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The editors of Chromatography would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015. [...]

The editors of Chromatography would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2014:[...]

N-methyl-D-aspartate (NMDA) receptors, which are widely present in the central nervous system, have also been found to be up-regulated in a variety of cancer cells and tumors and they can play active roles in cancer cell growth regulation. NMDA receptor antagonists have been found to affect cancer cell viability and interfere with tumor growth. Moreover, cancer cells also have been shown to have elevated levels of some D-amino acids. Two human skin cell lines: Hs 895.T skin cancer and Hs 895.Sk skin normal cells were investigated. They were derived from the same patient to provide tumor and normal counterparts for comparative studies. The expression of specific NMDA receptors was confirmed for the first time in both skin cell lines. Dizocilpine (MK-801) and memantine, NMDA receptor channel blockers, were found to inhibit the growth of human skin cells by reducing or stopping NMDA receptor activity. Addition of D-Ser, D-Ala, or D-Asp, however, significantly reversed the antiproliferative effect on the human skin cells triggered by MK-801 or memantine. Even more interesting was the finding that the specific intracellular composition of a few relatively uncommon amino acids was selectively elevated in skin cancer cells when exposed to MK-801. It appears that a few specific and upregulated D-amino acids can reverse the drug-induced antiproliferative effect in skin cancer cells via the reactivation of NMDA receptors. This study provides a possible innovative anticancer therapy by acting on the D-amino acid pathway in cancer cells either blocking or activating their regulatory enzymes.

Pharmaceutical effluents are complex media containing hundreds of compounds including active ingredients, intermediate products and unknown impurities. Bringing an industrial wastewater treatment plant (WWTP) into compliance with European directives requires a thorough analysis of the effluent. In this study, we demonstrate how online comprehensive two-dimensional liquid chromatography (on-line LC × LC) hyphenated to high resolution mass spectrometry (HRMS) can be a powerful analytical methodology to monitoring the outlet water, by analysing the content of known molecules while characterising unknown compounds. Reversed phase liquid chromatography (RPLC) was used in both dimensions, with a penta-fluoro-phenyl silica-based column at neutral pH in the first dimension (1D) and a C18 column at acidic pH in the second one (2D). The conditions were optimized for a total analysis time of 60 min. The variability of both retention times and peak areas was evaluated. The average standard deviation on retention times was found to be less than 0.1 s in 2D. The relative standard deviation on peak area was about 7% for run-to-run analysis. This analytical approach, applied to the pharmaceutical effluents before (inlet) and after (outlet) wastewater treatment permitted to detect 240 compounds. These included 27 priority pharmaceutical products, 8 of which were of very high priority and their concentrations could be compared to target values. The comparison of 2D-LC and 1D-LC approaches clearly highlights the power of on-line RPLC x RPLC technique, which allows both targeted quantitative analysis and non-targeted qualitative analysis of pharmaceutical effluents.

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Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Power functions, pn(x)=[f(x)]n , are embedded in some modern chromatography detectors and software which not only alter the linear dynamic range of such detectors but also improve the cosmetic aspects of the chromatograms. These aspects include a reduction in baseline noise, improved peak symmetry, and better resolution. However, after raising the electronic output of a detector to a selected power (n > 1), the original peak area information is lost as are very small peaks. Recent advances in the peak processing protocol allow us to recover peak areas from overlapping peak areas, even in noisy environments using power functions. One of the primary protocol requirements of this approach was to have resolution factors ≥0.9. An increasing positive bias in the recovered area was observed as the resolution factors decreased below 0.9. In this work, we extend the capabilities of power function to lower resolution values. This bias is addressed by considering the increasing contributions in peak height coming from adjacent peaks. It is shown that the power function can now be used as long as the peak maxima are visible, making Rs = 0.5, the lower treatable resolution factor for two peaks of similar areas.

International audience; Differences in elution strength between the sample solvent and the mobile phase usually give rise to undesirable effects on the chromatographic separation, which may range from slight broadening to severe peak deformation or even splitting. In the most extreme case, the retention factor of the analyte at the head of the column is so small at the time of injection that part of the analyte goes through the column with very little interaction with the stationary phase and hence elutes very close to the column dead time. This phenomenon is known as breakthrough. Usually, during breakthrough, the retained peak displays a wide array of deformations and it is not rare that multiple retained peaks appear for a given injected analyte. However, under certain conditions, it has been demonstrated that these deleterious effects could fully disappear, leaving only one breakthrough peak and one symmetrical peak on the chromatogram. This so-called “total breakthrough” phenomenon was recently highlighted in the specific context of the 2D-LC separation of peptides but has yet to be explained. In the present paper, we describe the results of a comprehensive study aiming to better understand and define the conditions of emergence of both breakthrough and total breakthrough phenomena in liquid chromatography. The effects of a broad range of parameters, including the nature of the solute, the retention mechanism, the injection and elution conditions, the column temperature, and the injected sample concentration on the occurrence of both phenomena were investigated. While breakthrough was found to occur for all studied compounds, it appears that the presence of positive charges on the molecule is a prerequisite for observing a total breakthrough phenomenon. Among all the parameters investigated in this work, only the injection conditions and the analyte retention were found to be impactful on the onset of both phenomena. This finding allowed us to suggest one necessary and sufficient condition, relying on the injection of critical volumes to observe each respective phenomenon. These critical volumes only depend on the column dead volume and the retention factor of the analyte in the injection solvent.

International audience; Quil-A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil-A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two-dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off-line configuration using basic instrumentation was promoted. Hence, reversed-phase liquid chromatography × reversed-phase liquid chromatography and hydrophilic interaction chromatography × reversed-phase liquid chromatography methods with ultraviolet and single-quadrupole mass spectrometry detection were kinetically optimized. The reversed-phase liquid chromatography × reversed-phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed-phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed-phase liquid chromatography × reversed-phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed-phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.

We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (2D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.

In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is well-known to give rise to a large retention space coverage when dealing with ionisable compounds. However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension, and thus the separation quality and the method sensitivity. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension (2D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy, which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in 2D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM. Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM). However, despite the presence of breakthrough, peak intensities were approximately 7-fold higher with Total Breakthrough Strategy than with flow splitting.

International audience; Twelve new azole compounds were synthesized through an ene reaction involving methylidene heterocycles and phenylmaleimide, producing four oxazoles, five thiazoles, and one pyridine derivative, and ethyl glyoxylate for an oxazole and a thiazole compound. The twelve azoles have a stereogenic center in their structure. Hence, a method to separate the enantiomeric pairs, must be provided if any further study of chemical and pharmacological importance of these compounds is to be accomplished. Six chiral stationary phases were assayed: four were based on macrocyclic glycopeptide selectors and two on linear carbohydrates, i.e., derivatized maltodextrin and amylose. The enantiomers of the entire set of new chiral azole compounds were separated using three different mobile phase elution modes: normal phase, polar organic, and reversed phase. The most effective chiral stationary phase was the MaltoShell column, which was able to separate ten of the twelve compounds in one elution mode or another. Structural similarities in the newly synthesized oxazoles provided some insights into possible chiral recognition mechanisms.

International audience; On-line comprehensive two-dimensional liquid chromatography is a powerful technique for the separation of highly complex samples. Due to the addition of the second dimension of separation, impressive peak capacities can be obtained within a reasonable analysis time compared to one-dimensional liquid chromatography. In online comprehensive two-dimensional liquid chromatography, the separation power is maximized by selecting two separation dimensions as orthogonal as possible, which most often requires the combination of different mobile phases and stationary phases. The online transfer of a given solvent from the first dimension to the second dimension may cause severe injection effects in the second dimension, mostly due to solvent strength mismatch. Those injection effects may include peak broadening, peak distortion, peak splitting or breakthrough phenomenon. They are often found to reduce significantly the peak capacity and the peak intensity. To overcome such effects, arising specifically in online comprehensive two-dimensional liquid chromatography, different methods have been developed over the years. In this review, we focused on the most recently reported ones. A critical discussion, supported by a theoretical approach, gives an overview of their advantages and drawbacks.

The dried aerial parts of the plant Artemisia glabella Kar. et Kir. contain about 1.1% of the very important sesquiterpene lactone arglabin. This compound can be extracted by supercritical CO2 giving a 20 time concentrated extract containing about 22% arglabin. The CO2 extract can be efficiently treated by hydrostatic countercurrent chromatography (CCC) able to produce in a single run 95% pure arglabin. Both mobile and stationary phase are liquid in CCC. The biphasic waterless liquid system heptane:ethyl acetate:acetonitrile 2:1:2 v/v/v was used to separate arglabin from its two major co-extracted compounds: argolide and eucalyptol. A three-factor Box–Behnken design was used to optimize the CCC preparative purification of the CO2 extract by a FCPC-5L instrument. Injected amount, mobile phase flow rate, and rotor rotation speed were used to optimize arglabin productivity. An optimized run was able to treat 90 g of extract producing 20 g of 95% pure arglabin in 50 min, a theoretical productivity of 400 mg/min.

From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.

Aim. To study pharmacokinetic parameters of the potential active pharmaceutical ingredient (API) morpholinium 2-((4-(2-methoxyphenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate in the blood plasma of rats when administered intraperitoneally.Results and discussion. The HPLC-MS method of determination of morpholinium 2-((4-(2-methoxy)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate in the blood plasma of non-linear rats has been adopted to study of pharmacokinetic parameters of the compound. It has been proven that high levels of API extraction from the blood plasma when using methanol for protein precipitation were observed. The measurement of the concentration of the drug substance studied in the blood plasma at different time intervals after injection allowed us to plot the pharmacokinetic curve and calculate the pharmacokinetic parameters.Experimental part. The Agilent 1260 Infinity liquid chromatography system consisted of a degasser, binary pump, autosampler, column thermostat, diode-matrix detector, single-quadrupole mass spectrometric detector (Agilent 6120). The pharmacokinetic study was performed in sexually mature white nonlinear rats of two sexes. The active pharmaceutical ingredient morpholinium 2-((4-(2-methoxy)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate, was administered in the isotonic solution intraperitoneally in the dose of 50 mg/kg.Conclusions. The pharmacokinetic curve has been plotted, and the pharmacokinetic parameters of morpholinium 2-((4-(2-methoxy)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate in the blood plasma when administered intraperitoneally have been calculated.Received: 30.06.2020Revised: 08.08.2020Accepted: 27.08.2020, Мета. Вивчити фармакокінетику потенційного активного фармацевтичного інгредієнту (АФІ) морфоліній 2-((4-(2-метоксифеніл)-5-(піридин-4-іл)-4H-1,2,4-триазол-3-іл)тіо)ацетату в плазмі кровi щурів при внутрішньочеревному введенні.Результати та їх обговорення. ВЕРХ-МС методику визначення морфоліній 2-((4-(2-метоксифеніл)-5-(піридин-4-іл)-4H-1,2,4-триазол-3-іл)тіо)ацетату в плазмі крові нелінійних щурів було адаптовано для дослідження фармакокінетики. Доведено, що при використанні метанолу для осадження білків спостерігаються високі показники вилучення АФІ з плазми крові. Вимірювання концентрації досліджуваної лікарської речовини в плазмі крові на різних інтервалах часу після введення дало змогу побудувати фармакокінетичну криву та розрахувати відповідні фармакокінетичні параметри.Експериментальна частина. Рідинно-хроматографічна система Agilent 1260 Infinity складалася з дегазатора, бінарного насосу, автосамплера, термостату колонки, діодно-матричного детектора, одноквадрупольного мас-спектрометричного детектора (Agilent 6120). Дослідження фармакокінетики проведене на статевозрілих білих нелінійних щурах обох статей. Активний фармацевтичний інгредієнт, морфоліній 2-((4-(2-метоксифеніл)-5-(піридин-4-іл)-4H-1,2,4-триазол-3-іл)тіо)ацетат вводили в ізотонічному розчині внутрішньочеревно при дозуванні 50 мг/кг.Висновки. Побудовано фармакокінетичну криву та розраховано фармакокінетичні параметри морфоліній 2-((4-(2-метоксифеніл)-5-(піридин-4-іл)-4H-1,2,4-триазол-3-іл)тіо)ацетату в плазмі крові при його внутрішньочеревному введенні.Received: 30.06.2020 Revised: 08.08.2020 Accepted: 27.08.2020

International audience; 8 The method development of liquid-liquid chromatography, either countercurrent 9 chromatography or centrifugal partition chromatography, is slowed down by the selection of 10 the biphasic solvent system that constitutes its column. This paper introduces a classification 11 of 19 solvent systems, including the most popular systems based on heptane/ethyl 12 acetate/methanol/water, some non-aqueous systems and some greener systems. This 13 classification is based on Abraham descriptors determined through the partition coefficients 14 of 43 probes. Among 21 determined models, nine of them allow an accurate prediction of 15 partition coefficients from solute descriptors and another ten provide a description of the 16 chromatographic interactions at the 5% significance level. A graphical tool (spider diagram) is 17 built for the comparison of the chromatographic columns previously characterized with the 18 solvation parameter model. The position of a solvent system in this spider diagram relates to 19 the interactions at stake, thus the selection of columns offering similar or orthogonal 20 interactions is facilitated, with no previous knowledge of the solute required. This semi-21 empirical strategy cannot fully predict the retention behavior but can judiciously orientate the 22 user towards a limited number of solvent systems to be experimentally tested. 23 24

In the present work, we have investigated the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC) for the separation of peptides in on-line HILIC x RPLC. This combination usually leads to significant solvent strength mismatch, since a weak solvent in HILIC becomes a strong solvent in RPLC. This may result in band broadening, peak distortion, and breakthrough phenomena. Our focus was directed towards the reduction of band broadening and peak distortion. The conditions of the emergence of breakthrough could be investigated with high resolution mass spectrometry (HRMS) detection. The importance of both the injection volume and the difference in composition between injection and elution solvents was highlighted. Reported strategies to avoid bad peak shapes mostly rely either on flow splitting to limit the injection volume, or on on-line dilution. Here, we propose an alternative approach which consists in injecting large volumes in the second dimension. In this case, no flow-splitting nor dilution prior to the second dimension is required. Our results show that above a certain critical injected volume, depending on both the compound and the elution conditions, narrow and symmetrical peaks can be obtained, despite the persistence of breakthrough. As a result, the injected volume in the second dimension must be larger than the largest critical volume. This counter-intuitive approach was applied for the on-line HILIC x RPLC-UV-HRMS analysis of a complex tryptic digest sample. A peak capacity close to 1500 could be achieved in 30 min, which is two-fold higher than in RPLC x RPLC within the same analysis time.

Dicationic ionic liquids (DILs) are more and more accepted as a new class of high temperature and polar stationary phases for gas chromatography (GC). This study deals with the effect of seven different fluorosulfonyl derivatized anions associated with two dications: 1,3-di(3-methylimidazolium)-2-methylpropane [2mC3(mim)2], and 1,3-di(3-methylimidazolium)-isobutene [i-eneC4(mim)2]. Thermophysical properties of the 14 synthesized DILs were evaluated in terms of melting point, viscosity, and thermal stability. The optimal physicochemical properties of 13 DILs allowed preparing 13 GC capillary columns. Accordingly, the polarity and selectivity of the DILs were evaluated by determining the Rohrschneider-McReynolds constants and the equivalent chain lengths of C18 fatty acid methyl esters (FAMEs). The symmetrical fluoroalkylsulfonyl and the trifluorosulfonate anions seem to produce the most polar DILs. Compared to classical polyethyleneglycol phases, the DILs showed substantially decreased retention of apolar compounds and a much stronger retention of the polar ones. Unique selectivities were observed with unsaturated FAMEs, polyaromatic hydrocarbons, and bacterial specific FAMEs. The two applications presented included a biodiesel and bacterial FAME analyses.

International audience; The molten organic salts with melting point below 100°C, commonly called ionic liquids (ILs) have found numerous uses in separation sciences due to their exceptional properties as non molecular solvents, namely, a negligible vapor pressure, a high thermal stability, and unique solvating properties due to polarity and their ionic character of molten salts. Other properties, such as viscosity, boiling point, water solubility, and electrochemical window, are adjustable playing with which anion is associated with which cation. This review focuses on recent development of the uses of ILs in separation techniques actualizing our 2008 article (same authors, J. Chromatogr. A, 1184 (2008) 6-18) focusing on alkyl methylimidazolium salts. These developments include the use of ILs in nuclear waste reprocessing, highly thermally stable ILs that allowed for the introduction of polar gas chromatography capillary columns able to work at temperature never seen before (passing 300°C), the use of ILs in liquid chromatography and capillary electrophoresis, and the introduction of tailor-made ILs for mass spectrometry detection of trace anions at the few femtogram level. The recently introduced deep eutectic solvents are not exactly ILs, they are related enough so that their properties and uses in countercurrent chromatography are presented.

The analysis of complex samples is facilitated by the emergence of two-dimensional liquid chromatography. Despite optimization efforts to reach tremendous peak capacities, the separation of neutral compounds remains limited. Indeed, most combinations of chromatographic modes suffer either from a lack of orthogonality or from serious solvent incompatibilities. The two-dimensional separation involving a combination of liquid chromatography and supercritical fluid chromatography seems to offer new opportunities for the separation of ionizable and neutral solutes. This review highlights the orthogonality the combination can offer thanks to a wide range of stationary phases. Injection effects occurring in SFC, that may drastically reduce the performances of online LC × SFC are also discussed in details. Finally, despite net improvement in SFC instrumentation in the recent years, instrumental limitations still have to be overcome. While this review highlights the potential of LC and SFC combination and its complementarity with 2D-LC, it also demonstrates that its development requires the joint efforts of researchers and instrumental suppliers before comprehensive LC × SFC gets adopted by the scientific community.

International audience; The characterization of complex mixtures is a challenging issue for the development of innovative processes dedicated to biofuels and bio-products production. The huge number of compounds present in biomass fast pyrolysis oils combined with the large diversity of chemical functions represent a bottleneck as regards analytical technique development. For the extensive characterization of complex samples, supercritical fluid chromatography (SFC) can be alternative to usual separation techniques such as gas (GC) or liquid chromatography (LC). In this study, an approach is proposed to define the best conditions for the SFC separation of a fast pyrolysis bio-oil. This approach was based on SFC data obtained directly from the bio-oil itself instead of selecting model compounds as usually done. SFC conditions were optimized by using three specific, easy-to-use and quantitative criteria aiming at maximizing the separation power. Polar stationary phases (ethylpyridine bonded silica) associated to a mix of acetonitrile and water as polarity modifier provided the best results, with more than 120 peaks detected in SFC-UV.

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