Development of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase

Development of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase Jisun Lee, Suraj Adhikari, Wonjae Lee, Hye-Ran Yoon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1957831/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chiral discrimination of thyroxine (T 4 ) enantiomers was performed using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) on a chiral crown ether-derived ChiroSil SCA(-) column. The different composition of mobile phases and the effect of column oven temperatures were investigated and the optimum chromatographic separation with respect to resolution and analysis time was achieved using a mixture of 60% methanol/water (v/v) with 0.1% formic acid at 40°C having a flow rate of 1.4 mL min − 1 . The thermodynamic data from van’t Hoff plots of temperature experiments revealed that the enantioseparation was enthalpically favored process. The method was validated in the concentration range of 0.5–100 µg mL − 1 for both enantiomers and proved to be rapid, precise, sensitive, and selective method for the enantiodiscrimination of T 4 under the optimized conditions. The calibration curves of both D- and L-T 4 showed an excellent linearity with coefficient of determination (R 2 ) > 0.9997. The developed chiral method was successfully applied for a quantitative assay to check the enantiomeric purities of the six pharmaceutical formulations of levothyroxine sodium tablets and the enantiomeric impurities identified were in the range of 0.11–0.29%. This method could be applied for the determination of enantiomeric purity on pharmaceuticals and also for the monitoring of thyroid hormone levels. Chiral stationary phase Crown ether chiral column Enantiomer resolution Thyroxine UHPLC-MS/MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Current trends in chiral analysis are focused on developing the improved enantioselective analytical methods that have faster and higher enantiomer resolution power with good efficiency for optical purity testing in commercially available pharmaceuticals [ 1 – 3 ]. Thyroxine (3,3’,5,5’-tetraiodothyronine; T 4 ), one of the major thyroid hormones (THs) that are responsible for the regulation of normal development, growth and metabolism in humans and animals, are secreted by the thyroid gland into the circulatory system [ 4 – 9 ]. Thyroxine affects most of the organs in body’s system including liver, heart, bone, kidney, intestine and central nervous system, which means proper thyroxine levels are vital for key functioning of these organ and normal health [ 7 ]. However, thyroxine is a chiral amino acid type molecule and exists as enantiomers (D-T 4 and L-T 4 ), which upon the interaction with the enantioselective environment of our living system can produce different biological, pharmacological and therapeutic effects [ 5 , 8 – 10 ]. L-T 4 , the naturally occurring thyroid hormone, as active pharmaceutical ingredient in pharmaceutical formulations, is a drug of choice for treating hypothyroidism, simple non-endemic goiters, and chronic lymphocytic thyroiditis worldwide and taken by millions of people [ 9 – 11 ]. On the other hand, D-T 4 has no marked influence on the metabolic rate enhancement but it alters the level of lipogenic enzymes in the liver and reduces the serum cholesterol significantly by increasing the rate of degradation and oxidation of cholesterol [ 5 , 6 , 9 ]. The use of D-T 4 was discontinued as sudden tragic deaths were believed to occur in hyperlipidemic patients treated with D-T 4 [ 5 , 12 ]. It is therefore important to determine D- and L-T 4 in commercially available drugs to ensure the proper dose present in the medicament for efficient therapeutic effects. Only a few chiral stationary phases (CSPs) derived from ovomucoid protein, antibiotic avoparcin, quinine, teicoplanin and chiral crown ether were employed for the chiral discrimination and resolution of T 4 enantiomers in liquid chromatography [ 5 , 6 , 13 – 16 ]. In crown ether type CSPs, the complexes formed between protonated amino acids under acidic conditions and chiral crown ethers has been proposed to be essential for the chiral recognition and discrimination [ 17 , 18 ]. There has been no exploration of crown ether type CSP ChiroSil SCA(-) derived from (-)-18-crown-6-tetracarboxylic acid for the direct enantiodiscrimination of T 4 using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Therefore, in this present research, we have developed a validated UPLC-MS/MS enantioselective analytical method that is rapid, sensitive and specific for the detection, separation and determination of the enantiomers of T 4 on a chiral crown ether-type ChiroSil SCA(-) column. Experimental Chemicals and Reagents Racemic T 4 , D- and L-T 4 , were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-MS grade, methanol, trifluoroacetic acid (TFA) and formic acid (FA) for the mobile phase optimization were purchased from Daejung Co. (Siheung, South Korea). Deionized purified water was generated using Milli-Q water system from Merck Millipore (Burlington, MA, USA). Six commercial formulations of levothyroxine sodium tablets were procured from Bukwang Pharm, Samnam, Dalim Biotech (Seoul, South Korea) and Abbott (Abbott Park, IL, USA). Human plasma was obtained from Hanyang University Hospital (Seoul, Korea). Liquid Chromatography and Tandem Mass Spectrometry The UHPLC-MS/MS system comprised of a highly sensitive Shimadzu UHPLC Nexera X2 LC-30AD system hyphenated to a triple quadrupole mass spectrometer LC-MS 8040 (Shimadzu Corporation, Kyoto, Japan). The LC system was composed on one Nexera X2 LC-30AD quaternary pump, a column oven (CTO-20AC) and a model DGU-20A5R degasser. The MS system consisted of an electrospray ionization interface and a triple quadrupole (QqQ) mass analyzer. The samples were kept in the autosampler (Nexera X2 SIL-30 AC) at 4 °C. Stereoselective separation of T 4 enantiomers was performed using a ChiroSil SCA(-) (250 × 4.6 mm, I.D., 5 µm) chiral crown ether column (RS Technologies, Daejon, South Korea). Optimized mobile phase condition of 60% methanol/water (v/v) with 0.1% formic acid was used at a flow rate of 1.4 mL min -1 for effective enantioseparation and enantiomeric purity checking. The column temperature was set at 40 °C and overall run time was 10 min. The injection volume was set at 1μL and delivered to the MS. Ultrapure nitrogen (99.95 %) was used for nebulizing and drying gas and generated from nitrogen generator. Multiple reaction monitoring (MRM) was performed for detection in the MS/MS system, with m/z transitions of 777.60>731.70 for DL-T 4 and 150.90>135.10 for L-phenylglycine (internal standard, IS) in positive ion electrospray mode. Data was collected and analyzed by Lab Solution (Shimadzu, Japan). Sample Preparation For UHPLC sample preparation, each commercially available levothyroxine sodium tablets was pulverized and the powder was suspended in 2 mL methanol containing 10 mM H 2 SO 4 . Ultrasonication was performed at room temperature for 10 min and 1 mL of mixed solution was moved to microtube and centrifuged at 13,500 rpm for 5 min. Afterwards, the solutions were filtered through a 0.2 µm membrane filter (Advantec, Japan) prior to injection and injected three times to ensure reproducibility and precision of the results. Standard Solutions for Calibration and Quality Control Samples The working standards were prepared by further serial dilution of the stock solution with methanol to produce different concentrations for constructing the calibration curve. The seven concentrations of the final working standards used were 0.5, 1, 5, 10, 40, 80, 100 μg mL -1 . For QC sample, 100 μL of the plasma specimen was withdrawn into an Eppendorf tube and 50 μL of L-phenylglycine (4 μg mL -1 ) added as the IS. Each concentration level was analyzed in triplicate. Calibration curve equations and the corresponding coefficients of determination were obtained for each enantiomer in the plasma. Three quality control (QC) solutions (10, 40, and 80 µg mL -1 ) were prepared for precision and accuracy testing and were stored at -20 °C until the analyses . Method Validation The proposed enantioselective analytical method was validated as per the U.S. Food and Drug Administration (FDA) bioanalytical method validation guidelines [19]. Parameters such as limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision were established to evaluate this UPLC-MS/MS method. Results And Discussion Method Development and Optimization of Mobile Phase Conditions The UPLC and mass parameters should be carefully optimized to achieve good chromatographic behavior and appropriate ionization. In our study, baseline separation and determination of target T 4 enantiomers was obtained using UPLC-MS/MS method on chiral crown ether-derived ChiroSil SCA(-) column. Each respective peak observed was distinct, and no other peaks significantly interfering with the analytes was found. Due to the presence of carboxylic acid and amino group in the characteristic chemical structure, the T 4 analyte can be measured both in positive and negative ionization mode (ESI) [20]. However, better sensitivity and lower background for the T 4 analyte was found in ESI positive than in ESI negative ion acquisition mode. Therefore, ESI positive mode was employed to obtain good peak shape, enhanced resolution and greater MS signal intensity of the T 4 analyte. MS/MS fragmentation parameters were optimized by the direct infusion of T 4 analyte and adjusting the parameters for precursor ion until two stable product ions were obtained [21, 22]. The selection of optimum mobile phase system was equally critical parameter for the good enantioseparation and resolution [23]. Mobile phase and acid additives compositions were tested to find the optimal condition for appropriate separation and checking of the optical purity. Table 1 shows the resolution (R s ), separation (α) and retention factors that were determined for the mobile phases which contain the methanol/water compositions ranging from 60% to 100% with trifluoroacetic acid (TFA) and formic acid (FA) as acid additives at a flow rate of 1.4 mL min -1 . As the methanol content of mobile phase increases, the enantioselectivities of the analyte increases but the resolution decreases. It is worth noted from Table 1 that the resolution and retention of the analytes were related to the acidic character of the additives. TFA (pK a = 0.52) of the stronger acidic character gives rise to the shorter retention of the T 4 enantiomers than those of FA (pK a = 3.75) in the same methanol content of mobile phase. Therefore, in the various concentrations of TFA (0.01, 0.05, 0.1%) and FA content (0.1, 0.2, 0.4%) with the same methanol content in mobile phase, the increase of the concentration of acid additive in the mobile phase correspond to the shorter retention times of enantiomers. The typical four UPLC-MS/MS MRM chromatograms on different methanol and additives compositions for optimization of mobile phase are shown in Fig. 1. Although TFA showed shortest retention of T 4 enantiomers, the worst resolution and MS signal intensity [Fig. 1(D)] of T 4 enantiomers compared to those of FA [Fig. 1 (A, B and C)] was observed. Overall, the lowest concentration of FA in the mobile phase showed the highest MS intensity with improved the peak shape [Fig. 1(A)] and thus, the best results in this study were achieved using 60% methanol/water (v/v) with 0.1% FA. Effect of Column Temperature on Enantiomeric Separation and Investigation of Thermodynamic Parameters Enantioseparation process is significantly influenced by the temperature and it is very important to investigate the effect of column temperature as variations of this parameter can produce changes in retention, selectivity and also the resolution (R s ) [24]. In this study, the separation of T 4 enantiomers was studied with stepwise increase of column temperature ranging from 30~50 °C with 5 °C increments. Table 2 lists the column temperature effects on the chromatographic parameters during the discrimination of T 4 enantiomers. It was observed that t 0 (dead time), k, α and R s of both enantiomers decreased linearly as the temperature of column increased from 30 °C to 50 °C. The lowering temperature results in better selectivity and resolution but longer retention time, and wider peaks which are typical for an enthalpy driven separation [25]. The optimum temperature for the highest MS signal intensity with better peak shape for T 4 enantiomers was observed at 40 °C. The thermodynamic effects that act on retention and separation of T 4 enantiomers can be explained by the van’t Hoff equation [25, 26]. A van’t Hoff plot is constituted by either the logarithm of the retention (k) or separation (α) factor for two enantiomers versus the inverse of absolute temperature (K). The relationship between k and α with T is expressed by the following van’t Hoff equation [Eq.(1) and (2)] as: ln k = - ΔH°/RT + ΔS°/R + ln φ (1) ln α = - Δ(ΔH°)/RT + Δ(ΔS°)/R (2) where, ΔH o and ΔS o are the enthalpy change and entropy change, respectively, when the analyte transfers from the mobile phase to the stationary phase; Δ(ΔH o ) and Δ(ΔS o ) represent differential enthalpy and entropy, respectively; R is the universal gas constant (8.314 J mol -1 K -1 ); T is the absolute temperature; φ is the phase ratio of the column (the volume of the stationary phase divided by the volume of the mobile phase) [26]. The calculated thermodynamic parameters and van’t Hoff plots of ln k and ln α versus 1/T are presented in Table 3 and Fig. 2. If the enthalpy, entropy and φ changes are constant over experimental temperature changes, van’t Hoff plot (ln k vs. 1/T) should be linear. The slope and intercept are −ΔH o /R and ΔS o /R+lnφ, respectively. Linear plots of ln (k' 1 and k' 2 ) vs. 1/T were obtained with coefficient of determination (R 2 ) values of 0.995 and 0.997 and negative ΔH° and ΔS°, as shown in Fig. 2 (A). For ln α vs 1/T, the plot that characterize the separation was linear (Fig. 2 B) having negative Δ(ΔH°) and Δ(ΔS°) with R 2 value of 0.998. It is noted that T 4 analyte/CSP interactions process are similar for two enantiomers irrespective of temperature and the difference in the interaction shown by Δ(ΔH o ) (Table 3) is sufficient for the discrimination of the enantiomers. The negative values of Δ(ΔH°) and Δ(ΔS°) obtained indicated that the interaction process between each enantiomer and the stationary phase was an enthalpy controlled process. It was observed that the value of T iso (Table 3) obtained was higher than the experimental temperature range which reveal that the enantioselectivity and resolution increased when the temperature is decreased as previously clarified. The high values of Δ(ΔH°) (-14.03 kJ/mol) in our study hinted that the chiral discrimination occurs due to the efficient and strong hydrogen bonding interactions in transient diastereomeric complexes [27]. Also, the good negative value of Δ(ΔG°) (-1.77 kJ mol -1 , Table 3) in our study predicted the efficient binding between analyte and CSP [25]. Validation To evaluate the proposed chiral UHPLC-MS/MS assay in terms of linearity, precision, and accuracy, several concentrations of D- and L-T 4 from the stock solution spiked in human plasma were prepared. Linearity and Sensitivity Calibration curves were constructed by plotting the ratio of peak areas against the injected concentration of the T 4 enantiomers. The data obtained appear to have excellent linearity and reproducibility in the positive mode over the concentration range of 0.5–100 μg mL -1 for both D-T 4 and L-T 4 , with regression equations of y = 0.1587x – 0.1237 and y = 0.0784x – 0.0646, respectively. The high R 2 value of both enantiomers (>0.9997) indicated that there was good linearity and shown in Fig. 3. For sensitivity, LOD, the lowest concentration at which a method can discriminate enantiomers with a signal-noise ratio (S/N) of 3, was found to be 0.01 and 0.04 μg mL -1 for D- and L-T 4 , respectively. The corresponding LOQs at S/N = 10 were found to be 0.04 and 0.1 μg mL -1 , respectively. Accuracy and Precision Accuracy and precision were performed on the LQC, MQC and HQC (n=5) on four separate days. Accuracy was assessed by comparing the mean of detected analyte concentration with the theoretical concentration of QC samples and expressed as percentage. Intra-day precision was calculated from the ratio of the relative standard deviation (%RSD) to the appropriate mean value expressed in percent for five replicates (n=5). Inter-day precision, defined as %RSD of four different day validation analyses. The accuracy and precision in the intra-day assay was 98.69-100.33% (RSD: 2.9-6.7%) and 98.31-99.36% (RSD: 2.8-6.5%) for D-T 4 and L-T 4 , respectively, and the corresponding values in the inter-day assay were 98.69-100.46% (RSD: 4.3-6.1%) and 94.05-98.31% (RSD: 5.9-8.2%), as shown in Table 4. The accuracy and precision results presented in Table 4 confirmed the reproducibility of the proposed method and were in the acceptance range specified by the FDA bio-analytical method validation guidelines. Analysis of Pharmaceutical Formulations as Application of the Method This developed chiral UHPLC-MS/MS method was applied for the determination of the enantiomeric purities as a test for the applicability of the proposed method. The enantiomeric impurities as D-T 4 of the analyzed six commercialized pharmaceutical formulations of levothyroxine sodium tablets were 0.11-0.29%. Table 5 shows the average of the three measurements of the enantiomeric purities of L-T 4 of six levothyroxine sodium tablets. The representative chromatograms for the determination of the enantiomeric purity of L-T 4 of levothyroxine sodium (formulation A and B) are shown in Fig. 4. Conclusion In this study, UHPLC-ESI-MS/MS was employed to develop an enantioselective analytical method for the determination of T 4 enantiomers. This developed validated method using positive mode tandem mass detection, offered a rapid and efficient enantiomer separation and determination of T 4 enantiomers. It was also demonstrated that the ChiroSil SCA(-) chiral crown ether column is excellent for selective and sensitive analysis of T 4 enantiomers. Critical validation parameters, including linearity, sensitivity, accuracy and precision, were all within the acceptable limits. 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J Chromatogr A 1003:101–111 Wani DV, Rane VP, Mokale SN (2018) Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose-based chiral stationary phase. Chirality 30:284–292 Declarations Conflict of Interest The authors declare that there is no conflict of interest to report regarding the publication of this research article. Ethical Approval This article does not contain any studies related to human or animals. Informed Consent Informed consent was obtained from all individual participants included in the study. Author Contributions Experiment, data collection and analysis were performed by J. Lee. Interpretation of the data and writing of the original manuscript was done by S. Adhikari. Conceptualization, design, editing, reagents and supervision were contributed by W. Lee. Analysis tools, financial support, investigation and supervision were provided by H.-R.Yoon. Tables Table 1 Effect of mobile phase for the optimum condition of T 4 enantioseparation Mobile phase R s α D-T 4 L-T 4 k' 1 R t k' 2 R t 60% methanol /water (v/v) with 0.1% FA 3.60 1.98 1.81 6.148 3.60 10.091 70% methanol /water (v/v) with 0.1% FA 3.48 2.19 1.41 5.285 3.09 8.958 80% methanol /water (v/v) with 0.1% FA 3.20 2.34 1.49 5.46 3.49 9.825 90% methanol /water (v/v) with 0.1% FA 2.74 2.35 1.86 6.273 4.37 11.759 100% methanol with 0.1% FA 1.82 2.47 1.82 6.168 4.49 12.022 60% methanol /water (v/v) with 0.2% FA 3.73 1.99 1.64 5.787 3.26 9.324 60% methanol /water (v/v) with 0.4% FA 2.85 1.97 0.91 4.192 1.79 6.106 60% methanol/water (v/v) with 0.01% TFA 2.20 2.05 1.32 5.08 2.71 8.118 60% methanol/water (v/v) with 0.05% TFA 1.43 2.02 0.43 3.142 0.87 4.101 60% methanol/water (v/v) with 0.1% TFA 1.38 1.91 0.37 3.011 0.71 3.742 R s : Resolution factor, α: Separation factor, k ' : Retention factor, R t : Retention time , FA: Formic acid, TFA: Trifluoroacetic acid Table 2 Effect of column temperatures on chromatographic parameters Temperature (°C) t 0 α D-T 4 L-T 4 k' 1 k' 2 30 2.28 2.34 2.62 6.14 35 2.24 2.17 2.18 4.72 40 2.19 1.98 1.81 3.60 45 2.16 1.82 1.57 2.86 50 2.14 1.66 1.25 2.08 t 0 : Dead time, α: Separation factor, k ' : Retention factor, See the experimental section for chromatographic conditions Table 3 Thermodynamic parameters for T 4 enantiomers separated on ChiroSil SCA(-) CSP Enantiomers R 2 ΔH o (kJ mol -1 ) ΔS o (J mol -1 K -1 ) R 2 Δ(ΔH o ) (kJ mol -1 ) Δ ( Δ S o ) (J mol -1 K -1 ) Δ(ΔG°) 313K (kJ mol -1 ) T (iso) (°C) D-T 4 0.995 -23.80 -69.99 0.998 -14.03 -39.16 -1.77 85.27 L-T 4 0.997 -37.59 -108.39 Table 4 Intra-day and inter-day assay for accuracy and precision of the developed enantioselective method Intra-day (n=5) Concentration added ( μ g mL -1 ) Concentration detected (µg mL -1 ) Accuracy (%) Precision (RSD, %) D-T 4 10 9.87 98.69 6.4 40 40.10 100.24 2.9 80 80.26 100.33 6.7 L-T 4 10 9.83 98.31 6.5 40 39.74 99.36 3.5 80 79.39 99.24 2.8 Inter-day (n=5) Concentration added ( μ g mL -1 ) Concentration detected (µg mL -1 ) Accuracy (%) precision (RSD, %) D-T 4 10 9.87 98.69 6.1 40 36.12 100.11 4.3 80 80.37 100.46 5.4 L-T 4 10 9.83 98.31 8.2 40 37.62 94.05 5.9 80 76.36 95.45 7.5 See the experimental section for chromatographic conditions Table 5 Enantiomeric purities of six different commercial pharmaceutical formulations preparations available as levothyroxine sodium Formulations D:L ratio a RSD (%) A 0.11:99.89 0.021 B 0.14:99.86 0.036 C 0.15:99.85 0.017 D 0.16:99.84 0.040 E 0.29:99.71 0.077 F 0.15:99.85 0.017 a Average value of three replicates, RSD: Relative standard deviation, See the experimental section for chromatographic conditions Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1957831","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":129436511,"identity":"12312c2b-cf7d-475d-83eb-b0562979a889","order_by":0,"name":"Jisun Lee","email":"","orcid":"","institution":"Duksung Women’s University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jisun","middleName":"","lastName":"Lee","suffix":""},{"id":129436512,"identity":"8e0971ca-2905-4b63-b7ad-a087bf3a5240","order_by":1,"name":"Suraj Adhikari","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Suraj","middleName":"","lastName":"Adhikari","suffix":""},{"id":129436513,"identity":"338cdda4-beab-4aeb-aec8-c9504fc363fa","order_by":2,"name":"Wonjae Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYDACdiD+wHAAzjcgrIWZgYFxBslamHlI0mJwmPngY5uaO3LyM3IPMPyoYTA2byCohS3ZOOfYM2ODG3kJjD3HGMxkDhDQYnaYx0w6t+Fw4gaJHAMG3gYGGwlCDjM7zP/9tyVQy/wZOQaMf4nTwsPGzAjU0nAjx4AZaIsZQS32h9mMJXuOHTY2OPPG4LDMMQljglok25sffvhRc1hOvj3H8OGbGhvDGYS0oIADDAwE7RgFo2AUjIJRQAwAAKJnOzRg/uU+AAAAAElFTkSuQmCC","orcid":"","institution":"Chosun University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wonjae","middleName":"","lastName":"Lee","suffix":""},{"id":129436514,"identity":"ea7c7cf4-a62c-4c31-a642-a125692a37be","order_by":3,"name":"Hye-Ran Yoon","email":"","orcid":"","institution":"Duksung Women’s University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hye-Ran","middleName":"","lastName":"Yoon","suffix":""}],"badges":[],"createdAt":"2022-08-13 02:29:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1957831/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1957831/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25425063,"identity":"4b3f9873-cc43-4d8a-8e53-f9a3a95ba50c","added_by":"auto","created_at":"2022-08-19 16:50:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29786,"visible":true,"origin":"","legend":"\u003cp\u003eLC-MS/MS MRM chromatograms of D- and L-T\u003csub\u003e4\u003c/sub\u003e on different methanol and acid additives compositions (\u003cstrong\u003eA\u003c/strong\u003e) 60% methanol/water (v/v) with 0.1% FA, (\u003cstrong\u003eB\u003c/strong\u003e) 70% methanol/water (v/v) with 0.1% FA, (\u003cstrong\u003eC\u003c/strong\u003e) 60% methanol/water (v/v) with 0.4% FA, and (\u003cstrong\u003eD\u003c/strong\u003e) 60% methanol/water (v/v) with 0.1% TFA, IS: internal standard, L-phenylglycine\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1957831/v1/62a26f26efe28b8e81baea68.png"},{"id":25425524,"identity":"656496a5-243e-4dcf-962c-81a0f302d514","added_by":"auto","created_at":"2022-08-19 16:55:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12826,"visible":true,"origin":"","legend":"\u003cp\u003eThe van’t Hoff plot showing the correlation of ln k (\u003cstrong\u003eA\u003c/strong\u003e) and ln α (\u003cstrong\u003eB\u003c/strong\u003e) vs 1/T for T\u003csub\u003e4\u003c/sub\u003e enantiomers\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1957831/v1/2beed1ccdddb46f733af94a5.png"},{"id":25425064,"identity":"ca36c3b3-02f6-425e-8232-3f5e2e6ffffa","added_by":"auto","created_at":"2022-08-19 16:50:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15837,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves for D- and L-T\u003csub\u003e4\u003c/sub\u003e analyzed by UHPLC-MS/MS\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1957831/v1/7f5d6373132e78d149c2d051.png"},{"id":25425066,"identity":"5dbb7a9d-cb46-448a-990b-516341e130d3","added_by":"auto","created_at":"2022-08-19 16:50:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22061,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms showing enantiomeric separation of racemic T\u003csub\u003e4\u003c/sub\u003e (\u003cstrong\u003e1\u003c/strong\u003e) and L-T\u003csub\u003e4\u003c/sub\u003e of commercially available levothyroxine sodium formulations [A (D:L = 0.11:99.89) and B (D:L = 0.14:99.86)]. IS: Internal standard, L-phenylglycine, See the experimental section for chromatographic conditions\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-1957831/v1/7790fa170d8ac3451101d4ea.png"},{"id":25605363,"identity":"da2131e0-28cf-441e-9c96-653dcdb8d8d5","added_by":"auto","created_at":"2022-08-24 14:29:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":451456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1957831/v1/f30d0fb3-735c-49ab-b20f-ec9d5fb183f7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCurrent trends in chiral analysis are focused on developing the improved enantioselective analytical methods that have faster and higher enantiomer resolution power with good efficiency for optical purity testing in commercially available pharmaceuticals [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thyroxine (3,3\u0026rsquo;,5,5\u0026rsquo;-tetraiodothyronine; T\u003csub\u003e4\u003c/sub\u003e), one of the major thyroid hormones (THs) that are responsible for the regulation of normal development, growth and metabolism in humans and animals, are secreted by the thyroid gland into the circulatory system [\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thyroxine affects most of the organs in body\u0026rsquo;s system including liver, heart, bone, kidney, intestine and central nervous system, which means proper thyroxine levels are vital for key functioning of these organ and normal health [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, thyroxine is a chiral amino acid type molecule and exists as enantiomers (D-T\u003csub\u003e4\u003c/sub\u003e and L-T\u003csub\u003e4\u003c/sub\u003e), which upon the interaction with the enantioselective environment of our living system can produce different biological, pharmacological and therapeutic effects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. L-T\u003csub\u003e4\u003c/sub\u003e, the naturally occurring thyroid hormone, as active pharmaceutical ingredient in pharmaceutical formulations, is a drug of choice for treating hypothyroidism, simple non-endemic goiters, and chronic lymphocytic thyroiditis worldwide and taken by millions of people [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. On the other hand, D-T\u003csub\u003e4\u003c/sub\u003e has no marked influence on the metabolic rate enhancement but it alters the level of lipogenic enzymes in the liver and reduces the serum cholesterol significantly by increasing the rate of degradation and oxidation of cholesterol [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The use of D-T\u003csub\u003e4\u003c/sub\u003e was discontinued as sudden tragic deaths were believed to occur in hyperlipidemic patients treated with D-T\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is therefore important to determine D- and L-T\u003csub\u003e4\u003c/sub\u003e in commercially available drugs to ensure the proper dose present in the medicament for efficient therapeutic effects. Only a few chiral stationary phases (CSPs) derived from ovomucoid protein, antibiotic avoparcin, quinine, teicoplanin and chiral crown ether were employed for the chiral discrimination and resolution of T\u003csub\u003e4\u003c/sub\u003e enantiomers in liquid chromatography [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In crown ether type CSPs, the complexes formed between protonated amino acids under acidic conditions and chiral crown ethers has been proposed to be essential for the chiral recognition and discrimination [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. There has been no exploration of crown ether type CSP ChiroSil SCA(-) derived from (-)-18-crown-6-tetracarboxylic acid for the direct enantiodiscrimination of T\u003csub\u003e4\u003c/sub\u003e using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Therefore, in this present research, we have developed a validated UPLC-MS/MS enantioselective analytical method that is rapid, sensitive and specific for the detection, separation and determination of the enantiomers of T\u003csub\u003e4\u003c/sub\u003e on a chiral crown ether-type ChiroSil SCA(-) column.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003eChemicals and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRacemic T\u003csub\u003e4\u003c/sub\u003e, D- and L-T\u003csub\u003e4\u003c/sub\u003e, were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-MS grade, methanol, trifluoroacetic acid (TFA) and formic acid (FA) for the mobile phase optimization were purchased from Daejung Co. (Siheung, South Korea). Deionized purified water was generated using Milli-Q water system from Merck Millipore (Burlington, MA, USA). Six commercial formulations of levothyroxine sodium tablets were procured from Bukwang Pharm, Samnam, Dalim Biotech (Seoul, South Korea) and Abbott (Abbott Park, IL, USA). Human plasma was obtained from Hanyang University Hospital (Seoul, Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid Chromatography and Tandem Mass Spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UHPLC-MS/MS system comprised of a highly sensitive Shimadzu UHPLC Nexera X2 LC-30AD system hyphenated to a triple quadrupole mass spectrometer LC-MS 8040 (Shimadzu Corporation, Kyoto, Japan). The LC system was composed on one Nexera X2 LC-30AD quaternary pump, a column oven (CTO-20AC)\u0026nbsp;and a model DGU-20A5R degasser.\u0026nbsp;The MS system consisted of an electrospray ionization interface and a triple quadrupole (QqQ) mass analyzer. The samples were kept in the autosampler (Nexera X2 SIL-30 AC) at 4\u0026nbsp;\u0026deg;C. Stereoselective separation of T\u003csub\u003e4\u003c/sub\u003e enantiomers\u0026nbsp;was performed using a ChiroSil SCA(-) (250 \u0026times; 4.6 mm, I.D., 5\u0026nbsp;\u0026micro;m) chiral crown ether column (RS Technologies, Daejon, South Korea). Optimized mobile phase condition of 60% methanol/water (v/v) with 0.1% formic acid was used at a flow rate of 1.4 mL min\u003csup\u003e-1\u003c/sup\u003e for effective enantioseparation and enantiomeric purity checking. The column temperature was set at 40\u0026nbsp;\u0026deg;C and overall run time was 10 min. The injection volume was set at 1\u0026mu;L and delivered to the MS. Ultrapure nitrogen (99.95 %) was used for nebulizing and drying gas and generated from nitrogen generator. Multiple reaction monitoring (MRM) was performed for detection in the MS/MS system, with m/z transitions of 777.60\u0026gt;731.70 for DL-T\u003csub\u003e4\u003c/sub\u003e and 150.90\u0026gt;135.10 for L-phenylglycine (internal standard, IS) in positive\u0026nbsp;ion electrospray mode.\u0026nbsp;Data was collected and analyzed by Lab Solution (Shimadzu, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor UHPLC sample preparation, each commercially available levothyroxine sodium tablets was pulverized and the powder was suspended in 2 mL methanol containing 10 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Ultrasonication was performed at room temperature for 10 min and 1 mL of mixed solution was moved to microtube and centrifuged at 13,500 rpm for 5 min. Afterwards, the solutions were filtered through a 0.2 \u0026micro;m membrane filter (Advantec, Japan) prior to injection and injected three times to ensure reproducibility and precision of the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandard Solutions for Calibration and Quality Control Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe working standards were prepared by further serial dilution of the stock solution with methanol to produce different concentrations for constructing the calibration curve. The seven concentrations of the final working standards used were 0.5, 1, 5, 10, 40, 80, 100 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e. For QC sample, 100 \u0026mu;L of the plasma specimen was withdrawn into an Eppendorf tube and 50 \u0026mu;L of L-phenylglycine (4 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e) added as the IS. Each concentration level was analyzed in triplicate. Calibration curve equations and the corresponding coefficients of determination were obtained for each enantiomer in the plasma. Three quality control (QC) solutions (10, 40, and 80 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) were prepared for precision and accuracy testing and were stored at -20\u0026nbsp;\u0026deg;C until the analyses\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Validation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proposed enantioselective analytical method was validated as per the U.S. Food and Drug Administration (FDA) bioanalytical method validation guidelines [19]. Parameters such as limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision were established to evaluate this UPLC-MS/MS method.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eMethod Development and Optimization of Mobile Phase Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UPLC and mass parameters should be carefully optimized to achieve good chromatographic behavior and appropriate ionization. In our study, baseline separation and determination of target T\u003csub\u003e4\u003c/sub\u003e enantiomers was obtained using UPLC-MS/MS method on chiral crown ether-derived ChiroSil SCA(-) column.\u0026nbsp;Each respective peak observed was distinct, and no other peaks significantly interfering with the analytes was found.\u0026nbsp;Due to the presence of carboxylic acid and amino group in the characteristic chemical structure, the T\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eanalyte can be measured both in positive and negative ionization mode (ESI) [20]. However,\u0026nbsp;better\u0026nbsp;sensitivity\u0026nbsp;and lower background\u0026nbsp;for the T\u003csub\u003e4\u003c/sub\u003e analyte was found in ESI positive than in ESI negative ion acquisition mode. Therefore, ESI positive mode was employed to obtain good peak shape, enhanced resolution and greater MS signal intensity of the T\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eanalyte. MS/MS fragmentation parameters were optimized by the direct infusion of T\u003csub\u003e4\u003c/sub\u003e analyte and adjusting the parameters for precursor ion until two stable product ions were obtained [21, 22].\u0026nbsp;The selection of optimum mobile phase system was equally critical parameter\u0026nbsp;for the\u0026nbsp;good enantioseparation and resolution [23].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMobile phase and acid additives compositions were tested to find the optimal condition for appropriate separation and checking of the optical purity. Table 1 shows the resolution (R\u003csub\u003es\u003c/sub\u003e), separation (\u0026alpha;) and retention factors that were determined for the mobile phases which contain the methanol/water compositions ranging from 60% to 100% with\u0026nbsp;trifluoroacetic acid (TFA)\u0026nbsp;and formic acid (FA) as acid additives at a flow rate of 1.4 mL\u0026nbsp;min\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;As the methanol content of mobile phase increases, the enantioselectivities of the analyte increases but the resolution decreases. It is worth noted from Table 1 that the resolution and retention of the analytes were related to the acidic character of the additives. TFA (pK\u003csub\u003ea\u003c/sub\u003e = 0.52) of the stronger acidic character gives rise to the shorter retention of the T\u003csub\u003e4\u003c/sub\u003e enantiomers\u0026nbsp;than those of FA (pK\u003csub\u003ea\u003c/sub\u003e = 3.75)\u0026nbsp;in the same\u0026nbsp;methanol content of mobile phase. Therefore, in the various concentrations of TFA (0.01, 0.05, 0.1%) and FA content (0.1, 0.2, 0.4%) with the same methanol content in mobile phase, the increase of the concentration of acid additive in the mobile phase correspond to the shorter retention times of enantiomers. The typical four UPLC-MS/MS MRM chromatograms on different methanol and additives compositions for optimization of mobile phase are shown in Fig. 1. Although TFA showed shortest retention of T\u003csub\u003e4\u003c/sub\u003e enantiomers, the worst\u0026nbsp;resolution and MS signal intensity [Fig. 1(D)] of T\u003csub\u003e4\u003c/sub\u003e enantiomers\u0026nbsp;compared to those of\u0026nbsp;FA [Fig. 1 (A, B and C)]\u0026nbsp;was observed. Overall,\u0026nbsp;the lowest concentration of\u0026nbsp;FA\u0026nbsp;in the mobile phase showed the\u0026nbsp;highest MS intensity with improved the peak shape [Fig. 1(A)] and thus, the best results in this study were achieved using 60% methanol/water (v/v) with 0.1% FA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Column Temperature on Enantiomeric Separation and Investigation of Thermodynamic Parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnantioseparation process is significantly influenced by the temperature and it is very important to investigate the effect of column temperature as variations of this parameter can produce changes in retention, selectivity and also the resolution (R\u003csub\u003es\u003c/sub\u003e) [24]. In this study, the separation of T\u003csub\u003e4\u003c/sub\u003e enantiomers was studied with stepwise increase of column temperature ranging from 30~50\u0026nbsp;\u0026deg;C\u0026nbsp;with 5\u0026nbsp;\u0026deg;C\u0026nbsp;increments. Table 2 lists the column temperature effects on the chromatographic parameters during the discrimination of T\u003csub\u003e4\u003c/sub\u003e enantiomers. It was observed that t\u003csub\u003e0\u0026nbsp;\u003c/sub\u003e(dead time), k, \u0026alpha; and R\u003csub\u003es\u003c/sub\u003e of both enantiomers decreased linearly as the temperature of column increased from 30 \u0026deg;C to 50 \u0026deg;C. The lowering temperature results in better selectivity and resolution but longer retention time, and wider peaks which are typical for an enthalpy driven separation [25].\u0026nbsp;The optimum temperature for the\u0026nbsp;highest MS signal intensity with better peak shape for T\u003csub\u003e4\u003c/sub\u003e enantiomers was observed at\u0026nbsp;40\u0026nbsp;\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe thermodynamic effects that act on retention and separation of T\u003csub\u003e4\u003c/sub\u003e enantiomers\u003csub\u003e\u0026nbsp;\u003c/sub\u003ecan be explained by the van\u0026rsquo;t Hoff equation [25, 26]. A van\u0026rsquo;t Hoff plot is constituted by either the logarithm of the retention (k) or separation (\u0026alpha;) factor for two enantiomers versus the inverse of absolute temperature (K).\u0026nbsp;The relationship between k and\u0026nbsp;\u0026alpha;\u0026nbsp;with T is expressed by the following van\u0026rsquo;t Hoff equation [Eq.(1) and (2)] as:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eln k = - \u0026Delta;H\u0026deg;/RT + \u0026Delta;S\u0026deg;/R + ln \u0026phi; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(1)\u003c/p\u003e\n\u003cp\u003eln \u0026alpha; = - \u0026Delta;(\u0026Delta;H\u0026deg;)/RT + \u0026Delta;(\u0026Delta;S\u0026deg;)/R \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(2)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewhere, \u0026Delta;H\u003csup\u003eo\u003c/sup\u003e and \u0026Delta;S\u003csup\u003eo\u003c/sup\u003e are the enthalpy change and entropy change, respectively, when the analyte transfers from the mobile phase to the stationary phase;\u0026nbsp;\u0026Delta;(\u0026Delta;H\u003csup\u003eo\u003c/sup\u003e)\u0026nbsp;and\u0026nbsp;\u0026Delta;(\u0026Delta;S\u003csup\u003eo\u003c/sup\u003e)\u0026nbsp;represent differential enthalpy and entropy, respectively; R is the universal gas constant (8.314 J mol\u003csup\u003e-1\u003c/sup\u003e K\u003csup\u003e-1\u003c/sup\u003e); T is the absolute temperature; \u0026phi; is the phase ratio of the column (the volume of the stationary phase divided by the volume of the mobile phase) [26]. The calculated thermodynamic parameters and\u0026nbsp;van\u0026rsquo;t Hoff plots of ln k and ln \u0026alpha; versus 1/T are presented in Table 3 and Fig. 2. If the enthalpy, entropy and\u0026nbsp;\u0026phi;\u0026nbsp;changes are constant over experimental temperature changes, van\u0026rsquo;t Hoff plot (ln k vs. 1/T) should be linear. The slope and intercept are\u0026nbsp;\u0026minus;\u0026Delta;H\u003csup\u003eo\u003c/sup\u003e/R and\u0026nbsp;\u0026Delta;S\u003csup\u003eo\u003c/sup\u003e/R+ln\u0026phi;, respectively.\u0026nbsp;Linear plots of ln (k\u0026apos;\u003csub\u003e1\u003c/sub\u003e and\u0026nbsp;k\u0026apos;\u003csub\u003e2\u003c/sub\u003e)\u0026nbsp;vs. 1/T were obtained with\u0026nbsp;coefficient of determination\u0026nbsp;(R\u003csup\u003e2\u003c/sup\u003e) values of 0.995 and 0.997 and negative \u0026Delta;H\u0026deg; and \u0026Delta;S\u0026deg;, as shown in Fig. 2 (A). For ln\u0026nbsp;\u0026alpha;\u0026nbsp;vs 1/T, the plot that\u0026nbsp;characterize the separation was linear (Fig. 2 B)\u0026nbsp;having negative \u0026Delta;(\u0026Delta;H\u0026deg;) and \u0026Delta;(\u0026Delta;S\u0026deg;) with R\u003csup\u003e2\u003c/sup\u003e value of 0.998. It is noted that T\u003csub\u003e4\u003c/sub\u003e analyte/CSP interactions process are similar for two enantiomers irrespective of temperature and the difference in the interaction shown by \u0026Delta;(\u0026Delta;H\u003csup\u003eo\u003c/sup\u003e) (Table 3) is sufficient for the discrimination of the enantiomers. The negative values of\u0026nbsp;\u0026Delta;(\u0026Delta;H\u0026deg;) and \u0026Delta;(\u0026Delta;S\u0026deg;)\u0026nbsp;obtained indicated that the interaction process between each enantiomer and the stationary phase was an enthalpy controlled process. It was observed that the value of T\u003csub\u003eiso\u003c/sub\u003e (Table 3) obtained was higher than the experimental temperature range which reveal that the enantioselectivity and resolution increased when the temperature is decreased as previously clarified. The high values of \u0026Delta;(\u0026Delta;H\u0026deg;) (-14.03 kJ/mol) in our study hinted that the chiral discrimination occurs due to the\u0026nbsp;efficient and strong hydrogen bonding interactions in transient diastereomeric complexes [27]. Also, the good negative value of \u0026Delta;(\u0026Delta;G\u0026deg;) (-1.77 kJ mol\u003csup\u003e-1\u003c/sup\u003e, Table 3) in our study predicted the efficient binding between analyte and CSP [25].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the proposed chiral UHPLC-MS/MS assay in terms of linearity, precision, and accuracy, several concentrations of D- and L-T\u003csub\u003e4\u003c/sub\u003e from the stock solution spiked in human plasma were prepared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity and\u0026nbsp;Sensitivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCalibration curves were constructed by plotting the ratio of peak areas against the injected concentration of the T\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eenantiomers. The data obtained appear to have excellent linearity and reproducibility in the positive mode over the concentration range of 0.5\u0026ndash;100 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e for both D-T\u003csub\u003e4\u003c/sub\u003e and L-T\u003csub\u003e4\u003c/sub\u003e, with regression equations of y = 0.1587x \u0026ndash; 0.1237 and y = 0.0784x \u0026ndash; 0.0646, respectively. The high R\u003csup\u003e2\u003c/sup\u003e value of both enantiomers (\u0026gt;0.9997) indicated that there was good linearity and shown in Fig. 3. For sensitivity, LOD, the lowest concentration at which a method can discriminate enantiomers with a signal-noise ratio (S/N) of 3, was found to be 0.01 and 0.04 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e for D- and L-T\u003csub\u003e4\u003c/sub\u003e, respectively. The corresponding LOQs at S/N = 10 were found to be 0.04 and 0.1 \u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy and Precision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccuracy and precision were performed on the LQC, MQC and HQC (n=5) on four separate days. Accuracy was assessed by comparing the mean of detected analyte concentration with the theoretical concentration of QC samples and expressed as percentage. Intra-day precision was calculated from the ratio of the relative standard deviation (%RSD) to the appropriate mean value expressed in percent for five replicates (n=5). Inter-day precision, defined as %RSD of four different day validation analyses. The accuracy and precision in the intra-day assay was 98.69-100.33% (RSD: 2.9-6.7%) and 98.31-99.36% (RSD: 2.8-6.5%) for D-T\u003csub\u003e4\u003c/sub\u003e and L-T\u003csub\u003e4\u003c/sub\u003e, respectively, and the corresponding values in the inter-day assay were 98.69-100.46% (RSD: 4.3-6.1%) and 94.05-98.31%\u0026nbsp;(RSD: 5.9-8.2%), as shown in Table 4. The accuracy and precision results presented in Table 4 confirmed the reproducibility of the proposed method and were in the acceptance range specified by the FDA bio-analytical method validation guidelines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Pharmaceutical Formulations as Application of the Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis developed chiral UHPLC-MS/MS method was applied for the determination of the enantiomeric purities as a test for the applicability of the proposed method. The enantiomeric impurities as D-T\u003csub\u003e4\u003c/sub\u003e of the analyzed six commercialized pharmaceutical formulations of levothyroxine sodium tablets\u0026nbsp;were 0.11-0.29%.\u0026nbsp;Table 5 shows the average of the three measurements of the enantiomeric purities of L-T\u003csub\u003e4\u003c/sub\u003e of six levothyroxine sodium tablets. The representative chromatograms for the determination of the enantiomeric purity of L-T\u003csub\u003e4\u003c/sub\u003e of levothyroxine sodium (formulation A and B) are shown in Fig. 4.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, UHPLC-ESI-MS/MS was employed to develop an enantioselective analytical method for the determination of T\u003csub\u003e4\u003c/sub\u003e enantiomers. This developed validated method using positive mode tandem mass detection, offered a rapid and efficient enantiomer separation and determination of T\u003csub\u003e4\u003c/sub\u003e enantiomers. It was also demonstrated that the ChiroSil SCA(-) chiral crown ether column is excellent for selective and sensitive analysis of T\u003csub\u003e4\u003c/sub\u003e enantiomers. Critical validation parameters, including linearity, sensitivity, accuracy and precision, were all within the acceptable limits. This method can be applied to the determination of enantiomeric purity of commercially available levothyroxine sodium tablets, and is also expected to be useful in the determination of free thyroxine in plasma and clinical tests for accurate diagnosis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Shun Y, Hao Z, Hang S (2010) Chiral separation of pharmaceuticals by high performance liquid chromatography. Curr Pharm Anal 6:114\u0026ndash;130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNie Y, Liu X, Yang X, Zhao Z (2013) Recent application of chiral liquid chromatography-tandem mass spectrometric methods for enantiomeric pharmaceutical and biomedical determinations. J Chrom Sci 51:753\u0026ndash;763\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdhikari S, Paik MJ, Lee W (2018) Liquid chromatographic enantiomeric separation of chiral aliphatic amines using 2-hydroxynaphthaldehyde as a derivatizing agent on polysaccharide-derived chiral stationary phases. Chromatographia 81:1337\u0026ndash;1344\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYen PM (2001) Physiological and molecular basis of thyroid hormone action. Physiol Rev 81:1097\u0026ndash;1142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGika H, L\u0026auml;mmerhofer M, Papadoyannis I, Lindner W (2004) Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 800:193\u0026ndash;201\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeon SH, Kim M, Han HK, Lee W (2010) Direct enantiomer separation of thyroxine in pharmaceuticals using crown ether type chiral stationary phase. Arch Pharm Res 33:1419\u0026ndash;1423\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJansen HI, Bruinstroop E, Heijboer AC, Boelen A (2022) Biomarkers indicating tissue thyroid hormone status: ready to be implemented yet? J Endocrinol 253:21\u0026ndash;45\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Jia ZP, Hu XL, Xu LT, Li YM, Chen LR (2003) Determination of serum thyroxine enantiomers in patients by liquid chromatography with a chiral mobile phase. J Chromatogr B 785:353\u0026ndash;359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin D, Kumar AP, Song GC, Lee YI (2008) Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography-mass spectrometry with precolumn derivatization. Microchem J 88:62\u0026ndash;66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts CG, Ladenson PW (2004) Hypothyroidism. Lancet 363:793\u0026ndash;803\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFallahi P, Ferrari SM, Ruffilli I, Ragusa F, Biricotti M, Materazzi G, Miccoli P, Antonelli A (2017) Advancements in the treatment of hypothyroidism with L-T\u003csub\u003e4\u003c/sub\u003e liquid formulation or soft gel capsule: an update. Expert Opin Drug Deliv 14:647\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorman CA, Jiang NS, Ellefson RD, Elveback LR (1979) Comparative effectiveness of dextrothyroxine and levothyroxine in correcting hypothyroidism and lowering blood lipid levels in hypothyroid patients. J Clin Endocrinol Metab 49:1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbou-Basha, LI and Aboul-Enein, HY (1995) Enantiomeric separation and optical purity determination of thyroxine enantiomers in bulk and pharmaceutical formulations. Pharm Acta Helv 70:237\u0026ndash;240\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkborg-Ott KH, Kullman JP, Wang X, Gahm K, He L, Armstrong DW (1998) Evaluation of the macrocyclic antibiotic avoparcin as a new chiral selector for HPLC. Chirality 10:627\u0026ndash;660\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGondova\u0026acute; T, Petrovaj J, Sucha\u0026acute; M, Armstrong DW (2011) Stereoselective HPLC determination of thyroxine enantiomers in pharmaceuticals. J Liq Chromatogr Relat Technol 34:2304\u0026ndash;2314\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAboul-Enein HY, Ali I, Hyun MH, Cho YJ, Jin JS (2002) Effect of acidity on the enantiomeric resolution of thyroxine and tocainide by HPLC on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid column. J Biochem Biophys Methods 54:407\u0026ndash;413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyun MH (2015) Development of HPLC chiral stationary phases based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid and their applications. Chirality 27:576\u0026ndash;588\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdhikari S, Lee W (2018) Chiral separation using chiral crown ethers as chiral selectors in chirotechnology. J Pharm Inv 48:225\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBioanalytical method validation: guidance for industry, FDA-2013-D-1020-0039 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Stapleton HM (2010) Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 397:1831\u0026ndash;1839\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanoue R, Kume I, Yamamoto Y, Takaguchi K, Nomiyama K, Tanabe S, Kunisue T (2018) Determination of free thyroid hormones in animal serum/plasma using ultrafiltration in combination with ultra-fast liquid chromatography-tandem mass spectrometry. J Chromatogr A 1539:30\u0026ndash;40\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowerbank SL, Carlin MG, Dean JR (2019) A direct comparison of liquid chromatography-mass spectrometry with clinical routine testing immunoassay methods for the detection and quantification of thyroid hormones in blood serum. Anal Bioanal Chem 411:2839\u0026ndash;2853\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDossou KSS, Farcas E, Servais AC, Chiap P, Chankvetadze B, Crommen J, Fillet M (2012) Optimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases. J Chromatogr A 1234:56\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanella C, Ferretti R, Casulli A, Cirilli R (2019) Temperature and eluent composition effects on enantiomer separation of carvedilol by high-performance liquid chromatography on immobilized amylose-based chiral stationary phases. J Pharm Anal 9:324\u0026ndash;331\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsnin LD, Stepanova MV (2018) Van't Hoff analysis in chiral chromatography. J Sep Sci 41:1319\u0026ndash;1337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChester TL, Coym JW (2003) Effect of phase ratio on van\u0026rsquo;t Hoff analysis in reversed phase liquid chromatography, and phase-ratio-independent estimation of transfer enthalpy. J Chromatogr A 1003:101\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWani DV, Rane VP, Mokale SN (2018) Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose-based chiral stationary phase. Chirality 30:284\u0026ndash;292\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest to report regarding the publication of this research article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies related to human or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment, data collection and analysis were performed by J. Lee. Interpretation of the data and writing of the original manuscript was done by S. Adhikari. Conceptualization, design, editing, reagents and supervision were contributed by W. Lee. Analysis tools, financial support, investigation and supervision were provided by H.-R.Yoon.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eTable 1\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eEffect of mobile phase for the optimum condition of T\u003csub\u003e4\u003c/sub\u003e enantioseparation\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\n \u003ctable style=\"border: none;width:476.95pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:204.85pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eMobile phase\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:45.2pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csub\u003es\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:45.2pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026alpha;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:90.85pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:90.85pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.85pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eL-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:45.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:9.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ek\u0026apos;\u003csub\u003e1\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:9.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csub\u003et\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:9.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ek\u0026apos;\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:9.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csub\u003et\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.1% FA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.60\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.81\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.148\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.60\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10.091\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e70% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.1% FA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.48\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.41\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.285\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8.958\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.1% FA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.34\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9.825\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e90% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.1% FA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.74\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.86\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.273\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e11.759\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e100% methanol\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ewith\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.1% FA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.82\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.47\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.82\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.168\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e12.022\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.2% FA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.73\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.99\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.787\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9.324\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e/water (v/v) with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.4% FA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.85\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.97\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.192\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.79\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.106\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol/water (v/v) with 0.01% TFA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.32\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.08\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.71\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8.118\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;padding:.75pt .75pt 0in .75pt;height: 17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol/water (v/v) with 0.05% TFA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.142\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.87\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.101\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:204.85pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60% methanol/water (v/v) with 0.1% TFA\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.38\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.2pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.011\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.71\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.6pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.95pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.742\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp 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4.95pt;height:16.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:200%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003e2.14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:58.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:16.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:200%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003e1.66\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:58.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:16.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:200%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003e1.25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:58.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:16.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:200%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003e2.08\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u003cbr\u003e\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003et\u003csub\u003e0\u003c/sub\u003e: Dead time, \u0026alpha;: Separation factor, k\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026apos;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e: Retention factor, See the experimental section for chromatographic conditions\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eTable 3\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;Thermodynamic parameters for T\u003csub\u003e4\u003c/sub\u003e enantiomers separated on\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eChiroSil SCA(-) CSP\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\n \u003ctable style=\"border: none;width:527.55pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:71.65pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eEnantiomers\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:40.1pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:67.8pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026Delta;H\u003csup\u003eo\u003c/sup\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(kJ mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:59.25pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026Delta;S\u003csup\u003eo\u003c/sup\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(J mol\u003csup\u003e-1\u003c/sup\u003e K\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:45.2pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:55.65pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026Delta;(\u0026Delta;H\u003csup\u003eo\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(kJ mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:62.65pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026Delta;\u003c/span\u003e\u003cspan style='font-family:\"Times New 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Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026Delta;(\u0026Delta;G\u0026deg;)\u003csub\u003e313K\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(kJ mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:55.65pt;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eT\u003csub\u003e(iso)\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(\u0026deg;C)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:26.65pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"height:26.65pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:71.65pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.1pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.995\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.8pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-23.80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:59.25pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-69.99\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:45.2pt;border:none;border-bottom: solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.998\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:55.65pt;border:none;border-bottom: solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-14.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:62.65pt;border:none;border-bottom: solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-39.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:69.6pt;border:none;border-bottom: solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-1.77\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:55.65pt;border:none;border-bottom: solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e85.27\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:19.1pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:71.65pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eL-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.997\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-37.59\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:59.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:19.1pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-108.39\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:19.1pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:150%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable 4\u003c/span\u003e\u003c/strong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;Intra-day and inter-day assay for accuracy and precision of the developed enantioselective method\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:408.5pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:67.25pt;border:none;border-top:double black 2.25pt;padding:.75pt .75pt 0in .75pt;height:18.15pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:341.25pt;border-top:double black 2.25pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:18.15pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eIntra-day (n=5)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:67.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.1pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eConcentration\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eadded (\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026mu;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eg mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eConcentration\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003edetected (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAccuracy (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePrecision\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(RSD, %)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width:67.25pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.1pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9.87\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e98.69\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.5pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40.10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e100.24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80.26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e100.33\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width:67.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e39.74\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e99.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e79.39\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e99.24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:67.25pt;border:none;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:341.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eInter-day (n=5)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:67.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.1pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eConcentration\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eadded (\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026mu;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eg mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eConcentration\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003edetected (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAccuracy (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border-top:solid black 1.0pt;border-left: none;border-bottom:solid black 1.0pt;border-right:none;padding:.75pt .75pt 0in .75pt;height:27.25pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eprecision\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e(RSD, %)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width:67.25pt;border:none;padding:.75pt .75pt 0in .75pt;height:16.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e98.69\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border:none;padding:.75pt .75pt 0in .75pt;height:16.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e36.12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e100.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e100.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width:67.25pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eL-T\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9.83\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e98.31\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e37.62\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e94.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.1pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.95pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e76.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e95.45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:90.05pt;border:none;border-bottom:solid black 1.0pt;padding:.75pt .75pt 0in .75pt;height:17.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e7.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:150%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eSee the experimental section for chromatographic conditions\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:150%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable 5\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eEnantiomeric purities of six different commercial pharmaceutical formulations preparations available as levothyroxine sodium\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:left;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\n \u003ctable style=\"border: none;width:5.25in;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:1.75in;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFormulations\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:1.75in;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD:L ratio\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:1.75in;border-top:double windowtext 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 4.95pt 0in 4.95pt;height:26.65pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eRSD (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:26.65pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"height:26.65pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:1.75in;border:none;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;border:none;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.11:99.89\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;border:none;padding:0in 4.95pt 0in 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.14:99.86\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.036\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:15.6pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:1.75in;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp 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Roman\",serif;'\u003e0.017\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:15.6pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:1.75in;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eD\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.16:99.84\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.15:99.85\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.75in;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 4.95pt 0in 4.95pt;height:15.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.017\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"height:15.6pt;border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u003cbr\u003e\u003c/span\u003e\u003c/strong\u003e\u003csup\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ea\u003c/span\u003e\u003c/sup\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAverage value of three replicates, RSD: Relative standard deviation,\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eSee the experimental section for chromatographic conditions\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chiral stationary phase, Crown ether chiral column, Enantiomer resolution, Thyroxine, UHPLC-MS/MS","lastPublishedDoi":"10.21203/rs.3.rs-1957831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1957831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChiral discrimination of thyroxine (T\u003csub\u003e4\u003c/sub\u003e) enantiomers was performed using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) on a chiral crown ether-derived ChiroSil SCA(-) column. The different composition of mobile phases and the effect of column oven temperatures were investigated and the optimum chromatographic separation with respect to resolution and analysis time was achieved using a mixture of 60% methanol/water (v/v) with 0.1% formic acid at 40\u0026deg;C having a flow rate of 1.4 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The thermodynamic data from van\u0026rsquo;t Hoff plots of temperature experiments revealed that the enantioseparation was enthalpically favored process. The method was validated in the concentration range of 0.5\u0026ndash;100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for both enantiomers and proved to be rapid, precise, sensitive, and selective method for the enantiodiscrimination of T\u003csub\u003e4\u003c/sub\u003e under the optimized conditions. The calibration curves of both D- and L-T\u003csub\u003e4\u003c/sub\u003e showed an excellent linearity with coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;0.9997. The developed chiral method was successfully applied for a quantitative assay to check the enantiomeric purities of the six pharmaceutical formulations of levothyroxine sodium tablets and the enantiomeric impurities identified were in the range of 0.11\u0026ndash;0.29%. This method could be applied for the determination of enantiomeric purity on pharmaceuticals and also for the monitoring of thyroid hormone levels.\u003c/p\u003e","manuscriptTitle":"Development of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-19 16:50:43","doi":"10.21203/rs.3.rs-1957831/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a2650e9-64f7-4ba4-9c98-1435db2d0ba4","owner":[],"postedDate":"August 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-24T14:29:35+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-19 16:50:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1957831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1957831","identity":"rs-1957831","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}