyclodextrin concentration in the mobile phase and composition of the mobile phase on the migration distance of the solute zones is investigated. Although HPTLC and PPEC systems enable to separate D- and L-DNS- amino acids the electrophoretic effect in PPECsystem is responsible for different separation selectivity in the latter one in comparison to that in first one. The retention of L-DNS- amino acids is stronger regarding their D-antipodes with both separation techniques. The repeatability of the results from separation of DNS-Ala enantiomers with PPEC and HPTLC technique is compared. Slightly worse standard deviation and RSD values for more retained enantiomer, researched with PPEC, are characteristics of this electromigrational technique. In addition a new mode of β-cyclodextrin zone determination on the chromatographic plate is presented. Keywords: β-cyclodextrin, chiral mobile phase, enantiomer separation, DNS-amino acids, HPTLC, PPEC.

High-performance liquid chromatographic enantioseparation of cationic 1,2,3,4-tetrahydroisoquinoline analogs on Cinchona alkaloid-based zwitterionic chiral stationary phases.

The stereoisomers of 1,2,3,4-tetrahydroisoquinoline analogs were resolved for the first time by applying a polar ionic mobile phase on a quinine or a quinidine moiety fused with a chiral sulfonic acid-type chiral selector immobilized on silica [Chiralpak ZWIX(+)™ and Chiralpak ZWIX(-)™]. The effects of the nature and concentrations of the mobile phase components and additives and temperature on the retention and enantioseparation on the investigated chiral columns were studied. Experiments were performed in the temperature range 10-50 °C. Thermodynamic parameters were calculated from plots of ln α versus 1/T. The separations were generally enthalpy-controlled, but entropy-controlled separation was also observed below 30 °C. The enantiomer elution order was determined in some cases and was observed to be opposite on the ZWIX(+)™ and ZWIX(-)™ columns. Our results contribute to a better understanding of the enantiorecognition mechanism of chiral bases with chiral zwitterionic selectors.

Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites

It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC–HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC–HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC–HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC–HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.