James D Oliver
email: james.oliver@uws.edu.au

Marianne Gaborieau
email: m.gaborieau@uws.edu.au

Emily F Hilder
email: emily.hilder@utas.edu.au

Patrice Castignolles
email: p.castignolles@uws.edu.au

Simple and robust determination of monosaccharides in plant fibers in complex mixtures by capillary electrophoresis and high performance liquid chromatography

Keywords: monosaccharide, plant fiber, capillary electrophoresis, ion-exchange chromatography, photo-oxidation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Carbohydrates make up most of the living world around us. Their identification and quantification is generally sought after in many fields such as food and beverage analysis, plant analysis, fermentation studies and metabolism studies. Different separation techniques for mono-and disaccharides have been previously compared, mainly on model samples or diluted samples such as fruit juices or wine. However there is a need for a separation technique that can characterize "complex" samples with little sample preparation. These complex samples have a significant variation of carbohydrate type and concentration as well as significant levels of acids, bases, salts, amino acids and other cell debris. Dilute acid treatment of fiber is routinely used in biotechnology to break down hemicellulose fiber and exposes cellulose to enzymatic breakdown [1][2][3][4][5][6][7]. The characterization of these important samples is the focus of this manuscript. Preparation of these samples for analysis, i.e. matrix removal, is tedious and lowers the accuracy. Carbohydrates can be separated by high performance liquid chromatography (HPLC) using a number of modes [8]. Ion exchange resins in the calcium or lead form were first shown to separate carbohydrates without clear mention of the separation mechanism [9,10] which was later proven to be ligand exchange with a contribution of size exclusion [11]. Separation with ligand-exchange on ion exchange resins containing cations such as lead or calcium provide the same order of separation; however, in the case of lead, an improved resolution is observed for the common fiber sugars xylose and galactose. Separation on a cation-exchange resin in the hydrogen form with a sulfuric acid mobile phase elutes disaccharides first, followed by hexoses, pentoses then alcohols. The mechanism of separation for carbohydrates on this column has been shown by several groups but has not been definitively proven [12]. Separation with amino columns, as Hydrophilic Interaction Liquid Chromatography (HILIC), utilizes hydrophilic interactions between the sample and the amino-rich resin with pentoses eluting first, followed by hexoses, disaccharides then oligosaccharides in order of increasing oligomer units. HILIC is better adapted to small oligomers than monosaccharides [13]. HPLC can also be used in reversed phase mode (RP-HPLC) after multistep derivatization [14]. Each method has its own issues relating to co-elution, tedious sample preparation, salt intolerance or acid intolerance leading to incomplete separation and short column life [15]. Gas chromatography (GC) is also commonly used; however, it requires multistep derivatization of the carbohydrates [16][17][START_REF] Knapp | Handbook of analytical derivatization reactions Wiley[END_REF]. RP-HPLC and GC were recently compared for plant fiber analysis [START_REF] Villanueva-Suárez | [END_REF], and although the determination of a number of monosaccharides was accurate within 10 %, rhamnose and galactose were not resolved via HPLC.

CE is used and recognized in both research and industry as a viable technique for the separation of carbohydrates [20][START_REF] Hanrahan | Chemometric Methods in Capillary Electrophoresis[END_REF][START_REF] Klampfl | [END_REF]. GC and CE were recently compared for the separation of hydrolyzed wood samples [START_REF] Dahlman | [END_REF]. Although these methods required derivatization, both carbohydrates and uronic acids could be determined in the same run with CE. HILIC and CE were also compared for fruit juice samples for the detection of sucrose, glucose and fructose [24]. Both methods were not significantly different and showed good repeatability. Pre-column derivatization is avoided in this work to keep the method simple and robust and ensure no side-reaction occurs with proteins and lipids of complex samples (we follow as definition of a robust method "a method that can be applied to analytes in a wide variety of matrices" [START_REF] Harvey | Modern analytical chemistry[END_REF]). GC of carbohydrates is not possible without derivatization and this may be why it is not currently a common method to analyze plant fiber degradation products [16][17][START_REF] Knapp | Handbook of analytical derivatization reactions Wiley[END_REF]. Without derivatization or complex formation CE cannot separate carbohydrates below pH 9, as expected. Monosaccharides can be charged, separated and detected in borate buffer since they complex the borate [START_REF] Schmitt-Kopplin | [END_REF]27]. The detection suffers however from a poor sensitivity of the borate complex. Separation can also be achieved at pH 7.5 with micellar electrokinetic chromatography (MEKC) [27]; however, the method requires the addition of sodium dodecyl sulfate (SDS) surfactant that also interacts with proteins and lipids present in complex mixtures. Separation of underivatized sugars in CE is possible using an electrolyte with a pH above the pKa of the sugars, which is generally above pH 12. On this basis earlier methods demonstrated separations in strongly alkaline electrolytes with indirect or pulsed amperometric detection [28][29][30][31]. CE with indirect UV detection was shown to be a rapid, repeatable and sensitive method for carbohydrates, and has also shown quantitative recovery of carbohydrates in similar samples [30,32]. However, the limitations and possible artifacts of indirect detection or pre-derivatization for complex samples containing complex carbohydrate mixtures in conjunction with other compounds have never been investigated. More recently CE has been shown to separate up to 12 different mono-, disaccharides and sugar alcohols in direct detection without derivatization using a high pH buffer of sodium hydroxide and sodium phosphate. The direct detection was found to be unexpectedly possible through UV absorption at 270 nm by Rovio et al. [33] and the method was applied to plant fiber samples with a maximum concentration of 400 mgL -1 for single sugars [34]. An adapted adaoted version on of the method has been applied to forensic, pharmaceutical and beverage samples) [35,36]. While maximizing the detection sensitivity is an objective for some applications, plant degradation leads to complex mixtures but with relatively large quantities of monosaccharides, thus detection does not require the highest sensitivity. Rather for this application we have focused on the development and critical comparison of approaches to achieve the most robust and simple separation of carbohydrate mixtures from complex plant samples. The specific objectives were to adapt the separation of Rovio et al. to achieve high resolution separations at higher concentrations and to compare the optimized CE method to existing HPLC methods, specifically in terms of quantification.

Material and Methods

Materials

Water was of MilliQ quality (Millipore, Bedford, MA, USA). Fused-silica capillaries (50 m i.d., 360 m o.d.) were obtained from Polymicro (Phoenix, AZ, USA). Xylose ≥99% was obtained from Alfa Asear (Ward Hill, MA, USA). Dimethyl sulfoxide (DMSO), D+glucose ≥99.5%, D+galactose ≥99%, Lrhamnose monohydrate ≥99%, L-arabinose ≥99% and D+cellobiose ≥99% and acetonitrile (ACN) were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). Sodium hydroxide pellets (NaOH), disodium hydrogen phosphate powder (Na2HPO4), lactose, glacial acetic acid and sulfuric acid were obtained from Univar (Ingleburn, NSW, Australia).

Plant sample and standard preparation

Three cladodes (flattened paddle shaped stems) of the plant of Opuntia fiscus-indicia were obtained from the wild in Richmond, NSW, Australia in November 2010. They were immediately homogenized with water and then centrifuged at 3000 rpm for 30 min to isolate the fiber. The insoluble fraction was then dried to a constant weight at 75 °C and milled to fit through a 1 mm sieve and stored in an airtight container until sample preparation. A 5 mL solution of 0 to 4 % (v/v) sulfuric acid was loaded with 5 % (w/v) of dried fiber in a sealed glass tube and heated to 134 °C for 1 h as done in [1]. The sample was then filtered through a nylon 0.45 µm filter before analysis. The fiber standard was prepared by measuring 200 mg of each sugar (glucose, galactose, rhamnose, arabinose, mannose, xylose and cellobiose) in a 200 mL volumetric flask then filling to the mark with MilliQ water.

High performance liquid chromatography

All separations were performed on a Shimadzu 10A Series System with a RID-10A refractive index detector and SPD-M10Avp PDA detector (Shimadzu Scientific Instruments, Rydalmere, NSW, Australia). The injector was equipped with a 20 µL injection loop and rinsed with 200 µL of samples before injection. The different column sets were purchased from Bio-Rad (HPX-87H, HPX-87P and HPX-87C) (Hercules, California,USA) and Supelco (LC-NH2) (Sigma-Aldrich Castle Hill, NSW, Australia). Experimental conditions are given in Table 1

Capillary electrophoresis

Separations were performed on Agilent 7100 or 3D Capillary Electrophoresis systems (Agilent Technologies, Santa Clara, CA, USA) with a Diode Array Detector monitoring at 200 nm and 270 nm with a 10 nm bandwidth. The buffer preparation and separation was carried out as described by Rovio et al. [33]. Typically a capillary with a 60 cm total length (51.5 cm effective length), was filled with 130 mmol NaOH and 36 mmol of Na2HPO4 and an electric field of 16 kV ramped up over 2 min. The capillary was pre-treated prior to use by flushing with 1 M NaOH, 0.1 M NaOH and water for 20 min each. The sample was injected hydrodynamically by applying 34 mbar of pressure for 4 s (≈55.6 nL according to the Poiseuille law) followed by buffer in the same manner. Between each run, the capillary was flushed with 10 % (v/v) acetic acid for 5 min followed by water and then running buffer. After the last injection, the capillary was flushed 1 min with NaOH 1M, 10 min with water and 10 min with air. Dimethyl sulfoxide (DMSO, 1 µL/500 µL) was added to each sample to mark the electroosmotic flow (EOF) and 1 g•L -1 of lactose was added as an internal standard. The EOF was determined at 200 nm. Calibration curves (Figure S-4) were calculated from 5 concentrations between 0.125 gL -1 and 1 gL -1 ; each concentration level was determined from an average of 5 injections with lactose used as an internal standard. All electropherograms were corrected for the EOF by plotting the intensity against the electrophoretic mobility (mep) (Equation S-1). Integration was performed on signals at 270 nm with Origin Pro 8.5 (Northampton, MA, USA).

NMR spectroscopy

1 H nuclear magnetic resonance (NMR) spectra of 1 gL -1 glucose and sucrose in water with 130 mmolL -1 NaOH were recorded at 25 °C on a Bruker Avance 400 spectrometer (Bruker, Alexandria, NSW, Australia) operating at 400 MHz for 1 H, with a BBO probe, using WATERGATE water suppression. A 14 s 90°pulse was used for the first 1 H irradiation and 18 s 180°pulses were used for WATERGATE; 16 scans were recorded with a 5 s relaxation delay. The chemical shift scale was externally calibrated with the resonance of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0 ppm.

1 H NMR spectra of glucose samples (1 gL -1 with 130 mmol NaOH in D2O) irradiated with a HP 3D CE deuterium lamp (Agilent Technologies, Part number: 2140-0585) were recorded at room temperature on a Varian Mercury 2000 spectrometer (Palo Alto, CA, USA) operating at a 1 H Larmor frequency of 300 MHz. A 11 s 90°pulse was used for 1 H irradiation; a 4 s repetition delay was used and 2 to 64 scans were recorded. The chemical shift scale was externally calibrated with the resonance of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0 ppm.

Results and Discussion

Plant fiber was partially hydrolyzed and the composition was determined for the first time by both common liquid chromatography methods as well as free solution capillary electrophoresis with direct UV detection.

Comparison of common HPLC separations of monosaccharides and application to fiber analysis

HPLC is the dominant method for carbohydrate identification and quantification owing to its relatively high throughput (separation in typically 10-18 min) and ease of use. Ligand exchange with lead and calcium form resins, LC with hydrogen exchange resins as well as HILIC have different advantages and drawbacks, but none has led to a clear, robust separation. Fig. S-1 demonstrates the separation of different standards containing common fiber sugars with three commonly used columns for carbohydrate analysis, with the resolution and sensitivity listed in Table 2.

Ligand-exchange separation against calcium (Fig. S-1A and Table 2) led to the lowest number of resolved peaks for standards with complete co-elution of galactose, xylose and rhamnose, as documented previously [37]. The stronger complexation of the carbohydrates with lead provides higher resolution between all fiber sugars, in particular xylose and galactose (Fig. S-1B and Table 2), which is of high importance in fiber analysis and fermentation studies. Although rhamnose still coelutes with galactose, it is generally only found in trace amounts in the woody biomass that is typically investigated. The weakness of both columns is their inability to tolerate salt and acids. Fiber samples, after being pre-treated with sulfuric acid at high temperatures, require neutralization [38]. This can be achieved with calcium hydroxide or barium hydroxide due to the low solubility of barium sulfate and calcium sulfate; however, trace amounts of salts still rapidly displace calcium or lead. The use of hydroxide additives or hydroxide form resins also adds to the sample preparation. Deashing systems are also available; however, they significantly add to the cost and the cartridges have short lives. HILIC does provide resolution between the sugars of interest (Fig. One of the most widely used columns in the literature is the hydrogen exchange HPX-87H, due to its tolerance to low pH samples and its ability to detect acids, alcohols, as well as mono-and disaccharides [12]. Investigations of wood acid hydrolysates have been carried out on this column as the acidity of the sample can be tolerated by the column negating the need for a tedious neutralization step [38]. The issues with this approach are the co-elution of galactose and mannose, which are only trace amounts in wood, with xylose, the acidic mobile phase degrading some disaccharides such as sucrose and the co-elution of acids, ketones and aldoses from the plant degradation with the carbohydrates [12]. In the fiber sample (Fig. 1-A), peak 9 elutes close to cellobiose, however its UV spectrum reveals it is not cellobiose. In the case of more complex carbohydrate mixtures the HPX-87H column gives robust separations, but still co-elution (Fig. 1 and Table 2).

S-1C and

Separation of monosaccharides in fiber samples using capillary electrophoresis

The use of CE provides an alternative separation technique with a lower running cost. To run 50 samples via HPLC, the cost of the column, guard column with holder is larger than that of the running cost of capillary electrophoresis for the same purpose for a higher throughput of the latter (see Table S-3). The same standard and sample shown in Fig. 1 were separated via CE with direct detection and without any pre-derivatization (Fig. 2). The sample did not need neutralization before injection saving preparation time and cost. All the common fiber sugars are resolved with baseline resolution maintained for standard concentrations up to 250 mg.L -1 , after which glucose and rhamnose start to lose baseline resolution. The peaks can be identified by the electrophoretic mobility and the precision of the mobility is greatly improved by the use of an electro-osmotic flow marker as well as an internal standard (see Table S-2). The addition of an internal standard improved also the repeatability of the peak area which this study showed to be better than published values [34]. The calibration curves obtained with different capillaries, reprepared standard solutions and a different CE machine exhibit good reproducibility (Fig. S-4 and Table 3). One of the most significant benefits is the flexibility of the separation in comparison to all HPLC modes compared. Capillary length, buffer concentration and type can be optimized with minimal cost and time in CE compared to optimization of the eluent concentration and nature, and stationary phase in HPLC. The electrophoretic mobility is not only a parameter to enable the quantitative determination of the sugars but it also characterizes the molecule and relates to its structure [39][40][41]. The electrophoretic mobility of each sugar measured in this work (Table 3 as well as reproduced data in Table S-1) was lower than that published by Rovio et al. [33] and by Gürel et al. [30] (Table S-2). The latter used methanol (direct detection) and water (indirect detection) as electro-osmotic flow markers, respectively. Water and methanol have a low but significant effective charge at pH 12 and should not be used as electro-osmotic flow markers in these conditions. Consistent with this we found that when DMSO was used to mark the electro-osmotic flow, methanol had a non-zero electrophoretic mobility (Fig. S-3). The ramping of the separation voltage in the first 2 min from 0 to 16 kV of the run was taken into account in this calculation. b When the internal standard is equal to 1 and 'x' is the sugar concentration Table 3. Electrophoretic mobility (mep) with its relative standard deviation (RSD) and calibration of response at 270 nm with its correlation coefficient R 2 , for the sugars in our fiber standard (capillary of 66 cm total length).

The aim of this work was to quantify carbohydrates in the fiber sample by CE and HPLC with the determined sugar concentrations compared in Table 4 (and Table S-4). Separation and quantification of three sugars had been compared for HILIC and CE with indirect detection [24]. The repeatability showed a higher relative standard deviation (RSD) than our own results since no internal standard and limited flushes between injections were used in CE and the refractive index detection used in HPLC suffered from poor temperature control. The RSD of the HPLC system shows it to be a more precise system. However, mannose, galactose and xylose co-elute in HPLC while they can be quantified separately in CE (Table 2). Taking this into account the total concentration of these three sugars determined by HPLC is compared with the sum of the peaks determined in CE and the concentrations determined by HPLC are systematically lower than those determined by CE: 17 to 22 % lower on the total sugar content and up to 44.14 % lower on glucose. These separations have been fully reproduced with fresh solution and new capillaries and led to the same results shown on Table S-4. Lower sugar concentrations determined in HPLC may be attributed to non-quantitative recovery due to absorption of the sugars onto the stationary phase. Such incomplete recovery has been observed previously on this HPLC column once [42]. The incomplete recovery cannot be explained by limited sensitivity since the signal-to-noise ratio is above 1,000 for all peaks quantified on Table 2 on the fiber sample. It is to be noted that the capillary electrophoresis instrument we used leads to a more than sufficient sensitivity for fiber samples, but in other cases, such as trace detection, other instruments lead to higher sensitivity [43]. Fractions were collected after LC separation and injected in CE (Table S The relative difference Diff. is in % and is calculated as the difference of the concentrations determined by CE and HPLC divided by their average, for recovery only the higher values were used e Loss in HPLC is a comparison to a recovery study previously carried out with this column under these conditions [42]. The study found a loss of recovery for different sugars; the relevant ones are listed in this row.

f As mentioned previously, repeatability (as measured by RSD) is improved with the use of an internal standard, the values in this row show the RSD as measured previously by [34] for comparison.

Table 4. Comparison of the determined sugar concentration C (g•L -1 ), with their relative standard deviation RSD (%), by CE (capillary of 66 cm total length) and HPLC with HPX-87H column.

Investigation of the detection in capillary electrophoresis

Monosaccharides do not normally absorb UV at 270 nm however Rovio et al. proved that sensitive detection at this wavelength is obtained at pH 12.6 [33] with the mechanism for this detection of the sugars at 270 nm still under debate. The complex mechanism of CE direct detection at 270 nm, which has recently been pointed out by Sarazin et al. [START_REF] Harvey | Modern analytical chemistry[END_REF], was further investigated in this study. Rovio et al. hypothesized an alkaline degradation of the sugars with the formation of an enediolate absorbing at 270 nm that is unable to proceed to a carboxylic acid due to complexation with the sodium ion [33]. Sarazin et al. subsequently argued that such a mechanism would not be as universal as observed experimentally since it is impossible for sucrose. They proposed instead a photooxidation of the saccharides in the detection window [43]. This mechanism is consistent with the mechanism proposed by Gilbert et al. after monitoring the degradation of carbohydrates by hydroxyl radicals using Electron Spin Resonance spectroscopy (ESR) [44] . The reaction pathway is shown on Figure S-7. The difference we observe between the UV spectra of glucose and that of xylose and arabinose (Fig. 2 C-ii) confirms that there is a difference in the structure of the absorbing molecule between five and six carbon sugars.

In elucidating the actual detection mechanism a possible electrochemical reaction should be considered. Since a sucrose solution can be detected by CE in the absence of electric field as previously shown by Sarazin et al. [43], the role of the electric field in the detection of sugars at 270 nm was further investigated. Glucose (1 g•L -1 in water) was injected into a 51.5/60 cm capillary with 130 mmol NaOH as the background electrolyte. The migration of glucose was obtained first by applying voltage; it was independently obtained applying the same voltage for 2 min to allow for adequate mixing of the glucose with the electrolyte shown by the separation of a water peak, then applying a 42 mbar pressure to the capillary inlet for migration. Both types of migrations lead to detection at 270 nm (Fig. 3). However, the peak area is close to 4 times larger in the presence of electric field, demonstrating that the electric field plays a part in the detection (as well as the photoirradiation). Detection via enediolate formation as suggested by Rovio et al. [33] was further investigated in this study. A glucose sample (1 g•L -1 in 130 mmol NaOH) was injected into the CE with 130 mmol NaOH as the back ground electrolyte at regular intervals and migrated alternatively by voltage (Fig. 4, as well as pressure Fig. S-6). Over a period of 46 hours the intensity of the glucose peak decreased and some negatively charged products formed (Table S-6). The products do not have UV spectra distinctive from that of glucose, but a higher mobility (possibly explained by a lower friction through a smaller size). Migration by pressure reveals a decrease in the amount of compounds detected at 270 nm after more than 4 hours. Migration by voltage reveals that the usual glucose peak reduces in intensity decreasing in an apparent first order reaction with respect to glucose (Fig. S-5). After 4 hours, degradation products with higher electrophoretic mobility than glucose are separated and they are also detected at 270 nm. Alkaline degradation of glucose is documented in the literature [45][46][47] but it is important to note that the degradation is very slow compared to the residence time of the monosaccharides in alkaline conditions before the detection (30-40 min) even in the presence of electric field.

The alkaline degradation of glucose, as well as sucrose, in alkaline solution, was characterized by 1 H NMR using water suppression. No structural change was observed with glucose within the first 2 hours confirming the monitoring of this alkaline degradation by CE as described above. No structural change was observed with sucrose even after a week (Fig. S-9B), as predicted by Sarazin et al. [43]. The potential formation of enediolates is definitely too slow to explain the detection in CE at 270 nm, thus the hypothesis presented in Rovio's pioneering work cannot explain the detection at 270 nm: the photo-irradiation is necessary for these species to be detected. We further investigated the mechanism of the photoreaction by attempting the identification of the potential products by NMR.

NMR analysis can be quantitative in a deuterated solvent, since the water peak does not have to be suppressed. We first tested if the photochemical reaction still takes place by replacing water with deuterated water. When injected into the CE with 130 mmol NaOH in D2O, the electrophoretic mobility is 20 % lower than in H2O, consistent with the difference in viscosity of D2O and H2O [48], leading to difference in hydrodynamic friction. In both cases the peak absorbance was still at 270 nm (Fig. S-8). The detection mechanism is still possible in the absence of water; however, the peak area observed was five times smaller than that in H2O. The difference in injection volumes can only be a minor contribution to this difference, since the difference of viscosities for our hydrodynamic injections is only of the order of 20 % and the difference might rather be due to the NaOD in D2O being significantly less alkaline than sodium hydroxide in H2O. The sample of glucose (1 gL -1 ) in 130 mmol NaOH in D2O was then exposed directly to the HP 3D deuterium lamp (Scheme S-1) for a time significantly longer than the residence time in the CE detection window and then characterized by 1 H NMR with no water suppression (Fig. 5). After 60 min of irradiation a minor structural change was seen by 1 H NMR at 4 ppm. It is thus clear than on the detection timescale, only a minimal fraction of glucose is photo-oxidized (and the decomposition product absorbing at 270 nm has to be in minimal amount and thus highly UV-absorbing at 270 nm). Sarazin et al. [43] and our results suggest that detection in CE is made possible by a photo-oxidation similar to the one observed in sodium borate solutions at pH 9 [49] but the extent and rate of the reaction is not clear. In Sarazin et al.'s experiments the sugar was moved into the window with pressure and made stationary for the photo-reaction to proceed [43]. However our results have shown that the electric field plays an essential role in the detection. To investigate the photooxidation reaction in the presence of the electric field, glucose (1 g•L -1 in water) was injected into 130 mmol NaOH as for the usual separation. After the peak was detected, the voltage was inverted and the glucose band passed through the detection window a second time. The voltage was inverted again. This was repeated 15 times in total. The peak retained the same electrophoretic mobility; however, its area was not constant, increasing during the first 6 passes and then decreasing (Fig. 6). This variation in peak intensity with the residence time is qualitatively similar to the one observed by Sarazin et al. [43] in the absence of electric field. In our experiment, the band is in the dark for several minutes between each pass/photo-irradiation. The putative intermediates and mechanism proposed by Gilbert et al. and then Sarazin et al. cannot solely explain our results since all the proposed intermediates are unstable radicals with lifetimes significantly shorter than one minute. It is possible that some intermediates might accumulate under photo-irradiation and be stable enough to remain in the capillary for the next pass, consistent with the detection of minimal degradation products detected by NMR. The decrease of the signal intensity after the 6 th pass might be due to build-up of oxygen arising from side reaction from the UV degradation of water and consistent with a photo-oxidation process enhanced by the electric field, however could not be definitely confirmed from these experiments.

Conclusions

HILIC, ligand exchange and other LC methods conventionally used for carbohydrates provide an inadequate separation of all sugars in acid-hydrolyzed plant fiber and none of the columns examined provide both a robust and clear separation. In order to maintain separation on the calcium and lead form resins a tedious neutralizing step must be carried out. This study showed free solution CE to be a superior quantitative method to HPLC in terms of robustness and resolution, as well as recovery. In comparison to HPLC, CE has a significantly lower running cost, a higher throughput and a greater flexibility. The direct detection of deprotonated saccharides is unexpectedly possible at 270 nm but it is neither due enediolate formation nor to alkaline degradation, which is slow compared to the residence time in the detection window. The detection is likely due to an intermediate in a photooxidation process that we showed to be enhanced by the electric field. Despite a still controversial mechanism, the detection is robust (possible for example in deuterated solvent although with lower sensitivity), although the presence of oxygen might be of importance. CE is thus a robust and simple method to study polysaccharides degradation in general. Equations are given in Table 2.

S-6

Quantitative Comparison of HPLC and CE:

The comparison of CE and common HPLC is given on Table S The relative difference Diff. is in % and is calculated as the difference of the concentrations determine by CE and HPLC divided by their average.

Rovio's conditions (60 cm total length capillary) lead to some comigration of mannose, glucose and rhamnose. The capillary length was thus slightly increased (66 cm total) and a lower recovery in HPLC compared to CE was still noted (Table 3).

S-7

To investigate a potential loss in the LC system, fractions were collected after LC separation and analyzed by capillary electrophoresis. 20 µL of glucose standards (10 or 20 g⋅L -1 ) and fibre samples (equivalent to the 4% (v/v) acid sample) were injected on a HPX-87H column and the glucose peak was collected as one fraction after the RID detector. The fractions were then injected in CE for quantification of the glucose (as it had the highest difference between CE and HPLC). The fibre sample (same 4%(v/v) acid treated sample) was also diluted to the same concentration, injected directly into CE for quantification. The glucose fractions led to quantification of glucose by the CE 24 % lower than the original amount for the 20 g⋅L -1 standard while it is 15 % higher for the 15 g⋅L -1 standard. This is not consistent with the repeatability of both LC and CE quantifications. One explanation might be column bleeding. Common LC columns for sugars are a relatively old technology (prior to online light-scattering detection) and column bleeding is expected. The limited results we have are consistent with bleeding, with an overestimate of the glucose content as the injected amount decreases. On the fibre samples, RID detection on the HPLC quantified the samples at 2.49 g⋅L -1 , however the CE quantified the fractions at 3.3 g⋅L -1 (see table S-5). Incomplete separation in LC could lead to overestimate of the sugar amounts. The total peak area in LC treated with the different possible calibration curves lead to 3.9, 6.9 and 7.8 % variation for the 1%, 3% and 4% acid samples respectively in the total sugar amount quantified by LC. Deconvolution of the peak is thus not necessary to discard the hypothesis that the limited resolution could explain the lower recovery observed in LC than in CE: deconvolution would not significantly change the total amount of quantified sugars. Finally, our investigation of the detection mechanism in CE does not indicate any likelihood of overestimating of the sugar content in fibre samples. We conclude that a loss of carbohydrates in the LC system likely explains the difference between our CE and LC quantification. The fact that the difference is larger than the loss observed in LC in the literature might be explained by column leaching or to a lesser extent the incomplete separation in LC.

Investigation the CE detection mechanism:

Additional information about the investigation of the detection in CE is given below. The set-up used to produce the samples for Figure 5

Fig. 1 .

 1 Fig. 1. Separation of a fiber sample (A) and fiber standard (B) and using HPX-87H column 1: Cellobiose, 2: Glucose, 3: Galactose, 4: Xylose, 5: Rhamnose, 6: Arabinose, 7: Void volume, 8: Galacturonic Acid, 9: Unknown.

Fig. 2 .

 2 Fig. 2. Fiber standard 250 m•gL -1 (A) and sample (B) plotted with electrophoretic mobility and migration time (C-i). Separation by CE via Rovio et al.'s method [33]. 1: Cellobiose, 3: Galactose, 2: Glucose, 5: Rhamnose, 4: Arabinose, 6: Xylose and corresponding UV absorption spectra (C-ii) for Glucose (dashed line), xylose (solid line) and arabinose (dotted line).

Fig. 3 .

 3 Fig. 3. Migration of 1 g•L -1 of glucose into 130 mmol NaOH electrolyte by 16 kV electric field (solid line) and with voltage for 2 min followed by 42 mbar pressure (dashed line).

Fig. 4 .

 4 Fig. 4. Degradation of glucose in 130 mmol NaOH monitored by migration with voltage. The arrows indicate the evolution with increasing time (0 h: bold solid line, 1.5 h: bold dashed line, 4 h: solid line, 7 h: dotted line, 27 h: dashed line, 46 h: bold dotted line).

Fig. 5 .

 5 Fig. 5. 1 H NMR of glucose (1 g•L -1 with 130 mmol NaOH in D2O) before (A) and after irradiation with CE deuterium lamp for 5 min (B), 30 min (C) and 60 min (D). The arrows indicate the region in which new signals appear.

Fig. 6 .

 6 Fig. 6. Detection of glucose (1 g•L -1 ) in 130 mM NaOH with 16 kV separation. Each peak represents a pass of the sugar though the lamp, after which the voltage was inverted.

Fig. S- 3 .

 3 Fig. S-3. Comparison of electrophoretic mobility of DMSO (1) and methanol (2) in 130 mmol NaOH and 36 mmol Na2HPO4 , detected at 200 nm.

Fig. S- 4 .

 4 Fig. S-4. Calibration curves with standards, showing R 2 values (capillary of 60 cm total length).Equations are given in Table2.

  Scheme S-1. Set-up of CE photo-oxidation experiment.

Fig. S- 5 .

 5 Fig. S-5. Evolution of the area of the glucose peak monitored by CE for a solution of glucose 1 g.L -1 in 130 mM NaOH.

Fig S- 6 . 10 Fig. S- 7 .

 6107 Fig S-6. Degradation of glucose in 130 mmol NaOH monitored by migration with pressure. The arrows indicate the evolution with increasing time (0 h: bold solid line, 1.5 h: bold dashed line, 4 h: solid line, 7 h: dotted line, 27 h: dashed line, 46 h: bold dotted line).

Fig. S- 8 .

 8 Fig. S-8. Separation and detection of glucose (1 g•L -1 ) in 130 mmol NaOH in water (dotted line) and in D2O (solid line).

Fig. S- 9 . 1 H

 91 Fig. S-9. 1 H NMR of 1 g•L -1 glucose (A) in 130 mmol of NaOH after 2 hours (A-I) and 5 days (A-II) and of sucrose (B) in water (B-I), 130 mmol NaOH after 2 hours (B-II) and after 5 days (B-III)

Table 1 .

 1 . Each mobile phase was vacuum filtered through a 0.45 µm filter before use. Results were integrated via VP class 5.0 software from Shimadzu. Experimental conditions used in HPLC for the different columns

	Column	Resin Form	Mobile Phase	Temperature	Flow Rate
				Column RID
	HPX-87P	Lead	Water	80 °C	60 °C	0.6 mLmin -1
	HPX-87C	Calcium	Water	80 °C	60 °C	0.6 mLmin -1
	HPX-87H	Hydrogen	0.005 M H2SO4	60 °C	60 °C	0.6 mLmin -1
	LC-NH2	Amino	75:25 ACN:water 25 °C	40 °C	1.0 mLmin -1

Table 2 )

 2 , however, galactose is still not baseline resolved from the glucose. In this case ACN needs to be added to the sample prior to injection to decrease the eluent's strength which can lead to precipitation of compounds insoluble in ACN such as polysaccharides.

	Carbohydrate peaks		HPLC
		HPX-87C (Calcium	HPX-87P (Lead	HPX-87H (Hydrogen	LC-NH2 (Amino	CE
		Form)	Form)	Form)	Form)
	Glucose Galactose	1.19	2.48	0.88	<0.5	1.67
	Glucose Rhamnose	1.37	2.48	1.66	3.95	0.79
	Glucose Xylose	0.81	1.35	0.88	2.92	6.83
	Glucose Arabinose	3.22	4.14	1.89	1.62	3.39
	Glucose Mannose	NA	4.32	NA	NA	1.71
	Xylose Galactose	<0.5	1.04	<0.5	2.61	8.05
	Xylose Rhamnose	<0.5	1.04	0.69	1.07	6.05
	Xylose Arabinose	1.27	2.56	1.01	0.56	3.86
	Xylose Mannose	NA	2.95	NA	NA	5.76
	Arabinose Galactose	1.92	1.53	1.01	1.63	4.92
	Arabinose Rhamnose	1.56	1.53	<0.5	1.35	2.55
	Arabinose Mannose	NA	0.74	NA	NA	1.93
	Rhamnose Mannose	NA	2.06	NA	NA	0.83
	Rhamnose Galactose	<0.5	<0.5	0.69	3.39	2.40
	Galactose Mannose	NA	2.06	NA	NA	3.42
			Sensitivity
	SNR*
		11,000	1,500	10,000	2,100	1,800

'NA' denotes that one of the carbohydrate was not injected * Signal-to-noise ratios (SNR) are indicated for glucose with 2 significant digits.

Table 2 .

 2 Resolution values for consecutive peaks on each column and in CE.

  -5) but the results only indicate a possible additional influence of column bleeding in LC. CA is the original acid concentration of the sample before dilution (% v/v) b galactose, mannose and xylose co-elute in HPLC. Values based on the refractive index of xylose (max) and galactose (min) c arabinose and rhamnose co-elute in HPLC. Values based on the refractive index of arabinose (min) and rhamnose (max)

	System CA	Glucose		Rhamnose	Arabinose	A+R c		Mannose	Galactose	Xylose		G+M+X b	Total
		a																(gL -1 )
	d		C	RSD	C	RSD	C	RSD	C	RSD	C	RSD	C	RSD	C	RSD	C	RSD
	CE	1	3.58	2.4	1.21 8.61 3.95 1.13 5.16		0.59 8.90 2.76 1.65 2.10 4.27 5.45	14.19
	HPLC		2.82	0.84					4.18 to	2.06							4.17 to	0.91 11.75
									4.40								4.53
	Diff. d		24%						21% to								27% to	19%
									16%								18%
	CE	3	3.67	5.38	1.28 11.1	3.19 6.21 4.47		0.58 8.58 2.41 3.98 1.59 3.36 4.58	12.72
						5
	HPLC		2.58	0.49					3.92 to	2.22							3.63 to	0.88 10.69
									4.13								3.98
	Diff. d		35%						13% to								23% to	17%
									8%								14%
	CE	4	3.54	2.98	1.05 4.98 2.39 9.67 3.44		0.54 15.2	2.03 3.21 1.06 4.05 3.63	10.61
												9
	HPLC		2.26	0.91					2.89 to	2.70							2.82 to	0.73 8.45
									3.05								3.14
	Diff. d		44%						17% to								25% to	22%
									12%								14%
	Loss in HPLC e	9.9										14.3
	[42]
	Sample			4.3 to				20 to				8.1		2.3		3.7
	quantifi-			16				30				to 19		to 19		to 11
	cation
	without
	internal
	standard
	[34]

a

Table S - 4 .

 S4 -3 in terms of cost and in Table S-4 in terms of carbohydrates quantification. It is important to note that Table S-4 give the same comparison as in Table 4 but from different experiments reproducing Table 4 ones with a different capillary and solutions. The results of the analysis of HPLC fractions by CE are given on Table S-5. Comparison of the determined sugar concentration C (g•L -1 ), with their relative standard deviation RSD (%), by CE (capillary of 66 cm total length) and HPLC with HPX-87H column. Compared to Table 4, these separations have been fully reproduced with fresh solution and new capillaries. galactose, mannose and xylose co-elute in HPLC. Values based on the refractive index of Xylose and Galactose c

	Table S-3
	Estimate of the cost of the typical CE and HPLC separations ($ is for Australian dollar and prices are
	as for 2012).
		HPLC System					CE System
				HPX-87P HPX-87H	LC-NH2
		Column		$2,096	$2,096	$731	Capillary	$7.2
		Guard Column	$537	$517
		Guard Column Holder $848	$848	$355
		Total		$3,481	$3,461	$1,086 Total	$7.2
	System CA a Glucose Rhamnose Arabinose Mannose Galactose	Xylose	M+G+X b
		C RSD C	RSD C RSD C RSD C RSD	C	RSD	C	RSD
	CE	1 6.12 0.33 0.76 13.38 4.77 6.3 1.35 3.01 5.1 0.33 2.25 5.2	8.7
	HPLC	4.84 0.20 1.00 0.20 4.26 0.2			6.57 to	0.17
							7.51
	Diff. c	23	27	11			15
	CE	2 6.48 2.69 1.40 7.97 4.01 5.59 1.24 9.67 5.36 1.02 3.34 3.98 9.94
	HPLC	5.12 0.80 1.26 1.40 4.12 0.5			6.86 to	0.66
							7.83
	Diff. c	23	11	2.7			27
	CE	3 7.98 2.10 1.98 6.66 4.78 0.2 1.71 5.27 6.48 2.77 3.74 6.9 11.93
	HPLC	5.92 0.60 1.43 0.70 4.26 0.3			7.75 to	1.53
							8.82
	Diff c	30	32	11			43

a CA is the acid concentration of the sample (% v/v) b

Table S - 5 .

 S5 Fraction experiments to determine the loss in HPLC in comparison to CE: comparison of the concentrations of glucose injected in HPLC, Cinj, eluted from HPLC according to RID detection, CRID, and as determined from CE, CCE.

	Sample	Cinj (g•L -1 )	CCE (g•L -1 )	CRID (g•L -1 )
	Glucose pure in water (Fraction)	20.00 20.00 10.00 10.00	15.53 15.76 12.65 13.12	NA NA NA NA
		NA	3.13	2.49
	Fibre sample	NA	3.11	2.49
	(Fraction)	NA	3.43	2.49
		NA	3.81	2.49
	Fibre sample	NA	3.22	NA
	Direct Injection	NA	3.35	NA
				S-8

Table S - 6

 S6 Peak areas of glucose degrading in 130 mmolL -1 sodium hydroxide, separated by CE with 16 kV field.

			Connection to HP 3D CE
			HP 3D CE Lamp
	1 cm gap		Petri dish with
			sample, 1 mm deep
			Lift
	Foil insulated box
	Time (h) Peak 1	Peak 2	Peak 3
	0	8.10E-08	-	-
	1.5	7.10E-08	-	-
	4	4.80E-08	4.00E-09	-
	7	4.90E-08	6.60E-09	6.80E-10
	27	2.30E-08	8.90E-09	3.70E-09
	46	6.60E-09	3.70E-09	4.10E-09
				S-9

Acknowledgements

PC and MG thank the College of Health and Science of the University of Western Sydney for a College Equipment Grant for the purchase of the Agilent Capillary Electrophoresis and the School of Natural Sciences for Small Equipment grant. Support from the Australian Research Council is gratefully acknowledged: EFH is recipient of an ARC Future Fellowship (FT0990521). We thank Dr Michael Phillips, Dr Paul Peiris, Dr Mark Williams and Julie Markham (UWS), Mark Thomas, Dr Artaches (Tom) Kazarian, A/Prof Michael Breadmore, Dr James Horne (University of Tasmania), Dr Yohann Guillaneuf and Dr Jean-Louis Clement (Aix-Marseille University) for fruitful discussions.

Supporting Information for

Simple and robust separation of monosaccharides in complex mixtures by capillary electrophoresis with direct UV detection and high performance liquid chromatography

James D. Oliver, 1 Marianne Gaborieau, 2 Emily F. Hilder, 3 Patrice Castignolles*

High Performance Liquid Chromatography (HPLC):

The

Capillary Electrophoresis:

The electrophoretic mobility mep was determined using the following equation [1]:

where l is the capillary length to the detection window, L is the total length of the capillary, V is the voltage, tm is the migration time of the analyte, teo is the migration time of the neutral species. The ramping was taken into account by averaging the voltage at tm.

The electrophoretic mobility values are then given on Table 3 and on Table S b relative difference calculated as the difference of our corrected mobility and the literature mobility divided by the average of both mobilities. c A. Gürel, et al. [3]


Olivier Girard
email: oliv.girard@gmail.com

Paul S R Goods

Franck Brocherie

Elevating Sport Performance to New Heights With Innovative 'Live Low -Train High' Altitude Training

Keywords: altitude training, hypoxia, repeated-sprint training in hypoxia, local hypoxia, sprint interval training, normobaric hypoxia

Editorial on the Research Topic Elevating Sport Performance to New Heights With Innovative 'Live Low -Train High' Altitude Training

HISTORICAL PERSPECTIVE

Traditional altitude training, where athletes are exposed to chronic hypoxia for several weeks as a means of additional physiological stimulus, has a long-standing history [START_REF] Owen | A Preliminary evaluation of altitude training particularly as carried out by some members of the Olympic teams of Great Britain and of other European countries in 1972[END_REF]. The prevailing view with such an approach is that it elevates red blood cell count, increasing the amount of hemoglobin available to ferry oxygen (O 2 ) from lungs to muscles, and eventually boost physical performance [START_REF] Chapman | Defining the "dose" of altitude training: how high to live for optimal sea level performance enhancement[END_REF]. Debate into the effectiveness of altitude training continues with some authors strongly believing it increases endurance performance [START_REF] Millet | Is live hightrain low altitude training relevant for elite athletes? Flawed analysis from inaccurate data[END_REF], while others argue that the effects of chronic hypoxia are not conclusive [START_REF] Robach | The role of haemoglobin mass on VO(2)max following normobaric 'live high-train low' in endurance-trained athletes[END_REF]. Modalities employing intermittent hypoxia for shorter durations (minutes to hours) are also popular [START_REF] Mclean | Application of 'live low-train high' for enhancing normoxic exercise performance in team sport athletes[END_REF], while only cause minor disruptions to the usual athlete daily lifestyle and training routine.

"Live Low-Train High" (LLTH) interventions have been implemented by Soviet scientists as early as in the 1930s [START_REF] Sirotinin | Effect of acclimatization to high mountain climates on adaptation to decreased atmospheric pressure in decompression chambers[END_REF]. Using altitude chambers or inhaled gas, LLTH methods were first implemented for altitude pre-acclimatization of pilots who flew in open cockpits at 5,000-6,000 m and for the treatment of a variety of clinical disorders [START_REF] Serebrovskaya | Intermittent hypoxia research in the former Soviet Union and the commonwealth of independent states: history and review of the concept and selected applications[END_REF]. For long, LLTH methods included two main hypoxic modalities corresponding to intermittent hypoxic exposure (IHE) at rest or combined with exercise (intermittent hypoxic training: IHT) [START_REF] Wilber | Application of altitude/hypoxic training by elite athletes[END_REF]. The consensus is that the use of IHE does not increase sea-level performance [START_REF] Bärtsch | Intermittent hypoxia at rest for improvement of athletic performance[END_REF], while the effects of IHT with respect to improving exercise capacity also remain elusive [START_REF] Faiss | Advancing hypoxic training in team sports: from intermittent hypoxic training to repeated sprint training in hypoxia[END_REF]

THIS RESEARCH TOPIC

The repeated-sprint training in hypoxia (RSH) paradigm requires the completion of maximal, short duration (typically ≤30 s) efforts interspersed with incomplete recovery periods (≤60 s) in hypoxic environment [START_REF] Brocherie | Effects of repeatedsprint Training in hypoxia on sea-level performance: a meta-analysis[END_REF]. This approach is now recognized as one LLTH method particularly useful for improving repeated-sprint performance in a wide range of sports, with the great majority of RSH studies having used moderately-trained individuals [START_REF] Brocherie | Effects of repeatedsprint Training in hypoxia on sea-level performance: a meta-analysis[END_REF]. For effective translation of sport science research to the field, it is crucial that research becomes "athlete and coach problem solving focused" [START_REF] Fullagar | The translation of sport science research to the field: a current opinion and overview on the perceptions of practitioners, researchers and coaches[END_REF]. Until now, our understanding of how RSH methods are effectively implemented in high-performance settings (i.e., during specific periods of the season) is limited [START_REF] Brechbuhl | Effects of repeated-sprint training in hypoxia on tennis-specific performance in welltrained players[END_REF][START_REF] Beard | Repeatedsprint training in hypoxia in international rugby union players[END_REF]. In International field hockey players (Malaysia national team), a 6-week "in season" runningbased RSH programme improved the succession of maximal treadmill efforts performed in hypoxia (James and Girard). In one case study, Faiss and Rapillard also reported the benefits of 150 repeated sprints at a simulated altitude of 3,300 m over 10 days in a professional cyclist. Advancing our understanding of RSH-induced adaptations also requires relevant research in sport-specific ecological test settings to be conducted. In this context, the effects of a 4-wks inwater swimming specific RSH program on freestyle swimming performance have been evaluated by Camacho-Cardenosa et al., but failed to induce further benefits than similar training in normoxia, possibly due to the selection of a sub-optimal hypoxic dose.

Other systemic or local LLTH methods based on the repetition of "all-out" efforts can be used to induce a potent physiological stimulus, up-regulate signaling pathways and eventually maximize performance outcomes. For instance, when hypoxia is induced by voluntary hypoventilation at low lung volume (VHL) [START_REF] Trincat | Repeated-sprint training in hypoxia induced by voluntary hypoventilation in swimming[END_REF], RSH can also ameliorate performance compared to training with unrestricted breathing, as demonstrated by Lapointe et al. in basketball players. These authors reported that, after 8 RSH-VHL sessions including changes of direction, gains may be attributed to greater muscle reoxygenation, enhanced muscle recruitment strategies, and improved K + regulation to attenuate the development of muscle fatigue, especially in type-II muscle fibers. Sprint-interval training (SIT) involves the repetition of long (∼30 s) "all-out" efforts and is an effective training strategy for upregulating mitochondrial biogenesis and exercise capacity [START_REF] Gibala | Sprinting toward fitness[END_REF]; however, the effect of additional hypoxia on these responses is uncertain. Takei et al. made the interesting observation that while six sessions of repeated Wingates, performed 3 times per week over 2 weeks, in either hypoxia or normoxia led to similar performance gains, more favorable blood lactate responses occurred in O 2 -deprived conditions. Finally, when compared to SIT alone, eight sessions of SIT immediately preceded by three cycles of bilateral occlusions (ischemic preconditioning: IPC) induce greater adaptations in cycling endurance performance that may be related to muscle perfusion and metabolic changes (Paradis-Deschênes et al.).

Across the Globe, an ever growing number of academic institutions, specialized clinics/hospitals (e.g., Aspetar Hospital), national sport institutes (e.g., Western Australia Institute of Sport), professional clubs (e.g., Manchester City Football Club) or national federations (e.g., France Rugby), and private gyms (e.g., The Altitude Centre) are now equipped with altitude simulation facilities. However, innovative LLTH methods such as resistance training in hypoxia can also be implemented in high performance centers located at moderate natural altitude ranging 1,800-2,400 m (e.g., Font Romeu in France or Sierra Nevada in Spain). In testing the viability of using mean propulsive velocity to adjust the load in the countermovement jump at a terrestrial altitude of 2,320 m, Rodríguez-Zamora et al. indicated that power-oriented exercises using intermittent hypobaric hypoxia allow athletes to lift higher loads, also evoking higher ratings of perceived exertion than at sea level.

MOVING FORWARD

Although an extensive number of LLTH studies have been published so far, several issues regarding the implementation of many innovative methods described in the updated panorama that we proposed (Girard et al.) and their physiological and performance consequences remain unresolved. The effectiveness of pre-acclimatization strategies for high altitude exposure should be explored [START_REF] Fulco | Effectiveness of preacclimatization strategies for high-altitude exposure[END_REF]. Moreover, very few studies have considered the de-acclimatization period or how LLTH methods can be used to extend the benefits associated with chronic hypoxia [START_REF] Hamlin | Hypoxic repeat sprint training improves rugby player's repeated sprint but not endurance performance[END_REF] and determined how various normobaric and hypobaric hypoxic methods can be best combined [e.g., live high train low and high; [START_REF] Brocherie | Live High-Train Low and High" hypoxic training improves team-sport performance[END_REF]]. Finally, some practitioners are starting to include hypoxic conditioning/rehabilitation for injured athletes or patients who cannot tolerate the mechanical stress associated with higher-intensity exercise [START_REF] Girard | Walking in hypoxia: an efficient treatment to lessen mechanical constraints and improve health in obese individuals?[END_REF]. Future studies are needed to refine the exercise characteristics and hypoxic dose needed to reduce the load while achieving a desired physiological response.

  . Our understanding of successful LLTH altitude training methods for athletes has significantly developed over the last decade (McLean et al., 2014). New technological advancements have prompted the development of innovative LLTH interventions, as summarized in an updated panorama of LLTH altitude training methods Girard et al..

'Live Low -Train High' Interventions

Frontiers in Sports and Active Living | www.frontiersin.org

August 2020 | Volume 2 | Article 108

AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.