Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS...

J. Sep. Sci. 2013, 36, 3141–3151 3141

Aurelie PeriatAlexandre Grand-GuillaumePerrenoud

Davy Guillarme

School of PharmaceuticalSciences, University of Geneva,University of Lausanne, Geneva,Switzerland

Received May 29, 2013Revised July 12, 2013Accepted July 12, 2013

Research Article

Evaluation of various chromatographicapproaches for the retention of hydrophiliccompounds and MS compatibility†

The goal of this study was to compare the performance of three separation techniques for theanalysis of 57 hydrophilic compounds. RPLC, hydrophilic interaction liquid chromatography(HILIC) and subcritical fluid chromatography (SFC) were tested. The comparison was basedon the retention, selectivity, peak shape (asymmetry and peak width) and MS sensitivity. Asexpected, RPLC had some obvious limitations for such classes of compounds, and on averagethe %ACN required to elute these hydrophilic substances was 4, 7, and 11% ACN at pH3, 6, and 9, respectively. However, a hybrid polar-embedded C18 phase with an appropriatemobile phase could represent a viable strategy for hydrophilic basic compounds with log Dgreater than –2 on average. HILIC and SFC were found to be more appropriate for analyzinga large majority of these hydrophilic analytes (∼60 and 70% of compounds eluted duringthe gradient in HILIC and SFC), while maintaining good MS sensitivity. Finally, this workdemonstrated the complementarity of the three analytical techniques and showed that theselection of a suitable strategy should mostly be based on physicochemical properties of theanalytes (pKa, log D, H-bonding capability, etc.).

Keywords: HILIC / MS sensitivity / Polar substances / RPLC / Subcritical fluidchromatographyDOI 10.1002/jssc.201300567

� Additional supporting information may be found in the online version of this articleat the publisher’s web-site

1 Introduction

A large variety of biologically active substances, includingpharmaceutical compounds, amino acids, hydrophilic pep-tides, neurotransmitters, oligosaccharides, carbohydrates,nucleotides and nucleosides, are highly hydrophilic and re-main difficult to analyze in LC. Normal phase LC is a suitableapproach for retaining polar substances but is environmen-tally unfriendly, and solubility issues may be observed [1, 2].Ion-exchange chromatography and ion-pairing chromatogra-phy are good alternatives but require significant amounts ofsalts or ion-pairing reagents, which are not compatible withMS detection [3]. For these reasons, some alternative solu-tions have been proposed over the last few years.

Due to hydrophobic interactions, a standard RPLC col-umn (C18 or C8) does not offer sufficient retention for hy-drophilic analytes. However, it has been demonstrated that

Correspondence: Dr. Davy Guillarme, School of PharmaceuticalSciences, University of Geneva, University of Lausanne, Boule-vard d’Yvoy 20, 1211 Geneva 4, SwitzerlandE-mail: [email protected]: +41-22-379-68-08

Abbreviations: BPR, backpressure regulator; 2-EP, 2-ethylpyridine; HILIC, hydrophilic interaction chromatography;PEEK, polyetheretherketone; SFC, supercritical/subcriticalfluid chromatography; UHPSFC, ultra high performance su-percritical/subcritical fluid chromatography

either polar end-capped C18 or shielded C18 phases contain-ing a polar group, such as carbamate, incorporated into thealkyl ligand could both improve the retention of hydrophilicanalytes through dipole–dipole or H-bond interactions [4, 5].In addition, the presence of a polar group increases the wetta-bility of the material, which makes such columns compatiblewith highly aqueous phases (up to 98–100% aqueous mobilephase) without the alkyl chain collapsing [6]. In addition tothe stationary phase, it is also possible to increase the reten-tion of polar compounds through the appropriate selectionof the mobile phase pH, since the retention of compoundsin their neutral form is enhanced in RPLC because of betteraffinity to the hydrophobic stationary phase [7, 8].

Hydrophilic interaction liquid chromatography (HILIC)is another well-established alternative for retaining hy-drophilic compounds. It involves the combination of a polarstationary phase and an aqueous/polar organic solvent mobilephase containing a large proportion (>60%) of organic modi-fiers (usually acetonitrile) [9,10]. The retention mechanism isprincipally based on the partition of a compound between awater-rich layer formed at the surface of the stationary phaseand the bulk mobile phase. Hydrogen bonding, dipole–dipole

†This paper is included in the virtual special issue HILICavailable at the Journal of Separation Science website.Colour Online: See the article online to view the figs 1–5 in colour.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1615-9314/homepage/virtual_special_issue__hilic.htm

3142 A. Periat et al. J. Sep. Sci. 2013, 36, 3141–3151

interactions and ion exchange are also involved in the inter-action mechanism [11–13].

Modern subcritical fluid chromatography (SFC) differsfrom other chromatographic techniques by the use of a mo-bile phase consisting of pressurized and heated CO2 and alimited proportion (up to 30–40%) of miscible polar organicmodifiers, such as methanol (MeOH). In the presence of acosolvent, the resulting fluid that is employed should be re-ferred to as “subcritical” instead of “supercritical.” However,regardless of the nature of the mobile phase in SFC, its realstate of matter is of little concern, as it was shown in the pastthat there was a continuum of properties when moving from asupercritical fluid to a liquid [14]. The supercritical/subcriticalmobile phase exhibits remarkable properties in terms of lowviscosity, high density and good diffusivity that allow one toperform fast and/or high-resolution analysis [15]. Today, SFChas become a popular and highly competitive technique forthe analysis of polar compounds by taking advantage of thegood chromatographic selectivity and retention provided bya normal phase-like separation mechanism [16, 17] and thehigh kinetic performance inherent to the use of a columnpacked with a fully porous sub-2 �m (UHPSFC, ultra highperformance supercritical fluid chromatography) [18] and su-perficially porous sub-3 �m material [19].

In the present work, the retention, selectivity and peakshape (asymmetry and peak width) of 57 hydrophilic com-pounds possessing log DpH3 values between –5 and 0 wereevaluated using three chromatographic approaches that wereMS compatible. The HILIC mode was evaluated using twostationary phases (bare silica and amide) at two pH condi-tions (3 and 6). RPLC using a polar-embedded C18 phase wastested at three pH conditions (3, 6, and 9). The SFC mode wasassessed with two columns (bare hybrid and 2-ethylpyridine).Finally, the MS sensitivity was also determined for a selectedgroup of ten representative hydrophilic compounds usingonly the best mobile phase and stationary phase conditionsfor each of the three analytical approaches.

2 Materials and methods

2.1 Chemical and reagents

Water was obtained from a Milli-Q Water Purification Sys-tem from Millipore (Bedford, MA, USA). Acetonitrile (ACN),methanol (MeOH), formic acid, and acetic acid were ofULC–MS grade and purchased from Biosolve (Valkenswaald,the Netherlands). Ammonium hydroxide was obtainedfrom Sigma–Fluka (Buchs, Switzerland). Pressurized liq-uid CO2 3.0 grade (99.9%) was purchased from PanGas(Dagmerstellen, Switzerland).

Ammonium formate buffer 20 mM (pH 3) was preparedwith an adapted volume of formic acid and pH was adjustedto 3.0 with ammonium hydroxide 28%. Ammonium acetatebuffer 20 mM (pH 6) was prepared with an adapted volumeof acetic acid and the pH was adjusted to 6.0 with ammo-nium hydroxide 28%. Ammonium formate buffer 20 mM

(pH 9) was prepared with an adapted volume of ammoniumhydroxide 28% and the pH was adjusted to 9.0 with formicacid.

2.2 Model hydrophilic compounds

Details and physicochemical properties of the 57 model com-pounds are summarized in the Supporting Information.

Stock solutions of each individual sample were preparedat 1 mg/mL in pure MeOH. Each compound was diluted inthe appropriate dissolution solvent (i.e. water for RPLC andACN for HILIC and SFC) to obtain concentrations between5 and 100 �g/mL, depending on the UV response [20].

2.3 Instrumentation and columns

2.3.1 UHPLC–UV system for RPLC and HILIC

experiments

The chromatographic experiments were performed using aWaters Acquity UPLCTM I-Class system (Milford, MA, USA)equipped with a binary solvent delivery pump, an autosam-pler and a UV diode-array detector. The system included aflow-through needle injection system with a 15 �L needleand a 0.5 �L UV flow cell. The dwell volume (Vd) was ex-perimentally measured as 110 �L, using a generic gradientfrom 0 to 100% B at 500 �L/min, where A was pure ACNand B was a mixture of ACN and 0.1% acetone. The acqui-sition rate and time constant of the UV detector were fixedat 20 Hz and 50 ms, respectively. Data acquisition, data han-dling and instrument control were performed with Empower2 (Waters).

2.3.2 UHPSFC–UV system for SFC experiments

The Waters Acquity UPC2 system was equipped with a bi-nary solvent delivery pump, an autosampler that includeda 10 �L loop employed in the partial loop injection mode,a column oven, a UV detector fitted with an 8 �L flow celland a backpressure regulator (BPR). The dwell volume of thesystem was measured at 440 �L, using the same approachas described in Section 2.3.1. The acquisition rate and timeconstant of the UV detector were fixed at 20 Hz and 50 ms,respectively. Data acquisition, data handling and instrumentcontrol were performed with Empower 3 (Waters).

2.3.3 UHPLC–MS system for RPLC and HILIC

experiments

The chromatographic experiments in RPLC–MS and HILIC–MS were performed using a Waters Acquity UPLCTM system.This instrument was equipped with a binary solvent man-ager, an autosampler with a 2 �L loop operating in the fullloop injection mode, and a column oven. This UHPLC system


J. Sep. Sci. 2013, 36, 3141–3151 Liquid Chromatography 3143

was hyphenated with a Waters TQD triple quadrupole massspectrometer fitted with a Z-spray ESI source. The experi-ments were performed with the ionization source operatingin the ESI positive mode, and selected reaction monitoring(SRM) mode was performed. Nitrogen was used as a dryinggas. The source temperature, cone gas flow, and source ex-tractor voltage were identical in both modes (120�C, 20 L/hand +3 V, respectively). The capillary voltage in HILIC and inRPLC was set at +3 and +2 kV, respectively. The desolvationgas temperature in HILIC and RPLC mode was set at 350 and450�C, respectively, and the nitrogen flow rate was adjustedto 600 and 800 L/h, respectively.

Cone voltages and collision energies were optimized foreach compound, and the optimal values are listed in Table S2of the Supporting Information. These parameters were not af-fected by the chromatographic mode and remained constantin HILIC, RPLC, and SFC. Finally, dwell times and interchan-nel delays were set to 15 and 5 ms, respectively, to achieve asufficient number of data points across the peaks [21]. Dataacquisition, data handling and instrument control were per-formed with Masslynx 4.1 (Waters).

2.3.4 UHPSFC–MS system for SFC experiments

Due to the high proportion of MeOH employed in the mo-bile phase to elute polar compounds, the term SFC employedin this study stands for “subcritical fluid chromatography”rather than “supercritical fluid chromatography.” UHPSFC–MS analysis was performed with the UPC2 system hyphen-ated with a triple quadrupole mass spectrometer, as describedin Sections 2.3.2 and 2.3.3. The hyphenation was achievedusing a pre-BPR flow splitter equipped with two serial zero-dead-volume tee-unions connected to the UV detector outletwith 41 cm, 175 �m id polyetheretherketone (PEEK) tubing.CO2-miscible sheath liquid (MeOH, MS grade), delivered bya Waters HPLC 515 make-up pump, was added and mixedto the chromatographic effluent in the upstream zero-dead-volume tee-union, while the downstream zero-dead-volumetee-union acted as a flow splitter. A fraction of the total flowwas directed from the union to the ESI source through a75 cm, 50 �m id PEEKsilTM transfer line, whereas the remain-ing mobile phase was directed to the BPR through a 127 cm,250 �m id PEEK connection. Experiments were carried out at40�C, the backpressure was fixed at 120 bar, and 0.3 mL/minMeOH was delivered by the make-up pump. The ESI sourceoperated in the positive mode, and the source temperature,cone gas flow, and source extractor voltage were set at 150�C,20 L/h, and +3 V, respectively. The capillary voltage was setat +1 kV, and the nitrogen desolvation gas temperature andflow rate were set 450�C and 1000 L/h, respectively. MS/MSdetection was performed using SRM mode. Data acquisition,data handling and instrument control were performed withMasslynx 4.1 (Waters).

2.3.5 Columns

The columns employed in this study were:

� A Zorbax HILIC Plus Rapid Resolution High Definition(50 × 2.1 mm, 1.8 �m) from Agilent technologies (Wald-bronn, Germany) and Acquity BEH amide column (50 ×2.1 mm, 1.7 �m) from Waters, in HILIC mode.

� An Acquity UPLC R© BEH Shield RP18 column (50 ×2.1 mm, 1.7 �m) from Waters, in RPLC mode.

� An Acquity UPC2 BEH (100 × 3.0 mm, 1.7 �m) and Ac-quity UPC2 BEH 2-EP (100 × 3.0 mm, 1.7 �m) both fromWaters, in SFC mode.

The column dead times were measured by injecting uracilin RPLC at 50:50 ACN/H2O; acenaphthene in HILIC at 80:20ACN/H2O; and acenaphthene in SFC at 20:80 MeOH/CO2.

2.4 Experimental procedure and data treatment

2.4.1 Retention and peak shape evaluation

The 57 compounds were individually injected (Vinj of 2 �Lin each case) in RPLC, HILIC and SFC modes, using UVdetection. In RPLC, the compounds were eluted from the sta-tionary phase at three different pH conditions (3, 6, and 9)using a monofunctional, fully end-capped, embedded polarC18 stationary phase [22]. The gradient profile was 2% ACNfor 1 min, followed by an increase up to 50% over 3.5 min.The flow rate was fixed at 500 �L/min and the column ovenset at 40�C. For HILIC analysis, it was previously demon-strated that the nature of the stationary phase and the mobilephase pH were the two most relevant parameters for tuningselectivity and retention [23]. Then, the samples were injectedindividually on the two stationary phases at two mobile phasepH conditions (3 and 6). The gradient profile was 95% ACNfor 1 min, followed by a linear decrease down to 60% ACN in2.55 min and then 0.7 min at 60% ACN. The flow rate wasalso maintained at 500 �L/min and column temperature setat 40�C. In SFC mode, two reference columns, namely, barehybrid and hybrid bonded with a 2-ethylpyridine (2-EP) moi-ety were employed. As reported elsewhere, these two typesof column chemistry provide good kinetic performance aswell as diverse selectivity and retention for a wide range ofcompounds [18,24]. To achieve a reasonable peak shape withbasic drugs, these two columns were used with 20 mM am-monium hydroxide in the organic mobile phase [24]. Thegradient profile was 2–40% MeOH in 3.75 min and then1 min at 40% MeOH. The flow rate was fixed at 1.5 mL/min,column oven and BPR were set at 40�C and 150 bar, respec-tively. Due to the high number of analytical measurements(57 compounds analyzed under nine different conditions),the experiments were not duplicated, since we observed fora few compounds that the RSD on retention time for threesuccessive injections was less than 0.5%, regardless of theanalytical conditions. This excellent result was attributed to asound adaptation of gradient reequilibrating time (3.2, 2, and4 min in SFC, RPLC, and HILIC, respectively).



The peak width at half height (W50%) and asymmetryat 10% were calculated by Empower software at a specificwavelength for each compound, as reported in Table S1 ofthe Supporting Information.

For comparison, the retention times of the hydrophiliccompounds were transformed into elution composition (Ce)using Eq. (1) for RPLC and SFC modes:

Ce = Ci +(

Cf − Ci

tgrad

)× (tr − tD − tiso) (1)

Where Ce corresponds to the percentage of ACN orMeOH required for eluting the analyte of interest; Ci andCf are the initial and final compositions of the gradient, re-spectively; tgrad is the gradient time; and tr, tD, and tiso are theretention time of the hydrophilic compound, system dwelltime, and duration of the initial isocratic step if it existed,respectively.

In the case of HILIC, Eq. (1) was modified to Eq. (2):

Ce = Ci −(

Ci − Cf

tgrad

)× (tr − tD − tiso) (2)

2.4.2 Comparison of MS sensitivity

To evaluate MS sensitivity in RPLC, HILIC, and SFC mode, arepresentative mixture of ten hydrophilic compounds, includ-ing hyoscyamine and sulpiride at 50 ng/mL, sulfapyridine,and MDMA at 100 ng/mL, 6-MAM at 250 ng/mL, atenolol,caffeine, hypoxanthine, and theophylline at 500 ng/mL, andadenosine at 1 �g/mL, was prepared. Triplicate analysis wasperformed in RPLC mode at pH 9, in HILIC mode at pH 6using bare silica, and in SFC mode using bare hybrid. Thegradients and mobile phase flow rates were similar to thosereported in Section 2.4.1. The average signal-to-noise ratiosand peak areas were determined for each individual com-pound in RPLC, HILIC, and SFC using Masslynx software.

3 Results and discussion

3.1 Selection of model compounds

A training set of 57 compounds possessing diverse physic-ochemical properties was selected to evaluate the retentionof hydrophilic probes using various chromatographic ap-proaches. Most of the selected analytes were drugs, and thefew remaining ones were biologically relevant (e.g. aminoacids, nucleotides/nucleosides). Only UV-active compoundswere employed because a universal detector, such as an evap-orative light scattering detector or a charged aerosol detector,was not available in our laboratory for SFC operation. Dueto this technical constraint, it was not possible to add impor-tant families of highly hydrophilic compounds such as sac-charides, aminoglycosides, or amino acids (except histidine,tyrosine, and tryptophan that were considered here).

As reported in Fig. S1 of the Supporting Information,55% of the model analytes possess acidic properties, with ei-ther phosphoric or carboxylic acid (pKa between 1 and 5) orphenol (pKa ranging from 7 to 10.6) moieties. Additionally,95% of the investigated molecules possess a basic pKa, mean-ing that there was a significant number of compounds withzwitterionic properties. For a few compounds (∼20%), thebasic pKa was below 6, while the average pKa values of theremaining basic compounds was 9 (corresponding mostly toan amino group).

Moreover, the estimated log D and log P values are re-ported in Fig. S2 of the Supporting Information to assess thelipophilicity of the model analytes. In our initial selection, wehave exclusively selected compounds possessing an estimatedlog DpH3 between –5 (histamine) and 0 (sulfapyridine). Theexperiments in RPLC are generally carried out in the presenceof 0.1% formic acid (pH 2.7), and it is well established thatsuch hydrophilic compounds may be hardly retained usingsuch conditions and a C18 material due to poor hydropho-bic interactions. Conversely, the estimated log P values, re-ported in Fig. S2 of the Supporting Information, ranged be-tween −2.27 (cytosine) and 2.25 (methamphetamine). Thisconfirms the higher lipophilicity of analytes under their neu-tral form. However, neutral conditions are often difficult toreach in chromatography. This difficulty is illustrated withmethamphetamine, which has a basic pKa of 10.4, therefore,a pH of ∼12.4 (99% neutral form) should be used, but thiswas not compatible with HILIC, RPLC, or SFC phases.

3.2 Comparison of the retentions achieved in HILIC,

RPLC, and SFC modes

The retention of the hydrophilic compounds was evaluatedusing three different analytical strategies, namely, HILIC,RPLC and SFC. Compared to conventional LC and SFC, itis important to mention that relatively short columns packedwith sub-2 �m fully porous particles and high linear veloc-ity were selected in combination with state-of-the-art LC andSFC systems. Using these conditions, the analysis time wasreduced to less than 5 min, and the performance of the sepa-ration techniques could be more easily compared.

Figure 1A summarizes the results obtained with the fourHILIC conditions (two columns at two pH values): the threeRPLC conditions (three different pH values) and the two SFCconditions (two different columns). As shown, it was possibleto elute ∼60 and 70% of the tested compounds during thelinear gradient in HILIC and SFC, respectively. In the case ofRPLC, the results strongly depend on the pH and only 25%of the hydrophilic analytes were eluted during the gradient atpH 3, and up to 56% were eluted at pH 6 and 9. During theseexperiments, there was a significant number of hydrophilicsubstances eluted during the initial isocratic step at 2% ACNin RPLC (60, 37, and 33% at pH 3, 6, and 9, respectively).In the case of SFC, the most hydrophilic compounds wereeluted during the final isocratic step at 40% MeOH (0–10%of the model compounds). Finally, there was also a number



Figure 1. (A) Percentages ofcompounds not eluted (blue),not retained (red), eluted dur-ing initial isocratic step (green),eluted during the gradient (pur-ple), and eluted during the fi-nal isocratic step (gray) for eachexperimental condition. (B) Per-centages of ACN required foreluting each compound in HILIC.(C) Percentages of ACN requiredfor eluting each compound inRPLC. (D) Percentages of MeOHrequired for eluting each com-pound in SFC. The horizontalmarks correspond to the av-erage Ce. Compounds not re-tained or not eluted were not in-cluded in 1B, 1C, and 1D.

of critical compounds that could not be eluted in the testedconditions from the SFC or HILIC column (∼20 and 12% oftested compounds, respectively) or that were not retained onthe HILIC or RPLC phases (∼5 and 11% of tested compounds,respectively).

Figure 1A illustrates that the three separation techniquescan be employed to analyze a large majority of the tested hy-drophilic analytes. This usefulness is obvious in the case ofHILIC, as this technique was originally developed for retain-ing polar substances. Conversely, SFC has been underusedfor such applications, as it was mostly employed in the pastfor analyzing lipophilic compounds, such as lipids or liposol-uble vitamins. As expected, RPLC at pH 3 was generally notsuitable for analyzing polar compounds (only 25% of the com-pounds were eluted during the gradient), but the use of RPLCwith an alkaline mobile phase (thanks to the hybrid stationaryphase) could represent a viable strategy for hydrophilic basiccompounds. It is finally important to notice that only onecompound among the 57 samples (histidine) could not beanalyzed with any of the tested conditions. This compoundhas one of the lowest log DpH3 values (∼ –5) and was noteluted under HILIC and SFC and not retained in RPLC. Thisdoes not mean that this compound cannot be analyzed inany of these three chromatographic modes, as only a limitednumber of analytical conditions were tested in the presentstudy.

3.2.1 Retention in HILIC mode

Figure 1B shows the distribution of compounds as a functionof Ce (Eq. (2)). The compounds that were not retained or noteluted were not included in this representation (about 15–20% of the tested analytes). The horizontal marks represent

the average percentage of ACN required to elute the analytes.From these marks, it appears that the amide phase was lessretentive than the bare silica one (∼86 versus 80% ACN). Thiswas attributed to the reduction of ionic interactions with theamide phase versus bare silica. The retention at pH 3 or 6remained almost identical on the amide phase. This resultwas due to (i) the neutral nature of the amide moiety and(ii) the low silanol activity, as the column was an inorganic–organic hybrid. Conversely, the retention was modified withthe bare silica at pH 3 and 6 (78 and 82% ACN), becausethe number of charged silanols was altered, which led to amodification of the ionic interactions.

As illustrated in Fig. 1B, the peaks were well distributedbetween 95 and 75% ACN with the amide phase, while therewere almost no peaks eluted between 75 and 60% ACN, and12% of the compounds that could not be eluted from thisphase (Fig. 1A). The behavior of the bare silica phase wasmuch more surprising; at pH 3, a vast majority of peaks wereeluted either at 95 or 74% ACN, while the peaks were slightlybetter distributed over the gradient at pH 6, but a majority ofthe compounds were strongly retained (eluted with < 75%ACN). Again, 12% of the compounds were not eluted fromthe bare silica phase for any pH (Fig. 1A). These compoundswere very polar (log D below –4) and the majority of themwere also not eluted from the amide phase. However, thisdoes not mean that these compounds cannot be analyzed inHILIC, as only a limited number of analytical conditions weretested in the present study. In conclusion, it appears that thediscrimination of the hydrophilic compounds was far betteron the amide phase and was particularly poor on the baresilica phase at pH 3.

Finally, the log DpH3 and log DpH6 values were plot-ted as a function of Ce for the two stationary phases. The



Figure 1. Continued.

correlation coefficients of these regression lines were low(ranging between 0.3 and 0.44), which means that the HILICretention was driven by not only hydrophilic partitioning butalso ionic interactions. Based on our results, it appears thatHILIC was particularly well suited for the analysis of hy-drophilic basic substances, while the retention of acidic andneutral compounds were lower, due to electrostatic repul-sions or a lack of ionic interaction, respectively.

3.2.2 Retention in RPLC mode

Figure 1C shows the Ce in RPLC at pH 3, 6 and 9 (calcu-lated with Eq. (1)). On average, the %ACN required to elutethese hydrophilic substances was 4, 7 and 11% ACN. Thisobservation was consistent with the physicochemical prop-erties of our analytes because there was a large majority ofbasic drugs. Then, retention increased with pH due to thehigher lipophilicity. There was a significant number of an-alytes eluted during the initial isocratic step at pH 3, whilethe compounds were better spread over the gradient at pH 6and pH 9. As an example, naloxone possessing a basic pKa ofabout 6.7 was eluted with 4 and 36% ACN at pH 3 and 9, re-spectively. Such behavior is well known in chromatography,and it is expected that basic substances were more retainedunder alkaline conditions. However, silica-based stationaryphases are often not stable in a large pH range, and only afew phases can withstand pH beyond 8. This instability wasnot observed with the hybrid phases employed in the cur-rent study [25]. On the other hand, the few acidic compoundsoffered lower retention under alkaline conditions.

Because the retention in RPLC was mostly based on hy-drophobic interactions, the log D values at pH 3, 6, and 9 wereplotted as a function of Ce at these different pH conditions.Again, the correlation coefficients were low (R2 between 0.2and 0.4). The poor correlation was explained by the fact thatthe amount and strength of hydrophobic interactions onlyhave a limited impact when analyzing polar compounds. Infact, the hydrophilic substances probably interact stronglywith the residual silanols of the stationary phase (ionic inter-actions or H bonds) and the polar-embedded group locatedbetween the silica matrix and the alkyl ligand (dipole–dipoleinteractions or H bonds). These secondary interactions alsoexplain why hydrophilic compounds can be retained on arelatively hydrophobic material.

In conclusion, RPLC could be well suited for the analy-sis of ionizable hydrophilic substances possessing log D ata given pH down to about –1 or –2. The pH can be easilycontrolled under RPLC conditions, leading to a possible in-crease of the lipophilicity of the compounds. However, withlog D below –2, retention becomes generally unacceptable inRPLC.

3.2.3 Retention in SFC mode

Figure 1D shows that the average Ce in SFC was equal to22 and 30% with the 2-EP and hybrid phase, respectively.Because the possible H-bonds and ionic interactions were



stronger between the hydrophilic compounds and the barehybrid material, the retention was higher on the latter mate-rial. As shown in Fig. 1D, the analyzed compounds were welldistributed over the gradient, from 5 to 34% MeOH on the2-EP phase and from 11 to 40% MeOH on the bare hybridphase.

Because polar stationary phases were employed in SFC, asignificant amount of MeOH (the polar modifier) was used toelute the hydrophilic analytes. As reported, up to 40% MeOHwas required to elute a few compounds, and some of themcould not be eluted from the SFC column. Under such ex-treme conditions, the kinetic advantage of SFC over RPLC isquestionable, as the mobile phase viscosity was much morecomparable to that of RPLC in the presence of a high percent-age of MeOH, leading to limited diffusion coefficients, loweroptimal linear velocity (uopt), higher pressure drop and longeranalysis time. Despite this limitation, SFC with a highly or-ganic mobile phase can be considered an attractive approachfor analyzing hydrophilic compounds.

When comparing the physicochemical properties of hy-drophilic compounds and their retention behavior, it was ob-served that H-bonding capability plays a key role in explainingretention. With the bare hybrid phase, ionic interactions be-tween the deprotonated silanols and the protonated moietiesof basic substances should occur. However, because ammo-nium hydroxide was included in the mobile phase, it actedas a competitor for active sites at the surface of the stationaryphase and masked the silanols. In this context, the predom-inant interaction between hydrophilic compounds and sta-tionary phase was H bonding between analytes and MeOHor ammonium hydroxide adsorbed at the surface of the barehybrid phase. With the 2-EP phase, �–� interactions signif-icantly influence the retention of aromatic compounds butmost importantly, the retention was found to be strongly re-lated to the H-bond acidity of the compound (because of theH-bond acceptor capability of the nitrogen atom in the pyri-dine group) [24]. With both stationary phases, the H-bondcharacter of hydrophilic compounds strongly contributes toretention increase. As a rule of thumb, if the hydrophilicmolecule has a high number of H-bond donor or acceptorgroups, it will be hardly eluted from the stationary phase. Thiswas for example the case of morphine glucuronide, pseudo-morphine, and codeine glucuronide, particularly on the barehybrid.

3.3 Comparison of peak shapes in HILIC, RPLC, and

SFC

3.3.1 Evaluation of peak asymmetry

In addition to retention, the peak shape (asymmetry andpeak width) of the hydrophilic compounds was also evalu-ated. Figure 2A summarizes the percentages of compoundswith asymmetry between 0.8 and 1.2 (common asymmetryrange for symmetrical peaks) and between 0.6 and 1.4 (ex-

Figure 2. (A) Percentages of compounds presenting asymmetrycomprised between 0.8 and 1.2 (blue) or between 0.6 and 1.4(red). (B) Percentages of compounds having asymmetry valuesbelow 0.5 (blue), between 0.5 and 2 (red) and superior to 2 (green).

tended range for symmetrical peaks [24]). The proportion ofsymmetrical peaks was relatively low, for all analytical con-ditions. For instance, less than 10% of the hydrophilic com-pounds offered an asymmetry between 0.8 and 1.2 in theRPLC, SFC, and HILIC conditions. Only the HILIC methodwith bare silica phase and pH 6 provides a more reasonablenumber of symmetrical peaks (∼25%). When extending theasymmetry range from 0.6 to 1.4, the number of symmet-rical peaks remained limited but could be increased up to25 and 50% for the SFC method (with a bare hybrid phase)and the HILIC method (with a bare silica phase and pH 6),respectively. As shown in Fig. 2A, the proportion of sym-metrical peaks in RPLC at pH 6 and 9 was particularly low.This behavior can be explained by the fact that the trainingset contained a large proportion of highly basic compounds,which remain charged even at pH 9. Then, the slow secondaryionic interactions between dissociated residual silanols andpositively charged basic analytes generated a significant tail-ing. In agreement with one of our recent studies [23], theHILIC condition with a bare silica phase and pH 6 workedfar better for giving a good peak shape than all the other con-ditions. This behavior could be attributed to the fact that mostof the silanols were neutral at pH 3, while a majority of themwas deprotonated at pH 6. Then, the basic substances of thetraining set of compounds should interact mostly throughionic interactions at pH 6, while there could be two com-petitive interactions mechanism at pH 3, leading to morepronounced tailing under acidic conditions.



Figure 3. Percentages of compounds with W50% below 0.02 min(blue), between 0.02 and 0.04 min (red), and higher than 0.04 min(green).

To better understand the asymmetry (As) differences be-tween the tested conditions, Fig. 2B illustrates the numberof peaks with a severe fronting (As < 0.5) and severe tail-ing (As > 2). Peak fronting was only relevant in the case ofHILIC conditions with an amide phase and pH 3 (20% ofcompounds with As < 0.5). On the other hand, the amountof tailed peaks was very high in the case of SFC with a 2-EPphase (∼70% of peaks with As > 2) and relatively importantwith all the other techniques (between 33 and 50% of peakswith As > 2), except for HILIC when using the silica phaseat pH 6 (only 25% of peaks with As > 2). This result showsthe difficulty in achieving a suitable peak shape when analyz-ing hydrophilic compounds. This behavior is logical, as theinvestigated molecules have a significant number of H-bonddonors, H-bond acceptors, or ionizable groups, which makesthem highly hydrophilic. Then, the polar substances are ableto interact with the stationary phase mostly through slow in-teraction kinetics, such as ionic interactions or H bonding,leading to band broadening and peak tailing.

3.3.2 Evaluation of peak width

The peak widths at half height are reported in Fig. 3 forall the investigated analytical conditions and analytes. Threecategories of peaks were considered, namely, narrow peaks(W50% < 0.02 min), intermediate peaks (W50% between 0.02and 0.04 min), and broad peaks (W50% > 0.04 min). Whenoperating a column in the gradient mode, the peak widths athalf height in time units (�t) can be estimated by Eq. (3) [26]:

�t = t0√N

.(1 + ke) (3)

Where t0 is the column dead time, N is the isocratic ef-ficiency and ke is the “average” or “effective” value of k dur-ing gradient elution. ke can be expressed with the followingequation:

ke = 1

2.3 · b(4)

The b term is the gradient steepness, obtained by:

b = t0 · �� · S

tgrad(5)

Where tgrad is the gradient time, �� = %Bfinal–%Binitial

is the change in composition during the gradient and S is aconstant term that corresponds to the elution strength of theorganic modifier.

If the values of b (Eq. (5)) and t0 (Eq. (3)) were identical, �t

expressed by Eq. (3) only varied with the N value for a givenanalyte (constant S value). In the case of RPLC and HILIC, thesame column geometry and flow rate were employed, leadingto very close t0 and N values (on the maximum, a variationof 10% in t0 and 15% on N was noticed). Because the ratio of��/tgrad, also known as the gradient slope, remained constantbetween both modes, the peak widths were directly compara-ble. In SFC conditions, the column dimensions and mobilephase flow rate were different, corresponding to higher t0 andN values in SFC than in HILIC and RPLC. Conversely, theb value was kept constant, by adjusting the gradient slopein direct proportion to t0. Subsequently, the peaks in HILICand RPLC should be narrower than those achieved in SFC,in agreement with Eq. (3).

In HILIC, the peaks obtained on the bare silica phase weresharper than those obtained on the amide one, particularlyat pH 3 (>60 and <20% of narrow peaks with bare silicaand amide, respectively). This observation confirms that thenarrowest peaks in HILIC were obtained on the bare silicaphase. However, the discrimination of hydrophilic probeswith this stationary phase was particularly limited at pH 3, asillustrated in Fig. 1B.

In RPLC, the number of broad peaks was significantlyhigher under acidic pH conditions than under alkaline pHconditions (23, 16, and 5% at pH 3, 6, and 9, respectively).Because the peak widths could be directly compared betweenHILIC and RPLC, it was possible to conclude that the sharpestpeaks were obtained either in HILIC using bare silica and pH3 or in RPLC at pH 9. The lowest number of narrow peaks(W50% < 0.02 min) was achieved on the amide phase, in allpH conditions.

Finally, the peak widths achieved in SFC cannot be di-rectly compared with those of HILIC and RPLC, and Fig. 3demonstrates that the proportion of narrow peaks (W50% <

0.02 min) was less than 30% on both SFC phases. Figure 3also illustrates that the number of broad peaks was reducedon the silica phase compared with the 2-EP phase. This ob-servation may be related to the peak shape of basic drugs, asit was previously demonstrated that narrower and more sym-metrical peaks were systematically observed with the barehybrid phase compared with the 2-EP phase in presence of20 mM ammonium hydroxide.

3.3.3 Representative chromatograms obtained in

RPLC, SFC, and HILIC modes

Figure 4 shows some representative chromatograms obtainedin HILIC, RPLC, and SFC modes during this study. The



Figure 4. Representative chromatograms of hydrophilic com-pounds recorded with UV detection, in RPLC at pH9 (black), inHILIC at pH 6 with bare silica (red), and in SFC with bare hybrid(green). (A) Sulfapyridine at 286 nm, (B) adenosine at 260 nm, and(C) uracil at 260 nm. The gradient profiles flow rates and columndimensions are described in Section 2.

best analytical conditions in terms of retention, selectivity,peak width and asymmetry were selected for each separationapproach. HILIC employed a bare silica phase operating atpH 6, RPLC was performed at pH 9, and SFC was carriedout on the bare hybrid in the presence of 20 mM ammo-nium hydroxide. Three model compounds are presented inFig. 4A, B, and C, namely, sulfapyridine (basic pKa of 2.9,acidic pKa of 8.5), adenosine (basic pKa of 3.5), and uracil(acidic pKa of 9.7), respectively. Because sulfapyridine was

the least hydrophilic analyte of our set of analytes, it wassufficiently retained in RPLC and SFC, but its retentionwas too limited in HILIC. In the three chromatograms inFig. 4A, the peak shape was acceptable, probably because ofthe absence of ionic interactions. Adenosine was retained toa greater extent in HILIC and SFC due to its hydrophilicnature but was eluted during the initial isocratic step at 2%ACN in RPLC. This molecule was particularly well retainedin SFC due to a high H-bond donor and acceptor capability(i.e. five and nine H-bond donors and acceptors, respectively).The asymmetry in HILIC and SFC was >2, probably becauseof numerous H-bond interactions with the hydrophilic sta-tionary phases. Finally, uracil, which is generally employedas a zero-dead-volume marker in RPLC, was tested using thethree analytical techniques. This compound was not retainedin RPLC at pH 9 (eluted within the column dead volume).Its HILIC retention was limited (eluted during the initial iso-cratic step at 95% ACN) due to its inability to create ionicinteractions with the stationary phase and its reasonable po-larity. Conversely, uracil was easily analyzed under SFC con-ditions, and the peak was sharp. These three examples provethe usefulness and complementarity of HILIC, RPLC andSFC for analyzing hydrophilic compounds.

3.4 MS sensitivity in HILIC, RPLC, and SFC modes

In addition to retention, selectivity and peak shape, MS sensi-tivity was also evaluated with the three analytical techniquesbecause there are a significant number of applications deal-ing with the determination of polar compounds in environ-mental, food, or biological matrixes. For such applications,high sensitivity is required and could possibly be achieved byreplacing RPLC with HILIC or SFC.

Among the hydrophilic compounds, ten representativemodel analytes presenting log DpH3 between 0 and –3 were se-lected to investigate sensitivity in HILIC–MS, RPLC–MS, andSFC–MS. Due to the high selectivity of our MS/MS platformoperating in the SRM mode, the background noise was foundto be statistically equivalent among the three techniques andfor the ten compounds. It was thus neglected in the sensi-tivity evaluation. Peak areas were considered instead of peakheights, to consider some small variations in peak width. Thedata are reported in Table S3 of the Supporting Informa-tion. On average, the sensitivity was increased by a factor of5.0 in HILIC with bare silica at pH 6 compared with RPLC atpH 9. Among the compounds, only 6-MAM had important de-crease of sensitivity (50%) in HILIC compared with in RPLC,for the tested conditions. Conversely, the gain in sensitivitywas higher than 10 for adenosine and 20 for hypoxanthine. Asshown elsewhere, this behavior was explained by the differ-ence of acetonitrile content during the ionization step, lead-ing to a better analyte desolvation in HILIC [27] as well as apossible change in mobile phase apparent pH and analyte ap-parent pKa, which could influence compound ionization [28].Between SFC with bare hybrid and 20 mM ammoniumhydroxide and RPLC at pH 9, the average gain in sensitiv-ity was ∼7. However, the peak area enhancement was found



Figure 5. Representative chromatograms of hydrophilic com-pounds recorded in SRM, in RPLC mode at pH 9 (black), in HILICmode at pH 6 with bare silica (red) and in SFC with bare hybrid(green). (A) Hyoscamine (290 → 124.1), (B) sulfapyridine (250 →156), and (C) theophylline (181 → 124.1). The gradient profilesflow rates and column dimensions are described in Section 2.

to be much more compound dependent. A decrease of 10, 45,and 80% was noticed in the case of sulpiride, hyoscyamine,and 6-MAM, respectively. On the other hand, the sensitiv-ity was enhanced by a factor of 15, 24, and 27 with caffeine,theophylline and hypoxanthine, respectively. In SFC–MS, thesensitivity improvement was mostly explained by the absenceof water in the mobile phase, presenting deleterious effectsin ESI.

These observations were illustrated in Fig. 5, showingHILIC–MS, RPLC–MS, and SFC–MS chromatograms ob-tained with hyoscyamine (Fig. 5A), sulfapyridine (Fig. 5B),and theophylline (Fig. 5C). In the case of hyoscyamine,the peak heights were comparable between the three ana-lytical techniques, while the differences were much morepronounced for sulfapyridine and theophylline, as shownpreviously.

These results confirm that SFC and HILIC should bepreferentially selected instead of RPLC for the analysis ofpolar compounds where MS is the detector.

4 Conclusion

This study compares the performance of three chromato-graphic approaches, namely, HILIC, RPLC and SFC, for theanalysis of hydrophilic compounds. Various types of station-ary phases and/or mobile phases were tested with a trainingset of 57 analytes possessing log DpH3 values between –5 and0. The best analytical strategies were determined from theretention, selectivity, peak shape (peak width and asymme-try) and MS sensitivity achieved with the model hydrophiliccompounds.

The initial goal of the present study was to find a re-lationship between retention and structure under the threetested analytical techniques. However, due to the complex-ity of interaction mechanisms involved between hydrophilicsubstances and stationary phases in RPLC, HILIC and SFC,it was not possible to draw any putative conclusions on whichcompounds could be analyzed successfully with which ana-lytical conditions.

In the case of HILIC, the peak shape was improved withthe bare silica phase versus the amide one, but the discrim-ination power of hydrophilic probes was drastically reduced,particularly at pH 3. Consequently, bare silica at pH 6 wasfound to be the best starting point for the development ofpolar analytes using the HILIC method. In RPLC, a hybridpolar-embedded C18 phase could be suitable for retainingpolar compounds and achieving sharp peaks. However, themobile phase pH has to be selected according to the chemi-cal nature of the analytes to increase the analyte lipophilicityas much as possible. SFC was also found to be a promisingstrategy for retaining hydrophilic substances, but it has notbeen widely employed for this purpose in the past. As shownhere, the H-bond donor capability of the molecules was arelevant characteristic to determine whether a molecule canbe eluted from a polar SFC material. Retention was gener-ally more reasonable on the 2-EP phase, but the bare hybrid



provides sharper peaks. Therefore, both phases have to betested during an initial screening step. Finally, if MS is em-ployed as a detector, SFC and HILIC are preferable, as thegains in sensitivity were on average ∼5 and 7, respectively,compared to RPLC.

The authors wish to thank Dr. Julien Boccard, from the Uni-versity of Geneva, Switzerland, for his help in the preparation ofFigure 1B–D.

The authors have declared no conflict of interest.

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