SWATH-MS screening strategy for the determination of food dyes in spices by UHPLC-HRMS.

A multi-class wide-scope screening method for the detection and identification of artificial colours and illegal dyes in spices was developed for regulatory purposes. The screening was carried out by ultra-high performance liquid chromatography hyphenated with a quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) with sequential window acquisition of all theoretical fragment-ion spectra (SWATH) and was validated with forty-one compounds by spiking experiments in curry and paprika extracts. In order to detect and identify the compounds with a high level of confidence, a home-made tandem mass spectrometry (QTOF-MS/MS) database of approximately one hundred illegal dyes and artificial colours was created. The procedure was then used to screen field samples of spices and spice blends purchased from Swiss markets. Sudan IV, Sudan I, bixin (E160b) and Ponceau 4R (E124) were all detected among the eight non-compliant samples.

Optimized selection of liquid chromatography conditions for wide range analysis of natural compounds.

Plant secondary metabolites are an almost unlimited reservoir of potential bioactive compounds. In view of the wide chemical space covered by natural compounds, their comprehensive analysis requires multiple and complementary approaches. In this study, numerous chromatographic conditions were tested for the analysis of a set of 120 representative natural compounds covering a wide polarity range (18 log P units). The experiments were performed on 59 different conditions involving 29 RPLC and HILIC dedicated stationary phases, as well as more exotic mixed mode columns. The best RPLC and HILIC conditions were determined using Derringer's desirability functions, based on various criteria (i.e. retention, peak shape, distribution of compounds during the gradient…). After this first selection, only the most promising conditions were kept (19 in RPLC and 11 in HILIC). The selectivity complementarity among each chromatographic mode was assessed by principal component analysis (PCA) and hierarchical cluster analysis (HCA). In RPLC, a pentabromobenzyl (PBrBz) stationary phase was identified as particularly versatile and could constitute an elegant first intention screening column. Two additional conditions allowed to extend the range of natural compounds space that can be analyzed, while offering better selectivity for basic analytes (hybrid silica graft with C8 moiety operated at pH 9 (Hyb C8)) and acidic compounds (positively charged hybrid silica graft with pentafluorophenyl moiety (Hyb+ PFPh). Although less generic in terms of amenable compounds, an ion exchange/RP mixed mode stationary phase (MM TriP1) offered notably enhanced retention of more polar analytes under RPLC conditions. With these four conditions, 89% of the natural substances were detected by LC-MS with acceptable retentions and peak shapes. In HILIC, four acceptable and complementary conditions were also highlighted. Both Syncro-Z (zwitterionic HILIC phase) and Diol columns were found to offer balanced retention and selectivity for most of the polar compounds (log DpH3<1.0). These two columns could be advantageously complemented by hybrid Amide column operated at pH 3 and Amino stationary phase at pH 5, to further enhance both retention and selectivity of polar basic and acidic species, respectively.

Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals.

A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used to explore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various, highly-relevant samples were tested, including different insulins, interferon α-2b and trastuzumab. This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elution order and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while the kinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquely able to resolve several important glycoforms at the middle-up level of analysis (fragments of 25-100kDa). Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX. Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers several additional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) a reduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC to limit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility to couple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.

Evaluation of thermally pretreated silica stationary phases under hydrophilic interaction chromatography conditions.

Three novel hydrophilic interaction chromatography columns packed with bare silica 2.6 µm superficially porous particles were evaluated. These stationary phases undergo a different pretreatment temperature (400, 525, and 900°C) that might influence their kinetic performance and thermodynamic properties. In the first instance, we demonstrated that the performance of these columns was inferior to the commercial ones in the low plate count range (10 000 plates), but was more favorable for N values beyond 40 000 plates. Thanks to its high permeability and reasonable flow resistance (φ = 695), together with a minimum reduced heights equivalent to a theoretical plate value of only 2.4, the stationary phase pretreated at 400°C was particularly attractive for N > 70 000 plates with a remarkably low impedance value (E = 2488). In a second step, the impact of pretreatment temperature was evaluated using two mixtures of polar substances, namely nucleobases and derivatives, as well as nicotine and derivatives. Retentions and selectivities achieved on the tested stationary phases were appropriate, but selectivity differences were minor when modifying pretreatment temperature from 400 to 525°C. When we increased the pretreatment temperature up to 900°C, the surface chemistry was more seriously modified. Finally, the columns presented a good stability even at high temperature (70°C), especially for the phases pretreated at 400 and 525°C.

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.

Hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry: effect of electrospray ionization source geometry on sensitivity.

In this study, the influence of electrospray ionization (ESI) source design on the overall sensitivity achieved in hydrophilic interaction chromatography (HILIC) and reversed phase liquid chromatography (RPLC), was investigated. State-of-the-art triple quadrupole mass analyzers from AB Sciex, Agilent Technologies and Waters equipped with brand specific source geometries were tested with various mobile phase pH on 53 pharmaceutical compounds. The design of the ESI source showed to strongly influence the gain in sensitivity that can be achieved in HILIC compared to RPLC mode. The 6460 Triple Quadrupole LC/MS system from Agilent Technologies was particularly affected by mobile phase settings. Indeed, compared to RPLC conditions, 92% of the compounds had an increased signal-to-noise ratio at a flow rate of 300 µL/min in HILIC mode at pH 6, while this percentage dropped to only 7% at 1000 µL/min and pH 3. In contrast, the influence of flow rate and mobile phase pH on the gain in sensitivity between RPLC and HILIC was found very limited with the API 5000 LC/MS/MS system from AB Sciex, as only 15 to 36% of the tested compounds showed an enhanced sensitivity in HILIC mode. With the Xevo TQ-S instrument from Waters, superior sensitivity in HILIC was noticed for 85% of the compounds with optimal conditions (i.e., pH 3 and 1000 µL/min), whereas at sub-optimal conditions (i.e. pH 6 and 300 µL/min), it represented less than 50%. The gain in sensitivity observed in HILIC was found less significant with the recent LC-MS platforms used in this study than for old-generation instruments. Indeed, the improved ESI sources equipping the recent mass analyzers allow for enhanced evaporation efficiency, mainly for RPLC mobile phases containing high proportion of water and this even at high flow rates.

Retention modeling and method development in hydrophilic interaction chromatography.

In the present study, the possibility of retention modeling in the HILIC mode was investigated, testing several different literature relationships over a wide range of different analytical conditions (column chemistries and mobile phase pH) and using analytes possessing diverse physico-chemical properties. Furthermore, it was investigated how the retention prediction depends on the number of isocratic or gradient trial or initial scouting runs. The most promising set of scouting runs seems to be a combination of three isocratic runs (95, 90 and 70%ACN) and one gradient run (95 to 65%ACN in 10min), as the average prediction errors were lower than using six equally spaced isocratic runs and because it is common in Method development (MD) to perform at least one scouting gradient run in the screening step to find out the best column, temperature and pH conditions. Overall, the retention predictions were much less accurate in HILIC than what is usually experienced in RPLC. This has severe implications for MD, as it restricts the use of commercial software packages that require the simulation of the retention of every peak in the chromatogram. To overcome this problem, the recently proposed predictive elution window shifting and stretching (PEWS(2)) approach can be used. In this computer-assisted MD strategy, only an (approximate) prediction of the retention of the first and the last peak in the chromatogram is required to conduct a well-targeted trial-and-error search, with suggested search conditions uniformly covering the entire possible search and elution space. This strategy was used to optimize the separation of three representative pharmaceutical mixtures possessing diverse physico-chemical properties (pteridins, saccharides and cocktail of drugs/metabolites). All problems could be successfully handled in less than 2.5h of instrument time (including equilibration).

UHPLC determination of catechins for the quality control of green tea.

An ultra-high performance liquid chromatography (UHPLC) with UV detection method was developed for the fast quantitation of the most represented and biologically important green tea catechins and caffeine. UHPLC system was equipped with C18 analytical column (50mm×2.1mm, 1.8µm), utilizing a mobile phase composed of pH 2.5 triethanolamine phosphate buffer (0.1M) and acetonitrile in a gradient elution mode; under these conditions six major catechins and caffeine were separated in a 3min run. The method was fully validated in terms of precision, detection and quantification limits, linearity, accuracy, and it was applied to the identification and quantification of catechins and caffeine present in green tea infusions. In particular, commercially available green tea leaves samples of different geographical origin (Sencha, Ceylon Green and Lung Ching) were used for infusion preparations (water at 85°C for 15min). The selectivity of the developed UHPLC method was confirmed by comparison with UHPLC-MS/MS analysis. The recovery of the main six catechins and caffeine on the three analyzed commercial tea samples ranged from 94 to 108% (n=3). Limits of detection (LOD) were comprised in the range 0.1-0.4µgmL(-1). An orthogonal micellar electrokinetic (MEKC) method was applied for comparative purposes on selectivity and quantitative data. The combined use of the results obtained by the two techniques allowed for a fast confirmation on quantitative characterization of commercial samples.

Systematic comparison of sensitivity between hydrophilic interaction liquid chromatography and reversed phase liquid chromatography coupled with mass spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) appears as a promising strategy to increase sensitivity with electrospray ionization source (ESI/MS). In the present study, peak heights, background noises and signal-to-noise ratios (S/N) obtained with HILIC-MS/MS and RPLC-MS/MS conditions were systematically compared using a dataset of 56 basic drugs possessing diverse physico-chemical properties. Various mobile phase conditions were investigated, including different pH (3 and 6 in HILIC; 3, 6 and 9 in RPLC) and flow rates (300, 600 and 1000µL/min). The average gain in sensitivity obtained between HILIC and RPLC was equal to 7 and 10 at pH 3 and 6, respectively. However, this value was not reliable, since it was altered by a few compounds possessing an "extreme" behaviour (gain in sensitivity from 100-fold to >8000-fold better). Then, the median gain in sensitivity, equal to 4 in our case, whatever the pH, should be considered. For about 90% of the tested compounds and analytical conditions, the best S/N was systematically attained under HILIC mode. Thanks to PCA representation, it was shown that the basic compounds with pKa between 6 and 8 generally had the best sensitivity in HILIC at pH 6, while the best sensitivity for basic analytes possessing pKa higher than 8 was usually obtained in HILIC at pH 3. As previously reported, the sensitivity gain in HILIC vs. RPLC was explained by the difference in acetonitrile concentration at elution (in average 29% ACN in RPLC and 82% ACN in HILIC at pH 6) leading to better analytes' desolvation. However, it seems that this high proportion of solvent also favourably influenced the ionization by modifying pH and pKa. Indeed the weakest bases of our training set of compounds (pKa between 2 and 5) showed an unexpectedly strong gain in sensitivity, between 20 and 100-fold in comparison to RPLC. These results prove that the ionic character of analytes in solution (i.e., pKa and pH) and the ionization mechanism (i.e., proton transfer) also play an important role for explaining the sensitivity enhancement in HILIC.

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

The goal of this study was to compare the performance of three separation techniques for the analysis of 57 hydrophilic compounds. RPLC, hydrophilic interaction liquid chromatography (HILIC) and subcritical fluid chromatography (SFC) were tested. The comparison was based on the retention, selectivity, peak shape (asymmetry and peak width) and MS sensitivity. As expected, RPLC had some obvious limitations for such classes of compounds, and on average the %ACN required to elute these hydrophilic substances was 4, 7, and 11% ACN at pH 3, 6, and 9, respectively. However, a hybrid polar-embedded C18 phase with an appropriate mobile phase could represent a viable strategy for hydrophilic basic compounds with log D greater than -2 on average. HILIC and SFC were found to be more appropriate for analyzing a large majority of these hydrophilic analytes (~60 and 70% of compounds eluted during the gradient in HILIC and SFC), while maintaining good MS sensitivity. Finally, this work demonstrated the complementarity of the three analytical techniques and showed that the selection of a suitable strategy should mostly be based on physicochemical properties of the analytes (pKa, log D, H-bonding capability, etc.).

Screening of the most relevant parameters for method development in ultra-high performance hydrophilic interaction chromatography.

The goal of the present work was to provide some guidelines for method development in hydrophilic interaction chromatography (HILIC). For this purpose, a training set of 82 representative pharmaceutical compounds possessing diverse polarity and including acidic, basic and neutral properties was analyzed. All these drugs were injected on five short HILIC columns packed with sub-2µm particles and dedicated for UHPLC (ultra-high performance liquid chromatography) operation. Four different pH conditions ranging from pH 3 to 6 were tested at two ionic strengths (10 and 50mmol/L) and finally, the reference organic modifier in HILIC, namely acetonitrile was modified with small amounts of methanol or isopropanol. From these experiments and using multivariate data analysis, it is clear that the stationary phase was the most relevant parameters for tuning selectivity in HILIC, since the types of interactions (i.e. dipole-dipole, hydrogen bonding and ion exchange) with analytes strongly vary between columns. Among the selected phases, the diol phase was the less interesting one, in terms of selectivity and peak shape. The zwitterionic phase was attractive, as it allowed a better retention of acidic compounds. Finally, the bare silica phase was the most versatile HILIC column packed with sub-2µm particles in terms of retention, peak shape and selectivity. Mobile phase pH was the other important parameter to achieve an appropriate selectivity and retention, even if it remains always difficult to assess precisely the mobile phase pH, analyte pKa and silanols pKa, when working with more 70% acetonitrile. Finally, buffer ionic strength and organic modifier nature could be considered as secondary parameters for HILIC method development. In conclusion, screening four different columns packed with sub-2µm particles at two mobile phase pH, using a fast gradient seems to be a good generic approach for initial HILIC method development. The total time for such a screening was estimated at ∼1h, including reequilibration.