This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Article Cite This: ACS Omega 2019, 4, 19710−19715 http://pubs.acs.org/journal/acsodf Development and Validation of a New High-Performance Liquid Chromatography Method for the Simultaneous Quantification of Coenzyme Q10, Phosphatidylserine, and Vitamin C from a Cutting- Edge Liposomal Vehiculization Marta Ruiz-Garcia,†,‡,§ Pilar Peŕ ez-Lozano,*,†,∥ Deb́ ora Mercade-́ Frutoś ,† Anna Nardi-Ricart,† Marc Suñe-́ Pou,†,∥ Mary Cano-Sarabia,‡ Sonia Garcia-Jimeno,‡ Josep M. Suñe-́ Negre,†,∥ Daniel Maspoch,‡,⊥ and Encarna García-Montoya†,∥ † See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Pharmacy, Pharmaceutical Technology and Physicochemical Department, Faculty of Pharmacy, University of Barcelona, Barcelona 08028, Spain ‡ Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, Bellaterra 08193, Spain § Downloaded via 95.169.239.196 on May 20, 2020 at 11:49:15 (UTC). Vitae Health Innovation S.L., Barcelona 08160, Spain ∥ Pharmacotheraphy, Pharmacogenetics and Pharmaceutical Technology Research Group, IDIBELL-UB, Bellvitge Hospital, Hospitalet de Llobregat, Barcelona 08908, Spain ⊥ Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain * S Supporting Information ABSTRACT: A high-performance liquid chromatography (HPLC) method was developed to simultaneously quantify three widely used active substances such as coenzyme Q10, phosphatidylserine, and vitamin C. This new method optimizes current timing and costs in the analyses of these three active substances. Additionally, since the analyzed compounds were encapsulated on a cutting-edge liposomal formulation, further processing was necessary to be developed prior to HPLC analyses. The technique was studied and adequately validated in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) regarding selectivity, linearity, accuracy, precision, and robustness. After data treatment of results, linear regressions for all active substances showed an optimal linearity with a correlation coefficient of >0.999 in the concentration range between 70 to 130% of the liposomal formulation and less than a 3% relative standard deviation (RSD) in accuracy and precision. ■ INTRODUCTION During the past years, science is accomplishing huge advances membrane, while hydrophilic actives can also be encapsulated in its aqueous core. Among many other benefits, this has in drug discovery through modeling and synthesis toward the converted them in an extensively used and very versatile drug treatment of idiopathic diseases.1−4 However, even if delivery technology.8−12 promising molecules are identified and synthesized, its Herein, we describe a novel method of analysis for the effectiveness will be reduced or equal to zero if they are simultaneous quantification of three coencapsulated active unable to achieve the therapeutic target because of a poor substances with very similar characteristics of hydrophobicity absorption. It is estimated that, nowadays, 90% of the in a liposomal matrix, and also, we explain the pretreatment developed molecules on investigation are chemically lipophilic procedure for these liposomal samples prior to analyses. drugs or, in other words, insoluble drugs in water and unable Specifically, the coanalyzed active substances are the coenzyme to cross physiological barriers on oral administration.5,6 Q10 (CoQ10), 6-O-palmitoyl-L-ascorbic acid (the lipid-soluble To enhance drug bioavailability of lipophilic actives, the vitamin C), and phosphatidylserine (PS). These active liposomal technology of encapsulation has been a broadly substances are widely used in both basic research and in the reported vehicle during the past decades on the literature. Discovered by Bangham and Horne in 1964,7 this lipid Received: August 2, 2019 spherical structure allows the insoluble active substances in Accepted: October 10, 2019 water to get intertwined with the forming phospholipids of the Published: November 11, 2019 © 2019 American Chemical Society 19710 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715 ACS Omega Article Figure 1. Chemical structure of the analyzed compounds. (A) PS. (B) Vitamin C. (C) CoQ10. pharmaceutical, cosmetics, and food industry, thus having a strategy, and the RSD after the analyses of the all the vials was high interest and also a huge impact in the economic ambit. In 2.35% for CoQ10, 2.14% for vitamin C, and 2.74% for PS, thus the past 10 years, 5651 articles related with CoQ10 were showing optimal results of repeatability. published in scientific journals, 6969 articles related to PS, and Method Development. The designed HPLC method was 46,079 articles related with vitamin C, all of them with an meant to properly analyze the content of CoQ10, vitamin C, exponential tendency of increase on each year.13 Although and PS from a liposomal formulation. First, we assayed many articles have been published describing analytical different mixtures and proportions between methanol, methodologies of each substance in separate, to the best of isopropanol, acetonitrile, and water buffer. To choose an our knowledge, there is no published method offering the adequate buffer, we looked out to the pKa of the three simultaneous analysis of the three active substances, and even substances and selected buffer at a pH where all compounds less coencapsulated within the liposomal technology and the would be in an undissociated form, such as 0.02 M NH4AcO at additional elements, this means to be considered upon analysis. pH = 6.5. Figure 1 shows the molecular structure of every To the proper determination and quantification of the active active substance, while its corresponding pKa, partition substances, it is important to have clear absorption peaks, so coefficient, and molecular weight are shown in Table 1. The we can extrapolate the areas to the actual amount we have of each active substance in the preparation. Since the chemical Table 1. Chemical Properties To Be Considered for the nature of all the three actives is very similar, it is actually very Design of the Method15−17 tricky to elucidate the compounds in separated peaks and parameter PS vitamin C CoQ10 avoid them to overlap. Additionally, both coenzyme Q10 and vitamin C absorb at a wavelength of 275 nm, and all the three log P 10.55 6 10 substances absorb at 200 nm wavelength as well, thus making it MW (g/mol) 810.025 414.539 863.365 absolutely necessary to develop a methodology to separate the pKa 9.38; 1.47 4.36; −3 −4.7 peaks for its quantification. With combinations of the actives in separate, just one article of simultaneous analysis has been first chromatographic meticulously tested conditions showed published, reported by Temova-Rakuša et al.,14 with the us that for a favorable elution of CoQ10, a 100% flow of pure coanalysis of coenzyme Q10 and vitamin C. This article methanol or isopropanol is needed, and any mixture with water provides an interesting method for their quantification but not made its elution impossible with an optimal peak shape. On valid for the analysis of phosphatidylserine. In the Supporting the other hand, for the analysis of vitamin C, a mixture of Information (Figure S1), we attach a chromatogram of this organic solvent and water was needed for its proper elution, reported method with our three active substances, where any and PS was not eluted in any isocratic condition with an absorption peak can be observed for our third active substance, optimal peak shape, considering theoretical plate numbers (N) the phosphatidylserine, meaning that it is not a suitable and USP tailing factor. analytical method for this substance or for the simultaneous At this point of development, a gradient of NH4AcO buffer quantification of the three active substances. Therefore, our with isopropanol was extensively investigated at different method could be considered as a unique and interesting proportions and with different gradient duration times until we contribution with applications in pharmaceutical/cosmetic/ found an adequate combination that allowed the optimal food industry. elution of all the compounds during the same analysis. An After the design and development of the method, a complete example of the obtained chromatogram after last optimization validation study was meticulously accomplished following the is shown in Figure 2. ICH quality guidelines. It was also used and proved in different Method Validation. Before carrying through the vali- real circumstances such as long-term stability studies at dation assays, the suitability of the equipment with the method different conditions. ■ was tested. Table 2 shows RSD of retention times (RT), symmetry of the peak, USP tailing factor, capacity factor, and RESULTS AND DISCUSSION N, demonstrating consistency and appropriateness. The Sample Preparation Strategy. After some assays, results tabulated retention times of the six analyzed samples and demonstrated that it is necessary to remove the water from the symmetry analyses on each peak of the active substances can samples to allow the elution of substances at the chromato- be found in the Supporting Information (Scheme S2 and gram and to successfully analyze and quantify the peaks of the Figures S3−S5). active substances. First, with any water in the sample, it was After checking the suitability of the equipment and before almost impossible to correctly solve all the lipids. Second, starting any other validation step, it was necessary to being hydrophobic active substances with water, the peaks understand for how long samples are stable above at least were not eluting in a satisfactory peak form or were not eluting 95% of all active substances. It was determined that, to at all. Freeze-drying of samples was carried out. Afterwards, accomplish this requirement, liposomal samples could be twenty different lyophilized vials with the liposomal injected up to 3 h after its preparation in the HPLC vials formulation were injected and analyzed with the developed without significant degradation of any sample, while for the HPLC method to test the accuracy of the sample preparation standard solution, the time was shortened to 1 h. So, it is 19711 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715 ACS Omega Article Figure 2. Chromatographic exemplification of the developed method with the three analyzed active substances. The peak with an RT of 12.754 min corresponds to vitamin C, an RT of 14.892 min corresponds to PS, and an RT of 17.714 corresponds to CoQ10. The red line is λ = 275 nm, while the blue line is λ = 200 nm. Table 2. Suitability of the Equipment and Data Analyses parameter PS vitamin C CoQ10 retention times (min) 14.86 12.72 17.73 retention times (RSD %) 0.09 0.25 0.14 symmetry of peak 1.01 0.73 0.89 USP tailing factor 1.22 1.23 1.08 capacity factor 3.08 2.49 3.85 plate numbers (N) 40,891 33,327 67,904 highly recommended to analyze samples right after its solubilization in organic solvents and preparation for HPLC quantification. The precision of the system was also checked before any other validation step. After five consecutive injections of a 100% concentration of PS, the area scattered only in a 0.59%, being inside our acceptance criteria of ≤1.0%, thus confirming its reliability (see the Supporting Information). Selectivity was confirmed by overlapping the chromatograms of an analyzed sample with a standard solution and a third one with the rest of the substances (Figure 3). Additionally, the purity factor of peaks was studied. In all cases, purity was above the threshold limit, set at 990.000 (see the Supporting Information, Table S6 and FiguresS7 and S8). Through analyzing all seven concentrations between 130 and 70% of the standard solution, results demonstrated an adequate linearity in this concentration range. Data is summarized on Table 3, with RSD and correlation coefficients accomplishing our established limits. Table 3 also shows data regarding precision and accuracy. For the precision of the method evaluated in different days, we established a 2.8% RSD and a 3.7% for repeatability (intra- laboratory). Regarding accuracy, every recovery percentage after the study was included in our quality specifications. All Figure 3. Representative chromatograms. (A) Standard solution. (B) this data demonstrated excellent results with respect to the Mixture of the other lipids: PC, Chol, and Chol-PEG. (C) Liposomal validation of the method and indicated that this method is sample. precise and accurate. Table 4 details the percentage of recovery on the nine parameter resulted in some degree of retention time differ- analyses at different concentrations for evaluating accuracy. ences or peak areas. Every concentration resulted in less than a 2% RSD, and Table 5 indicates which parameter showed significant or ANOVA test showed nonsignificant differences on any active nonsignificant differences after deliberated changes upon its substance at any concentration. analysis with a one-way ANOVA test. On this table, although Lastly, robustness studies revealed that the method must be significant differences were shown in many of the deliberated controlled in terms of temperature, injection volume, wave- changes, RSD values were not in any case over 5% but with length detection, and flow since otherwise results could slightly wavelength for PS. Since λ = 200 nm is a wavelength where variate. Being a method with three analyzed compounds, each also solvents have certain absorbance, it is a critical parameter 19712 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715 ACS Omega Article Table 3. Summarized Data of Linearity, Precision, and Accuracy during the Validation of the Method linearity precision accuracy range correlation RF RSD standard solution, different days, RSD different analysts, recovery (%, max and min analytes (mg/mL) coefficient (%) RSD (%) (%) RSD (%) values) PS 1.008−0.543 0.999 1.82 1.97 2.80 3.18 101.35−98.25 CoQ10 0.312−0.168 0.999 1.01 0.97 2.45 1.89 101.92−98.30 vitamin C 0.260−0.140 1.000 0.89 1.96 2.80 1.89 101.89−98.28 Table 4. Detailed Results of Studying the Accuracy Table 7. Results of Stability Studies with the Developed Method after 3 Months parameter PS vitamin C CoQ10 recovery at 130% 100.21 ± 1.61 101.20 ± 0.93 100.21 ± 1.82 temperature condition PS vitamin C CoQ10 (%) 4 °C (%) 100.09 ± 0.31 95.27 ± 0.58 97.56 ± 0.20 recovery at 100% 98.42 ± 0.23 100.28 ± 1.83 100.17 ± 1.01 25 °C (%) 96.19 ± 0.22 85.57 ± 0.15 87.27 ± 0.23 (%) recovery at 70% (%) 98.48 ± 1.26 100.98 ± 0.80 99.71 ± 0.93 ANOVA test 0.98 0.67 0.88 method for the cosmetics, pharmaceutical, and food supple- (p-value) ment industry. Table 5. Summarized Statistics from ANOVA Test on Additionally, the formulation involving the liposomal Robustnessa technology may be a tricky challenge to allow a correct quantification and analyses, especially for very lipophilic active PS vitamin C CoQ10 substances to which the presence of water can signify a RSD RSD RSD complicated limitation for the analyses. The performed parameter p-value (%) p-value (%) p-value (%) methodology of sample treatment prior to its analyses may temperature 0.239 1.72 >0.01* 4.47 >0.01* 1.35 be as well an additional step of valuable interest. The concept injection 0.225 3.34 0.792 1.15 0.032* 0.70 may be applied to any other possible liposomal formulations volume with highly hydrophobic active substances. wavelength >0.01* 14.88 0.013* 1.85 0.135 0.75 In terms of validation of the method, following the ICH flow 0.695 2.17 >0.01* 7.22 0.341 0.92 guidelines, the suitability of the equipment, selectivity of the a * indicates that there are significant statistical differences. method, linearity, robustness, precision, accuracy, and stability of the solution during the analyses were tested with positive to be controlled. Specifically, a two-tailed T-student test was and encouraging results. carried out to compare wavelengths 197 nm versus 200 nm Lastly, with the purpose of verifying the applicability of the and 200 nm versus 203 nm. The first T-test contained highly method, a testing of the method with a real case was carried significant differences with a p−value of >0.01, but non- out. Samples subjected to stability studies were analyzed, and results were studied and interpreted, with excellent results. ■ significant differences were observed with a p−value of 0.80 between 200 and 203 nm, thus being 203 nm also a suitable wavelength to be used. EXPERIMENTAL SECTION Based on the standard deviation of the response and the Materials and Chemical Reagents. 6-O-Palmitoyl-L- slope, based on the calibration curve, LOD and LOQ were ascorbic acid (vitamin C; analytical standard, ≥99.0%), calculated for three analytes. Results are presented in Table 6. cholesterol-(polyethylene glycol-600) (Chol-PEG), and cho- lesterol (Chol) were obtained from Sigma-Aldrich (Darmstadt, Table 6. LOD and LOQ Results Germany). 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (phosphatidylserine or PS; analytical standard, ≥99.0%) parameter PS vitamin C CoQ10 and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (phosphati- LOD (mg/mL) 0.082 0.018 0.021 dylcholine or PC) were purchased from Lipoid GmbH LOQ (mg/mL) 0.249 0.054 0.063 (Switzerland). Coenzyme Q10 (CoQ10; United States Pharmacopeia (USP) reference standard quality, ≥99.0%) Applications of the Method. Once validated, the was obtained from Vitae Health Innovation S.L (Spain). proposed method was used to accurately evaluate the stability Ammonium acetate (NH4AcO) was acquired from Panreac of the three active substances encapsulated on the mentioned Quimica (Barcelona, Spain). liposomal formula after 3 months of storage at 4 and 25 °C. Instrumentation. Analyses were carried out with an This study verifies the applicability of the method in a real case HPLC system model 1100 HP from Agilent (Agilent study. Results are presented in Table 7, while an illustrative Technologies, Santa Clara, EEUU), composed of a quaternary chromatogram of each temperature is included in the pump, an automatic injector, a diode array detector, and a Supporting Information (Figures S9 and S10). ■ column oven where our column was placed, all controlled by a ChemStation software version B04.02. For the freeze-drying of CONCLUSIONS samples, a Telstar Cryodos−50 lyophilizator was used. The developed method demonstrated promising results on the Preparation of Liposomes. Liposomal formulations were simultaneous analyses of CoQ10, vitamin C, and PS. To the prepared using the thin-film hydration method with some best of our knowledge, this is the first reported method for the modifications.18 Briefly, PC, PS, Chol, Chol-PEG, and CoQ10 determination of these widely used active substances at the were dissolved in chloroform solutions (100 mg/mL) or same time. Thus, this method could result in an ideal analytic chloroform/ethanol 7:3 for vitamin C and mixed afterward at 19713 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715 ACS Omega Article the desired molar. In this case, the molar concentration was 79 Table 8. Detailed Gradient of the Developed Method mM, with 30.4% PS, 22.8% PC, 19.0% Chol, 15.3% vitamin C, time (min) solvent B (%) solvent A (%) 8.8% CoQ10, and 3.8% Chol-PEG. The organic solvent was removed under vacuum and nitrogen to afford a dry lipid film, 0 60 40 which was hydrated with an aqueous solution under vigorous 5 60 40 stirring for 15 min at 40 °C. Under these conditions, the stacks 10 0 100 of liquid crystalline lipid bilayers become fluid and swell, 18 0 100 resulting in their detachment during agitation and their self- 19 60 40 closure to form multilamellar large vesicles (MLVs). Small 28 60 40 unilamellar vesicles (SUVs) were obtained by homogenizing the MLV suspension using an extruder (Lipex Biomembranes, Canada) and a polycarbonate membrane (pore size, 200 nm). Sample Preparation Strategy. The treatment and A gradient method was developed and is detailed in Table 8. preparation of samples prior to HPLC analyses were essential Solvent A in the table is isopropanol, while Solvent B is a buffer for allowing the quantification of the lipidic active substances. solution of 0.02 M NH4AcO. Liposomal formulations have by definition the characteristic of Method Validation. The development of the method was including both water and water-insoluble compounds, and this followed by validation studies to ensure selectivity, linearity, could make any posterior analyses difficult. Since the presence robustness, precision, stability of the solution during the of water is a very unfavorable condition to dissolve and allow analyses, and accuracy of the method, following the ICH the flow of the substances through the column in an guidelines.19 Prior to any validation test, six injections of a appropriate way, a procedure of analysis including the standard solution were analyzed to ensure the suitability of the lyophilization of the samples was carried out: an scheme equipment. Retention times with less than 1.0% variations, illustrating the preparation process of the samples can be found capacity numbers greater than 2, and symmetry of peaks in the Supporting Information (Scheme S2). For the between 0.7 and 1.2, theoretical plate numbers (N) above lyophilization of samples, 1 mL of the liposomal solution was 2000 for any peak, and USP tailing factor smaller than 1.5 were pipetted on each vial and frozen afterward at −80 °C on an checked out. ultralow temperature freezer for 12 h. Once samples were Selectivity is the ability to assess unequivocally the analyte in completely frozen, they were freeze-dried for 4 days. the presence of other components, which may be expected to Lyophilization conditions of pressure and temperature were be present. For this study, both sample and standard solutions 1 mbar and −50 °C, respectively. were compared, thus confirming the complete separation of all Once samples were completely dried, 2 mL of CHCl3 was compounds with a purity peak test being carried out for each added to properly solubilize the very apolar active substances: active substance. Linearity was tested by preparing seven PS, vitamin C, and CoQ10. After 5 min of slight shaking and different concentrations per triplicate between 70 and 30% of only when the sample was completely dissolved, 10 mL of the standard solution at the usual working concentration, methanol was added, and the final solution was transferred into studying if the response is directly proportional within this 25 mL volumetric flasks. The vials were afterward cleaned with range. Response factor (RF) was the analyzed parameter on methanol, which was also transferred to the flasks, making up linearity, being the result of dividing peak areas by the to 25 mL with more methanol. Lastly, all solutions were corresponding concentration. Precision is the parameter that filtered with 0.45 μm PVDF filters, and HPLC vials were expresses the degree of scatter between a series of measures. In prepared. The final concentration of each active compound at this case, the precision of the method and system was the moment of injection to the HPLC system was 776 μg evaluated with the injection of nine standard solutions at mL−1 PS, 240 μg mL−1 CoQ10, and 200 μg mL−1 vitamin C. different concentrations and with different analysts and To validate the preparation procedure developed, 20 different days of work, studying its repeatability. Robustness lyophilized vials were injected, establishing an RSD limit of is a measure of the capacity to remain unaffected by small but 2.7%. deliberated variations in method parameters such as the Standard Solution Preparation. For the preparation of a standard solution, 19.4 mg of PS, 6 mg of CoQ10, and 5 mg of temperature of the column (± 3 °C), the analyzed wavelength vitamin C were accurately weighed and dissolved in the same (± 3 nm), the injection volume (± 5 μL), or modifications in way than in the sample preparation procedure, so the final peak the flow level of the pump (± 0.05 mL/min). This study is areas would be also the same both in standard solutions and necessary to understand the importance of controlling a sample solutions. determinate parameter. Stability of the active substances in the Chromatographic Conditions. For HPLC analyses, the HPLC vials once prepared is also vital to understand for how employed column was a C18 Zorbax Eclipse Plus 2.1 × 150 long the solution is stable before injecting. Lastly, the accuracy mm (5 μm), with a constant flow of 0.25 mL min−1 and a of the method was assessed to verify if any of the lipids of the controlled temperature of 25 °C. The injection volume was 10 liposome affect the analyses of the active substances. To this μL, and the detection wavelengths were λ = 275 nm for end, three standard solutions and the other lipids were vitamin C and CoQ10 andλ = 200 nm for PS since no other prepared at 130, 100, and 70% of the usual working absorbance was found after checking with the diode array concentration, and real concentrations were compared with accessory. After 19 min, all the compounds were already the theoretical ones. According to the American Association of eluted, but the stop time was fixed at 28 min to return to the Official Analytical Chemists (AOAC20), the limits for the RSD initial conditions and stabilize the equipment. The detailed (coefficient of variation) are established in consonance with proportions of the two employed solvents during the time are the concentration of the analyte in the sample, so it established shown in Table 8. a limit for the active substances of 2.7% RSD. 19714 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715 ACS Omega ■ Article ASSOCIATED CONTENT (10) Kaul, S.; Gulati, N.; Verma, D.; Mukherjee, S.; Nagaich, U. Role of Nanotechnology in Cosmeceuticals: A Review of Recent Advances. * S Supporting Information J. Pharm 2018, 1−19. The Supporting Information is available free of charge on the (11) Torchilin, V. P. Recent advances with liposomes as ACS Publications website at DOI: 10.1021/acsome- pharmaceutical carriers. Nat. Rev. Drug Discovery 2005, 4, 145−160. ga.9b02456. (12) Pattni, B. S.; Chupin, V. V.; Torchilin, V. P. New Developments in Liposomal Drug Delivery. Chem. Rev. 2015, 115, 10938−10966. Instrumentation details, statistical parameters explained (13) Results of coenzyme Q10 plus ubiquinone, vitamin c and in the text, graphical chromatograms from validation, phosphatidylserine, respectively; https://apps-webofknowledge-com. and graphical examples of real analyses (PDF). sire.ub.edu/UA_GeneralSearch_input.do?product=UA&search_ ■ mode=GeneralSearch&SID= F211r8c4dns9yyfCoSK&preferencesSaved (accessed Feb 12, 2019) AUTHOR INFORMATION (14) Temova-Rakuša, Ž .; Srečnik, E.; Roškar, R. Novel HPLC-UV Corresponding Author Method for Simultaneous Determination of Fat-soluble Vitamins and Coenzyme Q10 in Medicines and Supplements. Acta Chim. Slov. *E-mail :perezlo@ub.edu. 2017, 64, 523−529. ORCID (15) Drug Bank bioinformatics and cheminformatics data base; Pilar Pérez-Lozano: 0000-0001-6899-066X https://www.drugbank.ca/drugs/. (accessed March 26, 2019) Daniel Maspoch: 0000-0003-1325-9161 (16) PubChem chemistry data base; https://pubchem.ncbi.nlm.nih. gov/. (accessed March 26, 2019) Author Contributions (17) ChemAxon Ltd. Calculators and Predictors; https://chemaxon. The manuscript was written through contributions of all com/. (accessed July 8, 2019) authors. All authors have given approval to the final version of (18) Szoka, F., Jr.; Papahadjopoulos, D. Comparative Properties and the manuscript. Methods of Preparation of Lipid Vesicles (Liposomes). Annu. Rev. Biophys. Bioeng. 1980, 9, 467−508. Funding (19) International conference on harmonisation of technical require- This work was supported by Industrial Doctorates Grant ments for registration of pharmaceuticals for human use. ICH harmonised [grant number DI55, 2016], as well as the support to ICN2 by tripartite guideline. VALIDATION OF ANALYTICAL PROCE- the Severo Ochoa program of the Spanish Ministry of DURES: TEXT AND METHODOLOGY Q2 (R1). 2005. https:// Economy, Industry and Competitiveness (MINECO, grant ich.org/ (accessed March 26, 2019) no. SEV-2017-0706), and the funding by the CERCA (20) AOAC Guidelines for Single Laboratory Validation of Chemical program/Generalitat de Catalunya. Methods for Dietary Supplements and Botanicals. (n.d.). www.AOAC. org. (accessed July 8, 2019) Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The authors want to render thanks to the Analytical Department of Chemistry at the University of Barcelona to its advice during the development of the analytical method. ■ REFERENCES (1) Ganesan, A.; Coote, M. L.; Barakat, K. Molecular dynamics- driven drug discovery: leaping forward with confidence. Drug Discovery Today 2017, 22, 249−269. (2) Chen, H.; Engkvist, O.; Wang, Y.; Olivecrona, M.; Blaschke, T. The rise of deep learning in drug discovery. Drug Discovery Today 2018, 23, 1241−1250. (3) De Vivo, M.; Masetti, M.; Bottegoni, G.; Cavalli, A. Role of Molecular Dynamics and Related Methods in Drug Discovery. J. Med. Chem. 2016, 59, 4035−4061. (4) Taylor, A. 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Technol. 2018, 79, 106−115. 19715 DOI: 10.1021/acsomega.9b02456 ACS Omega 2019, 4, 19710−19715