Hyphenation of ultra high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 1. Study of the coupling parameters for the analysis of natural non-polar compounds

Journal of Chromatography A, 1509 (2017) 132–140

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Hyphenation of ultra high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 1. Study of the coupling parameters for the analysis of natural non-polar compounds Johanna Duval a , Cyril Colas a,c , Virginie Pecher b , Marion Poujol b , Jean-Franc¸ois Tranchant b , Eric Lesellier a,∗ a

Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France LVMH Recherche, 185 avenue de Verdun, F-45800 Saint-Jean-de-Braye, France c CNRS, CBM, UPR 4301, Univ. Orléans, F-45071 Orléans, France b

a r t i c l e

i n f o

Article history: Received 25 December 2016 Received in revised form 24 April 2017 Accepted 7 June 2017 Available online 8 June 2017 Keywords: Hyphenation Ultra high performance supercritical fluid chromatography High-resolution mass spectrometry Make-up location Make-up solvent Vegetable oil

a b s t r a c t An analytical method based on Ultra-High-Performance Supercritical Fluid Chromatography (UHPSFC) coupled with Atmospheric Pressure Chemical Ionization − High-resolution mass spectrometry (APCI-QTOF-HRMS) was developed for compounds screening from oily samples. The hyphenation was made using a commercial UHPLC device coupled to a CO2 pump in order to perform the chromatographic analysis. An adaptation of the injection system for compressible fluids was accomplished for this coupling: this modification of the injection sequence was achieved to prevent unusual variations of the injected volume related to the use of a compressible fluid. UHPSFC-HRMS hyphenation was optimized to enhance the response of the varied compounds from a seed extract (anthraquinones, free fatty acids, diacylglycerols, hydroxylated triacylglycerols and triacylglycerols). No split was used prior to the APCI ionization source, allowing introducing all the compounds in the spectrometer, ensuring a better sensitivity for minor compounds. The effects of a mechanical make-up (T-piece) added before this ionization source was discussed in terms of standard deviation of response, response intensity and fragmentation percentage. The location of the T-piece with regards to the backpressure regulator (BPR), the flow rate and the nature of the make-up solvent were studied. Results show that the effects of the studied parameters depend on the nature of the compounds, whereas the make-up addition favours the robustness of the mass response (quantitative aspect). © 2017 Elsevier B.V. All rights reserved.

1. Introduction In the same way as carbohydrates or proteins, lipids are vital constituents. They appeared in the first organism 3 billion years ago. This long duration, up to now, has provided complex evolutions resulting in unequalled and significant molecular diversity. As a consequence, this large molecular family is involved in many molecular pathways discovered by M. R Hokin, in 1953. He discovered the first relationship between the neuroactive agent addition (acetylcholine) and the phosphatidylinositol production [1]. Consequently, this discovery of lipid involvement in the cellular control has increased the scientific interest for these derivatives includ-

∗ Corresponding author. E-mail address: [email protected] (E. Lesellier). http://dx.doi.org/10.1016/j.chroma.2017.06.016 0021-9673/© 2017 Elsevier B.V. All rights reserved.

ing a large variety of compounds (fats, waxes, steroids, prenyls, prostanoids, monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), phospholipids, aminolipids, glycolipids) [2,3]. Although lipids are a wide family, TAG are the most abundant lipids in vegetable oils [4]. Generally, glycerides can be identified using Gas Chromatography (GC). However, the low volatility of glyceride is a real problem requiring derivatization reactions [5]. As a result, sample preparation steps before GC are quite laborious to identify these lipids. In high-performance liquid chromatography (HPLC), several modes have been used. In particular, Non-Aqueous Reverse-Phase liquid chromatography (NARP-LC) is widely used in the TAG separation. In NARP-LC, lipids are separated according to acyl chain lengths and the number of double-bonds which are used to calculate the partition number (PN) [4,5]. Hydrophilicinteraction liquid chromatography (HILIC) is also adapted for the lipid separation by classes according to their polarity and so usu-

J. Duval et al. / J. Chromatogr. A 1509 (2017) 132–140

ally not for non-polar lipids [5]. Silver-Ion Chromatography (SIC) is another analytical technique performed with ion-exchange material coated onto classical silica. In that case, the discrimination of compounds is ruled by the number and the distribution of doublebonds, but sic fails to separate compounds having different chain lengths for an identical double-bond number [5]. Since the years 2000s’, Ultra-High-Performance Liquid Chromatography (UHPLC) with sub-two micrometer particles columns are preferred in lipid analysis, because it offers shorter analysis time, an improvement of efficiency and a reduction of solvent consumption [6]. Fluid Chromatography (SFC) is an Supercritical environmentally-friendly alternative to LC, mainly to NARP-LC. The mobile phase is mainly composed of CO2 in super/subcritical conditions of pressure and temperature. CO2 specificities are a low viscosity and a high diffusivity compared to liquid mobile phases used in LC. By consequence, SFC may be done at high flow rates and so reduces the analysis time, or allows the coupling of several columns in series [7]. An improvement of the efficiency, i.e. the theoretical plate number, is thus achieved, showing the great potential of this separation technique [9]. Moreover, the use of fused-core or sub–2 ␮m particles in Ultra-High-Performance Supercritical Fluid Chromatography (UHPSFC) has demonstrated a great interest for screening methods for complex oily mixtures. As a matter of fact, packed-column Supercritical Fluid Chromatography (pSFC) often offers improved separations for lipids, as it has been successfully applied for TAG, tocopherols and tocotrienols, phospholipids, sterols, fatty acids or prenols [8,9]. UV detection of lipids can be complicated, because of the lack of chromophore of glycerides. Consequently, low detection wavelengths are required [9]. However, most LC solvents (such as methanol or isopropylalcohol) have a UV absorbance cut-off wavelength including around 200–220 nm thus LC-UV analysis of lipids is complicated. Fortunately, with SFC, the absorption of CO2 at these low wavelengths is weak, allowing a sensitive detection in UV at 210 nm. Besides, the evaporative light-scattering detector (ELSD) is well suited for the detection of numerous lipids [10–12]. However, with ELSD, a large number of standard compounds is required for the accurate identification in complex mixtures, and analysis times might thus be very lengthy and time-consuming. Nevertheless, the use of retention models can help in the accurate identification. Consequently, mass spectrometry (MS) offers an interesting approach to identify lipid compounds in a complex mixture [2,13]. The number of publications about the use of LC–MS for the lipid identification is great whereas in SFC-MS, few papers have been published. This lack of papers can be in part explained by several experimental difficulties that might occur by coupling SFC with MS. On the one hand, during the CO2 decompression after the back-pressure regulator (BPR), the CO2 density decreases, thus the solubilisation of compounds in CO2 decreases gradually until the possible precipitation of compounds in the interface coupling the column to the ionization source [14]. Additionally, during the CO2 decompression, carbon ice is produced after the BPR due to the cooling effect of the CO2 depressurisation. Consequently, heated interfaces should be used to avoid this drawback. Because lipids are rather non-polar compounds, Atmospheric Pressure Chemical Ionization (APCI) and Atmospheric Pressure Photoionization (APPI) should be favoured over the use of Electrospray (ESI) as ionization source. Besides, APCI/APPI can accommodate relatively high flow rates compared to ESI. SFC has a higher optimal flow rate than HPLC, typically 2–5 times higher [15]. By consequence, APCI/APPI sources should be a good choice to interface SFC and MS. For polar lipids, such as ceramides, phospholipids or for the carotenoids and their epoxidized forms, Bamba et al. have employed SFC-ESI–MS [16–21]. Lísa and Holˇcapek have also used SFC-ESI–MS for the analysis of a wide range of lipids including free fatty acids, glycerolipids, glycerophospholipids, sphingolipids,

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sterols, and prenols [9]. Advances in the SFC-APCI/APPI-MS detection have contributed to demonstrate the great potential of APCI in the field of lipid analysis. In 2002, Sandra et al. identified TAG in vegetable oils by SFC-APCI-MS [22]. In this experiment, a solution of silver nitrate in methanol was used as make-up fluid to produce silver-TAG ions. This addition is interesting for the separation of cis/trans-isomers in complex mixtures. Méjean et al. have used the SFC-MS hyphenation using APCI, ESI and APPI for the tocopherols and tocotrienols analysis in soybean oil. They reported that APPI source was more sensitive and robust [23]. The goal of the present work is the development and optimization of the UHPSFC-APCI-HRMS hyphenation for the study of an oily extract. This extract was obtained by Supercritical Fluid Extraction from Kniphofia uvaria seeds with pure CO2 , and mainly contains free fatty acids, di- and tri-glycerides and a few coloured anthraquinones, which are the bioactive compounds. Using stationary phases made of fused-core particles bonded with C18 chains, the separation performances both in terms of chromatographic efficiency and selectivity were suited for the separation of the varied studied compounds. After some modification of the injection sequence, due to the use of compressible fluid as mobile phase, the location of an additional make-up, and the nature of make-up solvents are studied for UHPSFC-APCI −HRMS. 2. Material and methods 2.1. Chemicals Isopropylalcohol (IPA) and formic acid (HCOOH) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) Methanol (MeOH) was purchased from Fisher Scientific (Elancourt, France). All solvents were of HPLC grade. For supercritical fluid extraction, the CO2 was from Linde (Munich, Germany). For analytical chromatography coupled to MS detection, CO2 was from Messer (Bad Soden, Germany) with a purity of 99.995%. 2.2. Sample preparation of the oily extract by Supercritical Fluid Extraction (SFE) Kniphofia uvaria seeds were collected by LVMH-Recherche. Seeds of Kniphofia uvaria (300 g) were grinded with a Braun blender of 1 L. Seeds were extracted by Supercritical Fluid Extraction (SFE) with a Separex SFE-500 apparatus with pure CO2 at 29 MPa and 60 ◦ C. The extraction time was a function of the CO2 mass percolated through the extraction cell. When the CO2 mass reached 3 kg, the extraction was stopped. The oil was covered and dried in vacuum with some milliliters of ethanol in order to facilitate the drying. The oil extract was stored at 4 ◦ C. For UHPSFC-HRMS analysis, the extract was diluted in a mixture of methanol and methylene chloride (1:1) (v:v) at 0.5 mg/mL (stock solution). Several dilution solutions were prepared by diluting the Kniphofia uvaria oily extract from 0.5 to 0.005 mg/mL. 2.3. UHPSFC-HRMS Analytical chromatographic separations were carried out using equipments from Jasco (Lisses, France) and Dionex (Germering, Germany). The UHPSFC-HRMS system used to achieve separation was composed of the Jasco 2080 CO2 -plus pump and a Dionex UltiMate 3000 RSLC system (binary pump, autosampler and thermostated column compartment). Identification of the compounds was achieved with an ultra-high-resolution separation performed by using seven coupled columns: six Kinetex C18 (Phenomenex, Le Pecq, France) and one Accucore C18 (Thermo, Les Ulis, France), all in dimensions of 150 × 4.6 mm, 2.7 ␮m, linked in series to achieve

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a very high theoretical plate number, whereas the APCI-MS optimization tests were performed with only two Kinetex C18 columns connected in series. The mobile phase was composed of CO2 : MeOH (90:10) (v: v) in isocratic elution. UHPSFC flow rate was set up at 1.2 mL/min. This flow rate, rather low for SFC, is due to the column number used which induces a high inlet pressure. The injected volume was equal to 5 ␮L. Columns were thermostated at 9 ◦ C, and the backpressure (regulated with BP-2080 Plus back pressure regulator from Jasco) was set at 10 MPa. A second HPLC pump (Dionex) was used to add the make-up solvent by the addition of a tee piece (void volume = 0.57 ␮L). The backpressure regulator exit (10 MPa) was connected to the MS detector by a PEEK capillary tube (length of 32 cm) with an internal diameter of 125 ␮m in order to limit additional dead volumes. MS detection was carried out using a maXis UHR-Q-TOF mass spectrometer (Bruker, Bremen, Germany) in positive ionization mode. Corona voltage was set at 5000 nA. The APCI source was heated at 450 ◦ C. The nebulizing and drying gas (N2 ) flows were respectively set at 0.04 MPa and 3.5 L/min. Mass spectra were recorded from 50 to 1400 m/z. The compounds were characterized (determination of molecular families) by the injection of different standards (TAG, DAG, FA, anthraquinones) in order to determine the elution order. The compounds were identified using the protonated molecular ion peak and fragments. In this SFC-APCI-HRMS system, in-source fragmentation was observed. The fragment peaks confirmed the molecular structure. The exact mass generated by the HRMS yielded a chemical formula provided by the Smart Formula algorithm from Data Analysis 4.0 software (Bruker).

3. Results and discussions 3.1. Preliminary studies In a recent paper [24], we presented the SFC-MS chromatograms of the CO2 extract of Kniphofia Uvaria seeds, obtained with seven C18 columns packed with superficially porous particles (total column length 75 cm). This great number of connected columns was possible because of the low viscosity of the mobile phases containing a high proportion of carbon dioxide. This long fused-core column achieved a high chromatographic efficiency, and 53 peaks were separated (Fig. 1), in which we identified common triacylglycerols (LnLL, LLL, PoLL, PLL, OLL, PPL, POL, OOL, SLL, PPO, OOO and SOL), hydroxylated triacylglycerols (LLL-OH, PLL-OH, OLL-OH, PPO-OH, OOL-OH, POL-OH), diglycerides (LLn, PoL, LL, PL, OL, PO, OO, OS), monoglycerides (MG), Free Fatty acid (FA) and anthraquinones (mainly rhein and aloe-emodin). The interpretation of this Fig. 1 by SFC-APCI-HRMS was reported in details in our previous study [24]. For TAG, the fragmentation pattern observed is similar to the one reported in HPLC [13]. For hydroxylated TAG, Fig. 2 shows the mass spectrum obtained for a compound having a m/z value equal to 895.7317. The different fragments are related to the loss of a water molecule, or of one L chain (C18:2) or L-OH chain (C18:2-hydroxy). This MS spectrum shows the presence of a fatty acid with two double-bonds and one hydroxyl group in this triacylglycerol. Hydroxylated fatty acids have been largely observed in castor oil, but the ricinoleic acid (12-hydroxy-9-octadecenoic acid) has only one double-bond. However, the presence of dimorphecolic acid (9hydroxy-10,12-octacecadienoic acid) was reported in seed oil of Dimorphotheca aurantiaca [25] or in Glechoma hederacea L. Labiatae [26], whereas coriolic acid (13-hydroxy-9,11,-octacecadienoic acid) was reported in Coriaria nepalensis [27]. We also found at 13.78 min a peak having a m/z value equal to 297.24, that fits to the formula C18 H33 O3 . Thus, the presence of a hydroxylated fatty acid

having two double-bonds is hypothesized. The presence of these hydroxylated lipids could be due to the action of a lipoxygenase during the germination of the plant, as it occurs for Barley seeds (Hordeum vulgare) [28]. 3.1.1. Injection performances studies To study the effects of several operating parameters on the APCIMS response, the column number was limited to two to reduce analysis duration. The UHPLC autosampler (pull loop – WPS-3000T PL RS) was adapted to the use of compressible fluids. As shown in Fig. 3A, the injection program was modified in order to switch from the UHPLC to UHPSFC mode: the UHPLC autosampler was not well-adapted for the UHPSFC analysis. Indeed, by using the UHPLC autosampler for these analyses, we had important variations (R.S.D > 30%) in terms of MS responses due to the depressurized CO2 (Fig. 3A – from step 1 to step 2). This last one discharged the sample in the system. By consequence, we did not inject and analyse the same desired amount of sample (Fig. 3A – step 3). We had to reprogram the autosampler. In UHPSFC mode (Fig. 3B), the valve goes from inject (under high pressure) (step 1) to load mode (under atmospheric pressure) (step 2). The carbon dioxide of the mobile phase remaining in the sample loop becomes gaseous. The autosampler cannot be used in partial loop-mode due to CO2 depressurization. However, it was used in full loop-mode after depressurization of the fluid into a waste. The depressurization time was fixed to 30 s to ensure that the depressurization was complete. Consequently, an additional step was added to avoid residual presence of CO2 in tubings. After depressurization, the UHPLC needle was set up above the vial to collect the sample (step 3). The sample completely fills the 5 ␮L-loop. The autosampler switched to inject mode (step 4), while the tilting, the analysis started. The efficiency of the adapted autosampler was evaluated with the analysis of solutions at different concentrations. Fig. 4 represents the area of several ions (protonated molecular ions or fragments) in function of the injected sample concentration. As noted in this figure, a proportionality relationship of peak area according to the analyte concentration was observed from 5 to 30 ppm. For most of these studied values of m/z, the determination coefficients (R2 ) were superior to 0.98. Three analyses in a row were performed with the complex matrix at 30 ppm. The standard deviation was around 5% of the MS peak areas. Thus, the UHPSFC/UHPLC–MS autosampler with the modified injection sequence adapted to compressible fluids gives a high degree of linearity for each new injection, together with improved repeatability. 3.1.2. Influence of a make-up solvent addition on analytical performances The transfer of compounds from SFC to MS often requires adapted systems, such as splitter or make-up pump, to favour relevant ionization without causing a loss in chromatographic performances [29,30] For the UHPSFC-HRMS hyphenation, a make-up solvent can be added to prevent experimental difficulties, i.e. low ionization recovery and compound precipitations. Two configurations have been tested as illustrated in Fig. 5. In these configurations, no effluent splitting was used, the total fluid was introduced into the MS. In the first configuration (Fig. 5A), no make-up was applied whereas in the second configuration (Fig. 5B, 5C), another HPLC pump was used to deliver a make-up solvent at a flow rate of 0.25 mL/min. Methanol was used as make-up solvent because it is already present in the mobile phase. For the first configuration (Fig. 5A), the standard deviation of area with the UHPSFC-ACPI-HRMS system includes six fragments and protonated molecular ions (m/z = 263.2344; 601.5129; 615.492; 617.5085; 877.719; 855.725) from FA, DAG and TAG.

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Fig. 1. BPC chromatogram of an extract from seeds of Kniphofia uvaria by SFC-APCI-HRMS. – Analytical conditions: 7 columns (six Kinetex C18 and one Accucore C18, each 150 × 4.6 mm, 2.7 ␮m); CO2 /MeOH (90/10, v/v), 9 ◦ C, Outlet pressure (BPR): 10 MPa; injected volume: 5 ␮L, SFC flow rate: 1 mL/min; APCI, Nebulizing gas: 0.04 MPa, Corona discharge: +6000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.0 L/min.

Fig. 2. MS spectrum of a hydroxylated triglyceride.

Fig. 3. The UHPLC autosampler with (A) no adaptation or (B) adapted autosampler for the use of compressible fluids. Sample in yellow; CO2 bubbles in black.

By using the configuration presented in Fig. 5A, the R.S.D. was of 5.3% (measured on a triplicate injection) indicating the rather good method performances (Table 1). Compared to the technical

overview [31], similar results were obtained (R.S.D > 10%). When we added a make-up solvent at 0.25 mL/min (MeOH) by using the configuration presented in Fig. 5B, the standard deviation (measured

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Fig. 4. Injection evaluation with the adapted autosampler in SFC-APCI-MS – Analytical conditions: 2 Kinetex C18 columns (each column 150 × 4.6 mm, 2.7 ␮m), Temperature: 9 ◦ C, Outlet pressure (BPR): 10 MPa, Injected volume: 5 ␮L, SFC flow rate: 1.2 mL/min, APCI, Nebulizing gas: 0.04 MPa, Corona discharge: +5000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.5 L/min.

Table 1 Measured area of fragment and protonated molecular ions with the adapted autosampler. Analytical conditions: 2 Kinetex C18 columns (150 × 4.6 mm, 2.7 ␮m), 9 ◦ C, 10 MPa, 5 ␮L, 1.2 mL/min, APCI, 0.04 MPa, +5000 nA, 450 ◦ C, 3.5 L/min. m/z

Type of ion

No make-up – R.S.D. (%)

With make-up (before BPR) – R.S.D. (%)

263.2344 617.5085 615.492 877.719 855.725 601.5129 Arithmetic mean

Fragment (FA) Pseudo-molecular (DAG) Pseudo-molecular (DAG) Pseudo-molecular (TAG) Pseudo-molecular (TAG) Fragment (TAG)

4.6 5.2 5.6 5.0 6.0 5.4 5.3

2.9 5.2 1.8 0.7 1.6 3.0 2.5

on a triplicate injection) decreased down to 2.5%. Consequently, the decrease of R.S.D. from 5.3% to 2.5% shows the positive effect of the addition of a make-up solvent. Probably, for a mobile phase containing only 10% of methanol and 90% carbon dioxide, the addition of more solvent through the make-up favours the uniformity of the stream exiting the BPR or/and improves the detection sensitivity (largest peak area), leading to a better reproducibility of MS results ionization. 3.2. Make-up solvent location for APCI studies

Fig. 5. Influence of the make-up solvent: (A) No make-up, (B) Addition of make-up solvent after the BPR and (C) Addition of make-up solvent before the BPR. Analytical conditions: 2 Kinetex C18 columns (each 150 × 4.6 mm, 2.7 ␮m), Temperature: 9 ◦ C, Outlet pressure (BPR): 10 MPa, Injected volume: 5 ␮L, SFC flow rate: 1.2 mL/min, APCI, Nebulizing gas: 0.04 MPa, Corona discharge: +5000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.5 L/min.

In this work, two configurations to introduce the make-up solvent between the chromatograph and the mass spectrometer were investigated (Fig. 5B and C). These two configurations were experimented because the mobile phase density strongly varies before and after the BPR. Before the BPR, the mobile phase is a homogeneous mixture composed of solvent (10% methanol) and CO2 under a pressure of 10 MPa (sub/supercritical conditions) which allows solubilizing analytes because of the high density of the fluid. However, after the BPR (at the atmospheric pressure), the mobile phase gradually becomes a heterogeneous mixture composed of gaseous CO2 and a small portion of liquid solvent. Consequently, the make-up solvent location with regards to the BPR could be an important parameter to control. Before the BPR, the density of the make-up solvent and the mobile phase are rather close,

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Fig. 6. Influence of the flow rate of MeOH as a make-up solvent located after the BPR on the MS response of compounds. Analytical conditions: 2 Kinetex C18 columns (150 × 4.6 mm, 2.7 ␮m), Temperature: 9 ◦ C, Outlet pressure (BPR): 10 MPa, Injected volume: 5 ␮L, SFC flow rate: 1.2 mL/min, APCI, Nebulizing gas: 0.04 MPa, Corona discharge: +5000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.5 L/min.

Fig. 7. Influence of the make-up solvent nature for the analysis of pseudomolecular ions of compounds in Kniphofia uvaria (0.5 mg/mL) in SFC-APCI-MS. Analytical conditions: 2 Kinetex C18 columns (150 × 4.6 mm, 2.7 ␮m), Temperature: 9 ◦ C, Outlet pressure (BPR): 10 MPa, Injected volume: 5 ␮L, SFC flow rate: 1.2 mL/min, Make-up at 0.25 mL/min located after the BPR, APCI, Nebulizing gas: 0.04 MPa, Corona discharge: + 5000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.5 L/min.

which favours the mixing of the two fluids. After the BPR, numerous changes could occur due to the fluid depressurisation. Firstly, the linear speed of the fluid dramatically increases because the carbon dioxide changes from compressed to gaseous state. It could modify the homogeneity of the mixing of the mobile phase with the make-up solvent. Secondly, this depressurisation would decrease the compound solubility which should be solubilised in the make-

up solvent, whatever their own polarity, in order to avoid their precipitation onto the inlet surface of the capillary going to the mass spectrometer inlet [32]. Whatever the location of the make-up, before or after the BPR (Fig. 5B, C), we obtained similar quantitative results, showing the same efficiency in the compound transfer from the column to the spectrometer, and on the ionization recovery in the source. This

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Fig. 8. Influence of the make-up solvent nature for the analysis of fragment ions of TAG in Kniphofia uvaria (0.5 mg/mL) in SFC-APCI-MS. Analytical conditions: 2 Kinetex C18 columns (150 × 4.6 mm, 2.7 ␮m), Temperature: 9 ◦ C, Outlet pressure (BPR): 10 MPa, Injected volume: 5 ␮L, SFC flow rate: 1.2 mL/min, Make-up at 0.25 mL/min located after the BPR, APCI, Nebulizing gas: 0.04 MPa, Corona discharge: +5000 nA, APCI temperature: 450 ◦ C, Dry gas: 3.5 L/min.

can be explained by the low internal diameter and length of the capillary located between the BPR and the inlet of the MS, which should avoid the mobile phase depressurization just after the BPR. Moreover, the transfer line was rinsed with IPA after each run to remove possible precipitation of analytes. No precipitation of analytes occurred in the transfer line whatever the make-up solvent position. For the rest of the study, we decided to work with a makeup solvent located after the BPR to avoid to turn off the hybrid system. As a matter of fact, when we flushed out the make-up pump (localised before the BPR), it was necessary to set off the SFC system. Other studies achieved in SFC with an ESI source by using a split after the BPR, have shown the effect of a make-up located before the BPR, which ensured a higher ESI–MS response intensity on six drugs, whereas the peak efficiency was rather independent of this additional make-up [30].

3.3. Influence of make-up solvent on MS responses 3.3.1. Effects of make-up solvent flow-rate The flow rate of the make-up was also evaluated. To achieve this study, the normalized areas were calculated as the ratio of the area of protonated molecular ion with make-up to the area of protonated molecular ion in the standard analysis without make-up solvent. So, a normalized area value superior to 100% indicates a positive influence on the MS response in APCI+ whereas a normalized area value inferior to 100% illustrates a negative influence of the addition of a make-up solvent on the MS response. As shown in Fig. 6, by increasing the flow of MeOH, not all compounds have the same MS response variations. So, the TAG and Hydroxylated TAG MS responses increased whereas FA and anthraquinones MS signals decreased with the increase of the methanol flow (make-up). The change in response for DAG (OL, LL) is rather negligible. The addition of methanol through the make-up is generally supposed to improve the MS response. This is observed for the triglycerides with an almost regular increase vs. the makeup flow rate. However, the MS signals for the free fatty acids and anthraquinones decrease when make-up flow rate increases. Kostiainen and Kauppila reported that LC–APCI–MS changes were highly

dependent on the differences in proton affinities (PAs) between solvent and analytes, which may explain the differences observed in the present case [33]. When looking at the results for a makeup flow rate equal to 0.5 mL/min, the increase in response seems higher for the compounds with more double-bonds, i.e. LLL/OLL vs OL/LL and L/O, showing that the protonation probably occurs onto the double bonds, as reported previously [22]. Whatever the phenomenon explaining the varied changes in response with regards to the studied compounds, one can conclude that the lack of make-up favours the detection of some compounds, which also have acidic or hydroxyl groups, whereas the make-up solvent addition improves the response of the compounds having no polar group (triglycerides).

3.3.2. Effects of make-up solvent nature Different make-up solvents were tested: MeOH, IPA and acidified methanol with 0.1% formic acid. The SFC mobile phase is composed of CO2 :Methanol (9:1). Due to its recognized properties for improving protonation of compounds in positive APCI, MeOH was selected. Secondly, IPA was chosen because it often allows a good solubilisation of hydrophobic compounds. Finally, MeOH + HCOOH was tested because the addition of an acid provides an important proton source. The results are presented in Fig. 7 illustrating the influence of make-up solvent nature in function of different molecular families at a flow rate of 0.25 mL/min (which is the medium value from the previous study, part 3.3.1). As shown in this figure, the anthraquinones MS responses were suppressed with the make-up solvent addition, whatever the nature of the additional solvent, assessing the previous conclusion on the addition of a make-up for these compounds. For the free fatty acids (FFA) family (linolenic and linoleic acids), a better MS response was also noticed when no make-up was used, whatever the solvent nature. Similar observations have been published: make-up solvent addition did not improve the detection of pharmaceutical compounds [34,35]. However, IPA provided the worst responses with regards to MeOH or MeOH + HCOOH. It may be that IPA is a better Lewis base than MeOH because the cation formed would be more stable

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Table 2 Evaluation of fragmentation yield in SFC-APCI-MS for TAG in Kniphofia uvaria (0.5 mg/mL) in SFC-APCI-MS. Analytical conditions: 2 Kinetex C18 columns (150 × 4.6 mm, 2.7 ␮m), 9 ◦ C, 10 MPa, 5 ␮L, 1.2 mL/min, Make-up at 0.25 mL/min located after the BPR, APCI, 0.04 MPa, +5000 nA, 450 ◦ C, 3.5 L/min.

Make-up solvent

TAG

LLL

PLL

Ratio No make-up MeOH IPA MeOH + 0.1%HCOOH

LLL/LL 0.5 0.8 1.0 1.0

PLL/PL 1.0 1.0 1.0 1.0

OLL PLL/LL 1.4 1.4 1.3 1.4

OLL/LL 0.7 1.1 1.2 1.2

(Stevenson’s rule) [36]. Besides, the PA value of IPA (796 kJ/mol) is greater than the PA value of MeOH (754 kJ/mol) [37,38]. Moreover, MeOH has a lower surface tension and so a lower surface energy compared to IPA: MeOH would allow better nebulization and provides a better source of protons for ionization [33]. For the DAG and TAG molecular families, the MeOH, MeOH + HCOOH and IPA additions improved the intensities of MS signal compared to the MS responses obtained without make-up solvent. As expected, the acidification of methanol provides a better detection than the use of pure MeOH, which could be due to the higher protonation of analytes. Besides, it is surprising to see how the MS signals of di- and tri-glycerides increased with the addition of IPA, due to the relative properties of solvents explained previously. The use of IPA may provide a better solubilisation of TAG into the mobile phase, favouring the ionization process when going into the APCI source, whereas the lower solubility of TAG in MeOH could lead to aggregates of TAG into the APCI source which reduce the ionization recovery. This phenomenon could be explained by different PAs values for TAG and DAG compared to FA and anthraquinones: most of TAG and DAG’s PAs would be higher, which could induce higher MS responses [34]. We have also investigated the MS signals for TAG and DAG fragments (Fig. 8). Similar observations were obtained than those observed for protonated molecular ions in Fig. 7. The use of MeOH + HCOOH and IPA as make-up solvents improves the MS signals of most of the fragments. This increase of MS signal could be due to a larger amount of protonated molecular ions formed, which were then fragmented in the ionization source. We studied the influence of make-up solvent (IPA and MeOH + HCOOH) on the generation of protonated molecular ions and fragments ions in order to know if the solvent make-up improved only the protonated molecular ion generation with a constant fragmentation yield or if the solvent make-up improved both the generation of protonated molecular ions and the ionization yield. For this work, we calculated the ratio of the protonated molecular ion area to the fragment ion area of TAG. Thus, if the ratio was a constant, the fragmentation yield was not influenced by the added make-up solvent. Otherwise, if the ratio value was different according to experiments, the make-up solvent had an effect on the fragmentation yield. The results are presented in Table 2. For each TAG determined, the value was constant meaning that the addition of a make-up solvent did not influence the fragmentation yield in the ionization source. As a general rule, for IPA and MeOH + HCOOH, the make-up solvent addition had an impact on the ion protonated molecular and fragments ions amount (ionization yield) in the ionization source but did not changed the fragmentation yield for TAG. 4. Conclusions In this work, a UHPSFC-APCI-QTOF-HRMS system was applied for the analysis of different compounds included in a vegetable oil. As a result, we have established an exhaustive analytical method without the need for sample purification. Coupling UHPSFC to HRMS can easily be performed without any modifications of ionization source commonly used in LC–MS. A

POL OLL/OL 0.4 0.7 0.8 1.0

POL/PO 1.2 1.1 1.1 1.3

OOL POL/PL 1.0 1.1 1.1 1.3

POL/OL 0.9 0.9 0.8 1.0

OOL/OL 0.9 0.9 0.9 0.9

OOL/OO 1.7 1.5 1.7 1.8

OOO

POO

OOO/OO 0.1 0.1 0.1 0.1

POO/OO 0.1 0.1 0.1 0.1

POO/OP 0.1 0.1 0.1 0.1

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