Optimization of Carob Products Preparation for Targeted LC-MS/MS Metabolomics Analysis

Carob (Ceratonia siliqua) is an exceptional source of significant bioactive compounds with great economic importance in the Mediterranean region, where it is widely cultivated. Carob fruit is used for the production of a variety of products and commodities such as powder, syrup, coffee, flour, cakes, and beverages. There is growing evidence of the beneficial effects of carob and the products made from it on a range of health problems. Therefore, metabolomics could be used to explore the nutrient-rich compounds of carob. Sample preparation is a crucial step in metabolomics-based analysis and has a great impact on the quality of the data obtained. Herein, sample preparation of carob syrup and powder was optimized, to enable highly efficient metabolomics-based HILIC-MS/MS analysis. Pooled powder and syrup samples were extracted under different conditions by adjusting pH, solvent type, and sample weight to solvent volume ratio (Wc/Vs). The metabolomics profiles obtained were evaluated using the established criteria of total area and number of maxima. It was observed that the Wc/Vs ratio of 1:2 resulted in the highest number of metabolites, regardless of solvent type or pH. Aqueous acetonitrile with a Wc/Vs ratio of 1:2 satisfied all established criteria for both carob syrup and powder samples. However, when the pH was adjusted, basic aqueous propanol 1:2 Wc/Vs and acidic aqueous acetonitrile 1:2 Wc/Vs provided the best results for syrup and powder, respectively. We strongly believe that the current study could support the standardization of the metabolomics sample preparation process to enable more efficient LC-MS/MS carob analysis.


Introduction
The carob tree has been cultivated in the Mediterranean region for centuries, making an important contribution to the society and economy of many European (e.g., Spain, Italy, Portugal, Greece, Cyprus, etc.) and Middle Eastern countries (e.g., Egypt, Tunisia, Morocco, etc.). Its popularity is mainly due to its excellent nutritional and medicinal properties, as it is considered a functional food associated with the Mediterranean diet [1]. In the last decade, the interest in the sustainability and valorization of the carob tree (fruits, pods, and leaves) has increased beyond its unique agricultural importance as a fire-and drought-resistant tree [2]. Therefore, in modern societies, where the circular economy and climate change are gaining emerging attention, the potential contribution of the carob tree to the United Nations Sustainable Development Goals (UN SDGs) is driven by the promotion of social (no poverty), health (well-being), and ecological (restoration and support of terrestrial ecosystems) balance; as a nutraceutical product, it supports UN SDGs 1-3, 5, 12, and 15 [3]. results even though operators occasionally underestimate this step [18]. The present work aims to determine the optimal protocol for the analysis of two carob matrices, namely syrup (liquid) and powder (solid), in order to identify the largest number of metabolites; towards this, the effects of extraction, pH adjustment, solvent, and sample weight to solvent volume ratio (Wc/Vs) were examined. To our knowledge, there is no other work on sample optimization of carob products using HILIC-MS/MS. The obtained extracts were combined, the EtOH was removed, and the residual aqueous phase was frozen and lyophilized.
The UAE was carried out in an ultrasonic device: 3 g dried powdered samples extracted with 100 mL of each of the four solvents by the ultrasonic device at 375 W for 10 min + filtration and, as for the ME, the EtOH was removed, and the residual aqueous phase was frozen and lyophilized. Enzymes of carob flours (1 g) were inactivated by boiling in H 2 O for 5 min. The slush was filtered through ten layers of gauze and the resultant liquid adjusted to pH 6.0 with NaOH, and then lyophilized.
1 g of dry mass extracted with 10 mL of boiling water for 5 min + centrifugation + filtration Dietary fiber, total phenols, pinitol and antioxidant activity [24] Comparison of the sugar levels in wheat flour and wholemeal wheat flour plant-based high-protein ingredients, e.g., carob high-protein ingredients (HPIs)
Syringe Terumo 2.5 mL (Tokyo, Japan) was used, and PTFE filters 0.22 µm were obtained from Millex-Merk (Darmdtsdt, Germany). The Misonix XL Sonicator Ultrasonic Cell Processor (Farmingdale, NY, USA) equipment and the CyberScan 1000 (Eutech instruments PTE LTD, Singapore) pH meter were used. Vortex-mixing and centrifugation were performed on an IKA Ms1 Mini Shaker Laboratory Vortex (Staufen, Germany) and on a Micro Centaur Plus, MSE (London, UK) centrifuge, respectively. The samples included Cypriot carob powder (n = 4) and syrup (n = 6) commercial products purchased from a local market in Cyprus. , adjustment of pH value (acidic, neutral, basic) was accessed by the addition of formic acid, ammonium formate, or ammonia, respectively. The measurement of pH value was performed for all aqueous solvent-mix extracts. Every mixture was vortex-mixed for 1 min followed by sonication for 15 min and centrifugation for 20 min at 4 • C (10,000× g). The obtained clear supernatants were filtered through PTFE 0.22 µm syringe filters and QC samples were prepared from filtrates to evaluate the system's analytical performance. One hundred and fifty microliters were evaporated to dryness under nitrogen stream, resuspended with 150 µL of the mobile phase, and finally transferred into 2 mL autosampler glass vials equipped with 200 µL microinserts, before being subjected to targeted LC-MS/MS analysis. The illustration of the sample preparation process is presented in Figure 1.

LC-MS/MS Analysis
As described in previous publications [29,30], carob extracts were analysed using a previously developed and validated targeted LC-MS/MS method performed in an ACQUITY UPLC H-Class chromatography system coupled to a Xevo TQD mass spectrometer (Waters Corporation, Milford, MA, USA), operating in both positive and negative mode [31]. The method includes 80 MRM channels for small polar metabolites. Briefly, the column was an Acquity BEH Amide (2.1 mm × 150 mm, 1. MS parameters were set as followed: capillary voltage: ±3500 V, desolvation temperature: 350 • C, desolvation flow: 650 L/h, and cone gas flow: 50 L/h. Cone voltage and collision energy were optimized for each analyte. A Quality Control (QC) sample was used throughout the analytical batches. QC samples were prepared by mixing equal volumes of all tested samples, for the respective analysis, either syrup or powder.. QC samples were analyzed 5 times at the beginning of the analytical batch, for system equilibration. Also, a standard mixture containing all analytes of interest was injected in the beginning of the analytical run. Indicative chromatograms of a real samples (syrup and powder products) were illustrated in Figure S1 (supplementary information).

LC-MS/MS Analysis
As described in previous publications [29,30], carob extracts were analysed using a previously developed and validated targeted LC-MS/MS method performed in an ACQUITY UPLC H-Class chromatography system coupled to a Xevo TQD mass spectrometer (Waters Corporation, Milford, MA, USA), operating in both positive and negative mode [31]. The method includes 80 MRM channels for small polar metabolites. Briefly, the column was an Acquity BEH Amide (2.1 mm × 150 mm, 1.7 µm), equipped with an Acquity UPLC Van-Guard pre-column (Waters, UK). The mobile phase consisted of (a) ACN:H2O 95:5 v/v and (b) ACN:H2O 30:70 v/v, both containing 10 mM ammonium formate.
MS parameters were set as followed: capillary voltage: ±3500 V, desolvation temperature: 350 °C, desolvation flow: 650 L/h, and cone gas flow: 50 L/h. Cone voltage and collision energy were optimized for each analyte.
A Quality Control (QC) sample was used throughout the analytical batches. QC samples were prepared by mixing equal volumes of all tested samples, for the respective analysis, either syrup or powder.. QC samples were analyzed 5 times at the beginning of the analytical batch, for system equilibration. Also, a standard mixture containing all analytes of interest was injected in the beginning of the analytical run. Indicative chromatograms of a real samples (syrup and powder products) were illustrated in Figure  S1 (supplementary information).
Regarding method validation, linearity of the method was determined using different calibration standards per analyte, ranging between 0.01-2 mg/L up to 5-95 mg/L, depending on the analyte. Intra-day precision ranged between 0.5-7% for syrup samples and between 0.4-4% for carob powder samples.

Data Handling-Statistics
LC-MS/MS data were collected and processed using MassLynx ® (Waters, Milford, Regarding method validation, linearity of the method was determined using different calibration standards per analyte, ranging between 0.01-2 mg/L up to 5-95 mg/L, depending on the analyte. Intra-day precision ranged between 0.5-7% for syrup samples and between 0.4-4% for carob powder samples.
Analytes for further statistical evaluation were selected based on the criteria of either existing in the 60% of the samples analyzed or presenting a relative standard deviation (RSD) < 30% in QC samples.
The selection of the optimized sample preparation protocol for either powder or syrup carob samples was based on the number of extracted peaks, the total area of maxima, classification of metabolites, and standard deviation of replicates. Microsoft Excel tools were used for illustration of results, while SIMCA 13.0 (Umetrics, Umea, Sweden) was used for the constructed principal component analysis (PCA) score plot in Unit Variance (UV) scaling.

Results and Discussion
In total, two replicates of the 20 different prepared carob samples of each matrix were analysed and further assessed the worth of commonly used organic solvents and their respective aqueous mixtures in two extraction ratios (Wc/Vs), as well as in three pH values. The aim was to define the optimal protocol for the analysis of both matrices, in order to fully cover the extracted metabolites. Sample preparation, a fundamental process prior to the analysis, plays a crucial role in the quality of the obtained results and the robustness of the methodology [30]. Carob and carob products are considered challenging matrices due to the physicochemical characteristics of raw carob fruit and processed carob products.
The criteria of the optimal Wc/Vs ratio for both analysed matrices were the total area (sum of peak areas) and the number of maxima (higher peak areas) [30]. Based on both criteria, for both carob products, the 1:2 Wc/Vs ratio was selected as it provided higher intensities and peak areas, as expected. Neither deterioration of the analytical system nor saturation of the detector (as possible obstacles) were observed for the dense extracts; thus, the last was chosen.
For the solvent selection in the 1:2 Wc/Vs ratio resulting from the previous step, acetone, ACN, MeOH, PropOH, ACN:H 2 O, MeOH:H 2 O, and PropOH:H 2 O were tested. The aforementioned solvents and solvent mixtures are easy to use, commonly available in analytical laboratories, less toxic, easy to evaporate, and suitable for one-step global analysis and for the extraction of polar and semi-polar analytes providing reproducible results. The aqueous organic solvents were evaluated to achieve enhanced extraction recovery of the metabolites. As illustrated in the bar plots for carob syrup (Figure 2), aqueous ACN 1:2 Wc/Vs presented the highest total area, followed by aqueous MeOH and aqueous PropOH, by an infinitesimal difference. The highest total peak area was also attributed to the highest number of maxima for aqueous ACN 1:2 Wc/Vs. Interestingly, while aqueous MeOH showed a higher total peak area compared to aqueous PropOH 1:2 Wc/Vs, the number of maxima presented the opposite trend. For carob powder, in Figure 3 it is observed that aqueous ACN 1:2 Wc/Vs presented the highest total peak area, as expected, due to the very large number of maxima. Although neat and aqueous MeOH 1:2 Wc/Vs and aqueous PropOH 1:2 Wc/Vs showed similar total peak areas, the aqueous PropOH 1:2 Wc/Vs was, notably, the only extraction solvent mixture that presented maximum peak areas. In both carob products, neat organic solvents indicated lower peak area values and a minor number of maximum peaks, compared to their respective aqueous mixtures. As expected, polar and semi-polar compounds were favorably extracted in the presence of an aqueous amount in the solvent mixture.   The last step for the sample optimization protocol was the pH evaluation in aqueous solvent mixtures. Carob is considered an acidic product, with a pH value close to 6.5, while the pH value of both tested products also ranged between 4.4 and 5.5 [32,33]. Thus, it was an interesting point to investigate, whereas pH adjustment of solvent mixtures would affect metabolites extraction of the acidic matrices. For syrup samples, independent of the aqueous extraction solvent, basic pH demonstrated higher total peak areas, as it probably favors the measured metabolites. Basic aqueous PropOH 1:2 Wc/Vs was the first choice based on both total peak area and number of metabolomic maxima (n = 14), followed by basic aqueous ACN 1:2 Wc/Vs (n = 7) and basic aqueous MeOH 1:2 Wc/Vs (n = 6).
Neutral pH conditions for all tested aqueous mixture solvents illustrated comparable results (Figure 4).  The last step for the sample optimization protocol was the pH evaluation in aqueous solvent mixtures. Carob is considered an acidic product, with a pH value close to 6.5, while the pH value of both tested products also ranged between 4.4 and 5.5 [32,33]. Thus, it was an interesting point to investigate, whereas pH adjustment of solvent mixtures would affect metabolites extraction of the acidic matrices. For syrup samples, independent of the aqueous extraction solvent, basic pH demonstrated higher total peak areas, as it probably favors the measured metabolites. Basic aqueous PropOH 1:2 Wc/Vs was the first choice based on both total peak area and number of metabolomic maxima (n = 14), followed by basic aqueous ACN 1:2 Wc/Vs (n = 7) and basic aqueous MeOH 1:2 Wc/Vs (n = 6). Neutral pH conditions for all tested aqueous mixture solvents illustrated comparable results (Figure 4).  In different pH conditions, aqueous ACN was the optimal extraction solvent mixture to extract carob powder metabolites for both assessed criteria ( Figure 5). Infinitesimal differences were observed among different aqueous ACN pH conditions, with acidic having a slight predominance. A similar trend was also observed between aqueous PropOH and aqueous MeOH pH conditions, which showed satisfactory total peak areas. In the same manner, for aqueous MeOH, the number of maxima was not obtained. In the constructed PCA score plot a clear separation of the matrices was observed ( Figure 6). Furthermore, the analysed samples were clustered by solvent mixture and propanol showed the lowest deviation in both matrices. Although a satisfying separation was observed, the effect of pH was milder compared to the solvent nature, as expected, since the solvent is determining factor in the sample preparation process. Validity of the constructed PCA scores plot model was based on the R2X and Q2 values. R2X was 0.731 and Q2 was 0.629, while CV-ANOVA was <0.05. In different pH conditions, aqueous ACN was the optimal extraction solvent mixture to extract carob powder metabolites for both assessed criteria ( Figure 5). Infinitesimal differences were observed among different aqueous ACN pH conditions, with acidic having a slight predominance. A similar trend was also observed between aqueous PropOH and aqueous MeOH pH conditions, which showed satisfactory total peak areas. In the same manner, for aqueous MeOH, the number of maxima was not obtained. In the constructed PCA score plot a clear separation of the matrices was observed ( Figure 6). Furthermore, the analysed samples were clustered by solvent mixture and propanol showed the lowest deviation in both matrices. Although a satisfying separation was observed, the effect of pH was milder compared to the solvent nature, as expected, since the solvent is determining factor in the sample preparation process. Validity of the constructed PCA scores plot model having a slight predominance. A similar trend was also observed between aqueous PropOH and aqueous MeOH pH conditions, which showed satisfactory total peak areas. In the same manner, for aqueous MeOH, the number of maxima was not obtained. In the constructed PCA score plot a clear separation of the matrices was observed ( Figure 6). Furthermore, the analysed samples were clustered by solvent mixture and propanol showed the lowest deviation in both matrices. Although a satisfying separation was observed, the effect of pH was milder compared to the solvent nature, as expected, since the solvent is determining factor in the sample preparation process. Validity of the constructed PCA scores plot model was based on the R2X and Q2 values. R2X was 0.731 and Q2 was 0.629, while CV-ANOVA was <0.05.  From the 80 metabolites included in the targeted metabolomics-based method, the detected metabolites in each carob product were categorized into eight classes, according to their chemical taxonomy (Table 2), based on Human Metabolome Database (HMDB) [34]. Table 2. Chemical categorization for all detected analytes in carob syrup and carob powder.
In syrup, 32 compounds were not detected, while 16 metabolites namely adenine, creatine, creatinine, cytidine, kynurenate, maltose, nicotinic acid, putrescine, pyruvic acid, serine, taurine, theobromine, threonine, tyrosine, xanthine, and γ-aminobutyric acid, did not satisfy the aforementioned criteria. The highest number of compounds were met in 5 tested conditions, namely basic aqueous PropOH, PropOH, MeOH, neutral aqueous MeOH, and neutral aqueous ACN, while the lowest was met in ACN. The majority of the extracted analytes belonged to organooxygen compounds carbohydrates and carbohydrate conjugates, followed by carboxyl acids and derivatives, amino acids, peptides, and analogs, both classes of high biological significance. Notably, cytidine was only extracted in neutral aqueous ACN protocol, while nicotinic acid was favorably extracted in neutral aqueous PropOH. Caffeine was not detected since carob is a non-caffeine product [6]. To our surprise, glycine, isoleucine, arginine, tryptophan, aspartic acid, methionine, glutamic acid, lysine, and histidine were not detected or were under the limit of detection (LOD) for the applied method, although it was expected to be present in carob syrup. A possible explanation may be attributed to the thermal process of carob syrup production [35]. The total numbers of detected metabolites in syrup, based on the criteria described in Section 2.4, is summarized in Table 3.
A greater number of compounds were detected in carob powder samples compared to syrup. Forty-three out of 80 analytes were excluded according to the discussed criteria. Neutral aqueous ACN favored the extraction of most analytes, while neat ACN solvent extracted the least. Guanine was only extracted with aqueous PropOH but was further excluded from statistical evaluation. As illustrated in Table 4, most of the extracted analytes belonged to the same chemical categorization.   This study attempted to provide an optimal sample preparation protocol for carob products. However, a single extraction sample preparation process for such diverse molecules with different physicochemical properties, is considered a challenging task. All criteria selected for the optimal conditions, number of extracted peaks, number of maxima, total area, and standard deviation of replicates, were chosen to provide a more global and, at the same time, selective approach limited for the specific carob analytes.
Solvent evaporation and reconstitution in the mobile phase in the sample preparation protocol were performed to secure comparable results among the studied methodologies. The optimal extraction sample weight to solvent volume ratio was selected upon the set criteria, although the high density of the obtained extract could affect the analytical system's performance (detector saturation, analytical column, source contamination, peak overlap). In an analysis of a large number of samples this should be taken into consideration.

Conclusions
Carob syrup and carob powder sample preparation were studied for the optimal extraction of nutrient polar and semi-polar metabolites using HILIC-MS/MS. To our knowledge, this is the first attempt where various parameters, namely the number of metabolites, the effects of extraction, pH adjustment, solvent, and sample weight to solvent volume ratio (Wc/Vs) were examined with the aim of polar profiling the two different carob matrices. Aqueous acetonitrile at 1:2 Wc/Vs satisfied the established criteria for both carob syrup and powder samples. Nevertheless, when the pH was modified, a ratio of 1:2 Wc/Vs in basic pH using aqueous PropOH as the extraction solvent presented the optimal results for syrup analysis, while for carob powder analysis, acidic aqueous acetonitrile 1:2 Wc/Vs would be the best possible choice to obtain a satisfactory number of metabolites and extraction recovery.
The current study suggests an optimal sample preparation protocol focused on small polar and semi-polar metabolites appropriate for HILIC-MS/MS analysis. The optimal process should be adapted to the specific needs and intentions of each study; thus, the suggested protocol should not be considered a universal approach. The optimal pa-rameters offered either a global approach or a more selective one, for the extraction of specific metabolites.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/metabo13050645/s1, Figure S1: Chromatograms of uridine, nicotinamide, trehalose, choline, betaine and benzoic acid of powder (A) and a syrup (B) products, after the extraction with the respective optimal conditions.