The goal of this study was to investigate the chromatographic behavior of selected flavonoids from their different subgroups (flavonols, flavanones, flavones and isoflavones) in hydrophilic interaction liquid chromatography (HILIC). Chromatographic measurements were made on two different HILIC columns: cross-linked DIOL (Luna HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as the content of acetonitrile and pH of an eluent were studied. On the ZIC column, the retention factors of flavonoids increased with decreasing water content in the mobile phase. The increase in pH of the aqueous component mainly affects the polarity of the analytes. DIOL stationary phase shows more or less apparent dual retention mechanism, HILIC at the acetonitrile (ACN) content ≥75% and reversed phase (RP) with lower content of organic modifier. In the presence of ammonium acetate in the mobile phase, the retention of flavonoids onto the DIOL column increases without change in the selectivity of the separations. The similar effect, but considerably smaller was observed for aglycones on the ZIC column. The retention of studied glycosides (hesperidin, rutin) decreases in the presence of salt in the mobile phase. The significantly higher mass spectrometry sensitivity was observed under HILIC conditions in comparison with the most often used RP LC due to much higher content of ACN in the mobile phase. Finally, under optimal chromatographic conditions, the method was validated and applied for the determination of flavonoids in chamomile (Matricaria chamomilla L.) infusion.

Introduction

The pharmaceutical and food industry is particularly interested in developing rapid procedures for flavonoid analysis due to their wide spectrum of action in human body (1). Reversed phase (RP) is the most popular chromatographic mode for this purpose and polar compounds elute first, followed by those of decreasing polarity (2, 3). The conditions mainly include the use of C18 columns and the mobile phase usually consisting of an aqueous solution of acid and an organic solvent (acetonitrile or methanol). However, analysis time with a conventional C18 column is usually long, thus, different strategies have been applied, such as the use of monoliths instead of porous particles (4) or the narrow-bore columns packed with very small particles (<2 μm) and mobile phase delivery systems operating at high back-pressures (5).

Hydrophilic interaction liquid chromatography (HILIC) is an interesting alternative for the analysis of polar substances or semipolar compounds, offering a complementary selectivity compared with reversed phase liquid chromatography (RP LC) (6, 7). HILIC uses the mobile phase composed of polar solvents, typically of acetonitrile and water, with a greater percentage of the organic solvent. In HILIC mode, the separation is based on the different distribution between the acetonitrile-rich mobile phase and the water-enriched layer adsorbed onto the polar stationary phase. However, retention mechanism is a mixed mode and the partitioning between these two phases may occur together with adsorption, ion-exchange, hydrogen bond formation, dipole–dipole, and other interactions could play roles in retention, depending on the particular conditions employed (811). The complexity of this mechanism can manifest itself in different selectivity of different HILIC phases. HILIC separations can offer additionally significant increase in sensitivity when interfaced to mass spectrometry with electrospray ionization (12, 13). Along with increasing popularity of HILIC this method has been the subject of several reviews in the recent years (1417).

In this work, HILIC mode was used to investigate chromatographic behavior of several flavonoids from their different subgroups, such as flavonols (quercetin, rutin), flavonones (hesperetin, hesperidin), flavones (apigenin, luteolin) and isoflavone—genistein. For this purpose, two silica-based HILIC stationary phases: cross-linked diol and zwitterionic sulfoalkylbetaine (ZIC) were studied. They have proved to be very useful for the HILIC separation of a variety of polar compounds (1821). The present work extends our earlier study with polymeric zwitterionic sulfoalkylbetaine phase (20) to select the best column for separation and determination of the tested biologically active compounds. Additionally, comparison was done between HILIC and RP columns.

Experimental

Reagents

The commercial standards of flavonoids as well as the other chemicals were purchased from Sigma (Steinheim, Germany). Acetonitrile (ACN) was of HPLC grade from Merck (Darmstadt, Germany). Ultrapure water from Milli-Q system (Millipore, Bedford, MA, USA) was used in all experiments. Stock solutions of flavonoids as well as their diluted mixtures were prepared in acetonitrile. The chemical structures of the analytes are presented in Figure 1.

Figure 1.

The chemical structures of the studied flavonoids.

Figure 1.

The chemical structures of the studied flavonoids.

Instrumentation

The chromatographic analysis was performed with the Shimadzu LC system consisting of binary pumps LC20-AD, degasser DGU-20A5, the column oven CTO-20AC, autosampler SIL-20AC and 3200 QTRAP Mass Spectrometer (Applied Biosystems/MDS SCIEX). An MS system was equipped with electrospray ionization source (ESI) operated in negative ion mode. ESI conditions were as follows: capillary temperature 450°C, a curtain gas at 0.3 MPa, an auxiliary gas at 0.3 MPa and the ionization mode source voltage 4.5 kV. Nitrogen was used as the curtain and auxiliary gas. For each compound, the optimum conditions of selected reaction monitoring (SRM) mode were determined in infusion mode (22). Standard solutions were infused into the electrospray source via the 50 μm i.d. PEEK capillary employing the Harward Apparatus pump at 10 μL/min. The continuous mass spectra were obtained by scanning m/z values from 50 to 650.

Chromatographic measurements were performed on two different polar stationary phases with specific functionalities. The structures of bonded ligands and properties of all columns used in the study are summarized in Table I. The mobile phases were prepared by mixing appropriate volumes of ACN and aqueous solution of 10 mM formic acid (pH 2.8) or water. Toluene was applied as the void time marker. The mobile phase was delivered at 0.2 mL min−1 in the isocratic mode. For the analysis of chamomile extract, 10 mM of ammonium acetate (pH 7) as eluent A and ACN as eluent B were applied in gradient mode. Addition of salt and gradient elution significantly shortened the retention time of the analytes. The mobile phase was delivered in gradient mode: 0–4 min 98%B, 6–7 min 90% B, 8–8.4 min 80% B, 8.4–12 min 50% B and 13–20 min 98% B. Additionally for comparison, two RP columns were used—typical fully porous Luna C18 (25 cm × 4.6 mm × 5 μm) and core-shell Kinetex C18 (100 × 2.1 mm × 2.6 μm) both from Phenomenex. The analytes were identified by comparing retention time and m/z values obtained from MS and MS2 with the mass spectra. Quantification of compounds was done from the calibration curves obtained in selected reaction monitoring (SRM) mode.

Table I.

Characteristics of Used Columns

Column Bonded group Dimension (mm) Silica particle size (μm) Pore diameter (Å) Surface area (m2 g−1
DIOL (Luna HILIC from Phenomenex) graphic 2.0 × 100 3.5 200 200 
ZIC-HILIC (zwitterionic from Merck) graphic 2.1 × 100 3.0 100 135 
Column Bonded group Dimension (mm) Silica particle size (μm) Pore diameter (Å) Surface area (m2 g−1
DIOL (Luna HILIC from Phenomenex) graphic 2.0 × 100 3.5 200 200 
ZIC-HILIC (zwitterionic from Merck) graphic 2.1 × 100 3.0 100 135 

Results

The effect of ACN content in the mobile phase on flavonoid retention behavior was investigated at two pH values (2.8 and 7). It should be noted that the pH values of the mobile phases refer to the aqueous portion. Empirical calculations reveal that for every 10% increase in ACN, the pH value of the aqueous ammonium acetate increases by ∼0.3 pH units (23). Similarly to the ionizable analytes and the residual silanols, the pKa values of weak acids decrease with increasing organic content. Because the pH shift for the used aqueous–organic mobile phase and pKa for our analytes in addition to organic modifier are almost proportional (24), as a compromise, the behavior of flavonoids was studied taking into consideration their aqueous pKa values, which are in the range of 6.4–9.5. The obtained retention factors were plotted against the organic solvent content in the range of 40–95% (v/v) as shown in Figure 2.

Figure 2.

The retention factor as a function of acetonitrile content and pH of the mobile phase for ZIC and DIOL columns.

Figure 2.

The retention factor as a function of acetonitrile content and pH of the mobile phase for ZIC and DIOL columns.

On the ZIC column, the retention factors increase with decreasing water content in the mobile phase. Particularly for rutin, the retention factors increase rapidly with the increase of ACN content in the mobile phase from 75 to 95% (v/v) at both studied pH values. On the DIOL column, U-shaped curves are obtained when plotting solute retention factors versus the ACN content at pH 2.8 and 7.0 (Figure 2). The chemically bonded diol phase demonstrates high polarity and does not contain ionizable groups, other than nonreacted residual silanols (25). This stationary phase shows more or less apparent dual retention mechanism, HILIC at the concentration of ACN higher than 80% and RP in mobile phases with lower content of organic modifier. The hydration estimation of the used two silica-based columns was attempted by comparing the chromatographic behavior of toluene (nonretained marker compound) as shown in Figure 3. For the DIOL column, the elution time of toluene has its lowest values at ∼75% ACN (v/v), while at lower or higher percentages this time rises. For the ZIC column, toluene elution time is almost constant in the studied content range of ACN. As for toluene tRDIOL > tRZIC, it seems that the aqueous layer is thinner on the diol-bonded phase and this compound has the greater opportunity to reach and react with the hydrophobic part (i.e., siloxanes, propyl spacers) of the chromatographic stationary phases (25). Taking into account the different hydration of phases, it would be expected that studied flavonoids are retained more strongly on the ZIC column than it was observed (Figure 2).

Figure 3.

The elution time of toluene on ZIC and DIOL columns as a function of acetonitrile content. Detection at 245 nm.

Figure 3.

The elution time of toluene on ZIC and DIOL columns as a function of acetonitrile content. Detection at 245 nm.

The effect of the presence of buffer salt in the mobile phase on the retention of flavonoids was also investigated for both columns. The mobile phase consisting of 95% ACN (v/v) and ammonium acetate at the concentration of 10 mM (in the aqueous phase) was applied. The results are presented in Figure 4. Retention of flavonoids increases in the presence of CH3COONH4, particularly for the DIOL column. However, there is no significant effect on the selectivity of the separations. On the ZIC column, retention time for rutin dropped significantly from above 120 min (under isocratic conditions) up to 30 min in the presence of salt.

Figure 4.

The effect of the presence of ammonium acetate on the retention factors for flavonoids on ZIC and DIOL columns. Eluent: 95% ACN (v/v), pH 7.0.

Figure 4.

The effect of the presence of ammonium acetate on the retention factors for flavonoids on ZIC and DIOL columns. Eluent: 95% ACN (v/v), pH 7.0.

The selectivity and resolution parameters presented in Table II give clues for the selection of studied flavonoid separation condition using both studied stationary phases. Generally, the ZIC-HILIC column provided higher retention for the investigated flavonoids in comparison with the DIOL column under the same elution conditions due to a more complicated retention mechanism. Better selectivity, presented as the ratio between the retention factor of two analyzed compounds, and higher separation efficiency were obtained for the ZIC column. The elution order was slightly different for both columns.

Table II.

Separation Parameters for Flavonoid Analysis Under Isocratic Conditions with ACN/Water (95%, v/v) as an Eluent

Compound Retention time, tR Bandwidths Asymmetry Efficiency, N m−1 Selectivity, α 
ZIC column 
 Hesperetin 2.07 0.525 2.92 861 – 
 Genistein 2.33 0.322 1.15 2,901 1.49 
 Apigenin 2.36 0.252 2.38 4,859 1.04 
 Luteolin 6.92 0.660 5.57 6,072 6.50 
 Hesperidin 9.42 0.201 2.33 121,680 1.46 
 Quercetin 13.60 7.71 5.65 172 1.53 
DIOL column 
 Hesperetin 2.22 0.301 1.45 3,014 – 
 Genistein 2.26 0.345 2.41 2,377 1.10 
 Hesperidin 2.39 0.414 2.39 3,960 1.29 
 Apigenin 2.47 0.328 1.35 3,142 1.14 
 Luteolin 2.64 0.300 3.76 4,290 1.26 
 Rutin 4.52 0.470 1.96 512 3.24 
 Quercetin 5.62 3.77 5.58 123 1.40 
Compound Retention time, tR Bandwidths Asymmetry Efficiency, N m−1 Selectivity, α 
ZIC column 
 Hesperetin 2.07 0.525 2.92 861 – 
 Genistein 2.33 0.322 1.15 2,901 1.49 
 Apigenin 2.36 0.252 2.38 4,859 1.04 
 Luteolin 6.92 0.660 5.57 6,072 6.50 
 Hesperidin 9.42 0.201 2.33 121,680 1.46 
 Quercetin 13.60 7.71 5.65 172 1.53 
DIOL column 
 Hesperetin 2.22 0.301 1.45 3,014 – 
 Genistein 2.26 0.345 2.41 2,377 1.10 
 Hesperidin 2.39 0.414 2.39 3,960 1.29 
 Apigenin 2.47 0.328 1.35 3,142 1.14 
 Luteolin 2.64 0.300 3.76 4,290 1.26 
 Rutin 4.52 0.470 1.96 512 3.24 
 Quercetin 5.62 3.77 5.58 123 1.40 
Figure 5.

Extracted ion chromatograms of apigenin on HILIC and RP columns under isocratic elution. Eluent for the HILIC column—ACN/H2O (95/5%, v/v), for DIOL-RP—ACN/H2O (45/55%, v/v) and for C18—ACN/H2O (20/80%, v/v).

Figure 5.

Extracted ion chromatograms of apigenin on HILIC and RP columns under isocratic elution. Eluent for the HILIC column—ACN/H2O (95/5%, v/v), for DIOL-RP—ACN/H2O (45/55%, v/v) and for C18—ACN/H2O (20/80%, v/v).

As RP-LC is the most popular chromatographic mode for the analysis of flavonoids, we compared it with HILIC conditions. Figure 5 presents the MS-extracted ion chromatograms of apigenin as a model compound obtained on different HILIC and RP columns under isocratic elution for comparison. Two RP columns were used for comparison—typical fully porous Luna C18 and core-shell Kinetex C18. The composition of the mobile phase was ACN/H2O (20/80, v/v) (20% ACN) to obtain similar retention factors. Significantly, higher sensitivity was observed under HILIC conditions due to much higher content of ACN in the mobile phase (95%, v/v). Apigenin eluted with much longer retention time from both RP columns with lower sensitivity in comparison with HILIC mode. This compound, as other flavonoids, showed dual U-shaped profile of the retention on the DIOL column (Figure 2). This column was also studied under RP conditions (45% ACN). The retention data of apigenin in both modes are similar but much lower sensitivity was observed in the DIOL–RP–LC (Figure 5).

The longest analysis duration and efficiency of separation was achieved on the ZIC column; therefore, this stationary phase underwent the validation process. To check the linearity of the detector response, a linear regression analysis of the peaks area versus concentration of the studied compounds was used. The linearity was determined by the square correlation coefficients of the calibration curves generated by three repeated injections of standard solutions at five concentration levels in the range of 1–25 mg L−1. All the compounds showed good linearity with regression coefficients ≥0.97. The limits of detection (s/n = 3) were 0.05 mg L−1 for quercetin and rutin and 0.01 mg L−1 for other flavonoids (5 μL of sample injection). The repeatability of peak areas and retention times were calculated by the relative standard deviation (RSD) of seven injections carried out on the same day. The RSD for the retention times of all peaks was <3%, and the coefficient variation for the peak area was <4%.

On this basis, the flavonoid analysis was performed in the extract of commercially available chamomile (Matricaria chamomilla L.). Infusion was carried out by pouring 2 g of dried plant in 50 mL of freshly boiled water and allowed to steep for 10 min at room temperature. The analysis was performed in triplicate, and the presence of constituents was confirmed by checking their MS–MS spectra. Gradient elution started from 98% (v/v) of ACN has been applied to elute all unknown compounds present in natural samples. Addition of ammonium acetate and gradient elution significantly shortened the retention time of rutin. Retention of hesperetin increased due to the content of ACN (98%, v/v) in the eluent composition. Apigenin, luteolin, hesperetin and rutin were detected in chamomile infusion from flavonoid compounds. The mean values, expressed in mg L−1, were 2.59 ± 0.13, 0.02 ± 0.09, 0.03 ± 0.002 and 1.95 ± 0.06 for apigenin, luteolin, hesperetin and rutin, respectively. Similar set of flavonoids in chamomile infusions were reported earlier (26).

Discussion

The retention mechanism in HILIC is still under debate, although it is commonly believed that it is driven mainly by the partitioning of solutes between the bulk mobile phase and the adsorbed water-rich layer at increasing concentration of acetonitrile. Water is adsorbed more strongly on the surface of the polar stationary phase, but it depends on the polarity and type of the stationary phase. Soukup and Jandera (27) investigated the excess adsorption of water on various stationary phases to compare the role of different functionalities and the role it may play in the HILIC retention mechanism. At full column saturation, the excess adsorbed water filled 10.5 and 45.3% for DIOL and ZIC-HILIC columns, respectively. These approximately correspond to the equivalent of 5 and 6, respectively, water layer coverage of the adsorbent surface. The more hydrophilic are the analytes, the more the partitioning equilibrium is shifted toward the adsorbed water layer on the stationary phase, and more analytes are retained, as it was observed for the ZIC column (Figure 2). Also other studies confirmed that the amount of adsorbed water plays an important role in the retention mechanism in HILIC (26, 28).

Sulfoalkylbetaine phase contains basic quaternary ammonium groups and acidic sulfonic groups that exist in a molar ratio of ∼1 : 1. Ideally, the zwitterionic functionality should have a zero net charge and therefore be neutral. Because of the short, but distinct charge separation within both functional groups, the phase can express weak electrostatic interactions, regardless of the mobile phase pH (29). However, Guo and Gaiki (6) found that the retention of sulfoalkylbetaine silica columns is the least affected by the pH of the other studied HILIC columns, such as a bare silica, aminopropyl and amide silica. The increase in pH mainly affects analyte polarity. Flavonoids at pH 7.0 are more or less dissociated and exist in the equilibrium between the charged and the neutral forms. Under these conditions, the changes in separation selectivity are observed and the order of elution agrees with their polarity. The interactions between the less polar compounds, such as hesperetin or apigenin, and the zwitterionic phase are very weak.

The chemically bonded diol phase demonstrates high polarity and does not contain ionizable groups, other than nonreacted residual silanols (27). The DIOL phase showed more or less apparent dual retention mechanism (HILIC and RP) depending on the content of acetonitrile. Similar U-shaped retention curves were also found on polyethylene glycol and diol-bonded stationary phases (30). In HILIC, the existence of a layer on the surface of the polar stationary phase is postulated, which is rich in water even at highly organic mobile phases (10, 31). The maximum excess of adsorbed water is connected with minimum excess of adsorbed ACN and the scale of this adsorption excess depends on the type of the bonded stationary phase (28).

The presence of ammonium acetate in the mobile phase can modulate the electrostatic interactions (both attractive and repulsive) between charged solutes and polar stationary phase. The addition of ammonium acetate caused a significant increase in the retention of flavonoids on the DIOL column. Since this stationary phase does not contain ionizable groups, this effect can be attributed to screening of the repulsive interactions between ionized solutes and residual silanols. Much smaller increases in retention were observed in the case of ZIC column. For more polar glycosides (hesperidin, quercetrin), the decrease of retention was observed. Probably, the competition of salt ions for the active sites on the stationary phase may reduce the electrostatic attraction. It has been postulated that the presence of higher salt might increase the volume of the immobilized liquid layer on the stationary phase, thus leading to stronger retention (32).

Better efficiency of separation was achieved on the ZIC column than on the DIOL stationary phase. Moreover, the shape of quercetin peak was much better for sulfobetaine stationary phase (bandwidth was two time smaller). The ZIC column shows sufficient selectivity for the separation of studied flavonoids (Table II), except for apigenin/genistein pair, probably due to similar chemical structures (they belong to flavones and isoflavones, respectively, subgroups). On the DIOL column, the peaks of flavonoids were unresolved (except luteolin and rutin) and only because of high resolution of the MS detector, they can be quantified. The comparison of separation parameters obtained for the ZIC column with bare silica-based material with similar zwitterionic stationary phase but covalently attached to porous polymer beads (p-ZIC) (20) shows that for all studied compounds (except rutin) shorter retention times were obtained for the ZIC column. This phase carries negative charges from deprotonated silanol groups and sulfobetaine from negatively charged sulfate groups. It is possible that the reduced retention is related to electrostatic repulsion of negatively charged flavonoid compounds. However, higher column efficiency was obtained for the p-ZIC column (20).

The comparison of the ZIC-HILIC column with RP columns, usually applied for separation of flavonoids, for apigenin as a model compound (Figure 5) showed that HILIC separation can offer a significant increase in sensitivity when interfaced to mass spectrometry with electrospray ionization.

Conclusion

Although HILIC has been around for several years, the popularity has increased only in the last decade. This is primarily in regard to the necessity to develop highly sensitive analyses for polar compounds in complex mixtures, facilitated by the extensive adoption of mass spectrometry as the detection method. Acetonitrile as the component of an eluent allowed to increase the sensitivity for flavonoid quantification. High organic extracts from common sample preparation, such as liquid–liquid extraction or solid phase extraction, can be directly injected into a HILIC system, in contrast to RP-LC. This can result in improved signal intensity and less robust assays.

Acknowledgments

The authors are thankful to the Structural Research Laboratory (SRL) at the Department of Chemistry of University of Warsaw for making HPLC-MS measurements possible. SRL has been established with financial support from European Regional Development Fund in the Sectorial Operational Programme “Improvement of the Competitiveness of Enterprises, years 2004–2005” project no: WPK_1/ 1.4.3./1/2004/72/72/165/2005/U.

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